BUTADIENE

DRAFT NCEA-W-0267
DO NOT CITE OR QUOTE January 1998

External Review Draft
Health Risk Assessment

of 1,3-Butadiene
NOTICE
THIS DOCUMENT IS A PRELIMINARY DRAFT. It has not been formally released by the
U.S. Environmental Protection Agency and should not at this stage be construed to represent
Agency policy. It is being circulated for comment on its technical accuracy and policy
implications.
National Center for Environmental Assessment

Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
DISCLAIMER
This document is an external draft for review purposes only and does not constitute U.S.
Environmental Protection Agency policy. Mention of trade names or commercial products does
not constitute endorsement or recommendation for use.
1/28/98 ii DRAFT--DO NOT CITE OR QUOTE

CONTENTS
LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii
LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xii
PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii
AUTHORS, CONTRIBUTORS, AND REVIEWERS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiv
1. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1
1.1. BACKGROUND . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1
1.2. SUMMARY OF PAST CARCINOGEN ASSESSMENTS OF 1,3-BUTADIENE . . 1-1
1.2.1. Summary of EPA’s Carcinogen Assessment (U.S. EPA, 1985) . . . . . . . . . . . . 1-2
1.2.2. Summary of IARC’s Evaluation of 1,3-Butadiene (IARC, 1986, 1992) . . . . . 1-5
1.2.3. Summary of the National Institute for Occupational Safety and Health
(NIOSH) Evaluation of 1,3-Butadiene (NIOSH, 1991a) . . . . . . . . . . . . . . . . . 1-7
1.2.4. California Air Resources Board (CARB, 1991) . . . . . . . . . . . . . . . . . . . . . . . . 1-8
1.2.5. Summary of Findings by U.S. Occupational Safety and Health
Administration (OSHA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-10
1.3. DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-11
2. OVERVIEW OF EXPOSURE TO 1,3-BUTADIENE . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1

2.1. PHYSICAL/CHEMICAL PROPERTIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1
2.2. PRODUCTION AND USE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1
2.2.1. Styrene-Butadiene Latex and Rubber Production . . . . . . . . . . . . . . . . . . . . . . 2-2
2.2.2. Polybutadiene Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3
2.2.3. Neoprene Rubber Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3
2.2.4. Acrylonitrile-Butadiene (ABS) Resin Production . . . . . . . . . . . . . . . . . . . . . . 2-3
2.2.5. Nitrile Elastomer Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3
2.2.6. Adiponitrile Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4
2.3. SOURCES AND EMISSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4
2.3.1. Mobile Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4
2.3.1.1. On-Road Mobile Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-5
2.3.1.2. Nonroad Mobile Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-5
2.3.1.3. Aircraft . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-6
2.3.2. Miscellaneous Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-6
2.3.2.1. Miscellaneous Chemical Production . . . . . . . . . . . . . . . . . . . . . . . . . 2-6
2.3.2.2. Secondary Lead Smelters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-6
2.3.2.3. Petroleum Refining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-7
2.3.3. Combustion Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-7
2.3.3.1. Tire Burning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-7
2.3.3.2. Biomass Burning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-7
2.4. AMBIENT CONCENTRATION OF 1,3-BUTADIENE . . . . . . . . . . . . . . . . . . . . . . 2-8
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CONTENTS (continued)
2.4.1. Air . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-8

2.4.1.1. Ambient Monitoring Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-8
2.4.1.2. Ambient Source Apportionment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-23
2.4.2. Indoor Exposure to 1,3-Butadiene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-23
2.4.3. Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-24
2.4.4. Food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-24
2.5. PATHWAYS OF EXPOSURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-24
3. METABOLISM AND PHARMACOKINETICS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1

3.1. OVERVIEW OF PHARMACOKINETIC STUDIES . . . . . . . . . . . . . . . . . . . . . . . . 3-1
3.1.1. Pathways Elucidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-2
3.1.2. Species Differences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-2
3.1.2.1. In Vitro Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-2
3.1.2.2. In Vivo Pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-18
3.2. MOLECULAR DOSIMETRY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-41
3.3. STRUCTURE-ACTIVITY RELATIONSHIPS . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-42
3.4. DISCUSSION AND CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-45
4. MUTAGENICITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-1
4.1. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-1
4.2. GENE MUTATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-1
4.3. CYTOGENETIC EFFECTSCHUMAN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-4
4.4. CYTOGENETIC EFFECTSCRODENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-5
4.5. SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6
5. REPRODUCTIVE AND DEVELOPMENTAL EFFECTS . . . . . . . . . . . . . . . . . . . . . . . . 5-1

5.1. REPRODUCTIVE EFFECTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-1
5.1.1. Carpenter et al., 1944 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-1
5.1.2. Owen et al., 1987; Owen and Glaister, 1990 . . . . . . . . . . . . . . . . . . . . . . . . . . 5-2
5.1.3. NTP, 1984 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-2
5.1.4. NTP, 1993 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-3
5.1.5. Hackett et al., 1988a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-5
5.1.6. Hackett et al., 1988b . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-7
5.1.7. Anderson et al., 1993 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-8
5.1.8. Adler et al., 1994 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-8
5.2. DEVELOPMENTAL EFFECTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-9
5.2.1. IISRP, 1982 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-9
5.2.2. Hackett et al., 1987a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-11
5.2.3. Hackett et al., 1987b . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-16
5.2.4. Anderson et al., 1993 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-23
5.3. STRUCTURE-ACTIVITY RELATIONSHIPS . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-24
5.3.1. NTP, 1986 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-24
5.3.2. Melnick et al., 1994 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-26
5.3.3. Doerr et al., 1996 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-26
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5.4. SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-27
6. TOXICITY IN ANIMALS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-1

6.1. SUBCHRONIC TOXICITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-1
6.2. CHRONIC TOXICITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-1
6.3. CARCINOGENICITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-8
6.3.1. 2-Year Study (NTP, 1993) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-8
6.3.2. 2-Year Stop-Exposure Study (NTP, 1993) . . . . . . . . . . . . . . . . . . . . . . . . . . 6-19
6.3.3. Summary of NTP (1993) Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-28
6.3.4. 1-Year Study (Irons et al., 1989; Irons, 1990) . . . . . . . . . . . . . . . . . . . . . . . . 6-28
6.4. RELATED COMPOUNDS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-29
6.5. DISCUSSION AND CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-30
7. EPIDEMIOLOGIC STUDIES OF CARCINOGENICITY . . . . . . . . . . . . . . . . . . . . . . . . 7-1

7.1. MONOMER PRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-1
7.1.1. Texaco Cohort . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-1
7.1.1.1. Downs et al., 1987: Mortality Among Workers at a
Butadiene Facility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-1
7.1.1.2. Divine, 1990: An Update on Mortality Among Workers at a
1,3-Butadiene FacilityCPreliminary Results . . . . . . . . . . . . . . . . . . . 7-4
7.1.1.3. Divine et al., 1993: Cancer Mortality Among Workers at a
Butadiene Production Facility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-6
7.1.1.4. Divine and Hartman, 1996: Mortality Update of Butadiene
Production Workers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-7
7.1.2. Shell Oil Refinery Cohort . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-8
7.1.2.1. Cowles et al., 1994: Mortality, Morbidity, and Hematological
Results From a Cohort of Long-Term Workers Involved in
1,3-Butadiene Monomer Production . . . . . . . . . . . . . . . . . . . . . . . . . 7-8
7.1.3. Union Carbide Cohort . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-9
7.1.3.1. Ward et al., 1995: Mortality Study of Workers in 1,3-Butadiene
Production Units Identified From a Chemical Workers Cohort
Ward et al., 1996c: Mortality Study of Workers Employed in
1,3-Butadiene Production Units Identified From a Large
Chemical Workers Cohort . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-9
7.2. POLYMER PRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-10
7.2.1. Cohort Identified by Johns Hopkins University (JHU) Investigators . . . . . . . 7-10
7.2.1.1. Matanoski and Schwartz, 1987: Mortality of Workers in
Styrene-Butadiene Polymer Production . . . . . . . . . . . . . . . . . . . . . . 7-10
7.2.1.2. Matanoski et al., 1989: Epidemiologic Data Related to Health
Effects of 1,3-Butadiene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-14
Matanoski et al., 1990: Mortality of a Cohort of Workers in the
Styrene-Butadiene Polymer Manufacturing Industry (1943-1982) . 7-14
7.2.1.3. Matanoski et al., 1989: Epidemiologic Data Related to
Health Effects of 1,3-Butadiene . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-16
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Santos-Burgoa et al., 1992: Lymphohematopoietic Cancer in

Styrene-Butadiene Polymerization Workers . . . . . . . . . . . . . . . . . . . 7-16
7.2.1.4. Matanoski et al., 1993: Cancer Epidemiology Among
Styrene-Butadiene Rubber Workers . . . . . . . . . . . . . . . . . . . . . . . . . 7-19
7.2.2. Cohort Identified by University of Alabama (UAB) Investigators . . . . . . . . . 7-20
7.2.2.1. Delzell et al., 1996: A Follow-Up Study of Synthetic
Rubber Workers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-20
7.2.2.2. Macaluso et al., 1996: Leukemia and Cumulative Exposure to
Butadiene, Styrene, and Benzene Among Workers in the
Synthetic Rubber Industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-23
7.3. SUMMARY AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-25
7.3.1. Monomer Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-31
7.3.2. Polymer Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-32
7.3.3. Relevant Methodologic Issues and Discussion . . . . . . . . . . . . . . . . . . . . . . . . 7-34
7.3.4. Criteria of Causal Inference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-37
8. PHARMACOKINETIC MODELING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-1

8.1. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-1
8.2. PBPK MODELS FOR 1,3-BUTADIENE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-2
8.2.1. Hattis and Wasson (1987) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-2
8.2.2. Hallenbeck (1992) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-4
8.2.3. Kohn and Melnick (1993) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-4
8.2.4. Johanson and Filser (1993) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-8
8.2.5. Evelo et al. (1993) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-12
8.2.6. Medinsky et al. (1994) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-15
8.3. SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-19
8.4. CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-21
9. QUANTITATIVE RISK ASSESSMENT FOR 1,3-BUTADIENE . . . . . . . . . . . . . . . . . . 9-1

9.1. EPIDEMIOLOGICALLY BASED CANCER RISK ASSESSMENT . . . . . . . . . . . . 9-1
9.1.1. Exposure-Response Modeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-1
9.1.2. Prediction of Lifetime Excess Risk of Leukemia . . . . . . . . . . . . . . . . . . . . . . . 9-3
9.1.3. Sources of Uncertainty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-10
9.1.4. Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-12
9.2. CANCER RISK ESTIMATES BASED ON RODENT BIOASSAYS . . . . . . . . . . . 9-15
9.2.1. Rat-Based Estimates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-15
9.2.2. Mouse-Based Estimates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-16
9.2.2.1. Quantal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-16
9.2.2.2. Time-to-Tumor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-19
9.2.3. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-24
9.3. REPRODUCTIVE AND DEVELOPMENTAL TOXICITY . . . . . . . . . . . . . . . . . . 9-27
9.3.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-27
9.3.2. Fetal Weight Modeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-31
9.3.3. Male-Mediated Developmental Toxicity Modeling . . . . . . . . . . . . . . . . . . . . 9-35
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9.3.4. Ovarian, Uterine, and Testicular Atrophy Modeling . . . . . . . . . . . . . . . . . . . 9-40

9.3.5. Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-45
9.4. REFERENCE CONCENTRATIONS FOR REPRODUCTIVE AND
DEVELOPMENTAL EFFECTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-46
9.4.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-46
9.4.2. Calculation of RfCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-46
9.4.3. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-49
9.4.4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-51
9.5. CONCLUSIONS ON QUANTITATIVE RISK ESTIMATES . . . . . . . . . . . . . . . . . 9-52
10. WEIGHT OF EVIDENCE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10-1

10.1. EVALUATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10-1
10.2. CONCLUSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10-1
11. RISK CHARACTERIZATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11-1

11.1. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11-1
11.2. EXPOSURE OVERVIEW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11-2
11.3. CANCER HAZARD ASSESSMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11-2
11.3.1. Human Evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11-2
11.3.2. Animal Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11-8
11.3.3. Other Supportive Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11-9
11.3.4. Classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11-9
11.4. QUANTITATIVE RISK ESTIMATION FOR CANCER . . . . . . . . . . . . . . . . 11-10
11.5. SUMMARY OF REPRODUCTIVE/DEVELOPMENTAL EFFECTS . . . . . . 11-13
11.6. QUANTITATIVE ESTIMATION (RfC) FOR REPRODUCTIVE/
DEVELOPMENTAL EFFECTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11-14
11.7. SPECIAL SUBPOPULATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11-15
11.7.1. Sensitive Subpopulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11-15
11.7.2. Highly Exposed Subpopulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11-16
11.8. FUTURE RESEARCH NEEDS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11-16
11.9. SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11-17
12. REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12-1
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LIST OF TABLES
1-1 Carcinogenicity assessments of 1,3-butadiene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-12
2-1A Summary of 1,3-butadiene ambient data from the EPA Aerometric Information
Retrieval System (AIRS) for 1988 to 1991 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-10
2-1B Summary of 1,3-butadiene ambient data from the EPA Aerometric Information
Retrieval System (AIRS) for 1992 to 1994 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-12
2-2 Summary of 1,3-butadiene ambient data from the Urban Air Toxics Monitoring
Program (UATMP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-16
2-3 Summary of outdoor urban data from the National Ambient Volatile Organic
Compounds (NAVOC) Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-18
2-4 Summary of air monitoring program results for 1,3-butadiene . . . . . . . . . . . . . . . . . 2-19
2-5 Summary of 1,3-butadiene ambient data from the EPA Aerometric Information
Retrieval System (AIRS) based on sampling locations . . . . . . . . . . . . . . . . . . . . . . . 2-21
2-6 Summary of 1,3-butadiene data from Table 2-2 based on sampling locations . . . . . 2-22
2-7 Summary of 1,3-butadiene data from Table 2-3 based on sampling locations . . . . . 2-22
2-8 Summary of the relative contributions to ambient 1,3-butadiene emissions given
as percent of total mg/yr . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-24
3-1 Metabolic pathways of 1,3-butadiene metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4

3-2 Species comparison of reaction rates for epoxidation, GSH conjugation, and
hydrolysis reactions involved in the metabolism of 1,3-butadiene . . . . . . . . . . . . . . 3-13
3-3 Rate constants for in vivo hepatic clearance of 1,3-butadiene and EB
(extrapolated from in vitro) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-19
3-4 Summary of closed-chamber inhalation studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-21
3-5 Toxicokinetic parameters for uptake and elimination of 1,3-butadiene in
mice and rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-23
3-6 Toxicokinetic parameters for the uptake and elimination of epoxybutene in
rats and mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-23
3-7 Summary of nose-only inhalation studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-24
3-8 Comparison of 1,3-butadiene, epoxybutene, and diepoxybutane blood
concentration data from different species of laboratory animals exposed to
1,3-butadiene by inhalation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-30
3-9 Tissue levels of epoxybutene and diepoxybutane (pmol/g tissue) in male rats
and male mice exposed by inhalation to 62.5 ppm 1,3-butadiene for 4 h . . . . . . . . . 3-32
3-10 Tissue levels of epoxybutene and diepoxybutane (pmol/g tissue) in male
and female rats exposed by inhalation to 62.5 ppm 1,3-butadiene for 6 h . . . . . . . . . 3-33
3-11 Excretion of 14C by monkeys exposed to 1,3-[14C]-butadiene . . . . . . . . . . . . . . . . . . 3-36
5-1 Reproductive tract lesions in female B6C3F1 mice exposed to 1,3-butadiene

by inhalation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-4
5-2 Reproductive tract lesions in male B6C3F1 mice exposed to 1,3-butadiene
by inhalation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-6
5-3 Maternal toxicity in Sprague-Dawley CD rats exposed to 1,3-butadiene
by inhalation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-10
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LIST OF TABLES (continued)
5-4 Developmental toxicity in Sprague-Dawley CD rats exposed to

5-5 Malformations and variations in Sprague-Dawley CD rats exposed to
5-6 Design of the developmental toxicity studies on 1,3-butadiene . . . . . . . . . . . . . . . . 5-14
5-7 Maternal toxicity in Sprague-Dawley CD rats exposed to 1,3-butadiene
by inhalation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-15
5-8 Developmental toxicity in Sprague-Dawley CD rats exposed to
5-9 Malformations and variations in Sprague-Dawley CD rats exposed to
5-10 Maternal toxicity in pregnant CD-1 mice exposed to 1,3-butadiene by inhalation . . 5-19
5-11 Developmental toxicity in CD-1 mice exposed to 1,3-butadiene by inhalation . . . . . 5-20
5-12 Malformations and variations in CD-1 mice exposed to 1,3-butadiene
by inhalation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-21
5-13 Reproductive and developmental toxicity of chemicals structurally
similar to 1,3-butadiene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-25
6-1 Survival of male and female B6C3F1 mice exposed to 1,3-butadiene by

inhalation for 103 weeks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-3
6-2 Incidence of hyperplasia in male and female B6C3F1 mice exposed to
1,3-butadiene by inhalation for 103 weeks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-7
6-3 Incidence of hyperplasia in male B6C3F1 mice exposed to 1,3-butadiene by
inhalation in the stop-exposure study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-8
6-4 Incidence of primary neoplasms in male B6C3F1 mice exposed to
1,3-butadiene by inhalation for 103 weeks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-10
6-5 Incidence of primary neoplasms in female B6C3F1 mice exposed to
1,3-butadiene by inhalation for 103 weeks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-12
1,3-butadiene by inhalation for 9 months and 15 months . . . . . . . . . . . . . . . . . . . . . 6-14
6-7 Incidence of primary neoplasms in female B6C3F1 mice exposed to
1,3-butadiene by inhalation for 9 months and 15 months . . . . . . . . . . . . . . . . . . . . . 6-15
1,3-butadiene by inhalation in the stop-exposure study . . . . . . . . . . . . . . . . . . . . . . 6-21
7-1 Epidemiologic studies of the health effects of exposure to 1,3-butadieneC

monomer production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-11
7-2 Epidemiologic studies of the health effects of exposure to 1,3-butadieneC
polymer production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-26
8-1 Parameter values used in the Hattis and Wasson (1987) PBPK model . . . . . . . . . . . . 8-3
8-2 Parameter values used in the Kohn and Melnick (1993) PBPK model . . . . . . . . . . . . 8-5
8-3 Parameter values used in the Johanson and Filser (1993) PBPK model . . . . . . . . . . 8-10
8-4 Parameter values used in the Evelo et al. (1993) PBPK model . . . . . . . . . . . . . . . . . 8-14
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8-5 Parameter values used in the Medinsky et al. (1993) PBPK model . . . . . . . . . . . . . . 8-17
9-1 Results from exposure-response models of continuous cumulative

exposure to 1,3-butadiene and styrene using alternative structural forms
reported by Delzell et al. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-3
9-2 Results from "final" square root exposure-response model of continuous
cumulative exposure to 1,3-butadiene reported by Delzell et al. . . . . . . . . . . . . . . . . . 9-4
9-3 Maximum likelihood estimates (MLEs) of excess risk with one-sided 95% upper
confidence limits (95% UCL) from several models reported by Delzell et al. (1995)
for continuous lifetime exposures to varying concentrations of 1,3-butadiene . . . . . . 9-9
9-4 MLEs of parts per million continuous exposure concentrations associated with
varying excess risk levels with one-sided 95% lower confidence limits (95% LCL)
based on relative rate results of several models reported by Delzell et al. (1995)
and U.S. population rates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-10
9-5 Maximum likelihood (ECp) and 95% lower-bound (LECp) estimates of
the continuous exposure concentrations associated with varying levels of
excess risk (p) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-11
9-6 Cancer potency (unit risk) estimates based on linear extrapolation from
the LEC01 or EC01 calculated from the models presented by Delzell et al. . . . . . . . . 9-15
9-7 Dose-response data for linearized multistage model . . . . . . . . . . . . . . . . . . . . . . . . . 9-17
9-8 Parameter estimates for multistage Weibull time-to-tumor model based on
female mouse tumor incidence, w/o 625 ppm group . . . . . . . . . . . . . . . . . . . . . . . . 9-21
9-9 Parameter estimates for multistage Weibull time-to-tumor model based on
male mouse tumor incidence, w/o 625 ppm group . . . . . . . . . . . . . . . . . . . . . . . . . . 9-21
9-10 Human unit cancer risk estimate (extra risk, computed for risks of 10-6)
based on female mouse tumor incidences, w/o 625 ppm group using
multistage Weibull time-to-tumor model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-22
9-11 Human unit cancer risk estimates (extra risk, computed for risks of 10-6)
based on male mouse tumor incidences, w/o 625 ppm group using
multistage Weibull time-to-tumor model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-22
9-12 Unit potency estimates (extra risk) summed across tumor sites . . . . . . . . . . . . . . . . 9-25
9-13 Prenatal (developmental) toxicity study (Hackett et al., 1987b) . . . . . . . . . . . . . . . . 9-28
9-14 Male-mediated developmental toxicity (Anderson et al., 1993, 1995) . . . . . . . . . . . 9-28
9-15 NTP chronic study (1993) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-29
9-16 Fetal weight modeling (LOAEL = 40 ppm) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-33
9-17 ECs and LECs for male-mediated developmental toxicity . . . . . . . . . . . . . . . . . . . . 9-36
9-18 ECs and LECs for ovarian, uterine, and testicular atrophy using the quantal
Weibull and log-logistic models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-41
9-19 Parameters for Weibull time-to-response model used to model reproductive
effects observed in mice based on ppm butadiene exposure . . . . . . . . . . . . . . . . . . . 9-44
9-20 Human benchmark 1,3-butadiene exposure concentrations calculated for
reproductive effects observed in mice using a Weibull time-to-response
model (extra risk) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-45
9-21 Points of departure and RfC calculations for reproductive and
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developmental effects of 1,3-butadiene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-48
11-1 Summary of epidemiologic studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11-4

11-2 Epidemiologic causality criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11-8
11-3 Estimates of upper bounds on human extra unit cancer risk (potency)
from continuous lifetime exposure to 1,3-butadiene based on animal
inhalation bioassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11-11
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LIST OF FIGURES
3-1 Some pathways in the metabolism of butadiene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-3

3-2 Two-compartment pharmacokinetic model for inhalation chamber . . . . . . . . . . . . . 3-34
6-1 Kaplan-Meier survival curves for male B6C3F1 mice exposed to

6-2 Kaplan-Meier survival curves for female B6C3F1 mice exposed to
6-3 Kaplan-Meier survival curves for male mice in the stop-exposure
inhalation study of 1,3-butadiene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-20
9-1 Excess risk and 95% upper confidence limit excess risk estimates based
on the multiplicative model reported by Delzell et al., 1995 . . . . . . . . . . . . . . . . . . . 9-6
on the power model reported by Delzell et al., 1995 . . . . . . . . . . . . . . . . . . . . . . . . . 9-6
on the linear excess relative rate model reported by Delzell et al., 1995 . . . . . . . . . . . 9-7
on the final square root model reported by Delzell et al., 1995 . . . . . . . . . . . . . . . . . . 9-7
on the square root model reported by Delzell et al., 1995 . . . . . . . . . . . . . . . . . . . . . . 9-8
9-6 Observed versus predicted dose (exposure) probability P(d) of fetal weight
reduction below the 10th percentile of controls using log-logistic model . . . . . . . . . 9-34
9-7 Observed versus predicted mean fetal weight per litter using continuous model . . . . 9-34
9-8 Observed versus predicted percent of mean fetal weights per litter less
than the 5th percentile of controls (P0 = 0.05) using hybrid model . . . . . . . . . . . . . . 9-35
9-9 Observed versus predicted mean number of implants (prenatal)
using log-linear model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-37
9-10 Observed versus predicted proportion of early and late deaths
per implantation (prenatal) using log-linear model . . . . . . . . . . . . . . . . . . . . . . . . . . 9-37
9-11 Observed versus predicted proportion of live implants (prenatal)
9-12 Observed versus predicted mean number of implants (postnatal)
9-13 Observed versus predicted proportion of post-implantation losses (postnatal)
9-14 Observed versus predicted mean litter size at birth using log-linear model . . . . . . . . 9-39
9-15 Observed versus predicted mean litter size at weaning using log-linear model . . . . . 9-40
9-16 Ovarian atrophy (groups 1-5) using log-logistic model . . . . . . . . . . . . . . . . . . . . . . 9-42
9-17 Uterine atrophy (groups 1-6) using quantal Weibull model . . . . . . . . . . . . . . . . . . . 9-43
9-18 Testicular atrophy (groups 1-6) using quantal Weibull model . . . . . . . . . . . . . . . . . 9-43
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PREFACE
This Health Risk Assessment of 1,3-Butadiene has been prepared to serve as a source
document for Agencywide use. This document was developed primarily for use by the U.S.
Environmental Protection Agency’s (EPA) Office of Mobile Sources (OMS) to support decision
making regarding the Air Toxic Rule’s Section 202L2 of the Clean Air Act Amendment. Since
OMS requested that this assessment focus on mutagenicity, carcinogenicity, and
reproductive/developmental effects, an evaluation of other health hazards has not been included.
This document, therefore, is not a comprehensive health assessment. The exposure information
included here is an overview of the ambient exposures and exposure to populations adjacent to
emission sources, without any actual exposure assessment as such.
In the development of this assessment, relevant scientific literature has been
incorporated from the period July 1, 1985, through January 31, 1997. Key studies have been
evaluated to qualitatively describe the mutagenicity, reproductive/developmental effects, and
carcinogenicity of 1,3-butadiene. The assessment also includes a summary, conclusions, and
risk characterization. Measures of dose-risk relationships relevant to ambient air exposures are
discussed so that the adverse health effects can be placed in perspective with possible exposure
levels.
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AUTHORS, CONTRIBUTORS, AND REVIEWERS
This document was prepared by the National Center for Environmental Assessment-
Washington Office (NCEA-W) of EPA’s Office of Research and Development. Sections of this
report were prepared by Oak Ridge National Laboratory (ORNL) under Interagency Agreement
No. DW89937638-01-0. Aparna M. Koppikar1 served as the Project Manager, providing overall
direction and technical assistance.
AUTHORS
Chapter 1: Aparna M. Koppikar, Kowetha Davidson2
Chapter 2: Chieh Wu1
Chapter 3: Kim Hoang1, Carol Forsyth2, and Robert Young2
Chapter 4: Lawrence Valcovic1
Chapter 5: Kowetha Davidson
Chapter 6: Rosmarie Faust2
Chapter 7: Aparna M. Koppikar
Chapter 8: Jennifer Jinot1, Carol Kimmel1
Chapter 9: Jennifer Jinot
Chapter 10: Aparna M. Koppikar
Chapter 11: Jennifer Jinot and Aparna M. Koppikar
CONTRIBUTORS
Thomas M. Crisp1
Dharm Singh1
Steven Bayard (now at OSHA)
Milton Siegal1
John Schaum1
Leslie Stayner3
Stephen Gilbert3
Randall Smith3
1
National Center for Environmental Assessment-Washington Office.
2
Oak Ridge National Laboratory.
3
National Institute for Occupational Safety and Health.
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AUTHORS, CONTRIBUTORS, AND REVIEWERS (continued)
REVIEWERS
David Bayliss (NCEA-W)
Robert Beliles (NCEA-W)
Pam Brodowicz (OMS)
Robert Bruce (NCEA-Cin)
James Cogliano (NCEA-W)
Richard Cook (OMS)
Michael Dellarco (NCEA-W)
Gary Kimmel (NCEA-W)
William Pepelko (NCEA-W)
The authors would like to acknowledge the contributions of several people who have
made this report possible:
Theresa Konoza of NCEA-W, who was responsible for coordinating and managing the
production effort.
The CDM Group, Inc., under the direction of Kay Marshall, who was responsible for
editing, word processing, and literature searches.
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1. INTRODUCTION
1.1. BACKGROUND
1,3-Butadiene (CH2=CH-CH=CH2, CAS No. 106-99-0) is a colorless gas produced by
three different processes: (1) oxidative dehydrogenation of n-butene (the Oxo-D or O-X-D
process), (2) catalytic dehydrogenation of n-butane and n-butene (the Houdry process), and (3)
recovery from the C4 coproduct (by-product) stream from the steam cracking process used to
manufacture ethylene (the ethylene coproduct process). This noncorrosive gas has a boiling point
of 4.4 C and a vapor pressure of 1,900 mm/Hg at 20 C (Kirshenbaum, 1978). 1,3-Butadiene is
highly volatile and has a low solubility in water; thus environmental release results primarily in
atmospheric contamination. Atmospheric destruction of 1,3-butadiene occurs primarily by
photoinitiated reactions. A significant amount of destruction also occurs by the gas phase
reaction with ozone and reaction with nitrate radicals at nighttime, particularly in urban areas
(U.S. DHHS, 1992). The major photooxidation products of 1,3-butadiene are acrolein and
formaldehyde (Maldotti et al., 1980).
Approximately 12 billion pounds of 1,3-butadiene are produced annually worldwide and 3
billion pounds in the United States (Morrow, 1990; USITC, 1990). It is used as an intermediate
in the production of polymers, elastomers, and other chemicals. The major uses of 1,3-butadiene
are in the manufacture of styrene-butadiene rubber (SBR) (synthetic rubber) and of thermoplastic
resins. Elastomers of butadiene are used in the manufacture of tires, footwear, sponges, hoses
and piping, luggage, packaging, and a variety of other molded products. In addition, 1,3-
butadiene is used as an intermediate to produce a variety of industrial chemicals, including the
fungicides captan and captfol. The primary way the 1,3-butadiene is released in the environment
is via emissions from gasoline- and diesel-powered vehicles and equipment. Minor releases occur
in production processes, tobacco smoke, gasoline vapors, and vapors from the burning of plastics
as well as rubber (Miller, 1978).
1.2. SUMMARY OF PAST CARCINOGEN RISK ASSESSMENTS OF 1,3-BUTADIENE

The purpose of this section is to review past carcinogen risk assessments of 1,3-butadiene.
It should be noted that the Toxicological Profile for 1,3-butadiene (ATSDR, 1992), profile of 1,3-
butadiene to set the threshhold limit value (TLV) (ACGIH, 1994), and 1,3-Butadiene OEL
Criteria Document by the European Center for Ecotoxicology and Toxicology of Chemicals
(1997) are not reviewed here as they are not risk assessments.
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1.2.1. Summary of EPA’s Carcinogen Assessment (U.S. EPA, 1985)
Pertinent studies reported before 1986 were reviewed in Mutagenicity and Carcinogenicity
Assessment of 1,3-Butadiene (U.S. EPA, 1985). This document was peer reviewed by experts in
the field, as well as in public sessions of the Environmental Health Committee of EPA’s Science
Advisory Board. The studies presented in the 1985 document will not be reviewed in the present
document but are briefly summarized below.
EPA reviewed six epidemiological studies, which included four retrospective cohort
mortality studies, one nested case-control study, and an industrial hygiene and hematologic cross-
sectional survey. The first cohort study involved 6,678 hourly workers in a rubber tire
manufacturing plant in Akron, Ohio (McMichael et al., 1974). The standard mortality ratios
(SMRs) were calculated using the 1968 U.S. male population as the reference. Cause-specific
mortality was evaluated for 16 different occupational title groups (work areas) within the plant.
This study was followed up by a nested case-control study involving 455 of the 1,983 deaths
recorded between 1968 and 1973 (McMichael et al., 1976). The second cohort study was
conducted in 8,938 male workers in a rubber plant also located in Akron, Ohio (Andjelkovich et
al., 1976, 1977). The 1976 study used the U.S. male population as the reference for calculating
the SMRs, whereas the entire cohort was used to calculate the SMRs for 28 different work areas
for the 1977 study. The third cohort study included 2,756 workers at two styrene-butadiene
rubber facilities in eastern Texas (Meinhardt et al., 1982). The sex, age, race, and calendar time
cause-specific rates of the U.S. population were used to calculate the SMRs. The last and most
comprehensive study was conducted in 13,920 workers at one Canadian and seven U.S. styrene-
butadiene rubber plants (Matanoski et al., 1982). The SMRs for black and white workers were
calculated separately. The cross-sectional survey was conducted on workers in the same styrene-
butadiene rubber plant studied by McMichael et al. (Checkoway and Williams, 1982). Blood
samples were obtained to evaluate hematology parameters. The survey was not designed to
evaluate mortality experience and did not contribute to cancer risk evaluation of 1,3-butadiene.
Of the five epidemiologic studies that evaluated cause-specific mortality, three cohort
studies demonstrated statistically significant excess mortality due to cancers of the lymphatic and
hematopoietic tissues (Andjelkovich et al., 1976; McMichael et al., 1976; Meinhardt et al., 1982).
The fourth cohort study by Matanoski et al. (1982) also showed increased leukemia, but failed to
achieve statistical significance. Lastly, the nested case-control study by McMichael et al. (1976)
showed statistically significant increased standardized risk ratios for cancers of the lymphatic and
hematopoietic tissues among workers with exposures of 5 years or more in one area of the plant
(synthetic rubber plant area), as compared with either all the other workers or the matched
controls. Statistically significant excess cancer mortality was also observed for gastrointestinal

tract, respiratory tract, central nervous system, prostate, testicles, and urinary bladder in one or
more studies. However, these excesses were not observed consistently across all the studies.
Although excess mortality due to cancers of the lymphatic and hematopoietic tissues was
observed consistently in all the evaluated studies, the methodologic limitations, such as too few
deaths from specific cancers to evaluate the causal association; exclusion of large portions of the
population due to lack of records; lack of adjustment for smoking; confounding by other
exposures such as benzene or styrene; and excess cancer mortality at other sites prompted EPA to
conclude that the evidence was inadequate for determining a causal association between exposure
to 1,3-butadiene and cancer in humans.
Two long-term animal studies presented strong evidence for the induction of cancers at
multiple anatomical sites in both rats (HLE, 1981) and mice (NTP, 1984). Sprague-Dawley rats
were exposed by inhalation to 1,3-butadiene at concentrations of 1,000 or 8,000 ppm 6 h/day, 5
d/week for 111 weeks and 105 weeks for males and females, respectively. Statistically significant
increased incidences in the following neoplasms were observed at one or both concentrations:
mammary gland tumors, thyroid follicular adenomas/carcinomas, and Zymbal gland carcinomas in
female rats and Leydig cell adenomas/carcinomas, pancreatic exocrine adenomas, and Zymbal
gland tumors in male rats. In addition, gliomas occurred in four high-dose male rats.
Nonneoplastic effects due to long-term exposure of rats to 1,3-butadiene included clinical signs of
toxicity, an increase in liver weight in both sexes, marked to severe nephropathy in 27% of the
high-dose male rats compared with 9% or 10% of the controls, and alveolar metaplasia in male
rats.
Among B6C3F1 mice exposed by inhalation to 1,3-butadiene at 625 or 1,250 ppm for 6
h/day, 5 d/week, neoplasms also developed at multiple anatomical sites; this study was terminated
at week 60 to 61 because of high mortality in the treated groups, primarily due to neoplasms.
There was an overall increase in the number of animals with primary neoplasms and animals with
multiple neoplasms. Neoplasms showing statistically significant increased incidences among both
male and female mice were as follows: malignant lymphomas, alveolar/bronchiolar
adenomas/carcinomas, hemangiosarcomas of the heart, and forestomach papillomas/carcinomas.
In addition, mammary gland acinar cell carcinomas, ovarian granulosa cell carcinomas, and
hepatocellular adenomas/carcinomas occurred in female mice. Nonneoplastic effects observed
were testicular atrophy, chronic inflammation, fibrosis, cartilaginous metaplasia, osseous
metaplasia, and atrophy of the sensory epithelium of the nasal cavity in male mice. Ovarian
atrophy was observed in female mice. Some discrepancies were noted for this study, but they
were not considered to pose a significant impact on the overall interpretation of the study.
EPA also reviewed data from metabolism and mutagenicity studies, concluding that
inhaled 1,3-butadiene is metabolized to mutagenic epoxide intermediates.

In addition, EPA reviewed the carcinogenicity of related compounds (4-vinyl-1-
cyclohexene, epoxybutene, dl-1,2:3,4-diepoxybutane, and meso-1,2:3,4-diepoxybutane). 4-Vinyl-
1-cyclohexene is carcinogenic in female mice (oral/gavage), based on increased incidences of
ovarian and adrenal gland neoplasms. Equivocal evidence was noted for malignant lymphomas
and alveolar/bronchiolar adenomas in male mice and clitoral gland neoplasms in female rats (NTP,
1986). Skin painting of mice with meso-1,2:3,4-diepoxybutane induced papillomas and squamous
cell carcinomas (Van Duuren et al., 1963), and subcutaneous injection with dl-1,2:3,4-
diepoxybutane caused fibrosarcomas in mice and rats (Van Duuren et al., 1966).
Based on the studies in mice and rats, EPA concluded that there was sufficient evidence
for carcinogenicity of 1,3-butadiene in animals. EPA also concluded that evidence from
metabolism, mutagenicity, and carcinogenicity studies suggests that 1,3-butadiene presents a
genetic risk to humans.
Two developmental toxicity studies were reviewed. One study (HLE, 1981) was
conducted using pregnant female Sprague-Dawley rats exposed to 200, 1,000, and 8,000 ppm 6
h/day on gestation days 6-15. Developmental effects included slightly decreased fetal weight and
mean crown-rump length and increased skeletal variations and malformations. The other study
(Carpenter et al., 1944) was inadequately reported.
EPA presented the following conclusion regarding the qualitative evaluation of the data
for 1,3-butadiene: “On the basis of sufficient evidence from studies in two species of rodents, and
inadequate epidemiologic data, 1,3-butadiene can be classified as a probable human carcinogen,
Group B2.” Using the classification scheme of the International Agency for Research on Cancer
(IARC), 1,3-butadiene would also be classified as a “probable” human carcinogen, Group 2B.
The linearized multistage model was used to calculate the maximum likelihood estimate
for the incremental risk for 1,3-butadiene based upon the National Toxicology Program mouse
data (NTP, 1984), the HLE (1981) rat data, and internal dosimetry derived from data on mice and
rats exposed to varying concentrations of 1,3-butadiene for 6 h. The upper-limit unit risk of 6.4 ×
10 1(ppm) 1 was a geometric mean of the values calculated for male and female mice
separately. The unit risk extrapolated to humans was 2.5 × 10 2(ppm) 1. This value was used to
predict human responses in the epidemiologic studies, which were then compared with the actual
responses. According to EPA, “. . . The comparisons were hampered by a scarcity of information
in the epidemiologic data concerning actual exposures, age distribution, and work histories. In
addition, because there was no consistent cancer response across all of the studies, the most
predominant response, cancer of the lymphatic and hematopoietic tissues, was chosen as being the
target for 1,3-butadiene. Based on the comparisons between the predicted and observed human
response, the extrapolated value from the mouse data was consistent with human response, but in
view of all the uncertainties and apparent inconsistencies in the epidemiologic data, a fairly wide

range of potency estimates and exposure scenarios would also be satisfactory. . . .” (U.S. EPA,
1985).
1.2.2. Summary of IARC’s Evaluation of 1,3-Butadiene (IARC, 1986, 1992)

IARC reported the first evaluation of 1,3-butadiene as a separate chemical in 1986 (IARC,
1986). In an earlier report (IARC, 1982), 1,3-butadiene was evaluated as a chemical used in the
rubber industry. IARC’s 1986 evaluation of the animal data consisted of the NTP (1984) study
using male and female B6C3F1 mice exposed to 625 or 1,250 ppm 1,3-butadiene for 60 or 61
weeks and an abstract description of the HLE (1981) study in rats exposed to 1,000 or 8,000 ppm
(Owen et al., 1985). The human data consisted only of a cohort study described by Meinhardt et
al. (1982) and a brief mention of the following studies of workers in the rubber industry that were
included in IARC’s evaluation of the rubber industry: Andjelkovich et al., 1976, 1977;
McMichael et al., 1976; and Monson and Nakano, 1976. The supporting evidence considered by
IARC consisted of absorption, distribution, metabolism, and excretion (ADME) data. The
genotoxicity data showed that 1,3-butadiene was mutagenic in S. typhimurium with metabolic
activation, and the metabolites (1,2-epoxybutene and 1,2:3,4-diepoxybutane) were mutagenic in
S. typhimurium without metabolic activation. IARC also evaluated data on acute, reproductive,
and developmental toxicity of 1,3-butadiene. IARC (1986) concluded that the supporting
evidence for genetic activity was “inadequate,” the evidence for carcinogenicity in experimental
animals was “sufficient,” and the evidence for carcinogenicity in humans was “inadequate” (Group
2B).
IARC reevaluated the data on 1,3-butadiene and reported the results in 1992. Additional
animal and human studies were available for evaluation. In addition to the first NTP (1984) study
in mice, IARC (1992) evaluated a more recent NTP study reported by Melnick et al. (1990a). In
this study, male and female B6C3F1 mice exposed by inhalation to 1,3-butadiene at concentrations
of 6.25 to 625 ppm for 2 years developed neoplasms at multiple sites and at all concentrations.
IARC also evaluated the published HLE (1981) long-term study showing tumors developing at
multiple sites in male and female Sprague-Dawley rats exposed to 1,000 or 8,000 ppm 1,3-
butadiene (Owen et al., 1987) and a comparative study in B6C3F1 and NIH Swiss mice examining
the role of endogenous retroviruses on the induction of lymphomas by 1,3-butadiene (Irons et al.,
1987). IARC also presented some evidence showing that the metabolites 1,3-epoxybutene and
1,2:3,4-diepoxybutane possessed carcinogenic activity.
Epidemiologic studies evaluated by IARC (1992) consisted primarily of the studies
published since 1982. The following studies were evaluated: (1) the mortality study conducted
by Meinhardt et al. (1982) of workers in two styrene-butadiene rubber plants, but not the most
recent update of this study by Lemen et al. (1990); (2) the mortality study by Downs et al. (1987)

of workers who manufactured 1,3-butadiene monomer and the most recent update of this study
by Divine (1990); (3) a mortality study by Matanoski et al. (1989) of workers in eight U.S. and
Canadian styrene-butadiene rubber plants (update of the study by Matanoski and Schwartz,
1987); (4) a nested case-control study of the 59 workers from the eight U.S. and Canadian plants
who died from lymphopoietic cancer (Santos-Burgoa, 1988; Matanoski et al., 1990); (5) a nested
case-control study of rubber workers dying from various types of cancer including
lymphohematopoietic cancer (McMichael et al., 1976); and (6) a population-based case-control
study of various types of cancers (excluding leukemia) conducted in Montreal, Canada
(Siemiatycki, 1991).
Supporting evidence evaluated by IARC (1992) included in vitro studies on the
metabolism of 1,3-butadiene using human liver and lung homogenates and comparative in vivo
and in vitro metabolism studies in mice, rats, and monkeys. A detailed discussion on in vivo and
in vitro genetic toxicity of 1,3-butadiene and metabolites (1,2-epoxybutene and 1,2:3,4-
diepoxybutane) was presented along with other available information on short-term toxicity and
nonneoplastic effects of 1,3-butadiene in humans and experimental animals.
IARC (1992) concluded that the evidence for the carcinogenicity of 1,3-butadiene in
humans is “limited” based on (1) a study showing an increased risk for lymphosarcoma and
reticulosarcoma among workers who manufacture 1,3-butadiene monomers; (2) a suggested
increased risk for leukemia among workers at one of two styrene-butadiene rubber plants studied;
(3) no increase of leukemia among the entire cohort of workers at eight U.S. and Canadian
styrene-butadiene rubber plants, but a significant risk of leukemia among a subgroup of
production workers; and (4) a large excess of lymphohematopoietic cancer nested among workers
exposed to 1,3-butadiene in styrene-butadiene rubber plants. IARC also concluded that the
evidence for the carcinogenicity of 1,3-butadiene in experimental animals was “sufficient” based
on tumor induction at multiple sites in mice and rats, the induction of neoplasms in mice at all
concentrations tested (6.25 to 1,250 ppm), the carcinogenicity of two metabolites of 1,3-
butadiene, and the detection of activated K-ras oncogenes in lymphomas, liver tumors, and lung
tumors induced by 1,3-butadiene. Evidence from metabolism and genetic toxicity studies
supported the conclusions of the carcinogenicity studies. IARC (1992) concluded that 1,3-
butadiene is probably carcinogenic to humans (Group 2A).
1.2.3. Summary of the National Institute for Occupational Safety and Health (NIOSH)
Evaluation of 1,3-Butadiene (NIOSH, 1991a)
NIOSH (1991a) conducted a qualitative and quantitative assessment of the carcinogenicity
of 1,3-butadiene. The evaluation of animal data focused on the studies that could be used for
quantitative assessment, namely the studies using Sprague-Dawley rats (Owen et al., 1987) and

B6C3F1 mice (NTP, 1984; Melnick et al., 1990a). The qualitative evaluation of the evidence
from human studies focused on the studies by Downs et al. (1987) and updated by Divine (1990);
Meinhardt et al. (1982) and updated by Lemen et al. (1990); Matanoski and Schwartz (1987) and
updated by Matanoski et al. (1990); and a case-control study of the lymphopoietic cancers
(Santos-Burgoa, 1988) from the Matanoski cohort. According to NIOSH, the results of this
nested case-control study “provide the strongest human evidence to date for an association
between 1,3-butadiene and the risk of lymphopoietic neoplasms, particularly leukemia.” NIOSH
concluded that overall the epidemiologic studies showed an increase in lymphopoietic neoplasms,
which is consistent with the induction of lymphomas in mice exposed to 1,3-butadiene. However,
NIOSH reported that the epidemiologic studies had certain limitations, such as the lack of
historical exposure levels, the inclusion of both support and production personnel whose exposure
would probably be minimal, and the inconsistent diagnosis of the different types of
lymphohematopoietic neoplasms.
NIOSH reported on metabolism, pharmacokinetics, and disposition studies; their
evaluation focused primarily on studies that provided data for estimating metabolic rates at low
concentrations and comparison of metabolic pathways and rates in different species (mice, rats,
monkeys, and humans). With respect to genetic toxicity, NIOSH did not focus on details of any
studies, but noted that 1,3-butadiene is mutagenic in Salmonella with metabolic activation,
whereas the metabolites are mutagenic without metabolic activation.
NIOSH (1991a) concluded that the present evaluation supports the conclusion of a
previous evaluation (NIOSH, 1984), which stated that “1,3-butadiene should be considered to
represent a potential human health hazard with respect to carcinogenicity.” The basis for the
conclusion was positive evidence of carcinogenicity in three long-term animal bioassays in two
species, positive evidence of mutagenicity and genotoxicity, and less conclusive epidemiologic
evidence of excess deaths from lymphopoietic neoplasms.
NIOSH used data from the study in B6C3F1 mice (Melnick et al., 1990a) for its
quantitative assessment because the lowest concentration (6.25 ppm) was similar to the proposed
OSHA standard of 2 ppm. Weibull’s one-, two-, and three-stage time-to-tumor models were used
to derive the maximum likelihood and 95% confidence limit estimates on excess risk. The models
were fit for the individual tumors for which the incidences were significantly higher in exposed
groups than in control groups of male and female mice. Hemangiosarcomas of the heart and
lymphomas were modeled as fatal lesions and all others as incidental lesions. The equivalent
human doses were calculated based on body weight to the three-fourths power (BW3/4) and
converted back to ppm exposures in the workplace for 45 years of exposure. The excess risk for
lifetime occupational exposure at 1 ppm was 305/10,000 based on lung neoplasms in females
(highest) and 0.03/10,000 based on heart hemangiosarcomas in females.

NIOSH (1991b) discussed the uncertainties associated with its assessment. The dose-
scaling method chosen and species differences in the metabolism of 1,3-butadiene were major
sources of uncertainty. Another source of uncertainty was the most relevant tumor site used to
predict human risk. The female lung was the most sensitive site, but based on the epidemiologic
evidence, lymphomas may be the most relevant neoplasms. Other sources of uncertainty were the
model selection: (1) whether the Weibull time-to-tumor model was the most appropriate and
which stage model to use, (2) the assumption regarding lethality of tumors and omission of the
high-dose group, and (3) estimation of the internal dose and the application of kinetic data.
1.2.4. California Air Resources Board (CARB, 1991)

The CARB (1991) evaluated the data on 1,3-butadiene and presented quantitative
estimates of the cancer risk from inhalation exposure to 1,3-butadiene in ambient air. The
literature review consisted of toxicokinetic data that focused on information presented by Bond et
al. (1986, 1987) for absorption and tissue distribution data and reports published between 1985
and 1991 for metabolism and excretion data. Acute, subchronic, and noncancer chronic toxicity
information was obtained from excerpts from EPA’s 1985 carcinogen assessment document, and
reproductive/developmental toxicity data and genetic toxicity data were reported from the
primary literature. Genetic toxicity data focused on mutation tests in S. typhimurium, DNA
alkylation studies, SCE and chromosome aberration tests, and various in vivo studies.
Animal carcinogenicity studies evaluated by CARB included the two NTP studies in mice
(NTP, 1984; Huff et al., 1985; Melnick et al., 1990a), the inhalation study in rats (Owen et al.,
1987), the role of retroviruses in 1,3-butadiene-induced carcinogenesis (Irons et al., 1987), and
the expression of oncogenes in tumors induced by 1,3-butadiene (Goodrow et al., 1990). Human
studies evaluated by CARB started with the 1976 study by McMichael et al. and continued
through the 1990 reports by Lemen et al., Divine, and Matanoski et al. CARB discussed several
factors that must be considered when interpreting the epidemiologic studies: (1) misclassification
of exposure—unexposed individuals classified as exposed would bias the results toward the null;
(2) exclusion of most highly exposed workers—studies in which the workers with the highest
potential exposure (World War II workers) are excluded would be less likely to see a significant
effect; (3) no dose-response effect—the lack of a positive association with duration of exposure
should not discredit the study because the most recent NTP animal study (Melnick et al., 1990a)
demonstrated that short-term exposure to a high concentration of 1,3-butadiene could be more
effective than long-term exposure to low concentrations; and (4) varying health endpoints—there
were inconsistencies in the subtypes of lymphopoietic and hematopoietic cancers observed in the
various studies, but nomenclature changed over time and there are probably close relationships
between the different subtypes. CARB presented four points of evidence for an association

between exposure to 1,3-butadiene and lymphopoietic and hematopoietic cancers in humans. The
first point is that the strongest effect was observed in the cohort involved in the production of 1,3-
butadiene monomer, and this cohort had the greatest potential for exposure to 1,3-butadiene in
the absence of styrene. The second is that the observations of cancers in cohorts having potential
exposure to styrene and 1,3-butadiene are consistent with the findings from the cohort from the
1,3-butadiene monomer production facility. The third point is presented in the case-control study
by Matanoski and Schwartz (1987) and the cellular study by Checkoway and Williams (1982) in
which both attributed the observed effects to 1,3-butadiene exposure and not to styrene exposure.
The fourth point is that the cancers observed in humans are consistent with those observed in the
mouse experiments. CARB concluded that “the epidemiological studies reported to date give
evidence for increased incidences of leukemia and/or lymphohematopoietic neoplasms resulting
from exposure to vapors in styrene-butadiene rubber plants or butadiene production plants.”
They further stated that the evidence for elevated rates of stomach and lung cancers is
inconclusive.
CARB conducted an extensive quantitative assessment of the risk from exposure to 1,3-
butadiene. The two mouse studies and the rat study were considered suitable for quantitative
evaluation. Dose estimations were based on experimental (applied) dose, continuous internal
dose, metabolized dose, target tissue dose, and molecular tissue dose. CARB used the retention
data from Bond et al. (1986) to estimate the daily dose adjusted for 7-day week exposures
(internal dose). The pharmacokinetics model of Hattis and Wasson (1987) was used to estimate
internal exposure to metabolites, namely butadiene monoepoxide (metabolized dose). The tissue
distribution data of Bond et al. (1986, 1987) were used to estimate the target tissue doses, which
were not used for risk estimation because the data were not reliable. Sufficient data on DNA
adducts were not available for deriving molecular tissue doses.
CARB fitted the experimental (applied dose), internal, and metabolized doses estimated
from the first mouse study (NTP, 1984) to the linearized multistage (Global 86) and the Weibull
time-to-tumor models; the cancer potency estimates derived using the linearized multistage model
and Weibull’s model gave similar results. The multistage model was used to derive cancer
potency values using the second mouse study (Melnick et al., 1990a) and the rat study (HLE,
1981). Cancer potency estimates were derived for each anatomical site separately and for the
total number of tumor-bearing animals with significantly increased tumors in both males and
females. The human cancer potency estimates, based on 70 years of continuous exposures,
derived from the first mouse study using the total significant tumors, the internal dose, and the
multistage model were 0.32 (ppm) 1 or 0.59 (mg/kg/day) 1 for male mice and 0.18 (ppm) 1 or
0.33 (mg/kg/day) 1 for female mice. Cancer potencies derived from applied doses were about 10-
fold lower, and those derived from metabolized doses were about 50% lower. The human cancer

potency estimates using the rat data (total significant tumors), internal dose, and the multistage
model were 1.8 × 10 3 (ppm) 1 or 8.4 × 10 3 (mg/kg/day) 1 for male rats and 3.5 × 10 3 (ppm) 1
or 1.6 × 10 2 (mg/kg/day) 1 for female rats. The estimates based on applied or metabolized doses
were much lower. The data from the second mouse study were analyzed extensively; CARB
concluded that the best human cancer potency estimates based on internal doses and estimated
using the multistage model (Global 86) were 0.37 (ppm) 1 or 3.4 (mg/kg/day) 1 derived for
alveolar/bronchiolar adenoma/carcinoma in female mice. The corresponding unit risk derived
from the second mouse study was 1.6 × 10 4 (µg/m3) 1 and the exposure for the risk at 10 6 was
6.0 × 10 3 µg/m3. From their cancer potency values, CARB estimated the lifetime extra risk
associated with exposure to 1 ppb 1,3-butadiene to range from 9.8 × 10 6 to 3.7 × 10 4, which
corresponds to 10 to 370 additional cases per 1 million individuals.
1.2.5. Summary of Findings by U.S. Occupational Safety and Health Administration

(OSHA)
The most recent analysis of health effects of 1,3-butadiene by a government entity is by
OSHA (1996). While the analysis in general is similar to that of NIOSH, OSHA incorporated a
recent update of the large SBR polymer retrospective follow-up study that had been started by
Matanowski et al. This update, Delzell et al (1995), included not only an additional period of
follow-up, but also a detailed exposure history for 1,3-butadiene, styrene, and benzene for more
than 15,000 employees who had worked in SBR and related activities at the eight study plants.
Delzell et al. concluded that “This study found a positive association between employment in the
SBR industry and leukemia. The internal consistency and precision of the results indicate that the
association is due to occupational exposure. The most likely causal agent is BD or a combination
of BD and [styrene]. Exposure to [benzene] did not explain the leukemia excess.” OSHA in its
analysis of the Delzell et al. and previous studies recognized these consistencies and similarly
concluded that “there is strong evidence that workplace exposure to BD poses an increased risk
of death from cancers of the lymphohematopoietic system. The epidemiologic findings
supplement the findings from the animal studies that demonstrate a dose response for multiple
tumors and particularly for lymphomas in mice exposed to BD” (OSHA, 1996, p. 56764).
OSHA also examined the evidence for reproductive and developmental effects. Analyzing
data from both the NTP I and the NTP II studies, OSHA noted the consistency and dose response
and concluded “that exposure to relatively low levels of BD resulted in the induction of ovarian
atrophy in mice...” (OSHA, 1996, p. 56765). For the total database on these and mutagenic
effects, OSHA concluded that “these animal studies taken as a whole, offer persuasive qualitative
evidence that BD exposure can adversely affect reproduction in both male and female rodents.
The Agency also notes that BD is “mutagenic in both somatic and germ cells” (p. 56767).

For quantitative risk assessment, OSHA’s analysis was very similar to that of NIOSH
(1991a) in its choice of data set (NTP II mouse study), model (multistage Weibull), treatment of
tumors (dose-response analysis on an individual basis), treatment of fatal vs. nonfatal in the time-
to-tumor analysis, choice of parsimonious model algorithm (fewest parameters of the multistage
model that provide an adequate fit to the data) and reporting of the ML estimates. The major
difference between the NIOSH and OSHA analyses was that OSHA used (mg/kg bw-day)
equivalence for species conversion instead of the BW3/4 conversion used by NIOSH. This change
of species conversion factors and some minor modifications relating to animal weights and
breathing rates decreased OSHA’s potency estimates by a factor of approximately 4 from the
NIOSH estimates. Based on the female mouse lung tumor response, the OSHA ML estimate of
potency was 8.1 × 10-3 (ppm)-1 for an occupational lifetime of exposure to 1 ppm, 5 days/week,
50 weeks/year for 45 years. If this potency estimate is extrapolated to be based on a 70-year
continuous lifetime exposure, the OSHA estimate would be approximately 36.7 × 10-3 (ppm)-1.
Based on the OSHA risk assessment, their permissible exposure limit was lowered from 1,000
ppm to 1 ppm with a 15-min short-term exposure limit.
1.3. DISCUSSION
Six different carcinogenicity assessments of 1,3-butadiene, done by five different agencies
in different time periods, are summarized in this chapter. The major conclusions of these
evaluations are presented in Table 1-1.
Although no apparent agreement is evident from the table among the five agencies’
assessments, in fact they are very similar. Both EPA (1985) and IARC (1986) conclude that the
carcinogenicity evidence in humans is inadequate and in animals is sufficient. But due to different
classification systems, they get different alphabetical assignments, i.e., B2 and 2B, which
correspond to “probable” and “possible” descriptors, thus appearing to be in disagreement with
each other. NIOSH and OSHA both use the dichotomous descriptors with “potential
occupational carcinogen” being the highest ranking.

Table 1-1. Carcinogenicity assessments of 1,3-butadiene
Agency Cancer classification
(year) Quantitative risk Remarks
EPA (1985) “B2-probable human Unit risk to humans—2.5 × Cancer classification

carcinogen” —based 10 1 (ppm) 1 based on NTP using EPA
on inadequate human (1984) mouse data. carcinogen
and sufficient animal assessment
evidence. guidelines.
IARC (1986) “2B-possible human No quantitative risk Cancer classification

carcinogen”—based presented. using IARC system.
on inadequate human
and sufficient animal
evidence.
IARC (1992) “2A-probable human No quantitative risk
carcinogen”—based presented.
on limited human and
sufficient animal
evidence.
NIOSH (1991a) “Potential human Range of excess risk at 1 OSHA cancer policy
health hazard with ppm is MLE of 305/10,000 classification system
respect to based on female mouse lung used.
carcinogenicity.” neoplasms to MLE of Quantitative risk is
0.03/10,000 based on heart for occupationally
hemangiosarcomas in exposed populations.
females. Data from
Melnick et al. (1990a) used
for this quantitation.
CARB (1991) No formal Human cancer potency No formal cancer

classification given based on mouse data from classification system
Melnick et al. (1990a) used.
range for 1 ppb Quantitative risk is
exposure—9.8 × 10 6 to 3.7 for general
× 10 4. population.
OSHA (1996) Potential Human cancer potency “Convincing evidence

occupational estimate based on female that BD is a probable
carcinogen mouse lung neoplasms. human carcinogen.”
MLE is 8.1 × 10 -3 (ppm)-1. Quantitative risk is
for occupationally
exposed populations.

OSHA, NIOSH, and CARB assessments all state that the human evidence is strongest for
an association between butadiene exposure and the occurrence of lymphohematopoietic cancers.
The same evidence is described as “limited” human evidence by IARC, which elevates the
classification of this compound to “2A-probable human carcinogen.” Furthermore, it should be
noted that the quantitative risk estimates appear to be different for OSHA/NIOSH and
EPA/CARB. NIOSH/OSHA quantitative risk estimates are for occupationally exposed
populations, while quantitative estimates of CARB are for general population (lifetime risk), even
though they are derived from the same animal data.
The apparent differences in these assessments thus can be explained by availability of the
studies at the time of evaluations, different cancer classification systems, and quantitative
assessments done for different purposes.

2. OVERVIEW OF EXPOSURE TO 1,3-BUTADIENE
The purpose of this chapter is to present an overview of how human exposure to 1,3-
butadiene occurs. The chapter summarizes physical/chemical properties, production/use,
sources/emissions, and ambient air data. Pathways of exposure are briefly described, but no
quantitative estimates of exposure levels and numbers of people exposed are presented.
2.1. PHYSICAL/CHEMICAL PROPERTIES

1,3-Butadiene (CH2=CH-CH=CH2, CAS No. 106-99-0) is a colorless gas with mildly
aromatic odor (Sax and Lewis, 1987). It is a noncorrosive gas and has a molecular weight of
54.09. Its boiling point is 4.4 C (Weast, 1989) and its vapor pressure is 1,790 mm Hg (239
kPa) at 20 C (Santodonato, 1985). It is easily liquefied with a density of 0.6211 g/ml at
20 C/liquefied (Kirshenbaum, 1978; Verschueren, 1983). It is soluble in ethanol, diethyl ether,
and organic solvents (Verschueren, 1983; Sax and Lewis, 1987; Budavari, 1989) and is also very
slightly soluble in water with a solubility of 735 mg/l at 20 C. 1,3-Butadiene has a flash point
of 76 C (Sax and Lewis, 1987) and is slowly dimerized to 4-vinyl-1-cyclohexene (U.S.
Occupational Safety and Health Administration, 1990) and may form peroxide upon exposure to
air (Kirshenbaum, 1978). Since 1,3-butadiene is a highly volatile gas, it is expected to partition
predominantly to the atmosphere and then undergo rapid destruction by photoinitiated reactions.
The reaction with photochemically produced hydroxyl radicals has a calculated half-life of
approximately 6 h and is expected to be the dominant pathway for atmospheric removal (U.S.
Department of Health and Human Services [DHHS], 1992). Destruction of atmospheric 1,3-
butadiene by the gas-phase reaction with ozone and by the nighttime reaction with nitrate
radicals in urban areas is also expected to be significant (U.S. DHHS, 1992). The major
photooxidation products of 1,3-butadiene are acrolein and formaldehyde (Maldotti et al. 1980).
There are limited data on the fate of 1,3-butadiene in soil or water. Based on its physical
properties, rapid volatilization of 1,3-butadiene from either soil or water to atmosphere is
expected to dominate over all other potential environmental processes. Studies performed with
pure cultures indicate that 1,3-butadiene may be susceptible to microbial attack. Based on
estimated values, 1,3-butadiene is not expected to adsorb significantly to soil or sediment, nor is
it expected to bioconcentrate in fish or aquatic organisms (U.S. DHHS, 1992).
2.2. PRODUCTION AND USE

1,3-Butadiene was first produced in 1886 by the pyrolysis of petroleum hydrocarbons
(Kirshenbaum, 1978). Commercial production of 1,3-butadiene started in the 1930s (Kosaric et
al., 1987) and has been produced by three processes: catalytic dehydrogenation of n-butane and

n-butene, oxidative dehydrogenation of n-butene, and recovery from the C4 coproduct (by-
product) stream from the steam cracking process used to manufacture ethylene. The ethylene
coproduct process accounts for approximately 95% of U.S. and 85% of worldwide production
(Morrow, 1990). Approximately 12 billion pounds of this gas are produced annually worldwide
and 3 billion pounds in the United States (Morrow, 1990; USITC, 1990).
1,3-Butadiene is used as an intermediate in the production of polymers, elastomers, and
other chemicals. The major uses of this chemical are in the manufacture of styrene-butadiene
rubber (synthetic rubber) and of thermoplastic resins. In 1990, 1,3-butadiene was used in the
United States for styrene-butadiene rubber (30%), polybutadiene rubber (20%), adiponitrile/
hexamethylenediamine (15%), styrene-butadiene latex (10%), neoprene rubber (5%),
acrylonitrile-butadiene-styrene resins (5%), exports (4%), nitrile rubber (3%), and other
(including specialty polymers) (8%) (Anon., 1991).
2.2.1. Styrene-Butadiene Latex and Rubber Production

Styrene-butadiene (SB) latex and rubber production is the major use for butadiene,
accounting for 40% of butadiene consumption. SB latex and rubber are used for a variety of
products, including automobile tires, textiles, paper, and adhesives.
The 1994 EPA report Locating and Estimating Air Emissions From Sources of 1,3-
Butadiene lists SB latex and rubber production as the major contributor to industrial butadiene
emissions (EPA, 1994a). About 74% of the industrial emissions are from SB latex and rubber
production. There are at least 26 facilities in the United States that produce SB latex and rubber
(SRI International, 1993).
As stated previously, butadiene has a very low water solubility and high vapor pressure;
thus, if it were released to an aqueous waste stream, it would immediately evaporate. It is then
logical to assume, and the data confirms that, the amount of butadiene found in secondary
sources such as waste water and solid waste is minimal or nonexistent. The majority of the
butadiene releases during industrial production occurs via process vents, so only emission factors
for process vents will be presented. The emission factors, as presented in the 1994 EPA report
for process vent butadiene released during SB latex and rubber production, range from 0.00024
to 94.34 lb butadiene emitted/ton produced (mean of 7.10) measured at 18 facilities (EPA,
1994a).

2.2.2. Polybutadiene Production
The second largest use for butadiene is in the production of polybutadiene, accounting
for over 20% of butadiene consumption. Polybutadiene is used in tire manufacturing and in the
high-impact resin industry. Four companies at five locations in the United States currently
produce polybutadiene. The estimate for process vent butadiene emissions from polybutadiene
production, as stated in the 1994 EPA report, ranges from 0.00008 to 36.06 lb butadiene
emitted/ton produced (mean of 6.14) measured at six facilities (EPA, 1994a).
2.2.3. Neoprene Rubber Production

Neoprene, or polychloroprene, rubber production accounts for 5% of butadiene
consumption. Neoprene rubber is primarily used in the automotive industry as belts, cables,
hoses, and wires. Three facilities currently produce neoprene, though only two use butadiene as
a raw material and the other starts with chloroprene. The two facilities identified in the 1994
EPA report that used butadiene as a raw material yield estimated process vent butadiene
emissions from neoprene production ranging from 0.32 to 6.78 lb butadiene emitted/ton
produced (mean of 4.04) (EPA, 1994a).
2.2.4. Acrylonitrile-Butadiene (ABS) Resin Production

ABS resins are used to make plastic components such as automotive parts, pipes and
fittings, appliances, telephones, and business machines, among many other uses. ABS
production accounts for 5% of butadiene consumption. Currently, there are 10 facilities that
produce ABS resin, only 6 of which use butadiene as a raw material. The estimate for process
vent butadiene emissions from ABS resin production ranges from 0.16 to 10.66 lb butadiene
emitted/ton produced (mean of 4.22) measured at three facilities (EPA, 1994a).
2.2.5. Nitrile Elastomer Production

Nitrile elastomer or nitrile-butyl rubber is a specialty elastomer known for its oil-,
solvent-, and chemical-resistant properties. Some uses include hoses, belting, and cable
manufacturing and seals and gaskets. Nitrile elastomer is produced at nine facilities in the
United States and accounts for about 5% of total butadiene consumption. The estimate for
process vent butadiene emissions from nitrile elastomer production ranges from 0.0004 to 17.80
lb butadiene emitted/ton produced measured at six facilities identified in the 1994 EPA report
(EPA, 1994a).

2.2.6. Adiponitrile Production
Adiponitrile (hexanedinitrile) is primarily an intermediate used in the production of
nylon 6,6. Three facilities produce adiponitrile, but only two of these facilities use butadiene in
production. This accounts for 12% of butadiene consumption. Despite the large usage of
butadiene in adiponitrile production, emissions appear to be fairly small. The estimate for
process vent butadiene emissions from adiponitrile production, based on actual emissions
reported at two facilities, is 0.12 lb butadiene emitted/ton produced (EPA, 1994a).
2.3. SOURCES AND EMISSION

1,3-Butadiene may be released to the environment as an intentional or fugitive emission
during its production, use, storage, transport, or disposal. Its sources and emission to the
environment can be classified as industrial production and use (1.6%), mobile sources (78.8%),
and other miscellaneous combustion sources (19.6%) (EPA, 1994a).
Industrial butadiene emissions arise from process vents, equipment leaks, and secondary
sources such as waste water treatment. Since butadiene released to aqueous systems or entering
treatment plants is likely to evaporate completely, all emissions of butadiene can be considered
air emissions. Actual reported emissions of butadiene are available through the Toxic Release
Inventory, and the relative contribution of butadiene production to the national butadiene
emissions is 0.2% (EPA, 1994a).
2.3.1. Mobile Sources

Butadiene is formed as a product of incomplete combustion of fossil fuels and has been
reported in the emissions from gasoline and diesel vehicles, as well as aircraft. Emissions of
butadiene from combustion sources are commonly represented as a weight percent of total
organic gas emissions. The relative contribution of mobile sources to the national butadiene
emissions is 78.8%, which includes both on-road and nonroad engines. Levels of butadiene in
gasoline and diesel fuel are expected to be insignificant since butadiene tends to form a varnish
that can be harmful to engines; therefore, refiners try to minimize the butadiene content. Since
butadiene is not a component of gasoline, it is not present in mobile source evaporative or
refueling emissions and will be found only in exhaust emissions (EPA, 1992).
It should be noted that a recent reevaluation by Nordlinder et al. (1996) of the Concawe
report (1987) found that the concentrations of 1,3-butadiene in gasoline vapors were much lower
than had been reported. Two analyses by Lofgren et al. (1991) and Ramnas et al. (1994) also
found negligible amounts of 1,3-butadiene in gasoline vapors. When they compared the
concentrations of benzene and butadiene in gasoline, they found concentrations to be 3%-5%

and <0.0005%, respectively. Based on these three reports, Nordlinder et al. (1996) concluded
that there is no significant amount of 1,3-butadiene present in gasoline vapors.
2.3.1.1. On-Road Mobile Sources

On-road mobile sources include the following classes of vehicles: light-duty gasoline
vehicles (LDGV), light-duty gasoline trucks, heavy-duty gasoline trucks, light-duty diesel
vehicles, light-duty diesel trucks, heavy-duty diesel trucks, and motorcycles. On-road mobile
sources account for 37.7% national butadiene emissions.
Although data on the butadiene content of motor vehicle exhaust were lacking until the
late 1980s, butadiene emissions from LDGV’s are now reasonably well understood. As
mentioned previously, butadiene is not a component of gasoline and is not present in evaporative
or refueling emissions; thus, only exhaust butadiene emissions are included. Butadiene has been
found to be removed effectively from motor vehicle exhaust by catalytic convertors (McCabe et
al., 1992). Thus, nearly all on-road motor vehicle butadiene emissions come from older,
noncatalyst vehicles, new vehicles with nonfunctional catalysts, the cold-start emissions from
catalyst vehicles, and diesel vehicles.
The emission factors calculated for all of the vehicles listed above range from 0.01 to
0.09 gm/mile (EPA, 1994b). A composite emission factor of 0.0156 gm/mile has been
calculated for the calendar year 1990 by the Office of Mobile Sources (OMS) using the
MOBILE model. The composite emission factor represents all vehicles classes and is based on
the percentage of total vehicle miles traveled (VMT) attributable to each vehicle class (EPA,
1993a).
2.3.1.2. Nonroad Mobile Sources

Nonroad mobile sources include mobile gasoline- and diesel-powered equipment and
vehicles and other equipment types. Types of equipment included in this category range from
construction, industrial, and agricultural equipment to small engines used in lawnmowers, chain
saws, and other gasoline-powered equipment. Nonroad vehicles include motorcycles,
snowmobiles, golf carts, and all-terrain vehicles (ATVs) used for off-road recreation and
recreational and commercial marine vessels. However, trains and aircraft are not generally
included in the nonroad vehicle category.
Generally, most nonroad engines are in use for many years and are noncatalyst engines.
The lack of a catalyst, in conjunction with the engine deterioration associated with increased
equipment age, may have profound effects on the amount of butadiene emitted. The emission
factors expected for the three major engines types in this categoryCgasoline-powered two-stroke

engines, gasoline-powered four-stroke engines, and diesel enginesCare generally higher (by a
minimum of a factor of 10) than the gasoline engines (EPA, 1991). The EPA 1994 draft denotes
that nonroad engines are expected to contribute 41% to the national butadiene emissions (EPA,
1994a).
2.3.1.3. Aircraft
Human exposure to aircraft emissions is considered to be limited to the emissions that
occur during aircraft landing and take-off (LTO). Airborne aircraft are assumed to fly at
sufficiently high altitudes that their emissions do not reach the surface. This assumption is likely
to be valid for butadiene because of its short atmospheric lifetime.
Butadiene has been reported in aircraft LTO emissions from military, commercial, and
general aviation. Based on the EPA SPECIATE database, the butadiene weight percents for
aircraft LTO hydrocarbon emissions range from 1.57% for general aviation (piston engines) to
1.89% from military aircraft (jet and piston engines). The 1994 EPA report estimates that 0.1%
of the national butadiene emissions is attributable to aircraft LTO (EPA, 1994a).
2.3.2. Miscellaneous Sources

This section contains an overview of the miscellaneous sources of butadiene emissions.
These sources have been grouped as miscellaneous chemical production, secondary lead
smelters, petroleum refining, and combustion sources (especially biomass burning). Emissions
from these sources can account for 19.6% of the national butadiene emissions.
2.3.2.1. Miscellaneous Chemical Production

The 1994 EPA report notes that butadiene is used to produce other elastomers and
plastics not mentioned previously, as well as pesticides and fungicides at 19 separate facilities in
the United States (EPA, 1994a). This process accounts for 8% of the butadiene use, but only
contributes 0.1% to the national average butadiene emissions. The emission factors for process
vent butadiene released during miscellaneous chemical production range from 0.03 to 440 lb
butadiene emitted/ton produced (product varies) measured at only four facilities.
2.3.2.2. Secondary Lead Smelters

Secondary lead smelting involves the reclamation of scrap automobile batteries to
produce elemental and lead alloys. There are 23 such facilities in the United States, most of
which are located near large population centers. The plastic and rubber components of the
battery are the source of the butadiene emissions, contributing 0.4% of the national butadiene

emissions. The 1994 EPA report lists uncontrolled butadiene emissions measured from a blast
furnace yielding an average emission factor of 0.79 lb/ton (EPA, 1994a).
2.3.2.3. Petroleum Refining

The 1992 Toxic Release Inventory contains the emission factor of 437,590 lb/year for
petroleum refining. Using this emission factor would make this source the fifth largest emitter
of butadiene, contributing 0.3% to the national butadiene emissions. Data are currently being
collected to determine the actual contribution of petroleum refining to butadiene emissions.
2.3.3. Combustion Sources

Butadiene is, as mentioned previously, a product of incomplete combustion and has been
reported in the emissions from gasoline and diesel vehicles, as well as aircraft. Butadiene is also
released during the combustion of tobacco, biomass, and automobile tires, although only the
latter two will be discussed in this section due to the scarcity of data.
2.3.3.1. Tire Burning

There are approximately 240 million tires discarded annually, of which only 25% are
recycled. The remaining tires are discarded in landfills, stockpiles, or illegal dumps (Lemieux
and DeMarini, 1992). Tires are combusted through accidental fires at stockpiles, illegal burning,
tire-to-energy facilities, cement kilns, tire manufacturing facilities, and as a supplemental fuel in
boilers. Butadiene is a major constituent of the tire manufacturing process and therefore it is
present in emissions from tire burning. Emission factors have been calculated for the open
burning of tires (EPA, 1992; Lemieux and DeMarini, 1992). These emission factors range from
234.28 lb/1,000 tons of chunk tires to 277.95 lb/1,000 tons of shredded tires. No emission factor
for butadiene from tire incineration has been located.
2.3.3.2. Biomass Burning

Biomass burning includes residential wood combustion in both fireplaces and wood
stoves, open burning such as the backyard burning of yard waste, slash burning, land
clearing/burning, agricultural burning, forest fires/prescribed burning, structural fires, and other
wildfires. Although these fires differ in many important characteristics, the fuels in all cases are
primarily composed of wood. The relative contribution of biomass burning to the overall
national butadiene emissions was calculated at 18.8% in the 1994 EPA report (EPA, 1994a).
Emission factor models based on field and laboratory data were developed by the U.S.
Forest Service (Peterson and Ward, 1989). These models incorporate variables such as fuel type
and combustion types (flaming or smoldering) and these models correlated butadiene emissions

with CO emissions to develop emission factors for biomass burning (Campbell and Mangino,
1994). The calculated emission factors range from 0.40 lb/ton of yard waste burned to 0.90
lb/ton for large wood burning in forest fires and prescribed burning.
Butadiene emissions have been reported from the combustion of wood (Sandberg et al.,
1975; Ward and Hao, 1992). The data of Ward and Hao (1992), in which both butadiene and
benzene were quantified from biomass burning, provides a butadiene:benzene ratio of 0.36 for
wood smoke.
2.4. AMBIENT CONCENTRATION OF 1,3-BUTADIENE
2.4.1. Air
In 1989, total emissions of 1,3-butadiene to the air in the United States were estimated at
approximately 2,512 tonnes from 158 locations; total land releases were estimated at 6.7 tonnes
(U.S. National Library of Medicine, 1991).
2.4.1.1. Ambient Monitoring Data

Several EPA databases exist that contain the results of various air toxics monitoring
programs. These programs have set up monitoring devices that are used to collect air samples
all over the United States over a period of months or years. Three of these programs/databases
contain data on 1,3-butadiene. This section summarizes the three monitoring programs and
presents annual average concentrations of 1,3-butadiene derived from these programs.
One of these programs is the Aerometric Information Retrieval System (AIRS), which
became operational in 1987 and uses a network of monitoring stations called the State and Local
Air Monitoring System (SLAMS) (EPA, 1989a). This network consists of monitoring stations
set up by every State in accordance with regulations promulgated in response to requirements of
the Clean Air Act. EPA’s Office of Air Quality Planning and Standards (OAQPS) administers
the AIRS program.
The AIRS program allows State and local agencies to submit local air pollution data and
also have access to national air pollution data (EPA, 1989a). EPA uses data from AIRS in order
to monitor the States’ progress in attaining air quality standards for ozone, carbon monoxide,
nitrogen oxides, sulfur oxides, and lead through the use of State Implementation Plans (SIPS).
In addition to containing information about each monitoring site, including the geographic
location of the site and who operates it, the AIRS program also contains extensive information
on the ambient levels of many toxic compounds. The AIRS database catalogs ambient air
pollution data from 18 to 55 monitors in 15 to 23 urban areas, depending on the pollutant. These
monitors collect a 24-h sample every 12 days. However, in some cases not every target

compound was detected in every sample. Where this occurred, half the minimum detection limit
was used in the averaging of the data for this summary. The annual average ppb for each site
was calculated using only those sites that provided four quarters of monitoring data. The cities
monitored and the average concentrations determined can be found in Table 2-1.
Another air monitoring program is the Urban Air Toxic Monitoring Program (UATMP),
which the EPA developed in 1987 to assist State and local agencies in determining the nature
and extent of urban air toxic pollution (McAlister et al., 1989, 1990, 1991; Wijnberg and Faoro,
1989). Data from the UATMP also is used in air toxic risk assessment models (EPA 1989b, c;
EPA 1990a, b). In 1989, the UATMP had 14 monitors in 12 urban areas, and in 1990, the
UATMP had 12 monitors in 11 urban areas, of which 9 also participated in the 1989 monitoring
program.
In 1989 and 1990, the UATMP network simultaneously monitored 37 nonmethane
organic compounds, selected metals, benzo(a)pyrene (1989 only), formaldehyde, acetaldehyde,
and acetone for a 24-h period once every 12 days. The UATMP database lists the data collected
from the monitoring network using two methods. In the first method, only the concentrations
above the detection limit of the compound are included in the data. In the second method, if the
concentration of a compound is below the detection limit, then one-half of the compound’s
detection limit is incorporated into the data. The second method was used because it seemed
more reasonable and allowed a greater number of samples to be averaged. Data collected in
1989 and 1990 were used in this summary. The cities monitored and the average concentrations
determined can be found in Table 2-2.
The monitoring data for the UATMP that were collected from 1991 to 1994 have not yet
been released as separate reports. The data collected in those 4 years were entered into and
reported as part of the 1991-1994 AIRS database.
The National Ambient Volatile Organic Compounds (NAVOC) Database contains
approximately 175,000 records on the concentrations of 320 volatile organic compounds (VOCs)
observed in 1-h air samples taken every 24 h between 1970 and 1987 (Shah and Heyerdahl,
1988; Hunt et al., 1988). However, only the most current NAVOC data, taken during 1987, is
used in this summary. In addition, samples that had nondetects of 1,3-butadiene were included
as one-half the detection limit in averaging the data for this summary. The NAVOC Database
includes air samples collected using indoor and outdoor monitoring devices. Personal monitors
were also used. The types of locations of outdoor monitoring sites included remote, rural,
suburban, and urban areas, as well as near specific point sources of VOCs. Indoor monitoring

Table 2-1A. Summary of 1,3-butadiene ambient data from the EPA Aerometric Information Retrieval System (AIRS) for 1988 to 1991
1/28/98
Average concentration (ppb) Sampling sites Land use of monitor location Number of samples
1988 1989 1990 1991 1988 1989 1990 1991
0.13 Washington, DC Commercial/Urban & Center City 7
0.29 Ft. Lauderdale, FL Commercial/Urban & Center City 29
0.14 Miami, FL Commercial/Urban & Center City 31
0.10 Chicago, IL Commercial/Urban & Center City 10
0.29 Chicago, IL Residential/Suburban 25

2-10
0.73 Chicago, IL Mobile/Urban & Center City 9
0.22 St. Clair Co., IL Industrial/Suburban 29

DRAFT--DO NOT CITE OR QUOTE
0.13 Wichita, KS Residential/Suburban 29
0.16 Wichita, KS Residential/Urban & Center City 8
0.44 Louisville, KY Commercial/Urban & Center City 6
0.43 Baton Rouge, LA Commercial/Urban & Center City 29

Table 2-1A. Summary of 1,3-butadiene ambient data from the EPA Aerometric Information Retrieval System (AIRS) for 1988 to 1991 (continued)
1/28/98
1988 1989 1990 1991 1988 1989 1990 1991

0.33 0.07 Detroit, MI Commercial/Urban & Center City 19 28
0.12 St. Louis, MO Commercial/Urban & Center City 28
0.23 Camden, NJ Residential/Suburban 29
0.26 Newark, NJ Industrial/Urban & Center City 9
0.20 Plainfield, NJ Residential/Suburban 9
2-11
0.25 New York, NY Residential/Urban & Center City 9

0.29 New York, NY Commercial/Urban & Center City 9
0.11 Dallas, TX Commercial/Urban & Center City 23
1.11 0.60 0.72 Houston, TX Residential/Suburban 6 30 4

0.47 Burlington, VT Commercial/Urban & Center City 6
0.20 0.11 Arlington Co., VA Commercial/Urban & Center City 13 18
0.16 0.12 Henrico Co., VA Residential/Suburban 21 12
0.22 0.11 Hampton, VA Residential/Suburban 14 22
0.13 0.06 Hopewell, VA Residential/Suburban 16 15
0.24 0.12 Roanoke, VA Residential/Suburban 14 22
0.67 ppb 0.26 ppb 0.29 ppb 0.10 ppb
1,3-Butadiene Average Concentration Across Sites by Year
1/28/98
Table 2-1B. Summary of 1,3-butadiene ambient data from the EPA Aerometric Information Retrieval System (AIRS) for 1992 to 1994
1992 1993 1994 1992 1993 1994
0.12 0.33 Jefferson Co., AL Residential/Rural 83 79
0.19 Jefferson Co., AL Residential/Rural 9
4.32 0.78 Tarrant City, AL Residential/Suburban 82 81
0.91 Tarrant City, AL Residential/Suburban 10
0.07 0.15 Shelby Co., AL Agricultural/Rural 50 78
0.30 0.30 Fresno, CA Residential/Suburban 30 31
0.54 Clovis, CA Residential/Urban & Center City 111
0.28 0.27 Bakersfield, CA Residential/Urban & Center City 30 9
0.35 Bakersfield, CA Commercial/Urban & Center City 23
2-12
0.63 Bakersfield, CA Commercial/Urban & Center City 105

0.65 0.53 Los Angeles, CA Residential/Urban & Center City 26 30
0.16 0.15 Roseville, CA Mobile/Suburban 23 31
0.41 Citris Heights, CA Residential/Suburban 6
0.53 Sacramento, CA Residential/Suburban 84

0.34 0.27 El Cajon, CA Commercial/Suburban 28 28
0.18 0.17 Simi Valley, CA Residential/Suburban 28 29
0.36 0.28 Washington, DC Commercial/Urban & Center City 10 16
0.32 0.46 Washington, DC Commercial/Urban & Center City 13 15
0.09 0.18 Chicago, IL Industrial/Urban & Center City 6 14
1/28/98
Table 2-1B. Summary of 1,3-butadiene ambient data from the EPA Aerometric Information Retrieval System (AIRS) for 1992 to 1994 (continued)
1992 1993 1994 1992 1993 1994
0.06 0.08 Lemont, IL Residential/Suburban 15 13
0.10 0.11 St. Clair Co., IL St. Louis Metro Area 10 12
0.17 0.26 Kansas City, KS Industrial/Urban & Center City 11 13
0.42 0.43 Baton Rouge, LA Commercial/Urban & Center City 14 8
0.15 0.22 Glen Burnie, MD Commercial/Suburban 60 54
0.25 0.26 Essex, MD Residential/Suburban 39 58
0.18 0.17 Baltimore, MD Residential/Suburban 56 59
0.09 Baltimore, MD Industrial/Suburban 21
0.10 0.12 Baltimore, MD Industrial/Urban & Center City 50 50
0.26 0.25 0.28 Baltimore, MD Residential/Urban & Center City 58 57 48
0.09 0.10 Baltimore, MD Industrial/Urban & Center City 39 48
2-13
0.45 0.05 Alma, MI Commercial/Rural 22 14

0.06 0.08 Portage, MI Industrial/Suburban 25 4
0.08 0.09 0.07 Midland, MI Commercial/Rural 56 61 60
0.07 0.07 0.07 Midland, MI Commercial/Rural 55 61 22
0.07 0.07 0.07 Midland, MI Industrial/Rural 55 61 61

0.07 0.07 0.07 Midland, MI Agricultural/Rural 55 61 61
0.07 0.08 0.07 Midland, MI Industrial/Rural 55 60 61
0.07 0.07 0.07 Midland, MI Residential/Suburban 28 31 11
0.07 0.07 0.07 Midland, MI Residential/Rural 28 30 11
0.72 0.08 Detroit, MI Commercial/Urban & Center City 24 15
1/28/98
1992 1993 1994 1992 1993 1994
0.26 0.49 Camden, NJ Residential/Suburban 14 13
0.63 0.73 Newark, NJ Industrial/Urban & Center City 8 9
0.72 0.79 Plainfield, NJ Residential/Suburban 8 8
0.70 Nassau Co., NY Commercial/Suburban 9
0.19 Philadelphia, PA Residential/Suburban 38
0.08 San Antonio, TX Residential/Suburban 21
0.24 0.37 Clute, TX Residential/Suburban 29 53
0.18 0.21 Brownsville, TX Commercial/Urban & Center City 7 15
0.29 0.37 Brownsville, TX Commercial/Urban & Center City 19 59
2-14
0.32 Dallas, TX Commercial/Urban & Center City 82

0.20 0.37 Dallas, TX Industrial/Rural 34 58
0.41 1.68 Odessa, TX Residential/Suburban 39 59
0.18 0.37 Midlothian, TX Agricultural/Rural 39 51
0.45 0.43 El Paso, TX Commercial/Urban & Center City 14 15

0.98 1.16 El Paso, TX Commercial/Urban & Center City 79 79
1.49 El Paso, TX Commercial/Urban & Center City 9
0.83 1.29 El Paso, TX Commercial/Suburban 8 4
0.21 0.38 El Paso, TX Residential/Suburban 22 58
0.29 0.22 1.54 Texas City, TX Residential/Urban & Center City 5 35 51
0.35 0.29 Texas City, TX Residential/Suburban 12 15
0.19 0.30 0.56 Harris Co., TX Agricultural/Suburban 5 28 59
1/28/98
1992 1993 1994 1992 1993 1994
0.44 0.81 Houston, TX Industrial/Suburban 39 54
0.24 0.64 Houston, TX Industrial/Suburban 33 58
0.05 0.39 0.73 Houston, TX Industrial/Suburban 6 37 58
0.56 Beaumont, TX Residential/Suburban 78
0.05 0.25 0.37 Beaumont, TX Residential/Suburban 7 36 59
0.10 0.23 0.44 Port Arthur, TX Residential/Suburban 6 45 57
0.32 0.43 Port Neches, TX Industrial/Suburban 18 58
5.95 Port Neches, TX Residential/Urban & Center City 23
5.22 6.11 Port Neches, TX Residential/Suburban 11 14
0.18 0.37 Corpus Christi, TX Commercial/Suburban 39 52
0.18 West Orange, TX Residential/Suburban 27
0.33 Smith Co., TX Mobile/Rural 32
2-15
0.20 0.57 Fort Worth, TX Commercial/Urban & Center City 80 77

0.80 Fort Worth, TX Commercial/Urban & Center City 8
0.06 Fort Worth, TX Commercial/Suburban 9
0.05 Grapevine, TX Residential/Urban & Center City 15
0.13 Austin, TX Commercial/Urban & Center City 21

0.31 0.49 Burlington, VT Commercial/Urban & Center City 13 15
0.15 0.44 Rutland, VT Commercial/Urban & Center City 13 15
0.08 0.09 Waterbury, VT Commercial/Suburban 14 15
0.16 ppb 0.40 ppb 0.59 ppb
1,3-Butadiene Average Concentration Across Sites by Year
Table 2-2. Summary of 1,3-butadiene ambient data from the Urban Air Toxics Monitoring Program (UATMP)
1/28/98
Average concentration (ppb)a Detected/total

Sampling sites Land use of monitor location Sampling time/frequency
1989 1990 1989 1990
0.39 0.36 Baton Rouge, LA Urban/Center City-Commercial 24-Hour/Every 12 Days 11/31 8/29
0.24 0.06 Chicago, IL Suburban-Residential 24-Hour/Every 12 Days 10/27 4/29
0.20 0.10 Camden, NJ Suburban-Residential 24-Hour/Every 12 Days 19/32 9/30
0.08 Dallas, TX Urban/Center City-Commercial 24-Hour/Every 12 Days 8/25
0.20 Fort Lauderdale, FL Urban/Center City-Commercial 24-Hour/Every 12 Days 18/31
0.60 0.47 Houston, TX Suburban-Residential 24-Hour/Every 12 Days 23/34 111/28
0.11 Miami, FL Urban/Center City Commercial 24-Hour/Every 12 Days 7/33
0.06 Pensacola, FL Suburban-Industrial 24-Hour/Every 12 Days 6/42
0.09 St. Louis, MO Urban/Center City-Commercial 24-Hour/Every 12 Days 12/30
2-16
0.20 0.06 Sauget, IL Suburban-Industrial 24-Hour/Every 12 Days 7/31 2/27

0.11 0.10 Washington, DC-1 Urban/Center City-Commercial 24-Hour/Every 12 Days 9/27 11/30
0.29 0.15 Washington, DC-2 Urban/Center City-Commercial 24-Hour/Every 12 Days 19/27 12/27
0.16 0.06 Wichita, KS-1 Urban/Center City-/Residential 24-Hour/Every 12 Days 10/31 1/30
0.09 Wichita, KS-2 Suburban-Residential 24-Hour/Every 12 Days 7/31

11.09 Port Neches, TX Suburban-Residential 24-Hour/Every 12 Days 24/28
0.10 Orlando, FL Urban/Center City-Commercial 24-Hour/Every 12 Days 8/28
0.06 Toledo, OH Suburban-Residential 24-Hour/Every 12 Days 4/21
0.21 1.02
1,3-Butadiene Average Concentration in ppbb
a
The arithmetic average concentration of all samples using half minimum detection limit (MDL) value for samples in which the compound was not found.
b
Calculated by averaging all 390 samples taken from 13 sites equally in 1989 and 349 samples from 12 sites in 1990.
sites consisted of nonindustrial workplaces and residential environments. Personal monitors also
are included in the indoor category. This database was an interim precursor to the air toxics
portion of AIRS. For this summary, only the outdoor urban data were used. The cities
monitored and the average concentrations determined can be found in Table 2-3.
Table 2-4 summarizes the average concentrations (in ppb) of 1,3-butadiene found at the
monitoring sites of each air monitoring program. The table also shows the total number of
observations for each average and the number of sites that monitored the compounds in each
program. For AIRS, the average concentrations of 1,3-butadiene are listed separately for 1987
through 1994. Some of the highest averages in the AIRS database were from suburban
residential sites in Houston and Port Neches, TX. Both of these cities have high point-source
emissions that could be affecting the monitor. The AIRS and UATMP data from Houston and
Port Neches, TX, were excluded to create alternate annual averages (ppb and µg/m3) for the
years 1988 through 1994 (where applicable) and are presented in Table 2-4. This alternate
annual average may be more representative of areas that are not near strong point sources.
Tables 2-5, 2-6, and 2-7 regroup and summarize Tables 2-2, 2-3, and 2-4 according to
the sampling locations, i.e., rural, suburban, or urban settings. The data obtained from Port
Neches, TX, were not included in these averages because of the elevated levels due to industrial
emissions.
It should be noted that methods of averaging the data are not consistent between the air
monitoring databases. Also, in the NAVOC monitoring network, samples were taken for 1 h in
a 24-h period while the other monitoring networks collected a 24-h air sample every 12 days.
It should also be noted that the ambient levels detected in these three databases are not
meant to be indicative of an individual’s actual exposure to 1,3-butadiene. Times and
concentrations in microenvironments other than the outdoors need to be taken into consideration,
i.e., accounting for integrated exposure.
In addition, the ambient levels include contributions from a variety of source categories.
Typically, ambient monitoring data are adjusted to represent the amount attributed to a particular
source using emissions inventory apportionment. The derivation of an urban annual average
exposure estimate for all mobile sources will be used for illustration purposes (EPA, 1993a).
The range of ambient data from Table 2-4 (using alternate annual averages when available) is
0.22 to 1.02 µg/m3 (0.10 to 0.46 ppb). When this range is adjusted by the estimated proportion
of the inventory that is contributed by mobile sources (78.7%) and for integrated exposure to
account for time spent indoors and outdoors, the range becomes 0.11 to 0.50 µg/m3 (0.05 to 0.23
ppb).

Table 2-3. Summary of outdoor urban data from the National Ambient Volatile Organic Compounds
1/28/98
(NAVOC) Database
Sample years Average Number of Sampling sites Land use of Number of

concentration used samples monitor location samples/ sampling
(ppb) time
9/22/87 to 10/4/87 0.30 2 Bakersfield, CA Urban 1/24-hours
9/16/87 to 9/28/87 0.35 2 Concord, CA Urban 1/24-hours
10/4/87 to 10/4/87 0.60 1 Fremont, CA Urban 1/24-hours
10/4/87 to 10/4/87 0.40 1 Richmond, CA Urban 1/24-hours
9/9/87 to 10/7/87 0.25 2 San Jose, CA Urban 1/24-hours
9/29/87 to 9/27/87 0.30 1 Stockton, CA Urban 1/24-hours
2-18
Overall 1,3-Butadiene average concentration: 0.344 ppba

a
Calculated by averaging all nine samples taken from six cities equally.
Table 2-4. Summary of air monitoring program results for 1,3-butadiene
Annual average Alternate annual averagea

ppb (µg/m3) ppb (µg/m3)
AIRS
1988 level
(ppb) 0.67(1.48) 0.46(1.02)b
# Obs. 18 12
# Sites 3 2
1989
(ppb) 0.23(0.57) 0.25(0.55)b
# Obs. 399 369
# Sites 30 29
1990
(ppb) 0.29(0.64) 0.21(0.46)b

# Obs. 101 97
# Sites 7 6
1991
(ppb) 0.10(0.22) ----c
# Obs. 117
# Sites 6
1992
(ppb) 0.16 (0.40) 0.16 (40)b

# Obs. 656 650
# Sites 20 19
1993
(ppb) 0.40(0.88) 0.32(0.71)d

# Obs. 2069 1,931
# Sites 64 59
1994
(ppb) 0.59(1.30) 0.42(0.92)d

# Obs. 2666 2,401
# Sites 70 64

Table 2-4. Summary of air monitoring program results for 1,3-butadiene
(continued)
Annual average Alternate annual averagea

ppb (µg/m3) ppb (µg/m3)
UATMP
1989
(ppb) 0.21 (0.46) ----e
# Obs. 390
# Sites 13
1990
(ppb) 1.02(2.25) 0.12(0.27)b
# Obs. 349 293
# Sites 12 10
NAVOC
1987
(ppb) 0.34(0.75)f no data
# Obs. 9
# Sites 6
a
Alternate averages do not include data from Houston and Port Neches, TX, due to impacts from
strong point sources.
b
Average ppb from all 4-quarter data sites, excluding Houston, TX.
c
Houston, TX, was not monitored during this 4-quarter period.
d
Average ppb from all sites, excluding Houston and Port Neches, TX.
e
Port Neches, Texas, was not monitored during this 4-quarter period.
f
All urban California sites.

Table 2-5. Summary of 1,3-butadiene ambient data from the EPA Aerometric Information Retrieval System (AIRS)
1/28/98
based on sampling locations
Rural area Suburban area Urban area
Total samples/ Total samples/ Total samples/

number of number of number of
Year Range Averagea locations Range Average locations Range Average locations
1988 --- 1.11 6/1 0.44 - 0.46 12/2

0.47
1989 0.13 - 0.60 0.25 151/12 0.10 - 0.27 237/18

0.73
1990 0.16 - 0.72 0.29 95/5 0.20 - 0.27 32/2

0.33
2-22
1991 0.06 - 0.12 0.10 71/4 0.07 - 0.09 46/2

0.11
1992 0.07-0.45 0.13 271/6 0.05 - 0.19 0.10 154/7 0.09 - 0.29 176/5
0.72
1993 0.05-0.20 0.10 494/10 0.06 - 4.32 0.41 864/31 0.08 - 0.31 580/21
0.98
1994 0.07-0.37 0.18 522/11 0.06 - 1.68 0.45 1135/32 0.05 - 0.62 780/24
1.54
a
1,3-Butadiene average concentration in ppb.
Table 2-6. Summary of 1,3-butadiene data from Table 2-2 based on sampling locations
1/28/98
Suburban Urban
Total samples/ Total samples/ number

Year Range Averagea number of locations Range Average of locations
1989 0.09 - 0.60 0.27 155/5 0.08 - 0.39 0.18 235/8
1990 0.06 - 0.47 0.14 177/6 0.06 - 0.36 0.15 144/5

a
b
The arithmetic average concentration of all samples using one-half minimum detection limit value for samples in which the compound was not found.
Table 2-7. Summary of 1,3-butadiene data from Table 2-3 based on sampling locations
2-23
Urban
Total samples/number
Year Range Averagea of locations
1987 0.25 - 0.60 0.37 9/6

a
2.4.1.2. Ambient Source Apportionment
There are three studies that attempt to apportion sources as to their contribution to
ambient levels of 1,3-butadiene. The studies assume that all emissions to the atmosphere
contribute proportionally to ambient concentration. These three studies are summarized in Table
2-8.
As observed in Table 2-8, the source apportionment conducted by Systems Applications
International for the American Automobile Manufacturers Association (Ligocki, 1993) contains
the category of biomass burning as a large part of the inventory. The weight percentage of
butadiene in TOG for emissions from residential wood combustion, open burning, forest fires,
and other burning that are used in this analysis are derived from a single estimate provided in
EPA’s SPECIATE database. An actual 1,3-butadiene TOG weight percentage for incineration of
wood was not found in the literature; therefore, the solid waste incineration TOG weight
percentage was used. There is a great deal of uncertainty connected with the 1,3-butadiene
emission estimates that were developed for the biomass burning as well as for emissions from
aircraft. Many of the limitations revolve around the lack of real-world data on actual 1,3-
butadiene emissions and exposures for the scenarios mentioned above, as well as the allocation of
these scenarios nationwide. The emissions from residential wood combustion and forest fires
vary by season and region of the country. The mobile source and stationary source emissions
would, for the most part, remain constant throughout much of the year.
2.4.2. Indoor Exposure to 1,3-Butadiene

Information on 1,3-butadiene concentrations in homes or public buildings is limited at
this time. Indoor concentrations of 1,3-butadiene are primarily dependent on the presence of
environmental tobacco smoke (ETS) (CARB, 1992). Several studies indicate that on the average
most individuals spend anywhere from about 60% to 70% (Robinson et al., 1989; EPA, 1993b) of
their time each day indoors at their residence. In addition, individuals also spend a lot of time at
indoor workplaces. This makes indoor air a major route of exposure to 1,3-butadiene for
individuals who are exposed to tobacco smoke. It is also apparent that the potential for indoor
exposure can exceed outdoor exposure if ETS is taken into consideration. Löfroth et al. (1989)
and Brunnemann et al. (1990) measured 1,3-butadiene emissions in sidestream smoke ranging
from 200 to 400 µg/cigarette and 1,3-butadiene levels in smoke-filled bars ranging from 2.7 to 19
µg/m3. Further research and measurements are needed to quantify typical indoor 1,3-butadiene
exposures.

Table 2-8. Summary of the relative contributions to ambient 1,3-butadiene
emissions given as percent of total mg/year
Mobile Stationary point Biomass

Study sourcesa and area sourcesb Aircraft burningc
EPA, 1994a 78.7 2.4 0.1 18.8

CARB, 1992 96d 4 Included under NDe
mobile sources
Ligocki, 1993 57 5 3 35
a
Mobile sources included on-road and off-road vehicles and generally excluded trains and aircraft.
b
Area and point sources generally included all manufacturing and industrial process, oil and gas production
facilities, commerce, residential fuel combustion, and other stationary fuel combustion.
c
Biomass burning includes residential wood combustion, incineration, and other biomass burning.
d
The CARB off-road apportionment of mobile sources includes trains and aircraft.
e
ND=not determined.
2.4.3. Water
Although 1,3-butadiene has been detected in drinking water in the United States (U.S.
EPA, 1978; Kraybill, 1980), it is not clear what happens to the chemical in the body (U.S.
DHHS, 1992). Total releases to ambient water in 1989 were estimated to be 65 tonnes (U.S.
National Library of Medicine, 1991).
2.4.4. Food
Certain cooking oils release butadiene on heating. For example, 1,3-butadiene emissions
are approximately 22-fold higher from unrefined Chinese rapeseed oil than from heated peanut
oil. Of three fatty acids tested, heated linolenic acid produced the greatest amount of 1,3-
butadiene. Although cooking oils in the U.S. are refined for purity, U.S. rapeseed oil (canola)
also emitted 1,3-butadiene (Shields et al., 1995). Also, levels of <0.2 µg/kg 1,3-butadiene were
found in retail soft margarine; the plastic tubs containing the margarine contained < 5-310 µg/kg
(Startin and Gilbert, 1984).
2.5. PATHWAYS OF EXPOSURE

The 1992 U.S. DHHS report states that although 1,3-butadiene undergoes rapid
destruction in the atmosphere, it is almost always present at very low concentrations in urban and
suburban areas. Automobile exhaust is a constant source of 1,3-butadiene release to the
atmosphere. Because of the compound’s presence in the atmosphere, the general population is
exposed to ppb levels of 1,3-butadiene through inhalation. Exposure to 1,3-butadiene may also

occur from the inhalation of cigarette smoke, or possibly the smoke from wood fires. Possible
ingestion of contaminated drinking water may also lead to low levels of exposure, although the
concentration of this compound in drinking water has not been well characterized. The levels of
1,3-butadiene in soil are not known. Elevated levels of exposure for the general population may
occur for those near its site of manufacture or facilities where it is made into polymeric materials.
Occupational exposure to 1,3-butadiene is expected to be limited to those working at
facilities that manufacture 1,3-butadiene or convert it into commercial polymers. Exposure by
inhalation is expected to be the dominant pathway for exposure.

3. METABOLISM AND PHARMACOKINETICS
The pharmacokinetics of 1,3-butadiene have been reviewed previously by the U.S.

Environmental Protection Agency (U.S. EPA, 1985) and the International Agency for Research
on Cancer (IARC, 1986). Data from both in vitro and in vivo studies on the toxic effects of 1,3-
butadiene have established that 1,3-butadiene metabolites, not the parent compound, cause these
toxic effects. Differences have been noted in the toxic responses to 1,3-butadiene among
laboratory species, and understanding the pharmacokinetics of 1,3-butadiene and its metabolites is
important in assessing the carcinogenic risk and evaluating other health effects associated with
exposure to this chemical. This chapter summarizes the recent research that has provided
information on the pharmacokinetics of 1,3-butadiene in several animal species and elucidates the
metabolism of 1,3-butadiene, via both in vitro and in vivo studies.
The chemical terminology and units used in the publications reviewed in this chapter have
been standardized for consistency. Epoxybutene (EB) is used for 1,3-butadiene monoepoxide,
1,3-butadiene monoxide, 1,2-epoxybutene-3, 1,2-epoxy-3-butene, vinyl oxirane, and 3,4-epoxy-1-
butene; diepoxybutane (DEB) is used for 1,2:3,4-diepoxybutane; and butene diol (BD) is used for
1,2-dihydroxybut-3-ene and 3-butene-1,2-diol.
3.1. OVERVIEW OF PHARMACOKINETIC STUDIES

In recent years, considerable data have been generated regarding the pharmacokinetics of
1,3-butadiene in various laboratory species. Although in vitro studies can elucidate possible
metabolic products and allow measurements of metabolic reaction kinetic constants under
controlled conditions, in vivo studies usually encompass several issues of pharmacokinetics and
provide an account of the total disposition of the exposed dose. For 1,3-butadiene, because of the
toxicity of the 1,3-butadiene metabolites, in vivo pharmacokinetic studies validated the existence
of these metabolites and their metabolic rates of activation and detoxification. Absorption of the
parent compound was often assessed either from its distribution in the tissue organs or blood or
from its excretion in urine, feces, and exhaled air. Absorption and excretion have also been
measured from the presence of 1,3-butadiene metabolites in blood, urine, feces, and exhaled air.
Species differences have been observed in the toxic effects of 1,3-butadiene in mice, rats, and
monkeys and are reflected in the in vitro metabolism and pharmacokinetics of 1,3-butadiene in
these species. This section summarizes the metabolic pathways of 1,3-butadiene disposition and
the species differences in 1,3-butadiene pharmacokinetics and metabolism from in vitro and in
vivo studies.

3.1.1. Pathways Elucidation
Several in vitro and in vivo studies have elucidated the metabolic pathways of 1,3-
butadiene metabolism as shown in Figure 3-1 and summarized in Table 3-1 (Himmelstein et al.,
1997). Results from in vitro studies show that 1,3-butadiene undergoes cytochrome P-450-
mediated biotransformation to the reactive metabolite epoxybutene, which has also been validated
from in vivo studies in rats, mice, and monkeys. Epoxybutene can be activated further to another
reactive metabolite, diepoxybutane, or detoxified by epoxide hydrolase to butene diol, as shown
by in vitro studies and detected in vivo via their glutathione (GSH) conjugates in rats, mice,
hamsters, monkeys, and humans. Further metabolism of these two metabolites can be mediated
by either the P-450 system or epoxide hydrolase, giving 1,2-dihydroxy-3,4-epoxybutane. The
detoxification of epoxybutene occurs by hydrolysis and GSH conjugation and is mediated by the
enzymes epoxide hydrolase and glutathione S-transferase (GST), respectively; these reactions
have been supported by both in vitro and in vivo studies. Epoxybutene can also form DNA and
hemoglobin (Hb) adducts in both rats and mice. Of greater significance is the identification of
crotonaldehyde, a DNA-reactive chemical and known mutagen, as a new product of the oxidative
metabolism of butadiene. Crotonaldehyde was formed by the tautomerization of 3-butenal
formed by chloroperoxidase-dependent oxidation of 1,3-butadiene and was not a metabolic
product of epoxybutene. 3-Butanal rapidly tautomerized to crotonaldehyde at room temperature,
which may explain its nondetection in in vitro studies. A possible pathway for the metabolism of
3-butene-1,2-diol, a secondary metabolite of 1,3-butadiene, is oxidative dehydrogenation
catalyzed by alcohol dehydrogenase. The production of GSH-epoxide conjugates, S-(2-hydroxy-
3-buten-1-yl)glutathione (compound I) and S-(1-hydroxy-3-buten-2-yl)glutathione (compound
II), was confirmed using human placental GST. While compound II is chemically stable,
compound I tautomerizes to a stable sulfrane. Because these compounds are of low reactivity
(including the stable sulfrane), this biotransformation pathway may represent a physiological
protective mechanism against the DNA reactivity of epoxybutene.
3.1.2. Species Differences

3.1.2.1. In Vitro Metabolism
Species differences for several reactions described in the previous section are shown by
measuring their in vitro reaction rates using microsomal and cytosolic preparations from several
organs. Himmelstein et al. (1997) gives a comprehensive summary of the in vitro methodology
and the studies that measure the reaction rates of the reactions included in the metabolic pathways
shown in Figure 3-1. Table 3-2 summarizes the reaction rates and rate constants obtained from
the main studies that compare these differences (modified from Himmelstein et
1/28/98 DRAFT--DO
3-3 NOT CITE OR QUOTE
1/28/98
Figure 3-1. Some pathways in the metabolism of butadiene. Numbers in parentheses represent specific metabolic reactions for which literature
references are given in Table 3-1. Reactions (1), (2), (6), and (11) are mediated by cytochrome P-450-dependent monooxygenases. Glutathione (GSH) is a
substrate in reactions (3), (7), (9), (13), and (15), which are mediated by glutathione S-transferase or occur spontaneously. A GSH conjugate of
reaction(s) is excreted in the urine as N-acetyl-S-(1-hydroxy-3-butenyl)-L-cysteine. Enzyme-mediated GSH conjugates from reaction (7) include S-(2-
hydroxy-3-buten-1-yl)glutathione and S-(1-hydroxy-3-buten-2-yl)glutathione, which are subsequently excreted in the urine as 1-hydroxy-2-(N-
acetylcysteinyl)-3-butene and 2-hydroxy-1-(N-acetylcysteinyl)-3-butene. The GSH conjugate of reaction (9) is excreted in the urine as 1,2-dihydroxy-4-
(N-acetylcysteinyl)-butane. A=S-(2-hydroxy-3,4-epoxybut-1-yl)glutathione; B=S-(1-hydroxy-3,4-epoxybut-2-yl)glutathione; C=S-(1,2,3-trihydroxybut-4-
yl)glutathione; D=S-(1,3,4-trihydroxybut-2-yl)glutathione. The enzyme-mediated or spontaneous formation of GSH conjugates for reaction 13 form A
and B, which are excreted in the urine as C and D, respectively. Reactions (10) and (14) are mediated through the pentose phosphate pathway. Reactions
(8), (12), and (16) are mediated by epoxide hydrolase or occur spontaneously.
Sources: Dahl et al., 1990; Laib et al., 1990; Elfarra et al., 1991; Himmelstein et al., 1997.
3-3
1/28/98 DRAFT--DO
3-4 NOT CITE OR QUOTE
1/28/98
Table 3-1. Metabolic pathways of 1,3-butadiene metabolism

Reaction Species and tissue Reference
(1)1,3-Butadiene EB
In vitro experiments
Wistar rat liver microsomes. (1a) Malvoisin et al. (1979a)
SD rat liver microsomes. (1b) Bolt et al. (1983)
B6C3F1 and NMRI mouse; SD and Wistar rat, rhesus (1c) Schmidt and Loeser (1985)
monkey, and human (n = 1) postmitochondrial lung and
liver fractions.
Liver microsomes of rats (strain not stated), mice (1d) Wistuba et al. (1989)
(strain not stated), and humans ( n = 4).
3-4
B6C3F1 mouse liver microsomes. (1e) Elfarra et al. (1991)

SD rat, B6C3F 1 mouse, and human (n = 12) liver and (1f) Csanády et al. (1992)
lung microsomes.
SD rat and B6C3F 1 mouse liver, lung, kidney, and testis (1g) Sharer et al. (1992)
microsomes.
Purified human myeloperoxidase from human (1h) Duescher and Elfarra (1992)
polymorphonuclear leukocytes and B6C3F 1 mouse liver
microsomes.
Rat (strain not stated) liver microsomes. (1i) Cheng and Ruth (1993)
SD rat, B6C3F 1 mouse, and human (n = 6) liver (1j) Duescher and Elfarra (1994)
microsomes.
B6C3F1 mouse bone marrow cells and cell lysates, (1k) Maniglier-Poulet et al. (1995)
human bone marrow cells, and purified human
myeloperoxidase.
1/28/98
Table 3-1. Metabolic pathways of 1,3-butadiene metabolism (continued)

In vivo inhalation experiments
Closed-chamber gas-uptake system SD rats. EB in exhaled breath. (1l) Bolt et al. (1983)
SD rats. Metabolic uptake rate from untreated rats (1m) Bolt et al. (1984)
compared with that of rats pretreated with Aroclor 1254
and P-450 inhibitor.
B6C3F1 mice. Metabolic uptake rate from untreated (1n) Kreiling et al. (1986a)
rats compared with that of rats pretreated with P-450
inhibitor.
SD rats and B6C3F 1 mice. EB in exhaled breath. (1o) Kreiling et al. (1987)
SD rats and B6C3F 1 mice, pretreated with (1p) Medinsky et al. (1994)
4-methylpyrazole (P-450 inhibitor). Metabolic uptake
rate from untreated rats compared with that of rats
pretreated with P-450 inhibitor.
3-6
Nose-only exposure system SD rats and B6C3F 1 mice. EB in blood. (1q) Bond et al. (1986)
Cynomolgus monkeys. EB in blood. (1r) Dahl et al. (1990)
SD rats and B6C3F 1 mice. 1,3-butadiene and EB in (1s) Himmelstein et al. (1994)
blood.
SD rats and B6C3F 1 mice. 1,3-butadiene and EB in (1t) Bechtold et al. (1995)
blood.
SD rats and B6C3F 1 mice. EB in liver and lung tissues. (1u) Himmelstein et al. (1995)
SD rats and B6C3F 1 mice. EB in blood, fat, heart, (1v) Thornton-Manning et al.
liver, lung, spleen, and thymus. (1995a)
Female and male SD rats. EB in blood, femur, fat, (1w) Thornton-Manning et al.
lung, and mammary tissue. (1995b)
1/28/98

(2) 1,3-Butadiene 3-butenal
EB and crotonaldehyde in reaction mixture. B6C3F1 mouse liver microsomes and purified fungal (2a) Elfarra et al. (1991)
Proposed 3-butenal as intermediate in enzyme, chloroperoxidase.
formation of crotonaldehyde.
3-Butenal detected at optimal pH of 6.0. Purified fungal enzyme, chloroperoxidase. (2b) Duescher and Elfarra (1993)
(3) 3-Butenal GSH conjugates

In vivo inhalation experiments: 3-butenal-
GSH conjugate:
N-acetyl-S-(1-hydroxy-3-butenyl)-L-cysteine. SD rats and B6C3F 1 mice. Urinary metabolite detected (3a) Nauhaus et al. (1996)
3-7
in mouse, not in rat.

(4) 3-Butenal crotonaldehyde
Same experiment as (2a) above. (4a) Elfarra et al. (1991)

B6C3F1 mouse and SD rat liver, lung, kidney, and testis (4b) Sharer et al. (1992)
microsomes. Crotonaldehyde detected in mouse but not
in rat.
Same experiment as (1h) above. (4c) Duescher and Elfarra (1992)
Same experiment as (1i) above. (4d) Cheng and Ruth (1993)
Crotonaldehyde identified as tautomerization Same experiment as (2b) above. (4e) Duescher and Elfarra (1993)
product of 3-butenal.
1/28/98

Human (n = 6), B6C3F 1 mouse, and SD rat liver (4f) Duescher and Elfarra (1994)
microsomes and microsomes derived from human -
lymphoblastoid cells containing cDNA-expressed CYP
1A1, 1A2, 2A6, 2B6, 2D6, 2D1, 3A4. Same
experiment as (1j) above.
(5) Crotonaldehyde acrolein + CO 2 No published literature supporting
this reaction in vitro or in vivo.
(6) EB DEB
Wistar rat liver microsomes. (6a) Malvoisin et al. (1979b)
Detected two stereoisomers of DEB. Wistar rat liver microsomes. (6b) Malvoisin and Roberfroid
(1982)
3-8
SD rat, B6C3F 1 mouse, and human liver (n = 12) and (6c) Csanády et al. (1992)
lung (n = 5) microsomes. Identified DEB only in mouse
liver microsomes.
Human microsomes containing cDNA-expressed CYP (6d) Seaton et al. (1995)

isozymes (1A1, 1A2, 2A6, 2D6, 2E1, 2F1, 3A4) and
SD rat, B6C3F 1 mouse and human (n = 10) liver
microsomes.
Same experiments as (1s) above. DEB in blood of (6e) Himmelstein et al. (1994)
mice, not rat.
Same experiments as (1t) above. (6f) Bechtold et al. (1995)
Same experiments as (1u) above. DEB in lungs of mice (6g) Himmelstein et al. (1995)
but not rat.
1/28/98

Same experiments as (1v) above. DEB in blood, heart, (6h) Thornton-Manning et al.
and lung, thymus, fat, spleen and liver, higher in mice (1995a)
than in rats.
Same experiments as (1w) above. DEB in blood, bone

marrow, fat, lung, mammary (female only), higher in (6i) Thornton-Manning et al.
females than in males. (1995b)
(7) EB GSH conjugates
Same experiment as (1b) above. (7a) Bolt et al. (1983)
SD rat, NMRI mouse, and human ( n = 1) liver (7b) Kreuzer et al. (1991)
3-9
microsomes and cytosol.

Purified human placental class GSH S-transferase. (7c) Sharer et al. (1991)
SD rat and B6C3F 1 mouse liver, lung, kidney, and testis (7d) Sharer et al. (1992)
cytosol. Same experiment as (1g) above.
SD rat, B6C3F 1 mouse, and human liver (n = 12) and

lung (n = 5) microsomes and cytosol. Same experiment (7e) Csanády et al. (1992)
as (1f) and (6c) above.
1/28/98

In vivo experiments: EB-GSH conjugates:
1-hydroxy-2-(N-acetylcysteinyl)-3-butene Inhalation exposure of EB to SD and F344/N rats, (7f) Sabourin et al. (1992)
and 2-hydroxy-1-(N-acetylcysteinyl)-3- B6C3F1 mice, Syrian hamsters, and cynomolgus
butene. monkeys. Urinary metabolites.
S-(2-hydroxy-3-buten-1-yl)glutathione. i.p. injection of EB to SD rats. Isolated and identified (7g) Sharer and Elfarra (1992)
regioisomeric GSH conjugates in bile of rats.
S-(2-hydroxy-3-buten-1-yl)-N-acetyl-L- SD rats and B6C3F 1 mice, by i.p. injection. Urinary (7h) Elfarra et al. (1995)
cysteine and S-(1-hydroxy-3-buten-2-yl)-N- metabolites.
acetyl-L-cysteine.
S-(1-hydroxymethyl)-2-propenyl)-L-cysteine Inhalation exposure of rats and mice. Same study as (7i) Nauhaus et al. (1996)
in mouse, not in rat. N-acetyl-S-(2- (3a) above. Urinary metabolites.
3-10
(hydroxymethyl)-2-propenyl)-L-cysteine and
N-acetyl-S-(2-hydroxy-3-butenyl)-L-cysteine
in both mouse and rats.
(8) EB BD
Wistar rat liver microsomes. (8a) Malvoisin and Roberfroid
(1982)
SD rat liver microsomes treated with inhibitor of (8b) Bolt et al. (1983)
epoxide hydrolase. Same experiment as (1b) and (7a)
above.
SD rat, NMRI mouse, and human ( n = 1) liver (8c) Kreuzer et al. (1991)
microsomes.
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Rat, mouse, and human lung and liver microsomes. (8d) Csanády et al. (1992)
Same study as (1f) and (6c) above.
Rat (strain not stated) liver microsomes. Same (8e) Cheng and Ruth (1993)
experiment as (4d) above.
In vivo inhalation experiments: BD-GSH

conjugates:
1,2-dihydroxy-4-(N-acetylcysteinyl)-butane. Same experiment as (7f) above. Urinary metabolites. (8f) Sabourin et al. (1992)
(BD necessary intermediate of this product.)
Human urinary clearance predominantly via F344/N rats, B6C3F 1 mice, and humans in (8g) Bechtold et al. (1994)
epoxide-hydrolase-mediated hydrolysis. occupational exposure. See (9b) below.
Same study as (3a) and (7i) above. (8h) Nauhaus et al. (1996)
3-11
(9) BD GSH conjugates

In vivo inhalation experiments: BD-GSH
conjugates:
1,2-dihydroxy-4-(N-acetylcysteinyl)-butane. Same experiments as (7f) and (8f) above. Urinary (9a) Sabourin et al. (1992)
metabolites in monkey, rat, hamster, and mouse.
1,2-dihydroxy-4-(N-acetylcysteinyl)-butane. Same experiments as (8g) above. Urinary metabolites (9b) Bechtold et al. (1994)
in humans.
N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine Same study as (3a), (7i), and (8h) above. Urinary (9c) Nauhaus et al. (1996)
in mouse and rat, N-acetyl-S-(1- metabolites.
(hydroxymethyl)-3,4-dihydroxypropyl)-L-
cysteine in mouse but not in rat.
1/28/98

(10) BD 1,3-dihydroxypropanone
Same study as (3a), (7i), (8h), and (9c) above. (10a) Nauhaus et al. (1996)
(11) BD 3,4-epoxy-1,2-butanediol
Wistar rat liver microsomes. Same experiment as (6b) (11a) Malvoisin and Roberfroid
above. (1982)
Rat (strain not stated) liver microsomes. Same (11b) Cheng and Ruth (1993)
experiments as (4d) and (8e) above.
(12) DEB 3,4-epoxy-1,2-butanediol
3-12
Human (n = 6), SD rat, and B6C3F 1 mouse liver and (12a) Boogaard and Bond (1996)
lung microsomes.
(13) DEB GSH conjugates

In vitro experiments: DEB-GSH
conjugates:
S-(2-hydroxy-3,4-epoxybut-1- Salmonella typhimurium TA1535 transfected with rat (13a) Thier et al. (1995)
yl)glutathione and S-(4-hydroxy-2,3- GSH S-transferase 5-5 cDNA.
epoxybut-1-yl)glutathione.
SD rat, B6C3F 1 mouse, and human (n = 6) liver cytosol (13b) Boogaard et al. (1996)
and SD rat and B6C3F 1 lung cytosol.
In vivo experiments: DEB-GSH conjugates:
N-acetyl-S-(1-(hydroxymethyl)-3,4- Same experiment as (3a), (7i), (8h), (9c), and (10a) (13c) Nauhaus et al. (1996)
dihydroxypropyl)-L-cysteine in mouse, but above. Urinary metabolites.
not in rat.
Table 3-2. Species comparison of reaction rates for epoxidation, GSH conjugation, and hydrolysis reactions
1/28/98
involved in the metabolism of 1,3-butadiene
Reaction and tissue Species Strain Reaction rate a KM Reference

or Vmax (mM)b (exposure concentration) c
(1) 1,3-Butadiene EB
Liver microsomes Mouse All 0.24-9.9 All studies with species

Rat All 0.36-45 differences data
Monkey
Human 0.121-22.8
Mouse B6C3F1, NMRI 0.24-0.40d (1c) Schmidt and Loeser (1985)

Rat SD, Wistar 0.08-0.1d (30,000 ppm 1,3-butadiene)
Monkey rhesus 0.73g
Human 0.12d
3-13
Mouse B6C3F1 2.6 2.0 (1f) Csanády et al. (1992)

Rat SD 0.59 3.74 (600-25,000 ppm 1,3-butadiene)
Monkey
Human 1.18 5.14
Mouse B6C3F1 6.4 (1g) Sharer et al. (1992)

Rat SD 3.0 (330,000-660,000 ppm 1,3-
Monkey butadiene)
Human
Mouse B6C3F1 9.2 160 (1j) Duescher and Elfarra (1994)

Rat SD 2.0 120 (30,000-660,000 ppm 1,3-
Monkey butadiene)
Human 10.4-22.8 200-400
1/28/98
Table 3-2. In vitro rates and rate constants for epoxidation, GSH conjugation, and hydrolysis reactions
involved in the metabolism of 1,3-butadiene (continued)

(µM)b (exposure concentration) c
Lung microsomes Mouse All 2.3-6.1 All studies
Rat All 0.16-1.5
Human 0.15
Mouse B6C3F1, NMRI 4.4-5.6e (1c) Schmidt and Loeser (1985)

Rat SD, Wistar 0.6-0.91e (30,000 ppm 1,3-butadiene)
Human
Mouse B6C3F1 2.3 5.01 (1f) Csanády et al. (1992)

Rat SD 0.16 7.75 (600-25,000 ppm 1,3-butadiene)
Human 0.15 2.0
3-15
Mouse B6C3F1 6.1 (1g) Sharer et al. (1992)

Rat SD 1.5 (330,000-660,000 ppm 1,3-
Human butadiene)
Kidney microsomes Mouse B6C3F1 23.8 (1g) Sharer et al. (1992)

Rat SD 0.5 (330,000-660,000 ppm 1,3-
butadiene)
(6) EB DEB
Liver microsomes Mouse B6C3F1 1.4 141 (6d) Seaton et al. (1995)
Rat SD 0.41 145 (5-1,000 µM EB)
Human 0.38-1.2 304-880
1/28/98

Liver cytosol Mouse B6C3F1 107 3,100 (7d) Sharer et al. (1992)
Rat SD 71 3,100 (2-10 mM EB)
Human
Mouse B6C3F1 500 35,300 (7e) Csanády et al. (1992)

Rat SD 241 13,800 (20-200 ppm EB)
Human 45.1 10,400
3-16 DRAFT--DO NOT CITE OR QUOTE
Lung cytosol Mouse B6C3F1 12 3,100 (7d) Sharer et al. (1992)

Rat SD 3 3,100 (2-10 mM EB)
Human
Mouse B6C3F1 273 36,500 (7e) Csanády et al. (1992)

Rat SD 44.2 17,400 (20-200 ppm EB)
Human 2.56 10-4 f
Kidney cytosol Mouse B6C3F1 16 3,100 (7d) Sharer et al. (1992)

Rat SD 7 3,100 (2-10 mM EB)
Testis cytosol Mouse B6C3F1 30 3,100 (7d) Sharer et al. (1992)

Rat SD 51 3,100 (2-10 mM EB)
1/28/98

Purified placental GSH Human 500 10,000 (7d) Sharer et al. (1992)
S-transferase
Spontaneousg 2.01 · 10-4
(8) EB BD
Liver microsomes Mouse NMRI 19 1,500 (8c) Kreuzer et al. (1991)

Rat SD 17 700 (30, 300, 3,000 ppm EB)
Human 14 500
3-17 DRAFT--DO NOT CITE OR QUOTE
Mouse B6C3F1 5.79 1,590 (8d) Csanády et al. (1992)

Rat SD 2.48 260 (20-200 ppm EB)
Human 9.2-58.1 240-1,650
Lung microsomes h Mouse B6C3F1 1.86 103 (8d) Csanády et al. (1992)
Rat SD 1.32 103 (20-200 ppm EB)
Human 3.19-7.55 103
Spontaneousg Human 7.75 · 10-4 (8d) Csanády et al. (1992)
(12) DEB hydrolysis products

Liver microsomes Mouse B6C3F1 32.0 8,100 (12a) Boogaard and Bond (1996)
Rat SD 52.9 2,760 (0.185-15 mM DEB)
Human 156 4,800
1/28/98

Lung microsomes Mouse 49.3 7,500 (12a) Boogaard and Bond (1996)
Rat 19.3 7,100 (0.558-5 mM EB)
Human 21.7 2,830

Liver cytosol Mouse B6C3F1 162 6,400 (13b) Boogaard et al. (1996)
Rat SD 186 24,000 (0.1-25 mM DEB)
Human 6.4 2,100
Lung cytosol Mouse 38.5 1,700 (13b) Boogaard et al. (1996)

Rat 17.1 4,200 (0.1-25 mM DEB)
1.65 · 10-3
3-18
(13b) Boogaard et al. (1996)

Spontaneousg (0.1-25 mM DEB)
a
Reaction rates are either from the reported reaction rates (units of nmol min-1 mg microsomal or cytosolic protein-1unless otherwise noted) or from maximum
reaction rate (Vmax in units of nmol min-1 mg microsomal or cytosolic protein-1 unless otherwise noted), when corresponding KMis given.
b
Concentration at one-half maximum reaction rate.

c
Numbers and letters in parentheses preceding reference refer to appropriate metabolic pathway shown in Figure 3-1 and summarized in Table 3-1.
d
Values reported were corrected from nmol min-1 g tissue-1 to nmol min-1 mg microsomal protein-1 assuming values of 11.6 (mouse), 16.8 (rat),
and 14.5 (human) mg microsomal protein g tissue-1 reported by Csanády et al. (1992).
e
Value has units of nmol min-1 g tissue-1.
f
None of the human lung cytosolic fractions displayed Michaelis-Menten reaction kinetics. The reactions are described by first-order kgsh with units
of L nmol min-1 mg protein-1.
g
Spontaneous conjugation rate of EB or DEB with GSH is described by first-order kgsh with units L min-1 mmol-1.
h
Hydrolysis of epoxybutene in lung microsomes is described as first-order khy with units of min-1 mg protein-1.
I
Spontaneous hydrolysis rate of epoxybutene or diepoxybutane in 0.1 M phosphate buffer is described as first-order khy with units of min-1.
Source: Modified from Himmelstein et al., 1997.

al., 1997). In general, the range of reaction rates or maximal reaction velocity (Vmax) for different
reactions in different tissues do not provide a clear pattern of species differences; in particular, the
range of values for human tissues spans the range of values for both rats and mice. However,
within any single study, for the oxidation of 1,3-butadiene to epoxybutene, the reaction rates of
liver and lung microsomes are higher in rats than in mice. Multiple cytochrome P-450 enzymes
are involved in the metabolism of 1,3-butadiene. For example, in human liver microsomes, the
metabolic oxidation of 1,3-butadiene to epoxybutene is principally mediated by P-450 isoenzymes
2A6 and 2E1. Biotransformation of 1,3-butadiene to the non-DNA-reactive butene diol is the
predominant pathway observed in in vitro metabolism studies that used hepatic microsomes from
rats and humans, and formation of the DNA-reactive diepoxybutane is relatively minor in these
species. However, the latter pathway is significant in mouse hepatic microsomes. 1,3-Butadiene
can also be metabolized to epoxybutene by human myeloperoxidase and by mouse and human
bone marrow cells.
In the Csanády et al. (1992) study, the authors also extrapolated the kinetic constants
obtained from in vitro experiments to equivalent in vivo rates by adjusting the in situ protein
content and organ weights across species, as shown in Table 3-3. However, for GST, Kohn and
Melnick (1993) pointed out that the rate constants should be adjusted to the mg cytosolic
protein/g liver instead of to the mg microsomal protein/g liver as done by Csanády et al. (1992).
The corrected values are also included in Table 3-3. These can all be used in pharmacokinetic
models as hepatic and lung metabolic clearance.
3.1.2.2. In Vivo Pharmacokinetics

In vivo pharmacokinetic studies examine absorption, distribution, metabolism, and/or
elimination. Most studies report results on several of these four components. Absorption is often
measured either by the distribution of 1,3-butadiene and/or its metabolites in tissue organs or by
the elimination of 1,3-butadiene metabolites in excreted urine, feces, and exhaled air. In vivo
metabolism studies include measurements of concentration profiles of the various metabolite
pools after exposure to butadiene. Metabolic kinetic constants are usually calculated from the
rate of formation of the metabolites or from the clearance rate evaluated from excretion data.
This section summarizes the in vivo pharmacokinetic studies. Because inhalation is the principal
route of exposure to 1,3-butadiene, most of the absorption data for the chemical have been
derived from inhalation exposure studies. Based on the blood:air partition coefficient for 1,3-
butadiene (0.603 in vitro; 0.645 in vivo), the passage of 1,3-butadiene from the air into the blood
is by simple diffusion (Carpenter et al., 1944).
1/28/98
Table 3-3. Rate constants for in vivo hepatic clearance of 1,3-butadiene and EBa
(extrapolated from in vitro)
Cytosolic Microsomal
Cytochrome P-450 Epoxide glutathione conjugation of First-order
monooxygenaseb hydrolaseb,c S-transferaseb EB with GSHd hydrolysis
Mouse 55.9 0.16 4.4 0.011 0.0028

0.55e
Rat 7.92 0.48 5.7 0.024 0.0022
Human 6.19 0.86 0.46 0.069 0.0014
a
Values are in units of L/h/kg.
b
In vivo Vmax values were calculated from in vitro Vmax (tables 3-1 through 3-3) and adjusted for interspecies differences in
microsomal and cytosolic protein concentrations and liver volume. Mouse, rat, and human liver microsomal concentrations
were 11.6, 16.8, and 14.5 mg/g liver, respectively. Mouse, rat, and human liver cytosolic concentrations were 11.6, 16.8, and 14.5 mg/g
3-20
liver, respectively. Mouse, rat, and human liver cytosolic concentrations were 82.8, 108, and 58 mg/g liver, respectively. Liver organ
volumes for mice, rats, and humans were 6.2, 5.0, and 3.1% of body weight, respectively. In vivo hepatic clearance values (V max/KM
expressed in L/h/kg) were estimated by dividing the in vivo V max values by the apparent in vitro KM’s for the reaction.
c
Modified according to Kohn and Melnick, 1993.
d
For nonenzymic hydrolysis and reaction with glutathione, in vivo clearance was calculated using the organ fractions in footnote b.
To estimate the in vivo clearance for reaction with glutathione, a concentration of 10 mM GSH was used.
e
Rate constant for metabolism of EB to DEB.
Sources: Csanády et al., 1992; Kohn and Melnick, 1993.

Two main in vivo inhalation systems are used to conduct inhalation studies. The first one
is the closed-system inhalation chamber, and the second one is the nose-only exposure inhalation
system. These studies are reviewed by Himmelstein et al. (1997) and summarized in Tables 3-4 to
3-6 for the closed inhalation chamber studies and Tables 3-7 to 3-10 for the nose-only inhalation
studies.
In the closed-system inhalation chamber study, rats or mice are placed in a desiccator jar
chamber. Two rats or up to eight mice per experiment are exposed to different initial 1,3-
butadiene chamber concentrations. Air samples from the desiccator are measured directly by gas
chromatography-mass spectrometry (GC-MS) through an air valve. With the use of a two-
compartment pharmacokinetic model (Filser and Bolt, 1981), shown in Figure 3-2, uptake and
clearance kinetic constants of 1,3-butadiene and epoxybutene can be evaluated, as shown in
Tables 3-5 and 3-6, which give the results of these studies. Because the metabolic elimination
rate constant (kel) cannot be determined accurately from the gas uptake studies, 1,3-butadiene and
epoxybutene were administered intraperitoneally to the mice and rats, and exhaled 1,3-butadiene
and epoxybutene concentrations were monitored in the chamber and used to evaluate kel (Bolt et
al., 1984). Tables 3-5 and 3-6 show that for both 1,3-butadiene and epoxybutene, uptake (k12V1)
and clearance (Cltot) in mice are about twofold greater than in rats. Although the exhalation rate
constant (k21) and metabolic elimination rate constant (kel) are comparable for 1,3-butadiene in
both mice and rats, mice exhaled epoxybutene about twice as much as rats (k21), whereas the
metabolic rate constant (kel) is about fivefold higher in rats than in mice (Laib et al., 1990). Under
these conditions, the steady-state epoxybutene concentration in mice is about sixfold that in rats
(Melnick and Huff, 1992; Himmelstein et al., 1994).
A second inhalation experimental system is the nose-only exposure, where exhaled breath
is sampled by placing the animals in plethysmography tubes. Additional blood and tissue samples
can also be obtained by sacrifice of the animals after different exposure durations. However,
while the air samples are measured at real time, all blood and tissue samples are subjected to some
time delay due to processing of the samples. These studies are summarized in Table 3-7
(modified from Himmelstein et al., 1997). Table 3-8 summarizes the results of the studies
showing that 1,3-butadiene and its epoxide metabolites (epoxybutene and diepoxybutane) have
been found in blood at different inhalation exposure concentrations to 1,3-butadiene in rats, mice,
and monkeys.
Thornton-Manning et al. (1995a) also examined the disposition of epoxybutene and
diepoxybutane in various tissues following nose-only inhalation exposure of male Sprague-
1/28/98 3-21 DRAFT-DO NOT CITE OR QUOTE

1/28/98
Table 3-4. Summary of closed-chamber inhalation studies

Reaction and reference a Description of experiment Finding
(1l) Bolt et al. (1983) Exposure of SD rats to butadiene at 6,000 to 7,000 Quantified epoxybutene in exhaled breath.
ppm initial concentration. Peak concentrations of epoxybutene were 2
to 4 ppm at 15 h after exposure.
(1m) Bolt et al. (1984) Exposure of SD rats to butadiene at initial Metabolic uptake rate = 220 µmol h 1
concentrations ranged from 90 to 12,000 ppm. kg 1 for untreated rats when the butadiene
chamber concentration was >1,500 ppm.
Pretreatment with Aroclor 1254 caused a
linear increase in the metabolic uptake rate
from 220 to 1,200 µmol h 1 kg 1; P-450
inhibitor diethyldithiocarbamate completely
inhibited metabolism.
3-22
(1n) Kreiling et al. (1986a) Exposure of B6C3F 1 mice to butadiene at initial Maximal metabolic uptake rate = 400
concentrations ranged from 10 to 5,000 ppm. µmol h 1 kg 1. Pretreatment of mice
with P-450 inhibitor
diethyldithiocarbamate completely
inhibited uptake of butadiene.

Filser and Bolt (1984) Exposure of SD rats to epoxybutene at initial Linear metabolic uptake occurred up to
concentration ranged from 500 to 5,000 ppm (not 5,000 ppm. V max > 2,600 umol · h -1 · kg-1
reported by author, estimated from Figure 3-2 in (see text for description of model used in
their paper). calculation).
1/28/98
Table 3-4. Summary of closed-chamber inhalation studies (continued)

Uptake of EB b
Kreiling et al. (1987) Exposure of B6C3F 1 mice to epoxybutene at initial Saturated metabolic uptake occurred
concentration ranging from 10 to 5,000 ppm between 100 and 500 ppm. V max = 350
(estimated from Figure 1 of Kreiling et al., 1986b). umol · h-1 · kg-1.
Abbreviations: ppm = parts per million; SD = Sprague-Dawley rat; Vmax = Michaelis-Menten enzyme kinetic constant expressing maximum metabolic rate.
a
Numbers and letters preceding reference refer to the appropriate metabolic pathway shown in Figure 3-1 and summarized in Table 3-1.
b
Reaction rates measured as uptake of epoxybutene, which could involve several reactions shown in Figure 3-1 and summarized in Table 3-1.

3-23
Table 3-5. Toxicokinetic parameters for uptake and elimination of
1,3-butadiene in mice and rats
Parameter (units) Mouse Rat Definition of parameter
K12V1 (mL/h) 10,280 5,750 Equilibrium constant between chamber volume

and test animals; first order, V1 V2.
K21 (h-1) 3.2 2.5 Equilibrium rate constant between chamber
volume and animals; first order, V2 V1.
Keq (NA) 2.7 2.3 Static equilibrium constant representing virtual
absence of metabolism.
Kst (NA) 1 0.5 Steady-state concentration; ratio of
concentration in animal to chamber
concentration.
kel (h-1) 7.6 8.8 First-order metabolic elimination rate constant.
Cltota,b (mL/h) 7,300 4,500 Total clearance of chemical from chamber.
Vmax (µmol/h/kg) 400 220 Maximum rate of metabolism of chemical.
a
Calculated for V1 .
b
Valid for linear range of metabolism (up to 1,000 ppm for both species).
NA = not applicable.
Source: Filser and Bolt, 1981; Kreiling et al., 1990.
Table 3-6. Toxicokinetic parameters for the uptake and elimination of

epoxybutene in rats and mice
Parameter (units) Mouse Rat Definition of parameter
k12V1 (mL/h) 33,500 13,800 Equilibrium constant between chamber volume

and test animals; first order, V1 V2.
K21 (h-1) 0.79 0.37 Equilibrium rate constant between chamber
volume and animals; first order, V2 V1.
Keq (NA) 42.5 37 Static equilibrium constant representing virtual
absence of metabolism.
Kst (NA) 10.2 1.16 Steady-state concentration; ratio of
concentration in animal to chamber
concentration.
kel (h-1) 2.3 11.5 First-order metabolic elimination rate constant.
Cltota,b (mL/hr) 24,900 13,400 Total clearance of chemical from chamber.
Vmax (µmol/h/kg) 350 >2,600 Maximum rate of metabolism of chemical.
Metabolic saturation (ppm) 500 >5,000 Concentration resulting in saturated
metabolism.
a
Calculated for V1 .
b
Valid for linear range of metabolism (up to 1,000 ppm for both species).
Source: Filser and Bolt, 1981; Kreiling et al., 1987; Laib et al., 1990.

1/28/98
Table 3-7. Summary of nose-only inhalation studies

Reaction and referencea Description of experiment Finding
(1q) Bond et al. (1986) Exposure of SD rats and B6C3F 1 mice to Epoxybutene tentatively identified in blood after 2, 4, or 6 h
1,3-butadiene (7, 70, or 1,000 ppm for of exposure. EB blood concentrations were 0.4 and 3.3 µM
up to 6 h). in rats exposed to 70 and 1,000 ppm 1,3-butadiene and 0.5,
1.6, and 13 µM in mice exposed to 7, 70, and 1,000 ppm,
respectively. Concentration of EB in blood of mice 2 to 3
times > rats.
(1r) Dahl et al. (1990) Exposure of cynomolgus monkeys to Blood samples collected at single time immediately after
1,3-butadiene (10, 300, or 8,000 ppm for exposure. Quantitation of EB in blood by vacuum-trap
2 h). distillation method. Results shown in Table 3-8. Total
butadiene metabolites in blood were 5 to 50 times lower in
monkey than in the mouse and 4 to 14 times lower than in the
rat (rat and mouse data were from Bond et al., 1986).
3-25
(1s) Himmelstein et al. (1994) Exposure of SD rats and B6C3F 1 mice to 1,3-Butadiene and EB pharmacokinetics characterized in
1,3-butadiene (62.5, 625, or 1,250 ppm blood by GC (butadiene) and GC-MS (EB). Butadiene
up to 6 h; blood collected at 2 to 6 h of steady-state concentrations (µM) ranged from 2.4 to 58
exposure and up to 30 min (mice) and 1.3 to 37 (rats). EB steady-state concentrations
postexposure). (µM) ranged from 0.56 to 8.6 (mice) and 0.07 to 1.3 (rats);
EB blood concentration in mice was 4 to 8 times > rats.
(1t) Bechtold et al. (1995) Exposure of SD rats and B6C3F 1 mice to Quantitated butadiene and EB in blood by GC-GC-MS.
1,3-butadiene (100 ppm for 4 h; blood Blood levels (µM) of butadiene were 4.1 (rat) and 2.9
collected at end of exposure). (mouse). Blood levels of EB (µM) were 0.10 (rat) and 0.38
(mouse).
(1u) Himmelstein et al. (1995) Exposure of SD rats and B6C3F 1 mice to Quantitated EB concentration by GC-MS in mouse lung 14
1,3-butadiene (625, 1,250, or 8,000 [rats times > rat lung, mouse liver 5 to 8 times > rat liver. Peak
only] ppm for 6 h; tissue samples concentrations of EB (nmol g tissue 1) during exposures
collected at 3 and 6 h of exposure and 6 were 2.6 to 3.7 (mouse lung), 0.16 to 1.3 (rat lung), 0.58 to
and 12 min postexposure). 0.93 (mouse liver), and 0.06 to 1.2 (rat liver).
1/28/98
Table 3-7. Summary of nose-only inhalation studies (continued)

(1v) Thornton-Manning et al. (1995a) Exposure of SD rats and B6C3F 1 mice to Quantitated EB by GC-GC-MS. Detection limits were
1,3-butadiene (62.5 ppm for 4 h; samples 0.031, 0.037, and 0.062 nmol g tissue 1 in blood, heart, and
collected at 2 and 4 h of exposure and at lung, respectively. EB concentrations were 3 to 74 times
0.5 and 1 h postexposure included blood, higher in tissues of mice compared with rats. EB was not
fat, heart, liver, lung, spleen, and detected in lung or liver of rats.
thymus).
(1w) Thornton-Manning et al. Exposure of female and male SD rats to Quantitated EB by GC-GC-MS. EB concentrations were
(1995b) butadiene (6.25 ppm for 6 h, samples similar for males and females. At 6 h exposure. Results
collected at end of exposure included summarized in Table 3-10.
blood, femur, fat, lung, and mammary
tissue).
(1y) Thornton-Manning et al. (1996) Exposure of female SD rats and female Quantitated EB by GC-GC-MS. EB levels were 5- and 1.6-
B6C3F1 mice after either a single fold higher in mammary tissue and 2- and 1.4-fold higher in
6 h (daily 6 h exposure for 10 days to fat tissue in rats and mice, respectively, after repeated
3-26
62.5 ppm 1,3-butadiene). exposures. DEB levels were 7.7 ± 2.2 and 12.5 ± 0.8 pmol/g
in fat of rats and 265 ± 19 and 191 ± 29 pmol/g in mammary
tissue of mice after single and repeated inhalation exposures,
respectively.
(3) 3-Butenal GSH conjugates
(3a) Nauhaus et al. (1996) Exposure of SD rats and B6C3F 1 mice to Quantitated the urinary metabolite N-acetyl-S-(1-hydroxy-3-
1,3-[13C]-butadiene (800 ppm up to 5 h; butenyl)-L-cysteine in mouse urine using 13C-NMR. This
urine collected during exposure and for metabolite represented 3.7% of total urinary metabolites
up to 20 h postexposure). excreted by mice but was not detected in rat urine.
(6) EB DEB
(6e) Himmelstein et al. (1994) Exposure of rats and mice to 1,3- Quantitated time course of DEB in blood of mice by GC-
butadiene. Same conditions as (1s) MS. Peak concentrations of DEB in the blood of mice were
above. 0.65, 1.9, and 2.5 µM after 6 h of exposure to 62.5, 625, or
1,250 ppm 1,3-butadiene; DEB not quantitated in rats.
Detection limit = 0.13 µM.
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(6f) Bechtold et al. (1995) Exposure of rats and mice to 1,3- Same finding as (6d) above. DEB detected in mouse and not
butadiene. Same conditions as (1t) rat blood; DEB concentration = 0.33 µM in mouse blood
above. after 4 h exposure to 100 ppm 1,3-butadiene. Detection limit
= 0.1 µM.
(6g) Himmelstein et al. (1995) Exposure of rats and mice to 1,3- Quantitated time course of DEB in lungs of mice by GC-MS.
butadiene. Same conditions as (1u) Peak concentrations in mice exposed to 625 and 1,250 ppm
above. 1,3-butadiene were 0.71 and 1.5 nmol g tissue 1,
respectively. Detection limit = 0.04 µM.
(6h) Thornton-Manning et al. (1995a) Exposure of rats and mice to 1,3- Quantitated DEB by GC-GC-MS. Detection limits were
butadiene. Same conditions as (1v) 0.0016, 0.031, and 0.026 nmol · g tissue -1 in blood, heart,
above. and lung, respectively. DEB concentrations were 40- to 163-
fold lower in rat tissues compared with mice. DEB was not
detected in liver of rats. Results summarized in Table 3-9.
3-27
(6i) Thornton-Manning et al. (1995b) Exposure of female and male SD rats to Quantitated DEB by GC-GC-MS. DEB concentrations were
1,3-butadiene. Same conditions as (1w) 3.6- to 7.1-fold greater in tissues of female rats compared
above. with tissues of male rats. Results summarized in Table 3-9.
(7f) Sabourin et al. (1992) Exposure of SD and F344/N rats, Products identified in urine included 1-hydroxy-2-( N-
B6C3F1 mice, Syrian hamsters, and acetylcysteinyl)-3-butene and 2-hydroxy-1-( N-

cynomolgus monkeys to 1,3-[ 14C]- acetylcysteinyl)-3-butene.
butadiene (8,000 ppm, 0.78 µCi/mmol).
(7i) Nauhaus et al. (1996) Exposure of rats and mice to 1,3- Quantitated urinary metabolites (as percentage of total 13C)
butadiene. Same study as (3a) above. using NMR. S-(1-(hydroxymethyl)-2-propenyl)-L-cysteine
(4.7%) present in mouse urine but not detected in rat urine.
N-acetyl-S-(2-(hydroxymethyl)-2-propenyl)-L-cysteine was
present in mouse (22%) and rat (53%) urine. N-acetyl-S-(2-
hydroxy-3-butenyl)-L-cysteine was present in mouse (44%)
and rat (18%) urine.
1/28/98

(8) EB BD
(8g) Bechtold et al. (1994) Exposure of F344/N rats and B6C3F 1 Predominant pathway for clearance of epoxybutene in
mice to 1,3-butadiene (11.7 ppm for 4 h humans is by epoxide hydrolase-mediated hydrolysis rather
by nose-only exposure). In vivo than direct conjugation with GSH. See (9b) below.
inhalation exposure of humans
occupationally exposed to 1,3-butadiene
(human study described in more details in
text).
(8h) Nauhaus et al. (1996) Exposure of rats and mice to 1,3- Quantitated BD in mouse and rat urine using 13C-NMR.
butadiene. Same study as (3a) and (7i) This metabolite represented 2.9% and 5.0% of total 13C-
above. metabolites excreted by mice and rats, respectively.
(9) 3-Butene BD
3-28
(9b) Bechtold et al. (1994) Same experiment as (8g) above. Quantitated 1,2-dihydroxy-4-( N-acetylcysteinyl)-butane in
human urine using isotope-dilution GC-MS. Metabolite
represents 97% of GSH conjugates derived directly from EB
(see (7g) above) or indirectly by epoxide hydrolase-mediated
hydrolysis and GSH conjugation of BD (see (8e) above).
Quantitated N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine in
(9c) Nauhaus et al. (1996) Exposure of rats and mice to 1,3- mouse (7.1%) and rat (26.4%) urine using 13C-NMR. N-
butadiene. Same study as (3a), (7i), and acetyl-S-(1-(hydroxymethyl)-3,4-dihydroxypropyl)-L-
(8h) above. cysteine occurred in mouse (7.1%) urine but was not
detected in rat urine. This latter metabolite was also
surmised to be a product of DEB as described in reaction 13
below.
1/28/98

(10) BD 1,3-dihydroxypropanone
(10a) Nauhaus et al. (1996) Exposure of rats and mice to 1,3- Quantitated 1,3-dihydroxypropanone in rat urine as 5.3% of
butadiene. Same study as (3a), (7i), total 13C-metabolites excreted. It was not detected in mouse
(8h), and (9c) above. urine. Transformation of DEB to 1,3-dihydroxypropanone
may also contribute to excretion of this compound (see
reaction 14 below). This reaction would also be expected to
contribute to the formation of CO 2.
(13c) Nauhaus et al. (1996) Exposure of rats and mice to 1,3- Quantitated N-acetyl-S-(1-hydroxymethyl)-3,4-
butadiene. Same study as (3a), (7i), dihydroxypropyl)-L-cysteine in mouse (7.1%) urine. This
(8h), (9c), and (10a) above. metabolite was not detected in rat urine and also may be
formed as a product of BD as described in study (9c) above.
3-29
(14) DEB 1,3-dihydroxypropanone

(14a) Nauhaus et al. (1996) Exposure of rats and mice to 1,3- Quantitated 1,3-dihydroxypropanone in rat urine as 5.3% of
butadiene. Same study as (3a), (7i), total 13C-metabolites excreted. It was not detected in mouse
(8h), (9c), (10a), and (13c) above. urine. Transformation of BD to 1,3-dihydroxypropanone
may also contribute to excretion of this compound (see study

(10a) above). This reaction would also be expected to
contribute to the formation of CO 2.
(15) 3,4-Epoxy-1,2-butanediol GSH No published literature supporting this GSH conjugates of epoxybutanediol have not been
conjugates reaction. quantitated in either in vitro or in vivo studies.
1/28/98

(17) Erythritol CO2 No published literature supporting this Bond et al. (1986) showed that CO 2 was exhaled in the
reaction. breath of SD rats and B6C3F 1 mice exposed to 1,3- [14C]-
butadiene, although the chemical reactions leading to CO 2
formation are unknown. Mass balance of 14CO2 from
exhaled breath and residual 14CO2 from carcass showed that
mice had greater uptake of butadiene than rats. Assumption
is made that CO 2 derives from erythritol. Dahl et al. (1991)
also measured 14CO2 in exhaled breath of monkeys exposed
to 1,3-[14C]-butadiene. Uptake and retention of 14C in mice >
rat > monkey.
Abbreviations: BD = 3-butene-1,2-diol; DEB = 1,2:3,4-diepoxybutane; CO2 = carbon dioxide; EB = 1,2-epoxy-3-butene; GC = gas chromatography; GSH = glutathione; MS =
mass spectrometry; NMR = nuclear magnetic resonance spectrometry; ppm = parts per million; SD = Sprague-Dawley rat.
a
Numbers and letters preceding reference refer to the appropriate metabolic pathway shown in Figure 3-1 and summarized in Table 3-1.
3-30

1/28/98
Table 3-8. Comparison of 1,3-butadiene, epoxybutene, and diepoxybutane blood concentration data from
different species of laboratory animals exposed to 1,3-butadiene by inhalation
Butadiene exposure Concentration of analyte in blood
concentration (ppm) (nM analyte · ppm-1)
Mean (SE)
Species Mean (SE)a 1,3-Butadiene EB DEB Reference
Mouse 7.83 (0.02) 72 (20) 68 (9) 8.5 (0.8) Bond et al. (1986)b
80 (0.2) 5.4 (0.6) 20 (2) 2.1 (0.2) Bond et al. (1986)
1,031 (13) 5.8 (0.6) 12.9 (0.5) 2.3 (0.3) Bond et al. (1986)
71 (7) 34 (4) 7.9 (1) 9.2 (1.7) Himmelstein et al. (1994)c
603 (44) 61 (7) 6.1 (0.8) 3.2 (0.4) Himmelstein et al. (1994)
3-31
1,282 (33) 45 (3) 6.7 (0.5) 2.0 (0.4) Himmelstein et al. (1994)
101 (4) 29 (5) 3.8 (0.6) Bechtold et al. (1995)d

93 (5) 3.5 (0.9)
Rat 73.9 (0.4) 1.8 (0.2) 5.4 (0.4) 1.1 (0.2) Bond et al. (1986)b
949 (12) 3.2 (0.2) 3.5 (0.4) 0.8 (0.1) Bond et al. (1986)
63 (2) 21 (1) 1.1 (0.2) Himmelstein et al. (1994)c,e
616 (8) 29 (1) 1.5 (0.1) Himmelstein et al. (1994)

1/28/98
Table 3-8. Comparison of 1,3-butadiene, epoxybutene, and diepoxybutane blood concentration data from
different species of laboratory animals exposed to 1,3-butadiene by inhalation
Butadiene exposure Concentration of analyte in blood
concentration (ppm) (nM analyte · ppm-1)
Mean (SE)a
Species Mean (SE) Butadiene EB DEB Reference
Rat 1,249 (3) 30 (1) 1 (0.1) Himmelstein et al. (1994)
7,938f 32 (1) 0.18 (0.01) Himmelstein et al. (1995)
97 (2) 42 (4) 0.6 (0.1) Bechtold et al. (1995)d
Monkey 10.1 (0.1) 0.8 (0.4) 0.16 (0.05) 0.19 (0.06) Dahl et al. (1990)f for EB
310 (10) 1.8 (1.3) 1.6 (0.9) 0.9 (0.5) Dahl et al. (1991)f,j for DEB
7,760 (170) 4.1 (0.5) 0.14 (0.06) 0.08 (0.03)

3-32
a
Standard error (SE) combines variation of butadiene exposure concentration and blood concentration data.
b
Pooled mean ± SE of samples (n = 9) collected after 2, 4, and 6 h of exposure; animals removed from 1,3-butadiene exposure and exhaled 1,3-butadiene before blood
was collected; 14C-labeled analytes were recovered by vacuum-line cryogenic distillation and quantitated by liquid scintillation counting.
c
Values are means ± SE (n = 6-33 samples) for blood collected between 2 and 6 h of exposure; animals continued to inhale 1,3-butadiene as blood was collected;
1,3-butadiene was quantitated by a vial headspace equilibrium technique using GC-flame ionization detection; detection limit = 0.3 µM.
d
Values are means ± SE (n = 6) for samples collected at 4 h of exposure; animals continued to inhale 1,3-butadiene as blood was collected; analytes were recovered
by vacuum-line cryogenic distillation and analyzed by GC-GC-MS; detection limit = 0.1 µM.
e
One exposure was conducted at this 1,3- butadiene concentration, therefore no SE reported.
f
Blood (n = 3) collected immediately following 2 h exposure using indwelling catheter; 1,3-butadiene recovered by vacuum-line cryogenic distillation and quantitated
as 14C-labeled equivalent using liquid scintillation counting.
g
EB and DEB were recovered by extraction into methylene chloride and quantitated by GC-MS; detection limits = 0.03 µM for EB and 0.13 µM for DEB.
h
Detection limits for EB and DEB = 0.02 µM and 0.01 µM, respectively.
i
The authors exposed the monkeys to 1,3-[14C]-butadiene and vacuum-line cryogenic distillation for this compound includes14C-labeled DEB, BD, and potentially other unidentified14C-
metabolites. Since this study looked at products resulting from several reactions, it was not included in Table 3-7 (which described single reactions in the pathways).

Table 3-9. Tissue levels of epoxybutene and diepoxybutane (pmol/g tissue)
in male rats and male mice exposed by inhalation to 62.5 ppm 1,3-butadiene
for 4 h
EB DEBa
Tissuea
Rats Mice Rats Mice
Blood 36 ± 7 295 ± 27 5±1 204 ± 15

Heart 40 ± 16 120 ± 15 3 ± 0.4 144 ± 16
Lung NDb 33 ± 9 0.7 ± 0.2c 114 ± 37
Liver ND 8±4 ND 20 ± 4
Fat 267 ± 14 1,302 ± 213 2.6 ± 0.4 98 ± 15
Spleen 7±6 40 ± 19 1.7 ± 0.5c 95 ± 12
Thymus 12.5 ± 3.2 104 ± 55 2.7 ± 0.7c 109 ± 19
Bone marrowd 0.2 ± 0.1 2.3 ± 1.5 ND 1.4 ± 0.3
a
Mean ± SE; n = 3 or 4.
b
ND = not detected; indicates that analyte was not detected or was not above control level.
c
Includes at least one ND value.
d
As mean pmol/mg protein ± SE.
Source: Modified from Thornton-Manning et al., 1995a .
Table 3-10. Tissue levels of epoxybutene and diepoxybutane (pmol/g tissue)

in male and female rats exposed by inhalation to 62.5 ppm 1,3-butadiene for 6 h
EB DEB
Tissuea
Males Females Males Females
Blood 25.9 ± 2.9 29.4 ± 2.0 2.4 ± 0.4 11.4 ± 1.7c

Femur 9.7, 9.3 10.4 ± 1.0 1.1, 1.8 7.1 ± 1.3c
Lung 12.7 ± 5.0 2.7 ± 4.3 1.4 ± 0.8b 4.8 ± 0.7c
Fat 175 ± 21 203 ± 13 1.1 ± 0.1 7.7 ± 1.3c

Mammary ND 57.4 ± 4 ND 10.5 ± 2.4c
a
n = 3, except for male femur, where n = 2.
b
One value was not detectable; instrument detection limit/2 was substituted to calculate the mean.
c
Statistically greater than male tissue value, p 0.05.
ND = not determined.
Source: Modified from Thornton-Manning et al., 1995b.

Figure 3-2. Two-compartment pharmacokinetic model for inhalation
chamber.
Source: Filser and Bolt, 1981.

Dawley rats and male B6C3F1 mice to 62.5 ppm 1,3-butadiene for 4 h, as described in Table 3-7,
with the results shown in Table 3-9. The same group of investigators (Thornton-Manning et al.,
1995b) also examined gender differences in the production and disposition of epoxybutene and
diepoxybutane by determining tissue concentrations of the two butadiene metabolites in male and
female Sprague-Dawley rats, as described in Table 3-7, with the results shown in Table 3-10. The
concentrations of epoxybutene did not differ significantly between male and female rats in any of
the tissues examined. The highest concentrations were observed in the fat tissues of both sexes.
Tissue levels of the diepoxybutane, however, were consistently greater in females than in males.
Blood diepoxybutane levels of female rats were 4.75-fold greater than those of male rats. The
greatest gender difference was in the levels of the diepoxybutane in fat tissue, with females having
a sevenfold greater tissue concentration than males. The mammary tissue of females also
contained relatively high levels of the diepoxybutane. The authors suggest that the greater
production of the highly mutagenic diepoxybutane in females may play a role in the increased
incidence of mammary tumors observed in a chronic carcinogenicity study with rats (Owen et al.,
1987).
Dahl et al. (1991) exposed cynomolgus monkeys to nose-only inhalation of 1,3-butadiene
and measured the levels of diepoxybutane and 3-butene-1,2-diol. Results for diepoxybutane are
included in Table 3-9 for comparison to 1,3-butadiene and epoxybutene levels measured in their
previous study (Dahl et al., 1990). Exhaled air and excreta were collected during exposure and
for 96 h after exposure and are summarized in Table 3-11.
Two in vivo studies provided data on the urinary excretion of butadine metabolites by
humans. In the first study (included in Table 3-7 and described in more detail here), Bechtold et
al. (1994) identified and measured two metabolites of 1,3-butadiene, 1,2-dihydroxy-4-(N-
acetylcysteinyl-S-)-butane (M-I) and 1-hydroxy-2-(N-acetylcysteinyl-S-)-3-butene (M-II) in the
urine of workers employed at the Texaco Chemical Co. in Port Neches, Texas, a 1,3-butadiene
extraction plant. The study population included (1) exposed employees who worked in two areas
(described as low- and high-exposure areas) with time-weighted average concentrations of 3 to 4
ppm 1,3-butadiene over the previous 6 months; (2) an intermediate exposure group spending
variable time periods in low- and high-exposure areas; (3) nonexposed employees who worked in
areas with historical time-weighted average concentrations of less than 0.1 ppm 1,3-butadiene;
and (4) outside controls who had no known exposure to 1,3-butadiene. Urine samples were
analyzed from 7, 3, 10, and 9 subjects, respectively, from the above four groups. The assay was
based on isotope-dilution GC-MS. After addition of deuterated internal standards, the
metabolites were isolated from urine samples by solid-phase extraction and selective precipitation.
M-I but not M-II could be readily identified and quantitated in the urine samples

Table 3-11. Excretion of 14C by monkeys exposed to 1,3-[14C]-butadienea
Uptake
Exposure
Exhalants Total metabolites
concentration
(ppm) CO2 Otherb Urine Feces recoveredc
10.1 1.5 ± 0.2 0.45 ± 0.9 ± 0.1 0.021 ± 0.005 2.88 ± 0.22
0.33
310 0.21 ± 0.04d 0.40 ± 0.8 ± 0.2 0.011 ± 0.003d 1.40 ± 0.42d
0.21
7760 0.08 ± 0.02d,e 1.00 ± 0.58 ± 0.002 ± 0.001d,e 1.65 ± 0.29d
0.35 0.06d
a
Values are mean percentage of total inhaled ± SE measured for 96 h after 2-h exposure.
b
Includes all material (except CO2) exhaled during the 2-h exposure and 96-h postexposure.
c
Mean ± SE of the sums of CO2, other, urine, and feces values for individual monkeys; does not include
residues, if any, in monkeys' bodies.
d
Significantly different from low-level exposure (p<0.05).
e
Significantly different from mid-level exposure (p<0.05).
Source: Dahl et al., 1991.
(limits of sensitivity for this assay, 100 ng/mL). The average values of M-I for exposed,
intermediately exposed, nonexposed, and outside control employees were 3,200 ± 1,600, 1,390±
550, 630 ± 190, and 320 ± 70 ng/mL, respectively. Although the levels of exposure for each
individual were not known, the urinary levels of M-I for the exposed groups were significantly
higher (p<0.05) compared with the outside control group. The implications of M-I in the urine
from individuals with no known exposure to 1,3-butadiene are not known.
In the second study that provided human data, Ward et al. (1996a) reported increased levels of
the urinary metabolite 1,2-dihydroxy-4-(N-acetyl-cysteinyl)-butane (a human urinary metabolite
also idenfified by Bechtold et al., 1994) and somatic mutations in workers at a styrene-butadiene
rubber plant. Exposure was assessed in workers from areas of higher exposures (reactor,
recovery, tank farm, laboratory) and lower exposure (blend,coagulation, bailers, shipping,
utilities, shops) using badge dosimeters; the concentration of the metabolite was measured in
urine; and the frequency of hprt mutant lymphocytes was determined
by autoradiography. The detection limit (0.25 ppm) was exceeded in 20/40 dosimeter readings
in the high-exposure group and in 0/20 readings in the low-exposure group. Sixteen high- and
nine low-exposure urine and blood samples were analyzed. Expressed as ng/mg creatinine,
metabolite concentrations were 2,363 ± 1,880 and 937 ± 583 (p<0.05), respectively, for the

high- and low-exposure groups. The respective mean mutant frequencies were 7.09 ± 5.2 × 10-6
and 2.26 ± 1.34 × 10-6 (p<0.05).
Other in vivo studies that further confirm the pathways shown in Figure 3-1 but use different
intermediate endpoints than those shown or with noninhalation exposure are described below.
Deutschman and Laib (1989) studied the effects of 1,3-butadiene exposure on nonprotein
sulfhydryl (NPSH) content of lung, heart, and liver tissue of rats and mice. In these experiments,
male B6C3F1 mice and Sprague-Dawley rats were exposed to 1,3-butadiene at concentrations of
10, 50, 100, 250, 500, 1,000, or 2,000 ppm for 7 h. For rats, a reduction ( 70%; significance
level not stated) occurred in liver NPSH for animals exposed to 1,000 to 2,000 ppm. A
reduction of approximately 20% was observed in lung NPSH of rats, and no appreciable
depletion of NPSH was observed for heart tissue of rats. For mice, depletion of hepatic NPSH
was observed at exposure concentrations of 100 to 250 ppm and declined to 20% of control for
the 2,000 ppm exposure group. Similarly, the NPSH content of mouse lung tissue also declined
by 80% to 90% at the two highest exposure levels. For heart NPSH content in mice, minor
declines were noted for exposure levels up to 500 ppm, but a rapid decrease was observed
between 1,000 and 2,000 ppm that resulted in an 75% depletion. Kreiling et al. (1988)
suggested that the greater susceptibility of mice to the carcinogenic effects of inhaled 1,3-
butadiene might reasonably be explained by the higher rate of formation of the epoxide
intermediate and its limited detoxification and subsequent accumulation in mice. The authors
applied the concentration-response data to the exposures used in earlier bioassays (HLE, 1981)
and noted that, for rats exposed chronically to 1,3-butadiene concentrations of 1,000 or 6,000
ppm, a daily hepatic NPSH depletion of about 25% and 60% and lung NPSH content depletion
of 20% and 30% for the low and high exposures, respectively, was calculated. However, these
values were based on assumptions that 1,3-butadiene metabolism and NPSH resynthesis
remained constant throughout the duration of the chronic exposure. Applying the same methods
and assumptions for mice, daily NPSH depletions for the low-exposure (625 ppm) and high-
exposure (1,250 ppm) levels, respectively, were estimated for liver (50% and 70%), lung (70%
and 90%), and heart (25% and 40%). In studies assessing the effects of 1,3-butadiene exposure
on NPSH content of various tissues, Deutschmann and Laib (1989) reported that depletion of
cardiac NPSH content in mice after inhalation exposure was an indicator of systemically available
epoxide intermediates of 1,3-butadiene that reach the heart by efferent blood flow from the lungs
or liver.
The reduction and/or depletion of NPSH content in mice is also indicative of saturation of
conjugation of the epoxide metabolites of 1,3-butadiene by glutathione. Glutathione conjugation
of epoxybutene and metabolism by glutathione S-transferase was shown by Malvoisin et al.
(1981) and Bolt et al. (1983), respectively, and a reduction in hepatic NPSH content in mice

exposed to 1,3-butadiene was shown by Kreiling et al. (1987). A more recent study by Kreiling
et al. (1988) suggested that glutathione conjugation may be important in the detoxification of this
reactive intermediate. Air-exposed control animals exhibited a moderate time-dependent
decrease in hepatic NPSH content, whereas those animals exposed to 1,3-butadiene exhibited a
significantly greater reduction in NPSH. After 7 h of exposure, the hepatic NPSH content in
mice was reduced to approximately 20% and was reduced further to about 4% after 15 h of
exposure. For Sprague-Dawley and Wistar rats, hepatic NPSH content was initially reduced to
80% and 65%, respectively, but remained stable thereafter. Concurrent with the significant
depletion of NPSH in the mice were signs of acute toxicity (specifics not noted); no toxicity was
observed in any of the rats tested. This experiment clearly demonstrates species variability in the
magnitude and time course of hepatic NPSH depletion after inhalation exposure to high
concentrations of 1,3-butadiene. Furthermore, the progressive decline in hepatic NPSH content
in mice correlates with a reduction in epoxide exhalation and a decline in 1,3-butadiene
metabolism. The accumulation of epoxide intermediates (epoxybutene and diepoxybutane) in
mice (Bond et al., 1986) is consistent with the observed depletion of hepatic NPSH in this
species and the increased metabolism (i.e., production of epoxide intermediates) observed for
mice.
Nauhaus et al. (1996) indicated that metabolites were detected in mouse urine that are also seen
following exposure to acrolein and acrylic acid, suggesting that these compounds may arise
directly from 1,3-butadiene oxidation or indirectly from further metabolism of crotonaldehyde.
Rats excreted 1,3-dihydroxypropanone, a metabolite that may be derived from hydrolysis of
diepoxybutane. Metabolites derived from diepoxybutane were similar in rats and mice when
expressed as a percentage of total metabolites; however, when normalized to body weight, the
amount of diepoxybutane-derived metabolites was four times greater in mouse urine than in rat
urine. The greater body burden of diepoxybutane in the mouse and the greater ability of rats to
detoxify diepoxybutane through hydrolysis may be related to the greater toxicity of 1,3-butadiene
in mice. The metabolites derived via reactive aldehyde intermediates in mice also suggest a role
of these aldehydes in the toxicity of 1,3-butadiene.
Following i.p. injection of 14.3 or 143 µmol/kg of epoxybutene, two glutathione conjugates, S-
(2-hydroxy-3-buten-1-yl)glutathione (I) and S-(1-hydroxy-3-buten-1-yl)glutathione (II), were
detected in the bile of rats (Sharer and Elfarra, 1992). At either dose, the amount of conjugates
excreted in 30 min was at least 85% of that excreted in 120 min. When the epoxybutene dose
was varied between 14.3 and 286 µmol/kg and the combined amounts of conjugates I and II
excreted in 60 min were determined, an apparent linear dose-relationship was obtained.
Saturation was not observed at these dose levels. Total conjugates excreted in 60 min averaged
7.6% ± 4.2% of the administered dose with approximately a 3:1 ratio of conjugates I:II.

Although the study showed that epoxybutene GSH conjugates are formed in vivo after
administration of epoxybutene, biliary excretion of GSH conjugates account for only a small
portion of the administered dose.
N-Acetylcysteine derivatives of the two glutathione conjugates of epoxybutene identified in the
bile of rats by Sharer and Elfarra (1992) were detected in the urine of rats and mice administered
with epoxybutene intraperitoneally (Elfarra et al., 1995). When rats were injected with
epoxybutene at doses ranging from 71.5 to 285 µmol/kg, the urinary excretion of S-(2-hydroxy-
3-buten-1-yl)-N-acetyl-L-cysteine (I) and S-(1-hydroxy-3-buten-2-yl)-N-acetyl-L-cysteine (II)
within 8 h of epoxybutene administration exhibited a linear dose-relationship; the total amount of
the two mercapturic acids combined averaged 17% ± 4%. No metabolites were detected in urine
samples collected 8 to 24 h after dosing. Mice excreted similar amounts of mercapturic acids
(26% ± 13%) at 285 µmol/kg within 24 h of dosing. However, at 143 and 71.5 µmol/kg,
excretion accounted for only 7% ± 3% and 9% ± 3% of the dose, respectively. Rats
preferentially excreted mercapturic acid II over I (approximate ratio 3:1), whereas mice
preferentially excreted mercapturic acid I over II (approximate ratio 1.85:1). The study showed
that at low exposure levels, rats excrete higher levels of epoxybutene mercapturic acids than
mice.
In summary, the inhalation studies show the uptake of 1,3-butadiene exhibits first-order kinetics
at exposure concentrations <1,000 ppm, but at higher concentrations, the process becomes
saturated and exhibits zero-order kinetics; mice exhibit saturation kinetics at lower exposure
concentrations than do rats. At exposure concentrations up to 1,800 ppm, the uptake of 1,3-
butadiene is approximately fourfold greater in mice than in rats. In addition, mice accumulate a
greater amount of 1,3-butadiene or its metabolites or both than do rats exposed similarly.
Limited data on monkeys indicate that the metabolic uptake rate is less than that for rats or mice.
After inhalation of 1,3-butadiene, mice appear to have greater levels of radioactivity (15- to 100-
fold greater at all time points after exposure) in all tissues than do rats exposed similarly, but no
significant qualitative differences have been observed regarding storage depots or target tissues.
However, immediately after a 2 h inhalation exposure, mice exhibited higher levels of 1,3-
butadiene metabolites (including the reactive epoxybutene) in the blood than did rats. A
comparison of butadiene epoxide levels in target tissues (blood, bone marrow, heart, lung, fat,
spleen, and thymus) of rats and mice following inhalation of low levels of 1,3-butadiene showed
consistently higher epoxide levels in mouse than in rat tissues. Other in vivo experiments
demonstrated gender differences in the production of butadiene metabolites in rats, with tissues
from female rats containing higher concentrations of the diepoxybutane than tissues from male
rats. The experiment also showed that the levels of epoxybutene were similar in males and
females. In vivo experiments have confirmed the role of cytochrome P-450 in the metabolic

activation of 1,3-butadiene observed in in vitro studies. Epoxybutene, a reactive intermediate,
may undergo epoxide hydrolase-mediated hydroxylation or conversion via P-450 to another
reactive intermediate, diepoxybutane. Conjugation with glutathione represents a detoxification
process.
Although 1,3-butadiene may be metabolized by microsomal cytochrome P-450 in both rats and
mice, species-related quantitative differences in the fate of inhaled 1,3-butadiene are well
documented. The greater susceptibility of mice to the carcinogenic effects of 1,3-butadiene may
be related to the higher rate of epoxybutene formation and the limited detoxification and, hence,
the greater accumulation of this reactive intermediate in this species. At concentrations >2,000
ppm, the metabolism of 1,3-butadiene follows saturation kinetics in both rats and mice, but the
rate of metabolism in mice is greater (about twice); furthermore, the metabolism of epoxybutene
is saturable in mice but not in rats. With increasing exposure concentration, the metabolic
capacity for epoxybutene becomes rate-limiting in mice but not in rats. Data available from
studies with nonhuman primates show that at low-exposure concentrations ( 10 ppm), the
steady-state tissue levels of reactive 1,3-butadiene metabolites are lower in monkeys than in rats
or mice. The lower uptake rate of inhaled 1,3-butadiene by monkeys suggests that, for
comparable exposures, monkeys will receive a lower internal dose of reactive butadiene
metabolites. The uptake and retention of 1,3-butadiene appears to be nonlinear in the
concentration ranges used in long-term exposure studies, and repeated exposures to 1,3-
butadiene do not appear to induce its metabolism.
1,3-Butadiene may be excreted via the respiratory tract, urine, or feces. The rate of 1,3-
butadiene excretion by rats and mice was shown to be unaffected by exposure concentration
(0.14 to 13,000 µg/L). Half-lifes for urinary excretion of radioactivity were similar for both rats
and mice (5.6 and 4.6 h, respectively), but fecal excretion was somewhat greater in rats (22 h)
than in mice (8.6 h). A shift to excretion of 1,3-butadiene-derived [14C] via the lungs was noted
for rats but not mice at high (13,000 µg/L) exposure concentrations. Approximately 2% of the
total inhaled dose was excreted as 14CO2 or in the urine of monkeys exposed for 2 h to 1,3-[14C]-
butadiene at concentrations ranging from 10 to 8,000 ppm. At the higher concentrations, the
proportion of CO2 decreased, whereas exhaled metabolites (diepoxybutane and butene diol)
increased. Elimination of radioactivity from the blood and tissues of rats and mice after
inhalation exposure to 1,3-[14C]-butadiene was biphasic; half-lifes for initial removal were 2 to 10
h and for slower elimination were 5 to 60 days. Excretion of epoxybutene via the lungs by rats
and mice also has been studied and notable differences between the species observed. For rats,
exhaled epoxybutene concentrations at 10 h attained a plateau of about 4 ppm and remained at
this level for >12 h. For mice, however, the plateau level was about 10 ppm but declined to 6
ppm at 15 h, a decline that coincided with signs of acute toxicity in the mice.

Studies on the urinary excretion of 1,3-butadiene metabolites in mice, rats, hamsters, monkeys,
and humans have shown that all these species predominantly produce two urinary metabolites,
1,2-dihydroxy-4-(N-acetylcysteinyl-S)-butane (M-I) and 1-hydroxy-2-(N-acetylcysteinyl-S)-3-
butene (M-II), but in different proportions. The M-II is a mercapturic acid formed by
conjugation of GSH with epoxybutene, while M-I is a mercapturic acid that appears to form by
GSH conjugation with butene diol, the hydrolysis product of diepoxybutane. M-I but not M-II
was also found in the urine of workers exposed to low levels of 1,3-butadiene.
3.2. MOLECULAR DOSIMETRY

In addition to data on absorption, metabolism, and excretion, a complete dosimetry model for
1,3-butadiene should incorporate information on molecular dosimetry, which links exposure to
some internal biomarkers of exposure. This last component is best evaluated by assessing adduct
formation.
The use of Hb adducts as biomarkers of exposure to 1,3-butadiene was investigated by Sun et al.
(1989a). In this study, male B6C3F1 mice and male Sprague-Dawley rats were injected
intraperitoneally with 1,3-[14C]-butadiene at doses of 1, 10, 100, or 1,000 µmol/kg, and adduct
formation was monitored. Hb adduct formation was linearly related to dose up to 100 µmol/kg
for both species. The Hb adducts accumulated linearly after repeated injections of 100 µmol/kg
for 3 days. The 1,3-butadiene-derived Hb adducts showed lifetimes of 24 and 65 days in mice
and rats, respectively, which correlates with the lifetimes of red blood cells. Assuming that
adduct formation is a function of the extent of 1,3-butadiene metabolism, the similarity in the
degree of Hb adduct formation between mice and rats does not reflect the species variability in
toxicity of this compound. Therefore, Hb adducts may not serve as accurate indicators of levels
of reactive metabolites in the blood and, thus, as indicators of toxicity. However, Hb adduct
formation may be useful as an indicator of 1,3-butadiene exposure.
Similar findings of exposure-dependent Hb adduct formation and stability of the adducts were
reported by Osterman-Golkar et al. (1991) for Wistar rats exposed to 1,3-butadiene at
concentrations of 250, 500, or 1,000 ppm, 6 h/day, 5 days/week for 2 weeks. In this study, the
Hb adduct formation also increased linearly with exposure up to the highest exposure level. The
investigators also concluded that Hb adducts were useful for assessing dosimetry of long-term
exposure to 1,3-butadiene.
Osterman-Golkar et al. (1996) studied Hb adducts in 17 workers exposed to 1,3-butadiene in a
petrochemical plant and nine referents employed at the same factory but not exposed to 1,3-
butadiene. Using stationary and personal monitoring devices, the ambient 1,3-butadiene level for
workers handling 1,3-butadiene containers was 11.2 ± 18.6 mg/m3 and 1.2 mg/m3 for
maintenance and laboratory workers. The Hb adduct measured was 2-hydroxy-3-butylvaline,

formed by reaction of N-terminal valine with carbon 1 in epoxybutene. Higher concentrations of
Hb adducts (0.16 ± 0.099 pmol/g) were recorded in the workers handling 1,3-butadiene
containers compared with those in maintenance, laboratory workers, and nine unexposed
controls ( 0.05 pmol/g).
Citti et al. (1984) conducted an in vitro study that examined the reactivity of epoxybutene
(referred to as epoxybutene by the authors) with isolated nucleosides and DNA. They reported
that two adducts were formed: 7-(2-hydroxy-3-buten-1-yl)guanine and 7-(1-hydroxy-3-buten-2-
yl)guanine. The authors indicated that the epoxide reacted similarly with either free DNA or
DNA-bonded deoxyguanosine and that the half-life of these adducts under physiological
conditions was 50 h.
Kreiling (1987) reported the in vivo formation of the DNA adduct 7-(1-hydroxy-3-buten-2-
yl)guanine in the liver of mice exposed to 1,3-[14C]-butadiene (exposure concentration and
duration not specified). No DNA adducts were detected in the livers of 1,3-butadiene-exposed
rats. Note that this adduct was one of two reported by Citti et al. (1984) for the in vitro reaction
of 3,4-epoxybutene with DNA and deoxyguanosine. Additional details were not available in the
abstract by Kreiling nor was additional information reported in later publications by Kreiling and
coworkers.
Jelitto et al. (1989) reported species-dependent differences in the in vivo formation of DNA
adducts by male B6C3F1 mice and male Sprague-Dawley rats exposed to 1,3-[14C]-butadiene at
concentrations of 250, 500, or 1,000 ppm for 7 h. Analysis (alkaline elution and comparison of
HPLC profiles with synthesized adduct standards) of liver DNA from the mice showed that two
adducts had been formed: 7-N-(1-hydroxy-3-buten-yl)guanine and 7-N-(2,3,4-
trihydroxybutyl)guanine, the latter being derived from diepoxybutane. These products were not
detected in rat liver DNA. Alkaline elution curves showed that protein-DNA and DNA-DNA
cross-linking occurred in mice, but not in rats, after a 7 h exposure to 1,3-butadiene at
concentrations of 250 ppm and above. These findings provide additional evidence at the
molecular level for explaining the difference in the carcinogenic response between mice and rats.
3.3. STRUCTURE-ACTIVITY RELATIONSHIPS

Studies by Del Monte (1985) and Dahl et al. (1987) have shown that the metabolism of
structurally related isoprene (2-methyl-butadiene) may be qualitatively similar to that of 1,3-
butadiene. Although the diepoxybutane metabolite of isoprene has been shown to be genotoxic
in Salmonella, data are unavailable regarding the carcinogenic potential of isoprene.
Del Monte et al. (1985) showed that mouse hepatic microsomal monooxygenases converted
isoprene to epoxides and diepoxides and that the biotransformation was inhibited by cytochrome
P-450 inhibitors such as CO, SKF 525-A, and metyrapone. Specifically, 3,4-epoxy-3-methyl-

butene and 3,4-epoxy-2-methyl-1-butene were major and minor metabolites, respectively, with
the latter representing about 20% of the former. The 3,4-epoxy-2-methyl-1-butene metabolite
was metabolized further in microsomal incubations to the mutagenic isoprene dioxide
(diepoxide). Data from these in vitro metabolism studies were used to calculate the KM and Vmax
for the production of the diepoxide. The resulting KM (mM) and Vmax (nmol diepoxide/mg
protein/min) values for diol production by microsomes from control, phenobarbital-induced, and
3-methylcholanthrene-induced mice were 0.24 and 1.7, 0.29 and 5.1, and 0.22 and 2.0,
respectively. The Vmax for the formation of the diepoxide was significantly increased (p<0.01) in
incubations using hepatic microsomes from phenobarbital-treated mice.
Gervasi and Longo (1990) provided additional information on the metabolism of in vitro
isoprene by hepatic microsomal preparations from rats, mice, rabbits, and hamsters. Hepatic
microsomal preparations from these species metabolized isoprene to epoxybutene, 3,4-epoxy-3-
methyl-1-butene, and 3,4-epoxy-2-methyl-1-butene. The former was the major metabolite and
was found to have a half-life of 85 min. Microsomal preparations from all species further
metabolized the 3,4-epoxy-2-methyl-1-butene to isoprene dioxide (2-methyl-1,2,3,4-
diepoxybutane), which was found to be mutagenic and to have alkylating ability. The KM (mM)
and Vmax (nmol/mg/protein/h) for the rat, mouse, rabbit, and hamster microsomal metabolism of
isoprene were 0.08 and 0.24, 0.09 and 1.79, 0.2 and 0.66, and 0.06 and 1.20, respectively.
Unlike 1,3-butadiene, isoprene exhibited the same pattern of metabolism in all species tested and
did not result in mutagenic epoxybutene intermediates.
In the study by Dahl et al. (1987), groups of 30 male F344 rats were exposed by nose-only
inhalation to [14C]isoprene at concentrations of 8.0, 266, 1,480, or 8,200 ppm for 6 h (5.5 h for
the highest exposure), and urine, feces, and exhalants were collected over a 66 h postexposure
period. During this period, >75% of the nonisoprene (metabolites) radioactivity was excreted in
the urine. Except for the highest exposure group where greater amounts of radioactivity were
excreted in the feces, a pattern of predominantly urinary excretion was consistent among the
various exposure groups. The half-life (mean ± SE) for urinary excretion of 14C was 10.2 ± 1.0 h
(range of 8.8 to 11.1 h). Generally, the concentration of metabolites in the blood increased with
exposure concentration and duration of exposure. The authors noted that 85% of the
radioactivity in the blood was associated with material of low volatility and that it probably
represented covalently bound metabolites, conjugates of isoprene metabolites, or tetrols. Only at
the two highest exposure concentrations were materials detected that possessed volatilities
matching those of isoprene and isoprene monoepoxides. The percentage of inhaled isoprene-
derived 14C present as diepoxide or diol in the blood remained fairly constant with time but
decreased with exposure concentration. Assessing the distribution of isoprene and its
metabolites in some animals of the 1,480 ppm exposure group revealed that the liver and blood

contained the majority of the radioactivity. Relatively large amounts of metabolites were present
in respiratory tract tissues after 20 min of exposure. The mutagenic metabolite, isoprene
diepoxide, was identified in all tissues examined and, in the blood, represented between 0.0018%
and 0.031% of the inhaled 14C label. Although exposure to high concentrations of 1,3-butadiene
result in CO2 as the major metabolite, this study suggested that the major route of excretion for
isoprene is in the urine. The authors noted, however, that this finding is tentative and may be the
result of a labeling artifact. Although no evidence for metabolic saturation was detected for the
isoprene concentrations used, the uptake and fate of inhaled isoprene are similar to that of
butadiene.
Peter et al. (1987) also studied the pharmacokinetics of isoprene in male Wistar rats and male
B6C3F1 mice. Animals were exposed in closed systems to concentrations as high as 4,000 ppm
for up to 10 h. At concentrations <300 ppm, the rate of metabolism was found to be directly
proportional to the isoprene concentration, but saturation of metabolism was detected at higher
concentrations. The Vmax for the metabolism of isoprene in rats and mice was 130 and 400
µmol/h/kg, respectively. Exhalation of the parent compound was approximately 15% and 25%
in rats and mice, respectively.
Chloroprene (2-chloro-butadiene) is also structurally similar to 1,3-butadiene. Studies have
shown that the biotransformation of chloroprene results in the formation of peroxides that may
interact with tissue thiols (Haley, 1978). Furthermore, cytochrome P-450 mixed-function
oxygenases may form an epoxide intermediate similar to that formed during 1,3-butadiene
metabolism.
In summary, in vitro metabolism studies have shown that the structurally similar isoprene is
metabolized in a similar fashion by several different species and that epoxybutene intermediates
are formed, one of which may be epoxidized further to a genotoxic diepoxybutane. In vivo
inhalation studies that used rats and mice exposed to isoprene showed that its uptake and fate are
similar to that of 1,3-butadiene and that a genotoxic diepoxybutane metabolite, but not a
genotoxic epoxybutene intermediate, is formed.
Preliminary data indicate that Hb adducts may be useful as biomarkers of exposure for 1,3-
butadiene exposure. Research efforts are focusing on dosimetry modeling for extrapolating from
high- to low-dose exposures and for interspecies extrapolation. Furthermore, on validation,
dosimetry models may be useful in predicting levels of 1,3-butadiene and its reactive metabolites
in various tissues.
3.4. DISCUSSION AND CONCLUSIONS

Species variability in the metabolism and disposition of 1,3-butadiene may explain, in part,
species variability in the toxicity of the compound. Current data indicate that the toxicity of 1,3-

butadiene depends on the metabolic activation to reactive intermediates such as epoxybutene and
diepoxybutane and that these biotransformation processes vary quantitatively and qualitatively
among species. The mutagenic epoxybutene and diepoxybutane metabolites have been shown to
occur in the blood of rats and mice exposed to 1,3-butadiene, and their concentrations are two-
to fivefold greater in the blood of mice. Limited data for humans have shown that liver
microsomes have a higher capacity for the formation of epoxybutene than do rodent liver
microsomes but that the metabolism of epoxybutene to 1,3-butadiene epoxide by human liver
microsomes was 20-fold greater than that observed in rat or mouse microsomes. These data
suggest that levels of this reactive intermediate in humans may be substantially less than in the
rodent species. The oxidation of epoxybutene to diepoxybutane (also a reactive metabolite)
appears to be negligible in humans and rats (formation of the non-DNA-reactive butene diol 1,2-
dihydroxybut-3-ene is the preferred pathway) and is substantial in mice. Study results have
shown species-related differences in the uptake and retention of inhaled 1,3-butadiene. Uptake
and retention by mice is greater than for rats, and saturation kinetics are observed in mice at
exposure concentrations of 500 ppm but not in rats at exposures as high as 5,000 ppm. These
differences may be used to support the hypothesis that the greater sensitivity of mice to the toxic
effects of 1,3-butadiene may be a function of a greater internal dose, greater production of
reactive metabolites, and lower detoxification potential.
Although the previous findings provide considerable insight into the understanding of 1,3-
butadiene toxicity, some researchers have indicated the need for examining additional, although
quantitatively minor, metabolic pathways (e.g., glutathione S-transferase-mediated detoxification
processes and formation of toxic metabolites such as butene diol and crotonaldehyde) and the
possible effects of pulse exposures on the metabolism and disposition of 1,3-butadiene.
Molecular dosimetry studies have also shown species-related differences in the formation of
various adducts. Additional work in this area will be useful in assessing these adducts as either
biomarkers of exposure or effects.
Dosimetry models are being developed or refined to extrapolate the relatively high exposures and
doses used in animal tests to the low exposure concentrations in human exposure situations.
These models will be especially useful in predicting blood and tissue concentrations of butadiene
metabolites.

4. MUTAGENICITY
4.1. INTRODUCTION
The mutagenic effects of 1,3-butadiene have been reviewed extensively (Rosenthal,
1985; de Meester, 1988; Arce et al., 1990; Norppa and Sorsa, 1993; Jacobson-Kram and
Rosenthal, 1995). The last of these reviewed publications through 1994 are on the genetic
effects associated with butadiene (and metabolites). There is extensive evidence that butadiene
and the two primary epoxide metabolites (epoxybutene and diepoxybutane) induce genotoxic
effects in a variety of in vitro and in vivo test systems. Most of the in vivo studies discussed in
the cited reviews were assays in mice and rats using cytogenetic endpoints, and the results
generally support the dichotomy in carcinogenic response where mice are more responsive than
rats. This review will focus on recently published studies performed in vivo (both somatic and
germ cell effects) with an emphasis on those studies providing information relative to the mode
of action of butadiene metabolites.
4.2. GENE MUTATIONS

Most of the earlier in vivo genotoxicity studies used cytogenetic endpoints (aberrations,
micronuclei, or sister chromatid exchange [SCE]). It is recognized that this reflected the dearth
of in vivo assays measuring gene mutations and limited the interpretation of in vitro versus in
vivo findings. The ability to detect mutations at the hprt locus obtained from T lymphocytes
from exposed mammals including mice, rats, monkeys, and humans provides an important step
in developing an understanding of chemically induced mutational processes. Cochrane and
Skopek (1993, 1994a) used B6C3F1 mice and human TK6 cells to evaluate the mutagenic
potential of butadiene and the two major metabolites. In the in vivo studies, mice were exposed
for 6 h/day, 5 days/week for 2 weeks to butadiene at 625 ppm. The induced hprt mutant
frequency was 6.2 × 10-6 compared with 1.2 × 10-6 from unexposed controls. For the
metabolites, mice received three daily intraperitoneal (i.p.) injections of 60, 80, or 100 mg/kg of
epoxybutene or 7, 14, or 21 mg/kg of diepoxybutane. Mutant frequencies in hprt from splenic T
cells were dose related for both metabolites, with maximal responses of 8.6 × 10-6 and 13 × 10-6
for epoxybutene and diepoxybutane, respectively. Similarly, they found diepoxybutane about
100 times more effective than epoxybutene when human lymphoblastoid TK6 cells were treated
in vitro.
In a recent meeting presentation, Meng et al. (1996) reported on a study in which both
mice and rats were exposed by inhalation to 1,250 ppm butadiene for 2 weeks (6 h/day, 5
days/week). Groups of animals were necropsied before exposure (controls) and weekly up to 10
weeks after the last exposure. The researchers measured hprt mutants in both spleen and thymus

using the T-cell cloning assay. Mutant frequencies in both tissues of both species increased for
several weeks and then declined. Maximal frequencies were: in thymus, 1.3 × 10-6 in mice (2
weeks) and 4.9 × 10-6 in rats (3 weeks); in spleen, 19.7 × 10-6 in mice (5 weeks) and 8.4 × 10-6 in
rats (4 weeks). They determined a relative mutagenic potency (RMP) as the ratio of cumulative
increase in mutant frequency in treated versus controls. For the spleen the RMP was 7.18 for
mice compared with 2.04 for rats.
Several recent studies have measured in vivo mutations using the phage lacI or lacZ
genes incorporated into a rodent genome. Recio and Goldsworthy (1995) summarized several
experiments in which male B6C3F1 lacI transgenic mice were exposed to 62.5, 625, and 1,250
ppm butadiene (6 h/day, 5 days/week) for 4 weeks. Two weeks after the last exposure, animals
were euthanized and DNA was extracted from bone marrow to be examined for lacI
mutagenesis. Mutations increased in a dose-response manner, reaching an apparent plateau at
625 ppm (about a fourfold increase above controls). Sequence analysis of lacI mutant colonies
from the 625 and 1,250 ppm groups indicated an increased frequency of point mutations at A:T
base pairs. These findings are consistent with those observed in butadiene-induced hprt mutant
T lymphocytes from B6C3F1 mice (Cochrane and Skopek, 1994b).
Several studies of genetic effects in exposed workers have recently been reported. Ward
et al. (1994, 1996b) measured the frequency of hprt mutations in lymphocytes of workers in a
butadiene production plant (two studies) and in a styrene-butadiene rubber plant. In the first
study exposure estimates were based on 8 h samples in two production areas and in a central
control area. Mean butadiene concentration in the production areas was 3.5 ppm, but the
majority of samples showed concentrations below 1 ppm; mean butadiene concentration in the
control was 0.03 ppm. Variant frequencies at the hprt locus in PHA-stimulated peripheral blood
T cells of a high exposure group were increased more than threefold compared with the low-
exposure and nonexposed groups. The eight individuals in the high-exposure group had hprt
variant frequencies varying from 0.94 × 10-6 to 8.98 × 10-6 and the variant frequency generally
correlated with the level of the metabolite dihydroxybutane in the urine. Whether the difference
was due to differences in exposure or genetic differences in metabolism cannot be ascertained
from the data. A second study was conducted in the same plant about 1 year later (Ward et al.,
1996b). Measured butadiene concentrations in personal samplers were markedly lower, 0.30 ±
0.59, 0.21 ± 0.21, and 0.12 ± 0.27 ppm in areas defined as high, medium, and low exposure (no
controls were reported for the second study). The corresponding hprt variant frequencies were
5.33 ± 3.76, 2.27 ± 0.99, and 2.14 ± 0.97 × 10-6, respectively. Individual data were not reported
for this study, but again there is a high standard deviation in the highly exposed group. The
Ward et al. (1996b) paper also reported preliminary results from workers in a styrene-butadiene
rubber plant. Workers were assigned to high (20 of 40 personal samplers exceeded the 0.25 ppm

detection limit and 11 had a concentration over 1 ppm) and low (none of 26 exceeded the
detection limit) exposure groups. In nonsmokers, the hprt variant frequencies were 7.47 ± 5.69
and 1.68 ± 0.85 × 10-6 for the high and low groups, respectively. While the variant frequency
for smokers in the high-exposure group (6.24 ± 4.37) was not different from nonsmokers, the
frequency for smokers in the low exposure group was about twice the nonsmoker group (3.42 ±
1.57). These preliminary findings with small sample sizes and no detail about smoking history
or other confounding factors raise several unanswerable questions. The autoradiographic
procedure for detecting hprt variants was used in these studies. The limitation of this method is
that it is not possible to distinguish between several independent mutations and a single mutation
giving rise to a clone of cells with the mutant phenotype. The procedure using the T lymphocyte
cloning assay and subsequent DNA sequence analysis of clones as described by Albertini et al.
(1982), and Recio et al. (1990) provide sufficient data for ascertaining independent mutational
events.
Hayes et al. (1996) employed the T cell cloning assay to detect mutant frequencies in
lymphocytes of workers in a rubber production factory. Butadiene levels were measured using
personal samplers during the 6-h work shift and expressed as 6-h time-weighted average. These
were supplemented with several grab samples. Three different work areas were identified:
initial distillation and recovery from dimethyl formamide, polymerization, and recovery, with
median air levels of 3.5, 1.0, and 1.1, respectively. The T cell cloning assay was performed
from postshift blood samples. Unexposed subjects were age and gender matched and a brief
questionnaire was administered. Tabular hprt mutant frequencies were presented grouped only
by gender and exposed versus unexposed. Mean mutant frequencies were somewhat higher in
females than males. Smoking (in males only) was not different in either group, but mutant
frequency did significantly increase with age. Mean mutant frequencies, raw and adjusted for
age, sex, cloning efficiency, and exposure status, were similar in exposed and nonexposed
workers. Adjusted mean frequency for total exposed workers was 18.0 × 10-6 compared with
13.6 × 10-6 for nonexposed workers.
In a third study, Tates et al. (1996) used the T cell cloning assay on blood samples
collected from workers in a butadiene plant in the Czech Republic. Workers were sampled in
1993 and 1994, but most of the blood samples from 1993 were lost to technical errors. A
detailed analysis was conducted on the later group of 19 exposed and 19 nonexposed workers
from other parts of the same plant. Personal samplers indicated a mean butadiene concentration
of 1.76 ppm, with individual samples ranging from 0.012 ppm to 19.77 ppm. The geometric
mean hprt mutant frequencies (adjusted for age, smoking, and cloning efficiency) were 7.10 ×
10-6 for exposed and 10.59 × 10-6 for the controls. The range of mutant frequencies among

individuals was similar for both groups and individual mutant frequencies in the exposed group
were not correlated with concentrations of butadiene detected in the personal samplers.
The results in both of the T cell cloning assay groups are clearly in conflict with the
Ward et al. (1994, 1996b) findings both for exposed versus nonexposed and for smokers versus
nonsmokers. A simple explanation would be that the increase in the autoradiographic assay was
due to clones of mutants having arisen from earlier mutations. Even if true, the increase is
clearly exposure related because 7 of the 8 exposed workers exhibited higher variant frequencies
than the highest of the nonexposed controls. As indicated by Hayes et al. (1996), there are many
differences between the two studies and currently no basis for rejecting either finding.
4.3. CYTOGENETIC EFFECTSCHUMAN

There have been four studies evaluating cytogenetic effects of exposed workers. Au et
al. (1995) measured chromosome aberration frequencies in blood samples of 10 exposed workers
and 10 matched controls from the same population used in the Ward et al. (1996b) study cited
above. They reported measurable, but not significant (p>0.1), increases in chromosome
aberrations and chromatid breaks. Also, cells were exposed to gamma-rays in G1 and
aberrations were measured in the subsequent metaphase. With this indirect measure of DNA
repair, chromatid breaks, deletions, and dicentrics were all significantly higher in cells from
butadiene-exposed workers.
Sorsa et al. (1994) investigated chromosomal damage in blood lymphocytes sampled in
1993 from workers in the factories described by Tates et al. (1996) above. Chromosome
aberrations, micronuclei, and sister chromatid exchange (SCE) frequencies were not elevated
above samples from unexposed persons. They did note that smoking had a slight effect in
micronuclei and SCE but not chromosome aberrations. Preliminary data measuring
chromosome aberrations and micronuclei in blood samples from the 1994 group of workers was
reported by Tates et al. (1996). The percentage of aberrant cells was significantly increased
(p<0.01) in exposed subjects; however, the frequency of micronuclei in lymphocytes was similar
in exposed and unexposed subjects. Evaluation of data for each subject would be required to
determine the basis for the apparent discrepancy of the results between the two years.
The role of glutathione S-transferase (GST) genes GSTM1 and GSTT1 enzymes in the
detoxification of butadiene metabolites has been evaluated by measuring the induction of SCE in
cultured human lymphocytes. Uuskula et al. (1995) found that SCE induction in lymphocytes
from GSTM1-null individuals was 31% higher than in lymphocytes from GSTM1-positive
individuals when treated with 50 or 250 µM 1,2-epoxy-3-butene. The same group (Norppa et
al., 1995) reported no difference in SCE induction among GSTM1 nulls and GSTM1-positive
lymphocytes when treated in vitro with diepoxybutane; however, they observed a 60% increase

in SCE in lymphocytes from GSTT1-null individuals when treated with 2 or 5 µM
diepoxybutane. Neither GSTM1 nor GSTT1 deficiency affected the induction of SCE by 250 or
500 µM of 3,4-epoxybutane-1,2-diol (Bernadini et al., 1996). In a separate study, Kelsey et al.
(1995) found that GSTT1 deficiency significantly increased the frequency of SCE induced by
diepoxybutane in lymphocyte cultures of workers exposed to butadiene. Hence while all three
epoxides of butadiene metabolism are effective inducers of SCE in cultured human lymphocytes,
there are differences in the role of at least two of the GST genes (GSTM1 and GSTT1) in the
detoxification of the three metabolites.
4.4. CYTOGENETIC EFFECTSCRODENT

Most of the rodent in vivo cytogenetic studies on butadieneCespecially in somatic
cellsChave been thoroughly treated in the reviews cited in the introduction of this chapter. In
those studies, positive results were reported for all cytogenetic endpoints studied in mice and
negative results were consistently reported in rats. Recent efforts have focused on cytogenetic
effects in germ cells of butadiene as well as effects of the two primary epoxides of butadiene.
Butadiene induced dominant lethal effects in studies of male mice exposed by inhalation
(Adler and Anderson,1994); the details are described in Chapter 4. That study was followed by
an experiment measuring heritable translocations induced in exposed males (Adler et al., 1995).
Males were exposed by inhalation to butadiene at 1,300 ppm for 5 days for 6 h/day. Offspring
were tested for translocations by both litter size and cytogenetic analysis of meiotic and somatic
cells. The translocation frequency from treated males was 2.7% compared with 0.05% for
historical controls.
Xiao and Tates (1995) evaluated the cytogenetic effects of 1,2-epoxybutene (EB) and
1,2:3,4-diepoxybutane (DEB) in both somatic and germ cells of mice and rats. Male animals of
both species received single i.p. injections of 40 or 80 mg/kg of EB. Animals were sacrificed at
various time intervals after treatment and spleen and testes were processed for scoring of
micronuclei. In splenocytes, EB was almost four times more effective in the mouse as in the rat.
In mouse germ cells, the incidence of micronuclei was similar to controls on days 1 and 3 after
exposure, but was significantly increased on day 14. In rats, EB was equally effective on days 1
to 3 (late spermatocytes) and day 20 (early spermatocytes) and the frequency of micronuclei at
80 mg/kg was slightly higher than that observed on day 14 in the mouse. For DEB, mice were
injected with 15 or 30 mg/kg and rats received single i.p. injections of 20, 30, or 40 mg/kg. In a
separate experiment, rats received 3 daily injections of 10 mg/kg. The response in splenocytes
was similar in both mice and rats at 30 mg/kg. In mouse germ cells, DEB increased the
frequency of micronuclei only on day 3 after treatment. Significant increases of micronuclei
were observed in rat germ cells at all doses and all time periods. The results in somatic cells are

consistent with all other reports of greater sensitivity in mice than in rats. This difference is
contradicted in germ cells with rats equally (or more) sensitive to micronuclei induction by both
EB and DEB. The authors offered no explanation for this, stating that more research is needed
to better understand the organ and species differences. It is noted that the strains of both species
are different from those used in most other endpoint measurements. The mice were F1 males of
a (102 × C3H) cross of parent stocks from Adler’s laboratory in Germany. The rats were Lewis
rats supplied by Harlan CPB, the Netherlands.
Sjöblom and Lähdetie (1996) used an in vitro meiotic micronucleus assay to examine the
effects of EB, DEB, and 1,2-dihydroxy-3,4-epoxybutane (diolEB) in seminiferous tubule
sections of male Sprague-Dawley rats. Tissue sections were cultured for 4 days with EB at 100,
500, or 1,000 mol/L; DEB at 5, 10, or 20 µmol/L; or diolEB at 10, 50, or 100 µmol/L. The
frequency of micronuclei was increased only by DEB and the increase was clearly dose-related.
That EB was not effective is contrasted with the findings of Xiao and Tates (1995) above. The
authors suggest that EB requires further metabolism by P450 enzymes, which they indicate does
not occur in rat testes microsomes.
4.5. SUMMARY
The studies cited here along with the many earlier genotoxicity studies discussed in the
cited reviews provide clear evidence that 1,3-butadiene is both mutagenic and clastogenic
through its metabolism, primarily due to the mono- and diepoxide. While the difunctional DEB
is clearly more effective than the monofunctional EB for most endpoints, it is not possible to
ascribe the effects observed to one or the other when animals are exposed to butadiene. Where
both have been studied, mice are more responsive than rats, except for the recent germ cell
studies. Whether this exception is strain specific (among or between species) can only be
answered with future work.
The role of GST is also clearly established for the genotoxic effects of butadiene in
human lymphocytes. That the two glutathione S-transferases (GSTM1 and GSTT1) react
differently with the three epoxide metabolites suggests that the relative concentrations of these
metabolites will vary depending on the individual’s genotype.

5. REPRODUCTIVE AND DEVELOPMENTAL EFFECTS
5.1. REPRODUCTIVE EFFECTS

Several reproductive toxicity studies for 1,3-butadiene have been undertaken, starting with
a study in rats, guinea pigs, rabbits, and dogs by Carpenter et al., 1944. Two studies by Owen
and coworkers were done in rats (Owen et al., 1987; Owen and Glaister, 1990). NTP conducted
two chronic reproductive toxicity studies in mice (NTP, 1984; 1993). Hackett and co-workers
undertook an acute “sperm head morphology” study in B6C3F1 mice (Hackett et al., 1988a) and
a dominant lethal study in CD-1 male mice (Hackett et al., 1988b). Dominant lethal studies, both
acute and subchronic, have also been done in CD-1 male mice (Anderson et al., 1993, 1995) and
in (102/E1xC3H/E1)F1 mice (Adler and Anderson, 1994).
5.1.1. Carpenter et al., 1944

Four groups, each consisting of 24 albino rats, 12 guinea pigs, 4 rabbits, and 1 dog, were
exposed to 0, 600, 2,300, or 6,700 ppm 1,3-butadiene 7.5 h/day, 6 days/week for 8 months in
546-L chambers. Except for the dogs, which were all female (only one in each group), the
animals were divided equally between the two sexes. Body weights were measured weekly; blood
was analyzed monthly; and urinalysis, blood chemistry, organ weights (kidney and liver), and
gross and histopathologic examinations were performed at termination. Males and females were
mated, but the authors did not indicate when this occurred relative to the treatment period. No
deaths were noted in the exposed animals. Terminal body weights in rats were reduced to 90.5%,
86.3%, and 81.2% in the 600, 2,300, and 6,700 ppm groups, respectively, relative to control body
weights. A similar trend was noted for male guinea pigs, and weights for dogs and rabbits
fluctuated. No effects on organ weights that could be attributed to exposure to 1,3-butadiene
were observed. There were no abnormal findings for hematology values or blood chemistry.
Microscopic lesions were not observed in the testes, ovaries, or other organs examined (heart,
kidney, skeletal muscle, pancreas, or spleen) except for the liver, in which mild, cloudy swelling
was noted in 68% of the animals exposed to 6,700 ppm.
Carpenter et al. (1944) provided a few results regarding fertility of rats, guinea pigs, and
rabbits exposed to 1,3-butadiene. Fertility, defined as the number of litters produced within a
given time, was reduced in rats, with 3.3, 2.7, 2.5, and 2.6 litters being produced by animals
exposed to 0, 600, 2,300, or 6,700 ppm, respectively. Because the results were not analyzed
statistically and other details regarding the duration of the mating periods were not presented, it is
not possible to conclude that 1,3-butadiene had an effect on fertility in rats. Furthermore, fertility
in rats was not affected by exposure to 1,3-butadiene when litter size (8.4 pups/litter at 600 ppm,
7.9 pups/litter at 2,300 ppm, and 7.8 pups/litter at 6,700 ppm) was used as the measure; the

average litter size of the two higher exposure groups was similar to that of the control group.
Two male and two female offspring from rats exposed to each concentration were exposed along
with the parents. According to the authors, the F1 controls and the 660-ppm group produced
three times as many pups as did the F1 groups exposed to 2,300 or 6,700 ppm. Too few animals
were used to adequately evaluate the fertility of the exposed offspring. Guinea pigs in each
exposure group produced 16, 13, 10, and 13 pups, respectively. Rabbits exposed to 600 or 2,300
ppm produced no pups, whereas the controls produced 24 pups and the 6,700 ppm group
produced 27 pups. Considering that the highest concentration had no effect on fertility in rabbits,
it is doubtful that the lack of fertility at the lower concentrations was due to exposure to
1,3-butadiene.
5.1.2. Owen et al., 1987; Owen and Glaister, 1990

This 2-year toxicological and carcinogenicity study is the same as the Hazleton
Laboratories Europe, Ltd. (HLE, 1981), study discussed previously by EPA (U.S. EPA, 1985).
Male and female CD strain (Sprague-Dawley derived) rats (110 of each sex per group) were
exposed by inhalation to 1,3-butadiene (99.2% purity) at target concentrations of 0, 1,000, or
8,000 ppm 6 h/day, 5 days/week for 105 (females) or 111 (males) weeks. Ten males and 10
females were killed at 52 weeks. The average weekly concentration of 4-vinyl-1-cyclohexene (a
1,3-butadiene dimer) was 413 ppm (v/v). A comprehensive postmortem examination, including
necropsy and histopathologic examination, was conducted of all gross lesions, all tissues from
control and high-exposure groups, and selected tissues from low-exposure groups. Nonneoplastic
lesions were not induced in reproductive organs in either male or female rats, although benign and
malignant mammary tumors, uterine sarcomas, and Leydig cell tumors were observed.
5.1.3. NTP, 1984

The first inhalation toxicological and carcinogenicity study conducted by the National
Toxicology Program (NTP, 1984) showed that, in addition to the numerous neoplasms induced
by high concentrations of 1,3-butadiene in male and female B6C3F1 mice, nonneoplastic lesions
also were induced in reproductive organs. Male and female mice were exposed to 0, 625, or
1,250 ppm 1,3-butadiene 6 h/day, 5 days/week and then killed after 60 or 61 weeks of exposure.
Among female mice, ovarian atrophy was seen in 40/45 (89%) mice exposed to 625 ppm and in
40/48 (83%) mice exposed to 1,250 ppm, compared with an incidence of only 2/49 (4%) in
control mice. Involution of the uterus, which was considered a manifestation of ovarian atrophy,
was seen in 7/46 (15%) and 14/49 (29%) mice exposed to 625 and 1,250 ppm, respectively,
compared with 0/49 control mice. Uterine involution was characterized by fewer and less
prominent endometrial glands. A low incidence of mammary gland neoplasms (acinar cell and

adenosquamous carcinomas) was induced by 1,3-butadiene; nonneoplastic mammary lesions were
not induced. Testicular atrophy was observed in 19/47 (40%) mice exposed to 625 ppm and in
11/48 (23%) mice exposed to 1,250 ppm compared with 0/50 control mice. Statistical analysis
showed that the increased incidences of the lesions in male and female mice were significant
(p<0.05) for all groups compared with their respective controls.
5.1.4. NTP, 1993

NTP (1993) conducted a second inhalation toxicological and carcinogenicity study in male
and female B6C3F1 mice exposed to lower concentrations of 1,3-butadiene. Concentrations were
0, 6.25, 20, 62.5, 200, or 625 ppm 1,3-butadiene for 6 h/day, 5 days/week for 103 weeks, with
interim evaluations at 9 and 15 months. Additional male mice were exposed to 200 ppm of 1,3-
butadiene for 40 weeks, 312 ppm for 52 weeks, or 625 ppm for 13 or 26 weeks followed by
observation for the remainder of the 2 years (stop-exposure protocol). It should be emphasized
that this study was designed to study neoplastic and general toxicological, rather than
reproductive, endpoints. Further details are presented in Chapter 6.
The effects of 1,3-butadiene on reproductive organs in female mice are presented in Table
5-1. Ovarian atrophy was seen in the 200 ppm and 625 ppm exposure groups sacrificed for the 9-
month interim evaluation. The atrophic ovaries were characterized by the absence of oocytes,
follicles, and corpora lutea. No occurrences of this lesion were noted in the lower exposure
groups. Hyperplasia of the germinal epithelium was observed in one animal exposed to 625 ppm
for 9 months. Germinal epithelial hyperplasia was described as prominent down growth of the
mesothelial surface into the parenchyma of the ovary, forming tubular and gland like structures.
At the 15-month interim evaluation, ovarian atrophy was observed in mice exposed to 20 ppm or
higher; the incidence at 62.5 ppm or higher was significant compared with concurrent controls.
Hyperplasia of the germinal epithelium was seen at 200 and 625 ppm at nonsignificant incidences.
Angiectasis (dilation of blood vessels) was seen in one mouse in the control group, one exposed
to 6.25 ppm, and two exposed to 200 ppm. The ovary, which was evaluated at 15 months in only
two female mice exposed to 625 ppm, was atrophic in both. Among female mice exposed to
1,3-butadiene for 2 years, ovarian atrophy was observed in all exposure groups at incidences that
were significantly elevated compared with controls. Therefore, using ovarian atrophy as an
endpoint of reproductive toxicity, a no-observed-adverse-effect level (NOAEL) could not be
defined in this mouse study. The incidence of angiectasis was significantly elevated only at 62.5
and 200 ppm, and the incidence of germinal epithelial hyperplasia was significantly elevated at 20
to 625 ppm. The occurrence of ovarian atrophy and germinal epithelial hyperplasia showed
significant dose-related trends, whereas ovarian

1/28/98
Table 5-1. Reproductive tract lesions in female B6C3F1 mice exposed to 1,3-butadiene by inhalation
Concentration (ppm)
Lesion 0 6.25 20 62.5 200 625
9-Month interim evaluation
Ovarya 10 — — 10 10 8
b
Atrophy 0 (0%) — — 0 (0%) 9 (90%) 8 (100%)b
Germinal 0 (0%) — — 0 (0%) 0 (0%) 1 (13%)
epithelial
hyperplasia
(NOS)
Uterusa 10 — — 10 10 8
Atrophyc 0 (0%) — — 0 (0%) 3 (30%) 6 (75%)
Ovarya 10 10 10 10 10 2
b b
Atrophy 0 (0%) 0 (0%) 1 (10%) 9 (90%) 7 (70%) 2 (100%)d
5-4
Germinal 0 (0%) 0 (0%) 0 (0%) 0 (0%) 3 (30%) 1 (50%)

epithelial
hyperplasia
Angiectasis 1 (10%) 1 (10%) 0 (0%) 0 (0%) 2 (20%) 0 (0%)
a
Uterus 10 1 10 10 10 2
Atrophyc 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 2 (100%)
2-Year studye
Ovarya 49 49 48 50 50 79
Atrophy 4 (8%) 19 (39%) 32 (67%) 42 (84%) 43 (86%) 69 (87%)
p<0.001 p<0.001 p<0.001 p<0.001 p<0.001 p<0.001
Germinal 2 (4%) 3 (6%) 8 (17%) 15 (30%) 15 (30%) 18 (23%)
epithelial p<0.001 p=0.460 p=0.017 p<0.001 p=0.010 p<0.001
hyperplasia
Angiectasis 4 (8%) 6 (12%) 3 (6%) 13 (26%) 14 (28%) 17 (22%)
p=0.259 p=0.366 p=0.606 p=0.017 p=0.021 p=0.425
Uterusa 50 49 50 49 50 78
c
Atrophy 1 (2%) 0 (0%) 1 (2%) 1 (2%) 8 (16%) 41 (53%)
a
Number of animals for which this site was examined microscopically.
b
p<0.01, pairwise comparison with controls by Fisher’s exact test.
c
Statistical tests were not conducted for these lesions.
d
p<0.05, pairwise comparison with controls by Fisher’s exact test.
e
p values for the statistical analysis (logistic regression test) for the 2-year study are presented; the value for the trend test is in the column for the control group, and the value for
pairwise comparisons of individual exposed group with the corresponding control group is in the column for the exposed groups.
Source: NTP, 1993.

angiectasis did not. Although the functional integrity of the female reproductive system was not
assessed, it can be assumed that animals without oocytes or follicles would be infertile and would
express reduced estrogenic and progestin secretory capacities.
Uterine atrophy was seen at the two highest concentrations at 9 months, but was seen only
at the highest concentration at the 15-month evaluation. After 2 years, the incidence of uterine
atrophy among mice exposed to 200 and 625 ppm did not increase relative to that observed at 9
months.
Data regarding the effect of 1,3-butadiene on the reproductive organs of male B6C3F1
mice are summarized in Table 5-2. The testes of males exposed to the highest concentration of
1,3-butadiene (625 ppm) were atrophic at the 9- and 15-month interim evaluations and at
termination of the 2-year study. Among male mice exposed to 1,3-butadiene in the stop-exposure
studies, testicular atrophy was observed in only five mice exposed to 200 ppm (40 weeks), five
exposed to 625 ppm (26 weeks), three exposed to 312 ppm (52 weeks), and three exposed to 625
ppm (13 weeks). It is not possible to determine if the lack of a more prominent response in mice
exposed to 625 ppm for 26 weeks was due to insufficient time for induction of testicular atrophy
or if atrophy had been induced during exposure and the lesion repaired before termination of the
stop-exposure study.
5.1.5. Hackett et al., 1988a

This sperm-head morphology study was conducted in B6C3F1 mice at Pacific Northwest
Laboratories for NTP as part of a series of studies to investigate the effects of 1,3-butadiene on
reproductive function. Twenty male B6C3F1 mice (12 to 13 weeks old) per group were exposed
to 1,3-butadiene (99.88% purity; 174 ± 13 ppm mean headspace dimer [4-vinyl-1-cyclohexene]
concentration) at concentrations of 0 (filtered air), 200, 1,000, or 5,000 ppm 6 h/day for 5
successive days. Measured concentrations (mean ± standard deviation [SD]) were 199 ± 6.12,
999 ± 22.6, and 4,980 ± 130 ppm. The animals were exposed in a 2.3 m3 stainless steel chamber
with a mixing volume of 1.7 m3. Positive controls received intraperitoneal injections of 167
mg/kg of ethyl methane sulfonate daily for 5 consecutive days. After exposure, the mice were
observed twice daily for mortality, morbidity, and signs of toxicity; body weights were determined
weekly. The mice were killed 5 weeks after exposure, weighed, and examined for gross lesions,
with particular emphasis on the reproductive tract. Sperm collected from the right epididymis
were examined for abnormal heads (blunt hook, banana, amorphous, pinhead, two heads/two
tails, short) and other abnormalities (primarily midpiece abnormalities).
Final body weights for the unexposed, treated, and positive control groups were similar,
and net body weight gain over the period of the experiment was also similar for all groups.
Piloerection and dyspnea were observed within the first 20 to 30 min after exposure in mice

1/28/98
Table 5-2. Reproductive tract lesions in male B6C3F1 mice exposed to 1,3-butadiene by inhalation
Concentration (ppm)
Lesion 0 6.25 20 62.5 200 625
Testesa 10 10 10 10 10 10
Absolute weight (g) 0.117 ± 0.002 0.117 ± 0.003 0.114 ± 0.003 0.103 ± 0.004b 0.102 ± 0.002b 0.059 ± 0.003b
Relative weight (mg/g 2.89 ± 0.06 2.92 ± 0.09 2.76 ± 0.09 2.87 ± 0.12 2.54 ± 0.05b 1.57 ± 0.03b
c
BW)
Atrophyd 0 (0%) Ce C C 0 (0%) 6 (60%)
Testesa 10 10 10 10 10 7
Absolute weight (g) 0.116 ± 0.003 0.113 ± 0.003 0.104 ± 0.004 0.112 ± 0.003 0.100 ± 0.003b 0.071 ± 0.004b
5-6
Relative weight (mg/g 2.62 ± 0.07 2.79 ± 0.08 2.48 ± 0.04 2.66 ± 0.07 2.39 ± 0.05f 1.80 ± 0.05b
BW)
Atrophyd 0 (0%) C 0 (0%) C 0 (0%) 4 (57%)

2-Year study
Testesa 50 50 50 48 49 72
Atrophyd 1 (2%) 3 (6%) 4 (8%) 2 (4%) 6 (12%) 53 (74%)
a
Number of animals for which this site was examined.
b
p 0.01, pairwise comparison with controls by Williams’ or Dunnett’s test.
c
BW = body weight.
d
Statistical tests were not conducted for these lesions.
e
Testes were not examined microscopically at this concentration.
f
p<0.05, pairwise comparison with controls by Williams’ or Dunnett’s test.
Source: NTP, 1993.

receiving 5,000 ppm; no signs of toxicity were noted for the other groups. Exposure-related
gross toxicity was not observed in the reproductive tract. The percentages of epididymal sperm
with normal morphology were 98.08%, 97.23% (p<0.05), and 96.34% (p<0.05) at 200, 1,000,
and 5,000 ppm, respectively, compared with 98.40% for controls; these values also showed a
significant exposure-related trend (p 0.05). The percentage of the following abnormalities were
significantly elevated compared with controls (p<0.05): blunt hooks at 5,000 ppm, bananas at
1,000 and 5,000 ppm, and pinheads at 1,000 ppm. Amorphous, two heads/two tails, and shorts
were not significantly elevated at any dose. The predominant types of abnormalities were the
banana followed by blunt hook and amorphous. The authors speculated that late spermatogonia
or early primary spermatocytes were sensitive to 1,3-butadiene. The authors also stated that
examining the sperm at only one time point following termination of exposure precluded a
determination of the stage of spermatogenesis affected by the chemical.
5.1.6. Hackett et al., 1988b

This dominant lethal study was conducted using proven breeder male CD-1 mice (20 per
group) exposed to 0, 200, 1,000, or 5,000 ppm 1,3-butadiene 6 h/day for 5 successive days.
Measured concentrations (mean ± SD) were 200 ± 5.73, 1,010 ± 13.9, and 5,000 ± 85.4 ppm,
respectively. The purity of the 1,3-butadiene was 99.88%, and the headspace dimer concentration
was 215 ± 49 ppm. For mating, one exposed or control male mouse was placed with two
unexposed female mice for 1 week for 8 successive weeks; the two females were replaced each
week. Male mice were sacrificed at termination of matings, and female mice were sacrificed 12
days after the last cohabitation day. The reproductive status, total number, position and status of
implantations, the number of early and late resorptions, and the number of live and dead fetuses
were recorded.
No animals died during the study, and body weights of the exposed groups were similar to
those of the control group. All males exposed to 1,3-butadiene were fertile during the 8-week
exposure period. During the first week of mating (postexposure week), the total number of dead
implants was significantly elevated for the group exposed only to 1,000 ppm (p 0.05) compared
with that of controls. Early resorptions accounted for most of the dead implants. In addition, the
percentage of dead implants relative to the total implants was significantly elevated in groups
exposed to 1,000 ppm (p 0.05), and the percentage of females with more than one intrauterine
death was significantly elevated in all exposed groups (p 0.05) relative to controls. During the
second postexposure week, the total number of dead implants was also significantly elevated at
200 and 1,000 ppm relative to controls. The percentage of dead implants and the percentage of
females with more than one intrauterine death were elevated, but not significantly. For
postexposure weeks 3, 5, 6, 7, and 8, the number of dead implantations, percentage of dead

implantations, and percentage of females with more than one intrauterine death in all exposed
groups were similar to those of controls (i.e., not statistically significant). For postexposure week
4, the percentage of dead implants (5,000 ppm) and the percentage of females with more than one
intrauterine death (200 and 5,000 ppm) were significantly reduced (p 0.05) relative to the control
value. However, the control values for these parameters were unusually high compared with
control values at other postexposure weeks. Thus, the significantly reduced values for treated
mice were probably not treatment related.
The results indicate that exposure to 1,3-butadiene may affect mature spermatozoa and
spermatids assessed by preimplantation deaths for postexposure weeks 1 and 2. Interpretation of
these results is complicated because the effects occurred in the 200 and 1,000 ppm groups but not
in the 5,000 ppm group, which showed no indications of toxicity.
5.1.7. Anderson et al., 1993

The ability of 1,3-butadiene to induce dominant lethal mutations in male mice following
acute and subchronic inhalation exposure was assessed by evaluating the number of dead implants
in females mated to exposed males. For acute exposures, male CD-1 mice were exposed to 0,
1,250, and 6,250 ppm 1,3-butadiene for 6 h; 5 days later, each male was mated to two females.
Males used for subchronic exposures were treated with 0, 12.5, or 1,250 ppm, 6 h/day, 5
days/week for 10 weeks. Following mating in both experiments, one female was killed on
gestation day (gd) 17 and the other was allowed to litter for evaluation of long-term effects on the
offspring. Results of long-term carcinogenic effects on the live offspring are not yet available.
The female killed on gd 17 was examined for number of live fetuses, number and type of
malformations in the fetuses, and number of postimplantation deaths. The only effect seen in the
acute study was a decrease (p 0.05) in the number of implantations in females mated to males
exposed to 1,250 ppm. In the subchronic study, females mated to males exposed to 12.5 ppm had
an increase in the number of late postimplantation deaths (p 0.01; both fetal and placental tissue
were present); females mated to males exposed to 1,250 ppm had a decrease in mean
implantations per dam (p 0.01) and an increase in both early (p 0.001; resorption) and late
postimplantation deaths (p 0.001). 1,3-Butadiene appears to affect the male germ cell line,
resulting in late postimplantation death of the fetuses. It is unknown whether the
mutations/alterations of the germ cells resulting in a reduction in live fetuses are due to an effect
on reproductive ability or a teratogenic effect resulting in death.
5.1.8. Adler et al., 1994

To assess the stage at which male germ cells are affected by 1,3-butadiene, male (102/E1
× C3H/E1)F1 mice were exposed by inhalation to 0 or 1,300 ppm, 6 h/day for 5 consecutive days.

Four hours after the end of exposure, each male was mated at a ratio of 1:2 to untreated virgin
females. Females judged bred by the presence of a vaginal plug were replaced with new females,
and mating continued for 4 consecutive weeks. Females were killed on gd 14 to 16 and examined
for numbers of live and dead implants. Exposure of male mice to 1,300 ppm resulted in an
increase in dead implants during the first to the third weeks of mating; however, statistical
significance (p 0.01) was reached only in the second week. When expressed as a percentage of
dominant lethals, a significant increase was seen in the second (12.4%, p 0.01) and third (5.5%,
p 0.05) weeks. Because of the time course for dominant lethal mutations to manifest as dead
implantations, 1,3-butadiene appears to induce dominant lethality in spermatozoa and late
spermatids.
5.2. DEVELOPMENTAL EFFECTS

The developmental toxicity study sponsored by the International Institute of Synthetic
Rubber Producers (IISRP, 1982) is the same as the Hazleton study discussed briefly in the 1985
EPA document (U.S. EPA, 1985). Hackett and coworkers also conducted two developmental
toxicity studies, one using rats (Hackett et al., 1987a) and one using mice (Hackett et al., 1987b).
The study using rats was conducted to confirm and extend the findings of the IISRP (1982) study
in rats, and the mouse study was conducted for comparison of a rodent species more sensitive
than the rat to the toxic effects of 1,3-butadiene.
5.2.1. IISRP, 1982

Female Sprague-Dawley CD rats were mated with male rats of the same strain (2f:1m) to
produce 138 sperm-positive females. Groups of mated females (220 to 266 g) were exposed by
inhalation to 1,3-butadiene at target concentrations of 0, 200, 1,000, or 8,000 ppm 6 h/day on gd
6 to 15 and killed on gd 20. Measured concentrations (mean ± SD) were 2.8 ± 1.2, 202 ± 14, 990
± 24, and 7,647 ± 375 ppm for 0, 200, 1,000, and 8,000 ppm, respectively. The animals were
exposed in stainless steel chambers. Twenty-four pregnant females were exposed to each
concentration of 1,3-butadiene, 40 were exposed to filtered air (controls), and 26 were given 250
mg acetylsalicylic acid/kg body weight by gavage on gd 6 to 15 (positive controls). The purity of
the 1,3-butadiene was not reported; the mean concentration of the dimer, 4-vinyl-1-cyclohexene,
was 108.6 ± 53.59, well below the target of 300. The rats were weighed on gd 0, 3, 6, 9, 12, 15,
18, and 20. Various parameters of maternal and developmental toxicity were evaluated and
analyzed using the litter as the statistical unit.
Maternal effects of 1,3-butadiene are summarized in Table 5-3. No animals died of
exposure to 1,3-butadiene. One animal exposed to 1,000 ppm was killed because of morbidity
unrelated to treatment. Clinical signs of toxicity were not observed in any group, and the

Table 5-3. Maternal toxicity in Sprague-Dawley CD rats exposed to
1,3-butadiene by inhalation
Concentration (ppm)
Parameter 0 200 1,000 8,000
No. dams assigned 40 24 24 24
No. of deaths 0 0 1a 0
No. pregnant (%) 90 91.7 100 95.8
Whole body weight (g)
Day 0 239 238 240 239
Day 20 362 357 355 347
Body weight gainb (g)
Days 0-6 24 24 23 23
Days 6-9 13 9 1c 1c
Days 9-12 16 13 14 11c
Days 12-15 15 15 16 15
Days 15-20 54 58 61 60
Gravid uterine weight (g) 63.9 61.1 66.5 62.8
Extragestational weightd (g) 297.9 296.2 280.8 283.9
Extragestational weight gaine (g) 59 58 49f 45f
Significant clinical signs None None None None
a
This animal was killed in moribund state on day 19; morbidity was not related to exposure to butadiene.
b
Determined from differences in group mean body weights reported for specific days of gestation.
c
p<0.01, compared with corresponding control; analysis of variance andt test.
d
Body weight on gd 20 minus gravid uterine weight.
e
Extragestational weight minus body weight on gd 0.
f
p<0.05, compared with corresponding control; analysis of variance andt test.
Source: IISRP, 1982.

pregnancy rates were similar in all groups. Terminal body weights showed a dose-related
decrease (no statistical analysis). Maternal body weight gain was markedly depressed in dams
exposed to 1,000 and 8,000 ppm, especially during gd 6 to 9; a significant decrease was also
noted during gd 9 to 12 in rats exposed to 8,000 ppm. During the later stages (gd 12 to 15 and
16 to 20), body weight gain was similar to controls. The gravid uterus and extragestational
weights were similar to controls, but extragestational weight gain was significantly depressed by
17% (p<0.05) in dams exposed to 1,000 ppm and by 24% in dams exposed to 8,000 ppm
(p<0.05). No effects were observed on other measures of maternal toxicity. Developmental
effects of 1,3-butadiene are summarized in Tables 5-4 and 5-5. Fetal body weight and
crown/rump length were significantly reduced at 8,000 ppm (p<0.05). The percentage of fetuses
with major skeletal defects was significantly elevated at 1,000 and 8,000 ppm, and minor skeletal
defects were significantly elevated only at the lowest concentration. The percentage of fetuses
showing minor external/visceral defects, predominantly subcutaneous hematomas, was
significantly elevated only at 1,000 ppm, but the percentage was similar in all three experimental
groups. The incidence of bilateral lens opacity was elevated at all concentrations but was
significantly elevated only at 8,000 ppm. The incidence of marked-to-severe wavy ribs and the
total number of abnormal ossifications and irregular ossification of the ribs were elevated at 8,000
ppm. The incidence of thoracic bipartite centers was elevated in all exposed groups; a dose-
response relationship was not observed. Other malformations and variations occurred at
incidences similar to those of controls or were not significantly elevated compared with controls.
5.2.2. Hackett et al., 1987a

For the experiment with rats, 208 female Sprague-Dawley CD rats and 108 male Sprague-
Dawley CD rats (all 7 to 8 weeks old) were used. The rats were mated by placing two females
with one male rat overnight for 5 consecutive nights or until a sperm-positive vaginal smear was
obtained; gd 0 was the day sperm were detected. Thirty sperm-positive female rats per group
were exposed to 0, 40, 200, or 1,000 ppm 1,3-butadiene (99.84% purity; 197 ± 6 ppm mean
headspace dimer concentration). The measured concentrations (mean ± SD) were 40.1 ± 0.62
(mean ± SD), 199.8 ± 2.61, and 1,005 ± 11.9 ppm, respectively. On gd 6 to 15, the females were
exposed for 6 h/day in stainless-steel chambers having a total volume of 2.3 m3 and a mixing
volume of 1.7 m3. The females were weighed 1 week before mating and on gd 0, 6, 11, 16, and
20 (the day of sacrifice). Various parameters of maternal and developmental toxicity were
evaluated. The experimental design is summarized in the upper section of Table 5-6.
The effects of inhalation exposure to 1,3-butadiene on maternal endpoints in rats are
summarized in Table 5-7. All females survived to the end of the study. No clinical signs of
toxicity were observed. Final body weights were similar to those of controls; body weight gain,

Table 5-4. Developmental toxicity in Sprague-Dawley CD rats exposed to
Concentration (ppm)
Parameter 0 200 1,000 8,000
No. pregnant (%) 90 91.7 100 95.8
No. litters with live fetuses 36 22 23 23
No. implantations/dama 13.0 12.8 14.1 13.8
Preimplantation loss (%) 15.4 17.1 11.5 12.4
Postimplantation loss (%) 3.6 6.0 4.9 7.3
Total no. resorptions 17 17 16 23
Early resorptions 16 13 16 20
Dead fetuses/litter 0 0 0 0
No. fetuses/no. litters examined 450/36 265/22 308/23 294/23
Fetal body weighta (g) 3.3 3.2 3.2 3.1b
Femalesa 3.2 3.1 3.1 3.0
Malesa 3.4 3.3 3.3 3.2
Crown/rump length (mm) 37.8 37.2 37.2 35.9c
Sex ratio (% males) 49.8 54.7 51 50
a
Mean values.
b
p<0.05, Wilcoxon test.
c
p<0.01, Wilcoxon test.

Table 5-5. Malformations and variations in Sprague-Dawley CD rats
exposed to 1,3-butadiene by inhalation
Concentration (ppm)
Parameter 0 200 1,000 8,000
Total no. fetuses/no. litters 450/36 265/22 308/23 294/23

examined
External/visceral defects
Minor defects 76 (16.9)a 63 (23.8) 75 (24.4)b 75 (23.3)
Major defects 0 0 0 2 (0.7)
Unilateral lens opacity 19 (4.2) 11 (4.2) 8 (2.6) 13 (4.4)

Bilateral lens opacity 18 (4.0) 24 (9.1) 30 (9.7) 31 (9.5)b
Bilateral ureter dilation 13 (2.9) 8 (3.0) 3 (1.6) 19 (6.5)
Skeletal defects
Minor defects 72 (23.2) 49 (26.9)c 45 (20.9) 43 (21.1)
Major defects 2 (0.6) 4 (2.2) 6 (2.8)b 12 (5.9)d
Any thoracic center (10-13) 4 (1.29) 11 (6.04)d 14 (6.51)d 8 (3.92)d

bipartite
Wavy ribs (marked to 2 (0.6) 4 (2.2) 3 (2.3) 7 (3.4)b

severe)
Wavy ribs (slight to 3 (1.6) 3 (1.7) 7 (3.3) 8 (3.9)
moderate)
Variations (abnormal ossification) 267 (85.9) 164 (90.1) 185 (84.0) 199 (97.5)c
Skull (occipital) 79 (25.4) 58 (31.9) 69 (32.1) 71 (34.8)

Skull (interparietal) 82 (26.4) 66 (36.3) 79 (36.7) 75 (36.7)
Sternebrae no. 6 152 (48.9) 107 (58.8) 126 (58.6) 147 (72.1)
Ribs 0 1 (0.55) 2 (0.93) 6 (2.9)b
Metacarpals 207 (66.6) 140 (76.9) 141 (65.6) 172 (84.3)
Phalanges 141 (45.3) 114 (62.6) 139 (64.6) 123 (60.3)
a
Numbers of fetuses affected; numbers in parentheses denote the percentage of affected fetuses/fetuses examined.
b
p<0.05, Fisher’s randomization test based on frequencies of affected litters.
c
p<0.05, Wilcoxon’s test.
d
p<0.01, Fisher’s randomization test based on frequencies of affected litters.

Table 5-6. Design of the developmental toxicity studies on 1,3-butadiene
Species/strain/route of Exposure No. of animals/ Gestation days of Gestation day of

exposure (ppm) group exposure sacrifice
Rat/ 0 30 6-15 20
Sprague-Dawley CD/
Inhalation 40 30 6-15 20
200 30 6-15 20
1,000 30 6-15 20
Mouse/ 0 32 6-15 18
CD-1/Inhalation
40 33 6-15 18
200 31 6-15 18
1,000 33 6-15 18
Source: Hackett et al., 1987a, b.

Table 5-7. Maternal toxicity in Sprague-Dawley CD rats exposed to
Concentration (ppm)
Parameter 0 40 200 1,000
No. of deaths 0 0 0 0
No. pregnant (%) 28 (93) 24 (80) 26 (87) 28 (93)
Day 0 242 ± 3.7a 239 ± 3.2 244 ± 3.0 242 ± 4.0
Day 20 362 ± 7.1 351 ± 5.9 369 ± 6.6 354 ± 6.1
Body weight gain (g)
Days 0-6 21.4 ± 1.6 21.1 ± 1.6 22.9 ± 1.3 20.1 ± 1.5
Days 6-11 25.5 ± 1.3 23.6 ± 1.3 26.6 ± 1.5 17.5 ± 1.9b
Days 11-16 29.2 ± 1.4 30.9 ± 1.7 31.7 ± 1.9 31.2 ± 2.1
Days 16-20 44.5 ± 1.8 36.7 ± 2.5 43.6 ± 2.3 43.2 ± 2.9
Gravid uterine weight (g) 73.0 ± 2.9 69.5 ± 3.5 73.9 ± 2.8 71.2 ± 4.1
Extragestational weightc (g) 289 ± 5.7 282 ± 3.9 295 ± 5.8 283 ± 3.5
Extragestational weight 47.6 ± 2.8 42.7 ± 2.2 50.9 ± 3.0 39.9 ± 3.5b
gaind (g)
Significant clinical signs None reported None reported None reported None reported
a
Mean ± standard error.
b
p<0.05, compared with corresponding control.
c
d
Source: Hackett et al., 1987a.

however, was reduced by about 30% (p<0.05) in the 1,000 ppm group during the first 5 days of
exposure (gd 6 to 11). From gd 11 to 20, body weight gain was not significantly different from
that of controls. The gravid uterine weights and extragestational weights (whole body weight
minus gravid uterine weight) were similar to those of controls, but extragestational weight gain
was significantly lower (16%; p<0.05) in dams exposed to 1,000 ppm than in control dams.
The overall pregnancy rates were similar among all groups, ranging from 80% among
dams exposed to 40 ppm to 93% among controls and dams exposed to 1,000 ppm (Table 5-7).
Fetal measures, including the numbers of implantations/dam, resorptions/litter, dead fetuses/litter,
fetal body weights, sex ratios, malformations, and variations, were not affected by exposure to
1,3-butadiene (Tables 5-8 and 5-9). Overall, no developmental toxicity was observed in rats
exposed to 40 to 1,000 ppm during gd 6 to 15; a slight maternal toxicity, manifested as reduced
extragestational weight gain, was observed at the 1,000 ppm level.
5.2.3. Hackett et al., 1987b

Because 1,3-butadiene is more toxic in mice than in rats, a study was also conducted in
CD-1 mice using a protocol similar to that used for the rats. Groups of 31 to 33 sperm-positive
females were exposed to 0 (filtered air), 40, 200, or 1,000 ppm 1,3-butadiene (99.88% purity; 338
± 72 ppm mean headspace dimer concentration), 6 h/day on gd 6 to 15 (Table 5-6, bottom
section). Measured concentrations were 39.9 ± 0.06, 199.8 ± 3.0, and 1,000 ± 13.1 ppm (mean ±
SD). The dams were weighed on gd 0, 6, 11, 16, and 18 (day of sacrifice).
The effects of 1,3-butadiene on maternal toxicity in CD-1 mice are summarized in Table 5-
10. Three animals exposed to 1,000 ppm showed signs of dehydration: two died on gd 15, and
early parturition occurred in the third. No other clinical signs of toxicity were observed.
Exposure-related decreases in whole body weights on gd 18, body weight gain during gd 11 to
16, gravid uterine weight, extragestational weight, and extragestational weight gain were
significantly reduced in the 1,000 ppm exposure group compared with controls. Whole body
weight gain during gd 11 to 16 and extragestational weight gain was also reduced in the 200 ppm
exposure group. None of these parameters were significantly affected in dams exposed to 40
ppm. The pregnancy rates in mice were uniformly low in all groups and unaffected by exposure
to 1,3-butadiene. The effects of 1,3-butadiene on various parameters of developmental toxicity in
CD-1 mice are summarized in Tables 5-11 and 5-12. More resorptions per litter were observed
among control dams than among exposed dams. Fetal body weights were reduced in all exposed
groups compared with controls, and the reduction showed a significant exposure-related trend.
The overall fetal body weights (males and females combined) were reduced by 4.5% at 40 ppm
(not significant), 15.7% at 200 ppm (p 0.05), and 22.4% at 1,000 ppm (p 0.05). Significant
differences from controls were seen at all treatment concentrations for fetal males and

Table 5-8. Developmental toxicity in Sprague-Dawley CD rats exposed to
Concentration (ppm)
Parameter 0 40 200 1,000
No. pregnant (%) 28 (93) 24 (80) 26 (87) 28 (93)
No. litters with live fetuses 28 24 26 27a
No. implantations/dam 14.4 ± 0.55b 14.0 ± 0.71 15.3 ± 0.45 14.8 ± 0.63
No. resorptions/litter 0.46 ± 0.17 0.58 ± 0.17 0.96 ± 0.26 0.67 ± 0.14
Early resorptions/litter 0.39 ± 0.15 0.54 ± 0.16 0.88 ± 0.25 0.63 ± 0.14
Fetal body weight (g) 3.49 ± 0.04 3.44 ± 0.05 3.40 ± 0.05 3.50 ± 0.06
Females 3.40 ± 0.05 3.36 ± 0.05 3.29 ± 0.06 3.38 ± 0.06
Males 3.59 ± 0.05 3.52 ± 0.05 3.51 ± 0.06 3.59 ± 0.06
Sex ratio (% males) 50.2 ± 2.281 52.5 ± 2.95 50.5 ± 2.77 52.5 ± 2.58
a
One rat had only one implant; this animal was excluded from statistical evaluations.
b

Table 5-9. Malformations and variations in Sprague-Dawley CD rats
exposed to 1,3-butadiene by inhalation
Concentration (ppm)
Parameter 0 40 200 1,000
No. fetal heads examined 196 161 185 191
Malformationsa
Generalized edema 1/1 3/1 1/1 3/1
Hydrocephalus --b 3/3 -- --
Meningoencephalocele -- -- -- 2/1
Missing rib -- -- 2/2 --
Variations
Low ear set -- -- 2/1 --
Hydroureter 36/17 35/15 39/14 31/12
Misaligned sternebrae -- -- 1/1 1/1
Extra rib -- 1/1 4/2 --
Reduced ossification
Skull 27/13 22/13 18/10 29/11
Sternebrae no. 1-4 60/15 48/13 95/21 66/15
Ribs 1/1 2/2 5/3 2/2
Thoracic vertebrae (centra) 109/23 97/21 75/21 81/25
Pelvis 9/7 -- 6/5 5/5
Phalanges 1/1 6/1 2/1 --
a
Expressed as number of fetuses/number of litters; includes only those findings occurring in more than one fetus
or at more than one concentration.
b
--, no malformations observed.

Table 5-10. Maternal toxicity in pregnant CD-1 mice exposed to
Concentration (ppm)
Parameter 0 40 200 1,000
No. of deaths 0 0 0 3
No. pregnant (%) 18 (56) 19 (57) 21 (68) 22 (67)
Day 0 28.4 ± 0.25a 28.3 ± 0.32 28.2 ± 0.32 28.4 ± 0.32
Day 18 54.9 ± 1.21b 55.4 ± 1.09 52.5 ± 1.01 50.8 ± 0.86c
Body weight gain (g)
Days 0-6 2.7 ± 0.3 3.0 ± 0.3 2.5 ± 0.2 2.3 ± 0.2
Days 6-11 5.5 ± 0.4 5.8 ± 0.3 5.6 ± 0.3 4.8 ± 0.3
Days 11-16 13.3 ± 0.6b 12.7 ± 0.4 11.4 ± 0.5c 10.6 ± 0.4c
Days 16-18 5.5 ± 0.3b 5.7 ± 0.3 4.7 ± 0.4 4.8 ± 0.3
Gravid uterine weight (g) 19.3 ± 1.00b 20.3 ± 0.80 18.0 ± 0.87 16.8 ± 0.67c
Extragestational weightd (g) 35.5 ± 0.48b 35.1 ± 0.44 34.5 ± 0.46 34.1 ± 0.36c
Extragestational weight gaine 7.60 ± 0.48b 6.99 ± 0.38 6.20 ± 0.38c 5.91 ± 0.28c
(g)
Significant clinical signs None None None Dehydration
a
b
p 0.05, significant linear trend.
c
p 0.05, pairwise comparison with corresponding control parameter.
d
e
Source: Hackett et al., 1987b.

Table 5-11. Developmental toxicity in CD-1 mice exposed to 1,3-butadiene
by inhalation
Concentration (ppm)
Parameter 0 40 200 1,000
No. pregnant (%) 18 (56) 19 (57) 21 (68) 22 (67)
No. litters with live fetuses 18 19 21 20
No. implantations/dam 12.7 ± 0.52 13.3 ± 0.44 13.0 ± 0.64 13.1 ± 0.43
No. resorptions/litter 1.06 ± 0.22 0.84 ± 0.22 0.67 ± 0.20 0.90 ± 0.19
Early resorptions 1.00 ± 0.23 0.58 ± 0.21 0.43 ± 0.13a 0.75 ± 0.16
No. fetuses/no. litters 11.7 ± 0.66 12.5 ± 0.52 12.3 ± 0.62 12.2 ± 0.51
examined
Fetal body weight (g) 1.34 ± 0.03b 1.28 ± 0.01 1.13 ± 0.02a 1.04 ± 0.03a
Females 1.30 ± 0.03b 1.25 ± 0.01 1.10 ± 0.02a 1.06 ± 0.02a
Males 1.38 ± 0.03b 1.31 ± 0.02a 1.13 ± 0.02a 1.06 ± 0.02a
Placental weight (mg) 86.8 ± 2.99b 85.4 ± 2.29 78.6 ± 3.24a 72.6 ± 1.88a
Females 83.1 ± 3.03b 80.9 ± 2.46 74.7 ± 3.52a 70.1 ± 2.33a
Males 89.3 ± 3.03b 89.5 ± 2.27 80.1 ± 2.35a 74.5 ± 1.81a
Sex ratio (% males) 51.6 ± 3.91 49.8 ± 3.06 51.5 ± 3.68 51.8 ± 3.29
a
p 0.05, pairwise comparison with corresponding control.
b
p 0.05, significant linear trend.

Table 5-12. Malformations and variations in CD-1 mice exposed to
Concentration (ppm)
Parameter 0 40 200 1,000
No. fetal heads examined 105 120 130 120
Malformationsa
Exencephalus 1/1 --b -- 2/2
Open eye 1/1 -- -- 1/1
Limb flexure 2/1 -- -- --
Fused sternebrae — -- — 2/2
Fused ribs -- 2/2 -- --
Variations
Pale 2/2 -- -- --
Hydroureter 2/2 6/3 -- --
Abnormal sternebraec,d 0.6 ± 0.9 0.4 ± 0.7 0.4 ± 0.8 0.8 ±1.3e
Misaligned 10/6 3/3 9/8 10/8

sternebrae
Ossification site between -- 1/1 1/1 3/3

sternebrae 5 and 6
Supernumerary ribsc,d 1.7 ± 2.3 1.6 ± 2.1 6.0 ± 3.6e 9.9 ± 3.0e
Supernumerary ribs 30/11 30/9 127/20 198/20

(total number)
Normal length 6/5 5/1 29/9 68/10
Rudimentary 13/6 19/8 81/20 120/16
Ossification site at 11/5 6/4 17/10 10/7

lumbar 1

Table 5-12. Malformations and variations in CD-1 mice exposed to
1,3-butadiene by inhalation (continued)
Concentration (ppm)
Parameter 0 40 200 1,000
Reduced ossification (all sites)c 1.7 ± 1.7 1.2 ± 1.5 2.7 ± 2.7 3.9 ± 2.6e
Skull -- -- 2/2 3/1
Sternebrae 31/13 20/9 57/16f 76/19f
Vertebrae (centra) -- 1/1 -- 1/1
Phalanges -- -- -- 2/16
a
Expressed as number of fetuses/number of litters; includes only those findings occurring in more than one fetus
or at more than one concentration.
b
--, no malformations or variations noted.
c
Mean percentage per litter (mean ± SD).
d
p<0.05, linear trend, orthogonal contrast test.
e
p<0.05, Tukey’s test.
f
p<0.05, Fisher exact test (fetal incidence).

at the two higher concentrations for females. Placental weights showed an effect similar to that of
fetal body weights (Table 5-11). Malformations occurred sporadically and at low frequencies in
all exposure groups (Table 5-12). The frequency of supernumerary ribs was greatly elevated at
200 and 1,000 ppm; 6% of the fetuses/litter were affected at 200 ppm (p<0.05) and 9.9% at 1,000
ppm (p<0.05) compared with 1.7% in controls and 1.6% in the 40 ppm exposure group (not
significant). There also was a marked increase in the total number of fetuses with supernumerary
ribs at the 200 and 1,000 ppm exposure levels. The frequency of reduced ossification of the
sternebrae was elevated at 200 (p<0.05) and 1,000 ppm (p<0.001) (Fisher exact test); the litter
incidence was elevated but not significantly. The percentages of reduced ossifications at all sites
and the percentages of abnormal sternebrae (misaligned, scrambled, or cleft) per litter were also
significantly elevated at 1,000 ppm (p<0.05). The percentages of supernumerary ribs and
abnormal sternebrae also showed significant linear trends.
These studies showed that inhalation exposure to 1,3-butadiene causes maternal toxicity,
manifested as reduced body weight gain, in the mouse at 200 and 1,000 ppm; therefore, the
NOAEL for maternal toxicity is 40 ppm. 1,3-Butadiene also caused developmental effects,
manifested by reduced fetal body weight and increased frequency of skeletal variations at 200 and
1,000 ppm. In addition, inhalation exposure to 1,3-butadiene during gestation caused a significant
reduction in body weight of male fetuses at 40 ppm. Therefore, a NOAEL for developmental
toxicity in CD-1 mice could not be obtained. Although 1,3-butadiene did not induce gross
malformations in the mouse fetus, the dose-related increases in supernumerary ribs and reduced
ossifications, particularly of the sternebrae, may indicate delayed or altered development and
should be cause for concern.
5.2.4. Anderson et al., 1993

The acute and subchronic effects of inhalation exposure to 1,3-butadiene in male mice on
fetal abnormalities were examined. For acute exposures, male CD-1 mice were exposed to 0,
1,250, and 6,250 ppm 1,3-butadiene for 6 h; 5 days later each male was mated to two females.
Males used for subchronic exposures were treated with 0, 12.5, or 1,250 ppm 6 h/day, 5
days/week for 10 weeks. Following mating in both experiments, one female was killed on gd 17
and the other was allowed to litter. The female killed on gd 17 was examined for number of live
fetuses, number and type of malformations in the fetuses, and number of postimplantation deaths.
No treatment-related abnormalities were observed in offspring of males treated on the acute
exposure protocol; one fetus from one control litter had a gastroschisis (fissure of abdominal
cavity), and one fetus from each of the two low-dose litters was a runt (body weight 67% of
mean litter weight). Following subchronic exposure of males to 1,3-butadiene, 7 of 306 fetuses
sired by males exposed to 12.5 ppm (p 0.05) and 3 of 406 fetuses sired by males exposed to

1,250 ppm were affected compared with 0 of 278 fetuses sired by control males. Abnormalities in
low-dose fetuses included four exencephalies, two runts ( 70% of mean litter weight), and one
fetus with blood in the amniotic sac. At the high-dose level, one hydrocephaly and two runts
( 75% of mean litter weight) were observed. The authors calculated statistical significance on a
fetal incidence basis rather than on a litter incidence basis. Because litter incidence rates were not
included in the data, it is not possible to discern whether the affected fetuses were only from one
or two litters or whether a high percentage of litters sired by exposed males were affected.
Therefore, this study is inadequate to assess the developmental toxicity of 1,3-butadiene following
exposure of males prior to mating.
5.3. STRUCTURE-ACTIVITY RELATIONSHIPS

Data on structure-activity relationship are summarized in Table 5-13.
5.3.1. NTP, 1986

The 1,3-butadiene dimer, 4-vinylcyclohexene, and its diepoxide, 4-vinyl-1-cyclohexene
diepoxide, have been tested in long-term toxicological and carcinogenicity studies in rats and
mice. The NTP study (1986) on 4-vinylcyclohexene used male and female F344 rats and male
and female B6C3F1 mice dosed by gavage with 0, 200, or 400 mg/kg 4-vinylcyclohexene in corn
oil 5 days/week for 105 weeks. No nonneoplastic lesions attributed to exposure to 4-
vinylcyclohexene were observed in the reproductive organs of male or female mice or rats, and
hence data are not presented in Table 5-13. The incidences of granulosa cell neoplasms, mixed
benign tumors, granulosa cell hyperplasia, and tubular cell hyperplasia were increased in female
mice. Tubular cell hyperplasia is a proliferative lesion originating in the germinal epithelium; the
hyperplastic cells invade the ovarian stroma forming tubular structures. The granulosa cell
hyperplasia is a morphological continuum with granulosa cell neoplasms and tubular hyperplasia
with mixed benign tumors and therefore should not be included with nonneoplastic lesions. The
authors noted that female mice treated with 1,200 mg/kg 4-vinylcyclohexene for 5 days/week for
13 weeks had reduced numbers of primary and mature Graafian follicles whether they survived
until termination (5/10) or died before termination (5/10).
In another NTP study (1989), male and female F344 rats and B6C3F1 mice were treated
topically with 4-vinyl-1-cyclohexene diepoxide 5 days/week for 13 weeks and 2 years. No
nonneoplastic lesions occurred in reproductive organs of rats. Female mice treated for 13 weeks,
however, showed evidence of diffuse ovarian atrophy in 10/10 animals that received 10 mg/mouse
(highest dose) and in 4/10 receiving 5 mg/mouse. Uterine atrophy was observed in 2/10 animals
that received 10 mg/kg. In the 2-year study, ovarian atrophy occurred in almost

1/28/98
Table 5-13. Reproductive and developmental toxicity of chemicals structurally similar to 1,3-butadiene
Chemical Species Dose Effects LOAEL Reference

4-vinyl-1- Male and 2.5, 5, 10 mg/kg Females NTP 1989
cyclohexene female topically 5 days/week for 13 weeks: 5 mg/kg: ovarian atrophy; 10 mg/kg: 5 mg/kg for 13 weeks
B6C3F1 mice 13 or 105 weeks uterine atrophy
105 weeks: 2.5 mg/kg: ovarian atrophy 2.5 mg/kg for 105 weeks
Males
105 weeks: 5 mg/kg: inflammation of epididymis 5 mg/kg for 105 weeks
Butadiene Female 0.005, 0.02, 0.09, 0.36, 1.43 mmol/kg: reduced ovarian and uterine 1.43 mmol/kg Doerr et al.,
monoepoxide B6C3F1 mice 1.43 mmol/kg weights; decreased follicular counts 1995, 1996
intraperitoneally once
daily for 30 days
Female none Doerr et al.,
Sprague- 1996
Dawley rats
Butadiene Female 0.002, 0.009, 0.036, 0.14, 0.14 mmol/kg: decreased ovarian and uterine 0.14 mmol/kg Doerr et al.,
diepoxide B6C3F1 mice 0.29 mmol/kg weights; decreased follicular counts 1995, 1996
intraperitoneally once
5-25
daily for 30 days

Female 0.14 mmol/kg: decreased ovarian weight 0.14 mmol/kg Doerr et al.,
Sprague- 0.29 mmol/kg: decreased uterine weights 1996
Dawley rats
Isoprene Male B6C3F1 70, 220, 700, 2,200, 13 and 26 weeks: 7,000 ppm: testicular atrophy 7,000 ppm Melnick et al.,
mice 7,000 ppm inhalation 6 1994

h/day, 5 days/week, 13 or
26 weeks
Male F344 13 weeks: none Melnick et al.,
rats 1994
26 weeks: 7,000 ppm: hyperplasia of interstitial 7,000 ppm for 26 weeks
cells
all groups treated with 2.5, 5, and 10 mg/mouse (43/49, 42/49, and 47/50, respectively, compared
with 12/50 for controls). Ovarian atrophy such as that in animals exposed to 1,3-butadiene was
characterized by a complete absence of follicles and corpora lutea. Tubular hyperplasia occurred
at a high incidence in all dose groups (35/49, 38/49, and 34/50, respectively, compared with 5/50
for controls). In male mice, subacute inflammation of the epididymis occurred in 0/50, 6/50, and
13/49, respectively, compared with 0/50 for controls.
5.3.2. Melnick et al., 1994

Differences in susceptibility between rats and mice were seen in inhalation studies with
isoprene, the 2-methyl analogue of 1,3-butadiene. Male F344 rats and B6C3F1 mice were
exposed to 0, 70, 220, 700, 2,200, and 7,000 ppm isoprene 6 h/day, 5 days/week for either 13
weeks or 26 weeks followed by a 26-week recovery period. After 13 weeks of exposure, no
effects were observed in rats at any concentration, but testicular atrophy occurred in mice at
7,000 ppm. Following 26 weeks of exposure, all treated rats in the 7,000 ppm group had
hyperplasia of the interstitial cells of the testis (p 0.01; 10/10 vs. 1/10 controls); however,
following the 26-week recovery, there was only a marginal increase (not significant) in benign
testicular tumors: 9/30 compared with 3/30 for controls. Mice also had an increase in the
incidence of testicular atrophy following 26 weeks of exposure to 7,000 ppm (p 0.05; 5/10 vs.
0/10 controls). After 26 weeks of recovery, mice had a slight increase (not significant) in
testicular atrophy at 7,000 ppm (3/29 compared with 0/29 for controls).
5.3.3. Doerr et al., 1996

This study tested the ovarian toxicity of the mono- and diepoxide metabolites of 1,3-
butadiene in mice and rats. Butadiene monoepoxide (0.005, 0.02, 0.09, 0.36, or 1.43 mmol/kg),
butadiene diepoxide (0.002, 0.009, 0.036, 0.14, or 0.29 mmol/kg), or vehicle (sesame oil) was
administered intraperitoneally once daily to female B6C3F1 mice and Sprague-Dawley rats for 30
days. Following day 30, animals were sacrificed by CO2 inhalation, the ovaries and uteri were
weighed, and the ovaries processed for histologic examination of preantral follicles. At the high
dose, the monoepoxide resulted in reduced ovarian and uterine weights (p 0.05) and decreased
follicular counts in mice; rats, however, were unaffected. The diepoxide resulted in decreased
ovarian weights (p 0.05) in mice and rats at 0.14 mmol/kg and decreased uterine weights
(p 0.05) in mice at 0.14 mmol/kg and in rats at 0.29 mmol/kg. Because organ weights were
given in a histogram, the absolute differences were not available; all significant reductions
appeared to be approximately 50% of the control values. The ED50 value was defined as the
effective dose that reduces the follicular number to 50% of control. In mice, ED50 values for the
monoepoxide were 0.29 and 0.40 mmol/kg and for the diepoxide were 0.1 and 0.14 mmol/kg for

small and growing follicles, respectively. However, in rats, only 32% of the follicular population
was depleted at the highest diepoxide concentration. Therefore, mice were more sensitive than
rats to the ovotoxic effects of the mono- and diepoxides of 1,3-butadiene, and the diepoxide was
the more potent ovotoxicant in both species.
Doerr et al. (1995) also studied the ovarian toxicity of 4-vinylcyclohexene and several
related olefins, including butadiene mono- and diepoxide. Mice were administered 1.43 mmol/kg
of the monoepoxide or 0.14 mmol/kg of the diepoxide once daily for 30 days. Following day 30,
the mice were killed and the ovaries removed and sectioned for histologic examination. Mean
follicle counts in mice treated with the monoepoxide were depleted 98% and 71% for small and
growing follicles, respectively, compared with controls. In mice treated with the diepoxide,
follicle counts were depleted 85% and 63% for small and growing follicles, respectively,
compared with controls. Structural analogs of vinylcyclohexene that contain only a single
unsaturated site (vinylcyclohexane, ethylcyclohexene, cyclohexene) and their monoepoxide
metabolites were not ovotoxic to mice. On the other hand, butadiene monoepoxide, butadiene
diepoxide, and isoprene were ovotoxic. The study showed a relationship between chemical
reactivity, as assessed by nicotinamide alkylation, and ovotoxicity with vinyldiepoxide and
butadiene diepoxide that was 3.5 to 10 times more reactive than their monoepoxide precursors
and other structurally related monoepoxides. It can be concluded that those compounds that are
metabolized to a diepoxide or are a diepoxide are ovotoxic.
5.4. SUMMARY AND CONCLUSIONS

Evidence has been presented showing that 1,3-butadiene induces reproductive and
developmental effects in rodents. Although the studies conducted by Carpenter et al. (1944)
examined reproductive toxicity in four different species (rat, guinea pig, rabbit, and dog), the
experimental protocol and the results obtained are inadequate for evaluating reproductive toxicity.
The three long-term toxicity studies conducted in Sprague-Dawley CD rats (Owen et al., 1987;
Owen and Glaister, 1990) and B6C3F1 mice (NTP, 1984, 1993) suggest that mice are much more
sensitive than rats to the reproductive effects of 1,3-butadiene. Reproductive toxicity was not
observed in either male or female rats exposed intermittently to 1,3-butadiene at concentrations
up to 8,000 ppm for 2 years. However, ovarian atrophy was observed in female mice exposed to
6.25 to 625 ppm 1,3-butadiene.
Ovarian atrophy occurred in 39% of 49 mice at 6.25 ppm (the lowest concentration
tested) only after exposure for 2 years, a time at which this condition is expected to appear in
aged animals due to normal senescence mechanisms; however, it occurred in a significantly
greater number of mice exposed to 1,3-butadiene than in control animals. Furthermore, ovarian
atrophy was observed as early as 9 months after exposure to 200 and 625 ppm and 15 months

after exposure to 62.5 ppm. Therefore, the dose-response relationship observed for ovarian
atrophy and the significant increase at the lowest dose relative to that seen in control animals of a
similar age is evidence for a causal relationship between ovarian atrophy and exposure to 1,3-
butadiene at 6.25 ppm.
Similar ovarian lesions have been observed in mice after exposure to the 1,3-butadiene
dimer, 4-vinylcyclohexene, administered by gavage for 13 weeks at 1,200 mg/kg for 5 days/week
(NTP, 1986) or its diepoxide, 4-vinyl-1-cyclohexene diepoxide, administered by topical
application for 13 weeks or 2 years (NTP, 1989). Rats administered 4-vinylcyclohexene or 4-
vinyl-1-cyclohexene diepoxide did not develop ovarian lesions, thus showing a species-specific
response similar to that after exposure to 1,3-butadiene.
Ovarian lesions induced by 1,3-butadiene, 4-vinylcyclohexene, or 4-vinyl-1-cyclohexene
diepoxide are characterized by the absence of oocytes, follicles, and corpora lutea. The functional
integrity of the reproductive system in animals exposed to 1,3-butadiene has not been tested, but
the severity of the ovarian lesion is indicative of reproductive dysfunction. Furthermore,
Maronpot (1987) compared the ovarian toxicity and carcinogenicity of eight chemicals tested by
NTP and concluded that the occurrence of ovarian lesions in a 90-day study may also indicate that
ovarian neoplasia would be induced upon continued treatment.
Uterine atrophy is probably due to the indirect action of 1,3-butadiene metabolites and the
consequent interruption of ovarian sex steroid stimulation of the uterus. Oocyte toxicity and
destruction of the follicular and subsequent luteal components of the ovary result in reduced
steroidogenesis by the ovary. It is well known that ovarian estrogens and progestins have a
uterotropic function in laboratory rodents and humans.
Testicular atrophy, as reflected by reduced testis weight following 1,3-butadiene exposure
for 9 and 15 months in male mice (NTP, 1993) indicates gonadal sensitivity in the male as well as
in the female. However, the ovary is more sensitive than the testis because ovarian atrophy
results at very low concentrations (6.25 ppm) of 1,3-butadiene compared with that seen in males
after 2 years of exposure. The sperm-head morphology study showed that male mice are affected
at concentrations 1,000 ppm (Hackett et al., 1988a), and the dominant lethal test showed that
male mice may be affected at 200 and 1,000 ppm (Hackett et al., 1988b), again indicating that
higher exposure concentrations are necessary to induce toxic effects in male mice than in female
mice. As observed for other effects of 1,3-butadiene, the reproductive organs of male rats are
more resistant than those of female mice exposed to 1,3-butadiene. This resistance in males may
be attributed, in part, to the blood-testis barrier. No homologous anatomical barrier has been
demonstrated in the Graafian follicle (Crisp, 1992). No adverse reproductive effects have been
observed in male rats at concentrations up to 8,000 ppm (Owen et al., 1987; Owen and Glaister,
1990).

Several studies indicate that 1,3-butadiene affects spermatozoa and spermatids as
determined by postimplantation deaths during the first 3 weeks after exposure. The data from
Hackett et al. (1988b) is equivocal because of a lack of the dose-response relationship, but the
studies by Anderson et al. (1993) and Adler et al. (1994) confirm the hypothesis. Further
evidence that 1,3-butadiene is a germ cell mutagen was presented in a comparison of the latter
two studies (Adler and Anderson, 1994). In the first experiment, the percentage of dominant
lethality observed following 10 weeks of exposure of males to 1,250 ppm was 28.1%. During the
acute exposure experiment, the sum of dominant lethality over the 3 weeks of mating was 23.1%.
The results are in close agreement despite differences in protocols such as exposure regimen,
strains of mice, and mating scheme. It appears that 1,3-butadiene affects spermatozoa and
spermatids because the effects observed after 10 weeks are representative of the last 3 weeks of
treatment and increasing the length of exposure did not add to the response.
The mechanism by which 1,3-butadiene induces ovarian lesions is not known, but it is
unlikely that 1,3-butadiene is a direct-acting reproductive toxicant. Direct-acting compounds act
as hormonal agonists or antagonists or chemically reactive compounds (such as alkylating agents),
which directly interfere with hormone-receptor interactions or interact with macromolecules
(Maronpot, 1987; Mattison et al., 1990). More likely, 1,3-butadiene is an indirect-acting
reproductive toxicant. Indirect-acting toxicants require metabolic activation to exert their toxic
effects, which then may proceed via mechanisms similar to those of direct-acting compounds, or
they may interfere with endocrine homeostasis (Mattison et al., 1990).
Developmental effects observed after exposure to 1,3-butadiene consisted primarily of
reduced fetal body weight and minor skeletal defects such as abnormal ossifications, abnormal
sternebrae, and supernumerary ribs. No gross malformations were produced. The pattern for
developmental effects induced by 1,3-butadiene was similar to that of reproductive effects, with
mice showing greater sensitivity than rats. This difference was also seen for maternal toxicity as
manifested by decreased weight gain (whole body and extragestational) in both rats and mice.
The NOAEL for maternal effects is 200 ppm in rats (IISRP, 1982; Hackett et al., 1987a) and 40
ppm for mice (Hackett et al., 1987b). While the IISRP (1982) study showed increased
frequencies for bipartite thoracic centers and minor skeletal defects combined at 200 ppm in rats,
the response is not clearly dose-related. Several developmental effects occurred at significantly
increased frequencies at 8,000 ppm and showed a dose-response relationship: major skeletal
defects combined, wavy ribs, and abnormal ossification of the ribs (IISRP, 1982). The results
from the IISRP (1982) study were not confirmed in the more recent study by Hackett et al.
(1987a), which showed no developmental toxicity in the same rat strain similarly exposed to
1,3-butadiene at concentrations up to 1,000 ppm. Therefore, the NOAEL for developmental
effects in rats is 1,000 ppm (IISRP, 1982; Hackett et al., 1987a). These independent observations

strengthen the evidence that a decreased maternal weight gain during early development might
contribute to subtle adverse effects (1) undetected by insensitive indices in the studies or (2) at a
later point in time in the F1 or subsequent generations. An NOAEL for developmental effects
could not be defined for the mouse, because male fetal body weight was decreased at 40 ppm in
the Hackett et al. (1987b) study and postimplantation loss was observed at 12.5 ppm in the
Anderson et al. (1993) study, the lowest concentrations tested.
Reproductive and developmental toxicity studies show species specificity for exposure to
1,3-butadiene in rats and mice. Like other toxicological effects induced by 1,3-butadiene, mice
are more sensitive than rats to the induction of reproductive and developmental effects.
Pharmacokinetic studies show that uptake of 1,3-butadiene is about four times greater in mice
than in rats at concentrations up to 1,000 ppm (Dahl et al., 1990) and about two times greater at
8,000 ppm (Dahl et al., 1991). These data indicate that the availability of 1,3-butadiene is greater
in mice than in rats at comparable exposure concentrations. Nose-only exposure of mice and rats
to 1,3-[14C]-butadiene resulted in greater or similar concentrations of radioactivity (expressed as
nM/g of tissue) in tissues of rats than those of mice under conditions in which the rats were
exposed to a 10-fold higher concentration of 1,3-butadiene (Bond et al., 1987). If tissue uptake
was expressed as 1,3-butadiene equivalents/µM inhaled 1,3-butadiene, however, radioactivity
levels were 15 to 100 times higher in mice. Mammary tissue, which had 4.6-fold higher
concentration in rats than in mice, was the only tissue analyzed that was relevant to evaluating
reproductive effects of 1,3-butadiene. Because male animals were used, subcutaneous fat, which
had similar levels of radioactivity as mammary tissue, probably contaminated the samples. The
ovary, uterus, and testis, which are targets for 1,3-butadiene, were not analyzed by Bond et al.
(1987). In a recent in vitro study, Sharer et al. (1992) showed that microsomes from the testes of
rats and mice are ineffective in forming butadiene monoxide, but the cytosol fraction was very
effective in forming glutathione conjugates. Therefore, it is unlikely that toxic effects on the
testes are due to metabolites formed within the testes but rather are due to metabolites formed
elsewhere, indicating that 1,3-butadiene is an indirect-acting reproductive toxicant in males.
Species-specific differences have also have been observed for the formation of
metabolites. The monoxide hydrolase (detoxification) pathway is favored in rat microsomes,
whereas the monooxygenase pathway is favored in mouse microsomes. Although these data do
not fully explain the species differences, they show that the basis of the difference may be related
to the greater availability of 1,3-butadiene in mice, the greater production of toxic intermediates,
and a lower capacity for detoxification of these intermediates.
No data are available regarding the reproductive or developmental effects of the
metabolites of 1,3-butadiene, 1,2-epoxybutene, and diepoxybutane. Mice are more sensitive than
rats to the ovotoxic effects of the mono- and diepoxides of 1,3-butadiene, and the diepoxide is the

more potent ovotoxicant in both species. Data regarding the 1,3-butadiene dimer 4-
vinylcyclohexene and its diepoxide provide evidence that 4-vinylcyclohexene induces ovarian and
uterine atrophy after treatment by gavage for 13 weeks with 10 mg/kg 5 days/week and that 4-
vinyl-1-cyclohexene diepoxide (2.5 to 10 mg/mouse) induces ovarian atrophy and tubular
hyperplasia in mice after topical treatment for 2 years. Subacute inflammation of the epididymis is
seen in male mice receiving 4-vinyl-1-cyclohexene (5 or 10 mg/mouse) topically for 2 years.
Ovarian neoplasms are induced in mice by 4-vinylcyclohexene and its diepoxide. However,
neither 4-vinylcyclohexene nor its diepoxide induce either neoplastic or nonneoplastic lesions in
the ovaries of rats.
In conclusion, the animal data show that there is a potential reproductive hazard to
humans upon exposure to 1,3-butadiene, with women being more sensitive than men. The
quantitative aspects of this assessment will require application of pharmacokinetic parameters
because humans may be less sensitive than mice (Chapters 3, 8). The animal data also show that
there is a potential for developmental effects in humans upon in utero exposure to 1,3-butadiene
and that these effects may occur at concentrations below those causing maternal effects (Section
9.3).

6. TOXICITY IN ANIMALS
This chapter updates the evaluation of animal studies published from 1985 through January
1997. The study by Owen et al. (1987) is not evaluated here because it was reviewed previously
in U.S. EPA (1985) as the NTP study (1984), which was subsequently published by Owen et al.
(1987).
6.1. SUBCHRONIC TOXICITY

Irons et al. (1986a, b) conducted studies to assess the potential of 1,3-butadiene to induce
myelotoxicity by exposing male B6C3F1 mice and male NIH Swiss mice to 1,250 ppm 1,3-
butadiene, 6 h/day, 5 days/week for 6 weeks. Treatment-related hematological changes included
decreases in red blood cell counts, total hemoglobin, and hematocrit and increases in mean cell
volume and circulating micronuclei in both strains of mice. The observed anemia was not
accompanied by significant alterations in mean corpuscular hemoglobin concentration, increases in
reticulocyte counts, or increases in the frequency of nucleated erythrocytes in peripheral blood.
These hematologic changes were considered to represent a macrocytic-megaloblastic anemia,
because they were accompanied by mild megaloblastic changes in bone marrow cells.
Exposure of male B6C3F1 mice to 1,250 ppm 1,3-butadiene for 6 h/day, 5 days/week, for
6 or 12 weeks did not produce any persistent effects on humoral or cell-mediated immunity
(Thurmond et al., 1986). Relative thymus weights were unaffected, but relative spleen weights were
decreased 20% and spleen cellularity was decreased 29% in exposed mice. Extramedullary
hematopoiesis and erythroid hyperplasia in exposed mice correlated with a twofold increase in
thymidine incorporation into spontaneously proliferating splenocytes. Although the number of IgM
antibody plaque-forming cells (PFC) per 106 splenocytes was unchanged, a 30% decrease in
PFC/spleen was observed. Proliferation of alloantigens was similar for 1,3-butadiene-exposed
splenocytes and controls. The mitogenic response of mature T lymphocytes to phytohemagglutinin
was significantly suppressed after exposure to 1,3-butadiene for 6 or 12 weeks.
6.2. CHRONIC TOXICITY

A 2-year chronic inhalation toxicity and carcinogenicity study on the effects of 1,3-butadiene
on B6C3F1 mice was conducted by NTP (1993). In this study, groups of 70 male and 70 female
mice were exposed by inhalation 6 hours/day, 5 days/week to 0, 6.25, 20, 62.5, or 200 ppm 1,3-
butadiene for periods up to 103 weeks; groups of 90 male and 90 female mice were similarly
exposed to 625 ppm 1,3-butadiene, which was the lowest exposure level in the previous NTP (1984)
study. The additional animals in the 625-ppm exposure group were included because high mortality
rates, observed previously at this exposure concentration, might interfere with the scheduled interim
evaluations. Interim evaluations were conducted at 9 and 15 months.
Mean body weight gains of male and female mice exposed to 6.25-625 ppm 1,3-butadiene
for 103 weeks were similar to those of controls. However, concentration-related decreases in
survival were seen in male and female mice exposed to concentrations $20 ppm 1,3-butadiene
(Table 6-1, Figures 6-1 and 6-2) primarily due to the development of malignant neoplasms. No
female mice exposed to 200 or 625 ppm or male mice exposed to 625 ppm survived to the end of
the study. Statistical analysis for the probability of survival was estimated using the Kaplan and
Meyer (1958) procedure; the method of Cox (1972) and Tarone’s (1975) life table test was used to
identify concentration-related trends.
At the 9- and 15-month interim evaluations, no clinical findings other than those associated
with lesion development and moribundity were observed. Some statistically significant organ
weight changes were observed at interim evaluations in male and female mice exposed to 1,3-
butadiene concentrations $62.5 ppm. Effects related to toxicity to reproductive organs are discussed
in Chapter 5.
Hematological indices measured at the interim evaluations showed significant (p#0.05)
decreases in erythrocyte counts, hemoglobin concentration, and packed cell volume in male mice
exposed to $62.5 ppm and in female mice exposed to $200 ppm at 9 months. Mean cell volume was
significantly increased in male mice exposed to 625 ppm and in females exposed to $200 ppm at
9 months. A similar profile of hematological changes was observed in male and female mice
exposed to 625 ppm for 15 months. Increases in the percentage of erythrocytes with Howell-Jolly
body inclusions and mean cell hemoglobin were seen at 9 and 15 months. At the 15-month interim
evaluation, males exposed to 625 ppm 1,3-butadiene had a significantly increased mean platelet
value, a finding that correlated with the development of neoplasms. Because these hematological
changes were not associated with increases in reticulocyte counts or in frequency of polychromatic
erythrocytes in peripheral blood, they were attributed to a partial or poorly regenerative bone
marrow response to decreased levels of circulating erythrocytes. There were no significant changes
in total serum enzyme activity of lactate dehydrogenase (LDH) or creatine kinase in mice evaluated
at 9 months. LDH values at the 15 month evaluation were increased in males and females exposed
to $200 ppm. At 625 ppm, LDH-1 and LDH-2 were decreased and LDH-5, the principal enzyme
in skeletal muscle and liver, was increased.
Table 6-1. Survival of male and female B6C3F1 mice exposed to
1,3-butadiene by inhalation for 103 weeks
Concentration (ppm)
0 6.25 20 62.5 200 625
Male
Animals initially in study 70 70 70 70 70 90
a
9-Month interim evaluation 10 10 10 10 10 10
a
Natural deaths 6 5 11 12 23 39
Moribund kills 9 6 15 15 23 33
Accidental deathsa 0 0 0 0 0 1
Missinga 0 0 0 1 0 0
b
Animals surviving until study 35 39 24 22 4 0
termination
Percent survival at end of studyc 70 78 49 46 8 0
d
Mean survival(days) 597 611 575 558 502 280
e
Survival analysis p<0.001 p=0.430N p=0.044 p=0.021 p<0.001 p<0.001
Female
Animals initially in study 70 70 70 70 70 90
a
a
Natural deaths 3 7 11 8 12 33
Moribund kills 10 10 14 31 37 46
a
Accidental deaths 0 0 1 0 1 1
Animals surviving until study 37 33 24 11 0 0
termination
Percent survival at end of studyc 74 66 50 23 0 0
d
Mean survival (days) 608 597 573 548 441 320
e
Survival analysis p<0.001 p=0.510 p=0.013 p<0.001 p<0.001 p<0.001
a
Censored from survival analyses.
b
Includes one animal that died during the last week of the study.
c
Kaplan-Meier determinations. Survival rates adjusted for interim evaluations, accidental deaths and missing animals.
d
Mean of all deaths (uncensored, censored, terminal sacrifice).
e
The result of the life table trend test (Tarone, 1975) is in the control column, and the results of the life pairwise comparisons
(Cox, 1972) with the controls are in the dosed columns. A negative trend or lower mortality in a dose group is indicated by N.
Source: NTP, 1993.

Histopathological effects observed at the 9-month evaluations included bone marrow atrophy
(depletion of cells) in 50% of males and in 13% of females at the highest concentration (625 ppm).
The atrophy increased in severity from mild depletion of hematopoietic cells at 9 months to marked
depletion in mice that died or were killed at or before 15 months. An increased incidence of bone
marrow hyperplasia and an increased incidence or severity of hematopoiesis of the spleen, liver, and
lung occurred in females exposed to the three highest concentrations ($62.5 ppm). Thymic necrosis
(atrophy) and decreased thymus weights were seen at the 9-month evaluation in males and females
exposed to 625 ppm. Thymic necrosis also
occurred in females exposed to 62.5 or 200 ppm.
In mice exposed to 1,3-butadiene for 103 weeks, nonneoplastic effects were observed in the
bone marrow, liver, testes, ovary, heart, upper respiratory tract, and various other organs.
Figure 6-1. Kaplan-Meier survival curves for male B6C3F1 mice exposed to 1,3-butadiene by
inhalation for 103 weeks.
Source: NTP, 1993.
Figure 6-2. Kaplan-Meier survival curves for female B6C3F1 mice exposed to 1,3-butadiene
by inhalation for 103 weeks.
Source: NTP, 1993.
Effects in reproductive organs are discussed in Chapter 5. Organ weights, hematological indices,
and serum chemistry were not evaluated at 103 weeks.
In the 2-year study, bone marrow atrophy was recorded in 50% of males and 14% of females
exposed to 625 ppm.
The incidence of liver necrosis was increased at the higher exposure concentrations in males
and females, occurring in 8%, 10%, 16%, 27%, 29%, and 26% of males and in 4%, 4%, 14%, 10%,
38%, and 21% of females exposed to 0 (controls), 6.25, 20, 62.5, 200, and 625 ppm, respectively.
Centrilobular hepatocellular necrosis of the liver was seen in 4% and 8% of males exposed to 62.5
and 625 ppm, respectively, and in 2%, 8%, and 9% of females exposed to 62.5, 200, and 625 ppm,
respectively. Hepatocellular necrosis was not seen in any of the concurrent controls. Liver necrosis
with no particular lobular distribution was found primarily in animals with malignant lymphoma
and hemangiosarcoma; centrilobular hepatocellular necrosis was often found in animals described
as anemic and in animals with atrial thrombi.
Myocardial mineralization, a lesion of unknown pathogenesis, occurred with increased
frequency in both sexes at 625 ppm (males, 27%; females, 14%), but was not observed in controls.
A low incidence was observed at the lower concentrations. Myocardial mineralization was also
observed in a separate stop-exposure study in which male mice were exposed to 312 ppm 1,3-
butadiene for 52 weeks or 625 ppm for 13 or 26 weeks, and observed for periods up to 103 weeks.
The incidence of myocardial mineralization for these three exposure groups was 12%, 18%, and
28%, respectively. Details of the stop-exposure study are presented in Section 3.3.
Minimal to mild olfactory epithelial atrophy occurred in females exposed to 625 ppm and
in males exposed to concentrations $20 ppm. However, the incidence in males exposed to 625 ppm
was lower than that seen in females. The olfactory epithelial lesions were unilateral at the lower
concentrations and bilateral at the higher concentrations. The lesions were similar to those seen in
the NTP (1984) study, but osseous or cartilaginous metaplasia was not observed. The investigators
considered olfactory nasal atrophy a possibly compound-related lesion.
Compared with controls, mice exposed to 1,3-butadiene exhibited increased incidences of
proliferative lesions (hyperplasia) in several organs, including the heart, lungs, forestomach, ovaries,
mammary gland, and Harderian gland (Table 6-2). Hyperplasia of the endothelium (cardiac blood
vessels), alveolar epithelium, forestomach epithelium (focal), germinal epithelium and granulosa
cells of the ovaries, mammary gland, and Harderian gland were all considered preneoplastic lesions.
Other preneoplastic lesions identified in the 2-year study were hepatocellular foci (basophilic, clear
cell, mixed cell, and eosinophilic) in female mice exposed to 1,3-butadiene. Hepatocellular foci
were observed in 16% of controls and in 29%, 38%, 24%, 10%, and 5% of females exposed to 6.25,
20, 62.5, 200, and 625 ppm, respectively. Hyperplastic lesions were also observed in separate
studies with male B6C3F1 mice using variable exposure and durations (stop-exposure experiments).
Hyperplasia in these studies occurred primarily in the endothelium (cardiac blood vessels), alveolar
epithelium, forestomach epithelium, and Harderian gland (Table 6-3).
1/28/98
Table 6-2. Incidence of hyperplasia in male and female B6C3F

1 mice exposed to
1,3-butadiene by inhalation for 103 weeks
Concentration (ppm)
Organ/tissue Sex
0 6.25 20 62.5 200 625
Heart, endothelium M 0/50 (0%) 1/49 (2%) 0/50 (0%) 2/48 (4%) 4/48 (8%) 5/73 (7%)
F 0/50 (0%) 2/50 (4%) 1/50 (2%) 4/49 (8%) 5/50 (10%) 8/80 (10%)
Lung, alveolar epithelium M 2/50 (4%) 9/50 (18%) 6/50 (12%) 13/49 (27%) 17/50 (34%) 12/73 (16%)
F 5/50 (10%) 5/50 (10%) 3/50 (6%) 9/50 (18%) 11/50 (22%) 11/78 (14%)
Forestomach, epithelium M 4/50 (8%) 3/50 (6%) 3/50 (6%) 5/48 (10%) 4/48 (18%) 40/72 (56%)
F 4/50 (8%) 5/49 (10%) 4/47 (9%) 7/48 (15%) 14/50 (28%) 47/79 (59%)
Ovary, germinal epithelium F 2/49 (4%) 3/49 (6%) 8/48 (17%) 15/50 (30%) 15/50 (30%) 19/79 (23%)
6-7
Ovary, granulosa cells F 1/49 (2%) 0/49 (0%) 2/48 (4%) 3/50 (6%) 3/50 (6%) 2/79 (3%)
Mammary gland F 2/50 (4%) 0/50 (0%) 2/50 (4%) 4/50 (8%) 7/50 (14%) 2/80 (3%)
Harderian gland M 1/50 (2%) 3/49 (6%) 4/50 (8%) 6/47 (13%) 8/47 (17%) 5/40 (13%)
F 1/50 (2%) 5/49 (10%) 9/48 (19%) 4/49 (8%) 4/49 (8%) 7/66 (11%)
Source: NTP, 1993.

Table 6-3. Incidence of hyperplasia in male B6C3F1 mice exposed to
1,3-butadiene by inhalation in the stop-exposure study
Concentration (duration of exposure)

Organ/tissue
0 ppm 200 ppm 312 ppm 625 ppm 625 ppm
(40 weeks) (52 weeks) (13 weeks) (26 weeks)
Heart, endothelium 0/50 (0%) 6/50 (12%) 3/50 (6%) 7/50 (14%) 7/50 (14%)
Lung, alveolar 2/50 (4%) 18/50 (36%) 14/50 (28%) 10/50 (20%) 11/50 (22%)
epithelium
Forestomach, epithelium 4/50 (8%) 10/48 (21%) 20/48 (42%) 8/50 (16%) 15/50 (30%)
Harderian gland 1/50 (2%) 4/48 (8%) 6/48 (13%) 3/42 (7%) 7/36 (19%)
Source: NTP, 1993.
6.3. CARCINOGENICITY
The first NTP mouse inhalation study of 1,3-butadiene was terminated early due to induction
of fatal neoplasms (NTP, 1984); therefore, a second study (NTP, 1993) was conducted to better
characterize the exposure-response relationship for neoplasms and nonneoplastic lesions induced
in mice by exposure to 1,3-butadiene for 2 years. The concentrations ranged from
100-fold lower (6.25 ppm) up to the lowest concentration (625 ppm) used in the first study. In
addition, stop-exposure studies were conducted to assess the relationship between concentration and
duration of exposure on the induction of neoplasms by 1,3-butadiene. Results of this study have
also been published by Melnick et al. (1990a, b, c) and Melnick and Huff (1992). Miller and
Boorman (1990) provided morphological descriptions of the neoplastic lesions induced in B6C3F1
mice by 1,3-butadiene. The results are presented here in two parts, 2-year study and stop-exposure
study.
6.3.1. 2-Year Study (NTP, 1993)

The details of the study design are described in Section 6.2. For neoplasms that were
considered to be lethal tumors, the tumor incidence was analyzed using the life table test, a survival-
adjusted procedure appropriate for rapidly lethal tumors (Cox, 1972; Tarone, 1975). For incidental
tumors (tumors discovered as a result of death from an unrelated cause), the primary statistical
method used was the logistic regression test. Alternate statistical methods included the Fisher exact
test and the Cochran-Armitage trend test (Armitage, 1971; Gart et al., 1979), analyses based on the
overall proportion of tumor-bearing animals. Tests of significance included pairwise comparisons
of each dose group and a test for an overall concentration-response trend.
Exposure of male and female mice to 1,3-butadiene induced a variety of common and
uncommon tumors at multiple sites. The incidences of primary neoplasms associated with exposure
to 1,3-butadiene (for the 2-year study) are presented in Tables 6-4 and 6-5. The percentage of
animals bearing malignant tumors increased from about 30% in the controls to nearly 90% in the
highest exposure group, 625 ppm. The results of interim evaluations for 9 months and 15 months
are presented in Tables 6-6 and 6-7.

As in the previous study (NTP, 1984), exposure of mice to 1,3-butadiene was associated with
the development of malignant lymphocytic lymphomas and to a lesser extent with histiocytic
sarcomas. The incidence of malignant lymphomas, particularly lymphocytic lymphomas, was
significantly increased in males and females exposed to 625 ppm and in females exposed to 20 and
200 ppm (survival-adjusted) compared with controls. In addition, there were significant exposure-
response trends (p<0.001) in both sexes. The lymphocytic lymphomas were well differentiated and
occurred as early as week 23, peaking before the 15-month interim evaluation. Many organs,
particularly the spleen, lymph nodes, liver, lung, and kidney, were affected in mice with
lymphocytic lymphoma; however, the thymus was involved in most mice and was the primary organ
affected in some. The lymphocytic lymphomas consisted of uniform populations of small- to
medium-sized lymphocytes, whereas the mixed and undifferentiated lymphomas generally consisted
of more heterogeneous populations of lymphocytes with pleomorphism and atypia. Other
histological types of malignant lymphomas (mixed and undifferentiated), commonly associated with
the spontaneous lymphomas in aging B6C3F1 mice, were seen at low incidence in some groups. The
incidences of histiocytic sarcoma were significantly increased in males and females exposed to 200
and 625 ppm and in males exposed to 62.5 ppm. The histiocytic sarcomas (previously referred to
as reticulum cell sarcomas or type A sarcomas) were large and monomorphic, with dark basophilic
nuclei and relatively abundant eosinophilic cytoplasm.
Hemangiosarcomas of the heart were observed in male (at $20 ppm) and female (at $62.5
ppm) mice exposed to 1,3-butadiene for 2 years. The incidences of hemangiosarcomas of the heart
were significantly increased in male mice exposed to $62.5 ppm and in female mice exposed to
$200 ppm. There was a significant exposure-response trend in both sexes. The cardiac
hemangiosarcomas were observed in all ventricular locations, but were more frequent in

Table 6-4. Incidence of primary neoplasms in male B6C3F1 mice exposed to 1,3-butadiene
by inhalation for 103 weeks
1/28/98
Concentration (ppm)
Target organ Neoplastic lesion
0 6.25 20 62.5 200 625
a
All organs Malignant lymphoma (histiocytic, 4/50 (8%) 2/50 (4%) 4/50 (8%) 6/50 (12%) 2/50 (4%) 51/73 (70%)
lymphocytic, mixed, NOS, or 9.8%b 5.1% 12.2% 17.7% 4.0% 95.4%
undifferentiated) p<0.001c p=0.302N p=0.528 p=0.238 p=0.627 p<0.001
Histiocytic sarcoma 0/50 (0%) 0/50 (0%) 4/50 (8%) 5/50 (10%) 7/50 (14%) 4/73 (5%)
0.0% 0.0% 10.6% 14.3% 31.9% 10.8%
p<0.001c p=0.051 p=0.021 p<0.001 p=0.043
Malignant lymphoma or histiocytic 4/50 (8%) 2/50 (4%) 8/50 (16%) 11/50 (22%) 9/50 (18%) 55/73 (75%)
sarcoma 9.8% 5.1% 21.5% 29.6% 34.7% 95.9%
p<0.001c p=0.302N p=0.118 p=0.022 p=0.005 p<0.001
Heart Hemangiosarcoma 0/50 (0%) 0/49 (0%) 1/50 (2%) 5/48 (10%) 20/48 (42%) 4/73 (5%)
0.0% 0.0% 3.4% 19.4% 93.3% 44.6%
p<0.001c NA p=0.451 p=0.011 p<0.001 p<0.001
Lungs Alveolar/bronchiolar adenoma 18/50 (36%) 20/50 (40%) 10/50 (20%) 25/49 (51%) 21/50 (42%) 3/73 (4%)
46.9% 47.3% 28.2% 74.2% 100.0% 59.4%
6-10
p=0.200d p=0.517 p=0.080N p=0.036 p=0.061 p=0.492

Alveolar/bronchiolar carcinoma or 5/50 (10%) 6/50 (12%) 11/50 (22%) 12/49 (24%) 22/50 (44%) 3/73 (4%)
adenocarcinoma 14.3% 15.4% 38.3% 42.9% 94.6% 59.4%
p<0.001c p=0.577 p=0.017 p=0.006 p<0.001 p<0.001
Alveolar/bronchiolar adenoma, 21/50 (42%) 23/50 (46%) 19/50 (38%) 31/49 (63%) 35/50 (70%) 3/73 (4%)
adenocarcinoma, or carcinoma 54.9% 54.5% 53.6% 87.9% 100.0% 59.4%
p<0.001c p=0.552N p=0.276 p<0.001 p<0.001 p<0.001
Forestomach Squamous cell papilloma 1/50 (2%) 0/50 (0%) 0/50 (0%) 1/50 (2%) 7/50 (14%) 2/73 (3%)
2.5% 0.0% 0.0% 4.5% 51.7% 40.0%
p<0.001d p=0.535N p=0.486N p=0.739 p=0.012 p=0.446
Squamous cell papilloma or 1/50 (2%) 0/50 (0%) 0/50 (0%) 1/50 (2%) 8/50 (16%) 4/73 (5%)
carcinoma 2.5% 0.0% 0.0% 4.5% 54.5% 51.8%
p<0.001c p=0.481N p=0.545N p=0.679 p<0.001 p<0.001
Liver Hepatocellular adenoma 13/50 (26%) 13/50 (26%) 19/50 (38%) 16/48 (33%) 23/48 (48%) 5/72 (7%)
32.1% 31.3% 52.1% 57.0% 92.2% 100.0%
p=0.042d p=0.552 p=0.158 p=0.261 p=0.008 p=0.253
Hepatocellular carcinoma 11/50 (22%) 16/50 (32%) 16/50 (32%) 17/48 (35%) 26/48 (54%) 1/72 (2%)
26.0% 36.6% 44.8% 58.3% 100.0% 50.0%
p=0.036d p=0.142 p=0.389 p=0.088 p<0.001 p=0.347
by inhalation for 103 weeks (continued)
1/28/98
Concentration (ppm)
0 6.25 20 62.5 200 625
Liver (cont.) Hepatocellular adenoma or 21/50 (42%) 23/50 (46%) 30/50 (60%) 25/48 (52%) 33/48 (69%) 5/72 (7%)
carcinoma 47.9% 53.0% 70.1% 79.2% 100.0% 100.0%
p=0.067d p=0.375 p=0.078 p=0.185 p=0.030 p=0.450
Harderian gland Adenoma 6/50 (12%) 7/50 (14%) 8/50 (16%) 19/50 (38%) 30/50 (60%) 6/73 (8%)
14.8% 17.3% 25.8% 63.4% 95.4% 100.0%
p<0.001d p=0.497 p=0.395 p<0.001 p<0.001 p=0.264
Carcinoma 0/50 (0%) 1/50 (2%) 1/50 (2%) 3/50 (6%) 2/50 (4%) 0/73 (0%)
0.0% 2.6% 4.2% 11.7% 6.3% 0.0%
p=0.720d p=0.522 p=0.425 p=0.086 p=0.352 NA
Adenoma or carcinoma 6/50 (12%) 7/50 (14%) 9/50 (18%) 20/50 (40%) 31/50 (62%) 6/73 (8%)
14.8% 17.3% 29.5% 64.9% 95.5% 100.0%
p<0.001d p=0.497 p=0.217 p<0.001 p<0.001 p=0.002
Preputial gland Carcinoma 0/50 (0%) 0/50 (0%) 0/50 (0%) 0/50 (0%) 5/50 (10%) 0/73 (0%)
6-11
0.0% 0.0% 0.0% 0.0% 45.7% 0.0%

p<0.001c NA NA NA p<0.001 NA
a
Overall rate: number of tumor-bearing animals/number of animals examined.
b
Survival-adjusted rate. Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality.
c
Life table test. Beneath the control incidence are the p values associated with the trend test. Beneath the dosed group incidence are the P values corresponding to pairwise comparison between
the control and dosed groups. The life table analysis regards neoplasms in animals dying prior to terminal kill as being (directly or indirectly) the cause of death.
d
Logistic regression test. This test regards the neoplasms as nonfatal.
NA = not applicable; no tumors in these groups.

NOS = not otherwise specified.
N = incidence in dose group is lower than in control group.
Source: NTP, 1993.
Table 6-5. Incidence of primary neoplasms in female B6C3F1 mice exposed to 1,3-butadiene by inhalation for 103 weeks
Concentration (ppm)
1/28/98

0 6.25 20 62.5 200 625
a
All organs Malignant lymphoma 6/50 (12%) 12/50 (24%) 11/50 (22%) 7/50 (14%) 9/50 (18%) 32/80
(lymphocytic, mixed, NOS, or 14.6%b 34% 38.7% 35.9% 39.7% (40%)
undifferentiated cell type) p<0.001c p=0.068 p=0.029 p=0.055 p<0.001 70.8%
p<0.001
Histiocytic sarcoma 3/50 (6%) 2/50 (4%) 7/50 (14%) 4/50 (8%) 7/50 (14%) 4/80 (5%)
6.9% 4.5% 20.0% 17.7% 28.1% 10.3%
p<0.001c p=0.518N p=0.077 p=0.195 p=0.002 p=0.038
Malignant lymphoma or 9/50 (18%) 14/50 (28%) 18/50 (36%) 11/50 (22%) 16/50 (32%) 36/80 (45%)
histiocytic sarcoma 20.5% 37.0% 52.1% 47.2% 56.7% 73.9%
p<0.001c p=0.136 p=0.005 p=0.021 p<0.001 p<0.001
Heart Hemangiosarcoma 0/50 (0%) 0/50 (0%) 0/50 (0%) 1/49 (2%) 21/50 (42%) 23/80 (29%)
0.0% 0.0% 0.0% 4.8% 100.0% 100.0%
p<0.001c NA NA p=0.392 p<0.001 p<0.001
Lungs Alveolar/bronchiolar adenoma 4/50 (8%) 11/50 (22%) 12/50 (24%) 17/50 (34%) 14/49 (29%) 17/78 (22%)
10.5% 30.9% 40.7% 64.8% 100.0% 100.0%
p=0.002d p=0.039 p=0.013 p<0.001 p=0.002 p=0.010
6-12
Alveolar/bronchiolar 0/50 (0%) 5/50 (10%) 11/50 (22%) 9/50 (18%) 19/49 (39%) 8/78 (10%)
adenocarcinoma or carcinoma 0.0% 13.3% 42.9% 40.8% 100.0% 100.0%
p<0.001c p=0.029 p<0.001 p<0.001 p<0.001 p<0.001
Alveolar/bronchiolar adenoma, 4/50 (8%) 15/50 (30%) 19/50 (38%) 24/50 (48%) 25/49 (51%) 22/78 (28%)
adenocarcinoma, or carcinoma 10.5% 39.5% 63.7% 78.5% 100.0% 100.0%

p<0.001c p=0.004 p<0.001 p<0.001 p<0.001 p<0.001
Forestomach Squamous cell papilloma 0/50 (0%) 0/50 (0%) 2/50 (4%) 1/50 (2%) 3/50 (6%) 16/80 (20%)
0.0% 0.0% 8.3% 9.1% 100.0% 100.0%
p<0.001d NA p=0.149 p=0.260 p=0.078 p=0.002
Squamous cell carcinoma 0/50 (0%) 0/50 (0%) 1/50 (2%) 1/50 (2%) 1/50 (2%) 6/80 (8%)
0.0% 0.0% 4.2% 8.3% 3.8% 70.5%
p<0.001c NA p=0.414 p=0.277 p=0.374 p<0.001
Squamous cell papilloma or 0/50 (0%) 0/50 (0%) 3/50 (6%) 2/50 (4%) 4/50 (8%) 22/80 (28%)
carcinoma 0.0% 0.0% 12.5% 16.7% 100.0% 100.0%
p<0.001c NA p=0.056 p=0.044 p=0.001 p<0.001
Liver Hepatocellular adenoma 11/49 (22%) 10/49 (20%) 9/50 (18%) 14/48 (28%) 15/50 (29%) 1/80 (1%)
29.7% 27.8% 30.3% 65.8% 89.0% 100.0%
p=0.599N p=0.531N p=0.519N p=0.025 p=0.009 p=0.505
Hepatocellular carcinoma 4/49 (8%) 6/49 (12%) 8/50 (16%) 9/50 (18%) 8/50 (16%) 1/80 (1%)
10.3% 14.5% 25.0% 39.9% 82.7% 12.5%
p=0.178d p=0.381 p=0.141 p=0.066 p=0.006 p=0.910
Table 6-5. Incidence of primary neoplasms in female B6C3F1 mice exposed to 1,3-butadiene
by inhalation for 103 weeks (continued)
1/28/98
Concentration (ppm)
0 6.25 20 62.5 200 625
Liver (cont.) Hepatocellular adenoma or 15/49 (31%) 14/49 (29%) 15/50 (30%) 19/50 (38%) 16/50 (32%) 2/80 (3%)
carcinoma 39.3% 34.3% 45.5% 74.8% 91.7% 100.0%
p=0.497d p=0.504N p=0.441 p=0.027 p=0.008 p=0.302
Ovary Benign granulosa cell tumor 1/49 (2%) 0/49 (0%) 1/48 (2%) 6/50 (12%) 6/50 (12%) 6/79 (8%)
2.8% 0.0% 3.2% 28.5% 100.0% 27.1%
p=0.030d p=0.517N p=0.735 p=0.026 p=0.020 p=0.303
Malignant granulosa cell tumor 0/49 (0%) 0/49 (0%) 0/48 (0%) 3/50 (6%) 2/50 (4%) 0/79 (0%)
0.0% 0.0% 0.0% 19.3% 54.2% 0.0%
p=0.068d NA NA p=0.046 p=0.037 NA
Benign or malignant granulosa cell 1/49 (2%) 0/49 (0%) 1/48 (2%) 9/50 (18%) 8/50 (16%) 6/79 (8%)
tumor 2.8% 0.0% 3.2% 42.9% 100.0% 27.1%
p=0.006d p=0.517N p=0.735 p=0.001 p=0.001 p=0.303
Mammary gland Adenoacanthoma 0/50 (0%) 1/50 (2%) 2/50 (4%) 6/50 (12%) 4/50 (8%) 0/80 (0%)
0.0% 2.9% 7.7% 32.5% 13.6% 0.0%
6-13
p=0.025c p=0.489 p=0.152 p<0.001 p=.021 NA

Carcinoma 0/50 (0%) 2/50 (4%) 2/50 (4%) 6/50 (12%) 11/50 (22%) 12/80 (15%)
0.0% 5.8% 5.7% 16.2% 39.1% 100.0%
p<0.001c p=0.221 p=0.192 p=0.008 p<0.001 p<0.001
Malignant mixed tumor 0/50 (0%) 0/50 (0%) 0/50 (0%) 0/50 (0%) 0/50 (0%) 4/80 (5%)
0.0% 0.0% 0.0% 0.0% 0.0% 29.4%
p<0.001c NA NA NA NA p=0.003
Harderian gland Adenoma 8/50 (16%) 10/50 (20%) 6/50 (12%) 15/50 (30%) 20/50 (40%) 9/80 (11%)
20.8% 29.2% 20.7% 61.0% 89.3% 45.2%
p=0.046d p=0.356 p=0.511N p=0.016 p=0.001 p=0.176
Carcinoma 0/50 (0%) 1/50 (2%) 1/50 (2%) 0/50 (0%) 1/50 (2%) 0/80 (0%)
0.0% 2.7% 2.3% 0.0% 50.0% 0.0%
p=0.873Nd p=0.493 p=0.631 NA p=0.085 NA
Adenoma or carcinoma 8/50 (16%) 10/50 (20%) 7/50 (14%) 15/50 (30%) 20/50 (40%) 9/80 (11%)
20.8% 29.2% 22.5% 61.0% 89.3% 45.2%
p=0.061d p=0.356 p=0.575N p=0.016 p=0.001 p=0.176
a
b
Overall rate; number of tumor-bearing animals/number of animals examined.
c
Life table test. Beneath the control incidence are the p values associated with the trend test. Beneath the dosed group incidence are the p values corresponding to pairwise comparison between
d
the control and dosed groups. The life table analysis regards neoplasm in animals dying prior to terminal kill as being (directly or indirectly) the cause of death.
Source: NTP, 1993.
by inhalation for 9 months and 15 months
1/28/98
Concentration (ppm)
0 6.25 20 62.5 200 625
All organs Malignant 9 months 1/10
lymphoma
(histiocytic,
lymphocytic,
mixed, NOS, or 15 months 2/7
undifferentiated)
Heart Hemangiosarcoma 9 months
15 months 1/10 3/7
Lungs Alveolar/ 9 months 1/10 1/1 ½ 0/10 2/10 3/10

bronchiolar
adenoma,
adenocarcinoma, 15 months 2/10 4/10 5/7
or carcinoma
6-14
Forestomach Squamous cell 9 months 1/10

papilloma or
carcinoma 15 months 1/10 3/7
Liver Hepatocellular 9 months 4/10 0/10 1/10 0/10 1/10 1/10

adenoma or
15 months 2/10 1/10 4/10 3/10 4/10 5/7
carcinoma
Harderian gland Adenoma or 9 months
carcinoma 15 months 2/10 4/10 3/10 3/7
a
Overall rate: number of tumor-bearing animals/number of animals examined.
b
c
Life table test. Beneath the control incidence are the p values associated with the trend test. Beneath the dosed group incidence are the p values corresponding to pairwise comparison
between the control and dosed groups. The life table analysis regards neoplasms in animals dying prior to terminal kill as being (directly or indirectly) the cause of death.
d

Source: NTP, 1993.

Table 6-7. Incidence of primary neoplasms in female B6C3F1 mice exposed to 1,3-butadiene
by inhalation for 9 months and 15 months
Concentration (ppm)
0 6.25 20 62.5 200 625
All organs Malignant lymphoma 9 months 1/8
(lymphocytic, mixed,
NOS, or undifferentiated 15 months 1/10 1/10 0/2
cell type)
Heart Hemangiosarcoma 9 months
15 months 1/10 2/2
Lungs Alveolar/bronchiolar 9 months 2/10 1/8
adenoma,
adenocarcinoma, or 15 months 3/10 3/10 ½
carcinoma
Forestomach Squamous cell papilloma 9 months
or carcinoma 15 months 1/10 2/10 ½
Liver Hepatocellular adenoma 9 months
or carcinoma 15 months 1/10 1/10 0/10 1/10 3/10 ½
Ovary Benign or malignant 9 months 1/10
granulosa cell tumor 15 months 1/10 4/10 ½
Mammary gland Adenoacanthoma, 9 months
adenocarcinoma,
carcinoma, or malignant 15 months 2/10 ½
mixed tumor
Harderian gland Adenoma or carcinoma 9 months 1/5
15 months 2/9 1/1 1/1 1/10 3/10 0/2
a
Overall rate; number of tumor-bearing animals/number of animals examined.
b
c
Life table test. Beneath the control incidence are the p values associated with the trend test. Beneath the dosed group incidence are the p values corresponding to pairwise comparison
between the control and dosed groups. The life table analysis regards neoplasm in animals dying prior to terminal kill as being (directly or indirectly) the cause of death.
d

Source: NTP, 1993.

the left ventricular wall. Typical hemangiosarcomas had solid foci of anaplastic, pleomorphic
spindle cells at the center with a loose arrangement at the periphery. They were occasionally
multifocal and frequently coexisted with foci of endothelial hyperplasia distant and separate from
the main neoplasm. Hemangiosarcomas of the heart are considered uncommon in untreated B6C3F1
mice (none were observed in 573 male and in 558 female historical controls in NTP inhalation
studies). In male mice, the lower incidence of cardiac hemangiosarcoma at 625 ppm compared with
that at 200 ppm, was attributed to the early mortality due to induction of lethal lymphocytic
lymphoma at 625 ppm. The time-to-tumor detection for all hemangiosarcomas of the heart ranged
from 682 days at 20 ppm to 289 days at 625 ppm for males and from 649 days at 20 ppm to 307
days at 625 ppm for females. When hemangiosarcomas occurred in multiple organs, the cardiac
neoplasms were usually designated as primary, because the incidence of hemangiosarcomas was
highest in the heart and the earliest lesions occurred in the heart. However, it could not be
determined with certainty if the hemangiosarcomas observed in other organs were metastases or
primary neoplasms. Subcutaneous, splenic, and hepatic hemangiosarcomas that were found in the
absence of cardiac hemangiosarcomas may reflect the development of spontaneous vascular
neoplasms known to occur in B6C3F1 mice.
Exposure of mice to 1,3-butadiene was also associated with an increased incidence of
pulmonary neoplasms in male and female mice. Although the incidence of alveolar/bronchiolar
adenomas was not significantly increased in male mice in the 2-year study, the combined incidences
of alveolar/bronchiolar adenocarcinomas and carcinomas and the combined incidences of the benign
and malignant pulmonary neoplasms were significantly increased at 62.5, 200, and 625 ppm. In
female mice, the incidences of the benign and malignant neoplasms analyzed separately or together
were significantly increased in all exposure groups compared with controls. Thus, even at 6.25
ppm, 1,3-butadiene was carcinogenic to female B6C3F1 mice. The lower incidence of lung
neoplasms at 625 ppm compared with the incidence at 200 ppm was attributed to the high rate of
early deaths due to the competing risks of lymphocytic lymphoma in female mice exposed to 625
ppm. There was a significant exposure-response trend for combined adenomas and carcinomas in
both sexes. The time-to-tumor detection for lung tumors combined ranged from 587 days at 6.25
ppm to 251 days at 625 ppm for males, and from 519 days at 6.25 ppm to 275 days at 625 ppm for
females. The spectrum of lung lesions ranged from alveolar epithelial hyperplasia (Section 3.2 of
this chapter) to adenomas, carcinomas, and adenocarcinomas. Histologically, the
alveolar/bronchiolar adenomas exhibited distortion of the alveolar structure due to the formation of
complex, irregular papillary patterns; the alveolar/bronchiolar carcinomas were similar, but
consisted of heterogeneous cell populations with various degrees of cellular pleomorphism and
atypia. The adenocarcinomas were larger, highly anaplastic neoplasms, often accompanied by
hemorrhage or necrosis.
In the forestomach, significant increases in squamous cell papillomas and carcinomas
combined were observed in male mice exposed to 200 ppm and in female mice exposed to 62.5
ppm compared with controls. There was a significant exposure-response trend for papillomas and
carcinomas combined in both sexes. The combined incidence of squamous cell papillomas and
carcinomas of the forestomach (males, 4/575 [0.7%]; females, 9/561 [1.6%]) for historical controls
suggests that these lesions are relatively uncommon in B6C3F1 mice.
Increased incidences of hepatocellular adenomas and carcinomas were also seen in 1,3-
butadiene-exposed mice (Tables 6-4 and 6-5). The hepatocellular adenomas were discrete, expansile
masses; the carcinomas were larger than the adenomas and consisted of markedly disorganized
hepatocytes. The low incidence of liver neoplasms observed in males and females at 625 ppm
probably reflects increased early deaths from malignant lymphoma. Hepatocellular adenomas and
carcinomas are common neoplasms in B6C3F1 mice, occurring in 196/572 (34%) of male and
87/558 (15.6%) of female historical controls in NTP inhalation studies. The data suggest that 1,3-
butadiene has only a weak tumorigenic effect in the livers of male and female mice. However, a
chemical-related effect is supported by the detection of an activated K-ras oncogene in liver
neoplasms obtained from mice exposed to 1,3-butadiene (Goodrow et al., 1990). According to
Reynolds et al. (1987), activated K-ras oncogene had never been detected in liver neoplasms from
untreated B6C3F1 mice.
Although a variety of neoplasms were seen in the ovaries of female mice, only benign and
malignant granulosa cell tumors were definitely attributed to exposure to 1,3-butadiene (Table 6-5).
The ovarian granulosa cell tumors varied from small benign tumors to large cystic tumors with thick
trabeculae and spaces filled with blood or clear fluid. The overall historical control incidence at
NTP for benign and malignant granulosa cell tumors each was 1/548 (0.2%).
Increased incidences of mammary gland neoplasms were seen in female mice exposed to
62.5 ppm 1,3-butadiene. Mammary tumors included adenoacanthomas, adenocarcinomas, and
malignant mixed tumors, the latter occurring only at 625 ppm. The mammary gland tumors
combined exhibited a significant exposure-response relationship. The adenoacanthomas were
considered variants of adenocarcinomas that have prominent squamous differentiation. The
malignant mixed tumors consisted of epithelial components arranged in glandlike structures and
anaplastic spindle-cell components. Mammary gland adenocarcinomas and adenoacanthomas were
considered uncommon in female B6C3F1 mice; the overall historical incidence at NTP was 21/561
(3.7%) for carcinomas and 1/561 (0.2%) for adenoacanthomas in female control mice.
The Harderian gland was identified as another site of 1,3-butadiene-induced neoplasms in
male and female mice (Tables 6-4 and 6-5), with significant exposure-related increases in adenomas
at 62.5 and 200 ppm and a low incidence of carcinomas in males exposed to 6.25, 20, 62.5, or 200
ppm. The low incidence of Harderian gland tumors at 625 ppm was attributed to early deaths due
to lymphocytic lymphoma which precluded the development of Harderian gland tumors. The
investigators noted that the occurrence of Harderian gland carcinomas in mice, particularly males,
is unusual. The overall incidence of Harderian gland carcinomas was 2/575 (0.3%) in male and
3/561 (0.5%) in female historical controls at NTP. The 2-year historical incidence of adenomas and
carcinomas (combined) of the Harderian gland for control groups in NTP inhalation studies was
25/575 (4.3%) for males and 13/561 (2.3%) for females.
Preputial gland carcinomas, also considered to be rare neoplasms in B6C3F1 mice, were seen
in five males (p<0.05) exposed to 200 ppm (none were reported in one survey of NTP historic
control data). These tumors were also thought to be exposure-related lesions. Some preputial
carcinomas were composed of large eosinophilic epithelial cells that were well differentiated; more
frequently, the carcinomas had necrotic cores and a thin layer of very anaplastic basophilic epithelial
cells that aggressively invaded surrounding tissue and blood vessels.
Renal tubule adenomas were seen in 2/50 females exposed to 200 ppm 1,3-butadiene and
in 1/50, 3/48, and 1/49 of males exposed to 6.25, 62.5, and 200 ppm, respectively. At the 15-month
evaluation, renal tubular adenoma occurred in 1/7 males exposed to 625 ppm. The historical
incidence of spontaneous renal tubule adenomas in untreated control groups in NTP inhalation
studies was 1/571 (0.2%) for males and 0/559 (0.0%) for females. Histologically, the renal tubule
adenomas contained multiple dilated tubules separated by thin connective tissue septa. These renal
lesions were probably related to exposure to 1,3-butadiene in males and possibly related to exposure
in females.
One neurofibrosarcoma of the subcutaneous tissue was observed in two females exposed to
625 ppm at the 15-month evaluation. In the 2-year study, the combined incidences of
neurofibrosarcomas and sarcomas of the subcutaneous tissue were significantly increased in female
mice exposed to 62.5 ppm (p=0.017), 200 ppm (p=0.002), and 625 ppm (p=0.013) by the life table
test. Subcutaneous tissue sarcomas (all types) were considered uncommon spontaneous neoplasms;
the historical incidence was 2/561 (0.4%) for female controls at NTP, suggesting that these
subcutaneous tissue neoplasms may have been exposure-related. The historical incidence for NTP
inhalation studies was not reported.
One adenoma and one carcinoma of the Zymbal’s gland were seen in females exposed to 625
ppm; one adenoma also occurred in a concurrent control male mouse, but none were reported in
historical controls. The report indicated that these Zymbal’s gland neoplasms may be related to 1,3-
butadiene exposure.
Carcinomas of the small intestine, another uncommon tumor in the B6C3F1 mouse, were
seen in two females exposed to 6.25 ppm and in one female exposed to 62.5 ppm. One carcinoma
each was seen in one male each exposed to 6.25, 20, or 62.5 ppm, and in two males exposed to 200
ppm. The relationship of these neoplasms to exposure to 1,3-butadiene could not be determined;
however, controls did not exhibit proliferative lesions of the intestine.
In supplemental analyses, the authors performed a "Poly-3" quantal response test (Bailer and
Portier, 1988; Portier and Bailer, 1989) as an alternative to the logistic regression analyses, whose
sensitivity was reduced by the decreased survival in the higher exposure groups. For tumor sites
related to butadiene exposure, the "Poly-3" test detected significant responses in some of the
exposure groups that had not been detected by the logistic regression analyses. The overall results
were consistent with those already presented.
The authors also fitted a modified Weibull model (Portier et al., 1986) to the "Poly-3"
survival-adjusted tumor rates to determine the shape parameters for the exposure-response
relationships. About half of the tumor sites associated with butadiene exposure had exposure-
response relationships consistent with a linear model (i.e., shape parameter of 1). Most of the other
tumor sites exhibited supralinear exposure-response relationships (i.e., steep slope in low-exposure
region; shape parameter significantly <1). These sites were the liver in male mice, the mammary
gland in females, and the Harderian gland and lung in both sexes. Only the malignant lymphoma
in males and heart hemangiosarcoma in females had a shape parameter significantly greater than 1,
suggestive of a sublinear exposure-response relationship.
6.3.2. 2-Year Stop-Exposure Study (NTP, 1993)

An additional study with B6C3F1 mice, referred to as "stop-exposure study" was conducted
to assess the relationship between exposure level and duration of exposure to outcome of 1,3-
butadiene carcinogenicity. Groups of 50 male mice were exposed 6 hours/day, 5 days/week at
concentrations of (a) 200 ppm for 40 weeks, (b) 625 ppm for 13 weeks, (c) 312 ppm for 52 weeks,
or (d) 625 ppm for 26 weeks. After the exposures were stopped, the animals were placed in control
chambers for the remainder of the 103-week studies. The total exposures to 1,3-butadiene
(concentration × duration of exposure) were approximately 8,000 ppm weeks for groups exposed
to 200 ppm for 40 weeks or 625 ppm for 13 weeks; the total exposures were approximately 16,000
ppm week for groups exposed to 312 ppm for 52 weeks or 625 ppm for 26 weeks. No additional
controls were included for these studies, because they were run concurrently with the 2-year studies.
Using the stop-exposure protocol, inhalation exposure to 1,3-butadiene had no effect on
mean body weights. However, exposure to 1,3-butadiene markedly reduced survival in all stop-
exposure groups as a result of the development of neoplasms, particularly malignant lymphomas
and hemangiosarcomas of the heart (Figure 6-3). A comparison of the two groups receiving total
exposures of 8,000 ppm weeks showed that the survival of mice exposed to 625 ppm (13 weeks) was
similar to that of mice exposed to 200 ppm (40 weeks). By contrast, the groups exposed to 16,000
ppm weeks, survival of mice exposed to 625 ppm (26 weeks) was significantly lower than that of
mice exposed to 312 ppm (52 weeks).
Neoplasms induced in the stop-exposure studies are summarized in Table 6-8. Overall, the
data show that exposure of male mice to 1,3-butadiene using the stop-exposure protocol induced
neoplasms at the same sites as those observed in the 2-year study.
Figure
6 - 3 .
Kaplan-
Meier
survival
curves
f o r
m a l e
mice in the stop-exposure inhalation study of 1,3-butadiene.
Source: NTP, 1993.
Table 6-8. Incidence of primary neoplasms in male B6C3F 1 mice exposed to 1,3-butadiene by
inhalation in the stop-exposure study

1/28/98
Parameters 0 ppm 200 ppm 625 ppm 312 ppm 625 ppm
Duration of exposures (weeks) 103 40 13 52 26
Total exposure (ppm weeks) 0 8,000 8,000 16,000 16,000
Hematopoietic Lymphocytic malignant lymphoma 2/50a (4%) 6/50 (12%) 17/50 (34%) 4/50 (8%) 30/50 (60%)
4.7%b 26.7% 35.8% 100.0% 81.5%
-- p=0.033c p<0.001 p=0.034 p<0.001
Lymphoma (mixed or NOS) 2/50 (4%) 2/50 (4%) 5/50 (10%) 4/50 (8%) 3/50 (6%)
5.3% 7.8% 34.8% 58.0% 43.3%
-- p=0.382c p=0.010 p=0.005 p=0.002
Histiocytic sarcoma 0/50 (0%) 5/50 (10%) 2/50 (4%) 7/50 (14%) 2/50 (4%)
0.0% 21.3% 28.9% 43.0% 15.6%
-- p=0.006c p=0.011 p<0.001 p=0.036
Malignant lymphoma or histiocytic 4/50 (8%) 13/50 (26%) 24/50 (48%) 15/50 (30%) 35/50 (70%)
sarcoma 9.8% 46.8% 72.1% 100.0% 91.2%
-- p<0.001c p<0.001 p<0.001 p<0.001
6-19
Malignant lymphoma (lymphocytic, mixed, 4/50 (8%) 8/50 (16%) 22/50 (44%) 8/50 (16%) 33/50 (66%)
or NOS) 9.8% 32.4% 58.2% 100.0% 89.5%
-- p=0.023c p<0.001 p<0.001 p<0.001
Heart Hemangiosarcoma 0/50 (0%) 15/50 (30%) 7/50 (14%) 33/50 (66%) 13/50 (26%)
0.0% 76.2% 61.8% 100.0% 100.0%
-- p<0.001c p<0.001 p<0.001 p<0.001
Lungs Alveolar/bronchiolar adenoma 18/50 (36%) 24/50 (48%) 17/50 (34%) 26/50 (52%) 12/50 (24%)
46.9% 94.3% 85.3% 100.0% 100.0%
-- p=0.015d p=0.044 p=0.001 p<0.001
Alveolar/bronchiolar 5/50 (10%) 22/50 (44%) 18/50 (36%) 16/50 (32%) 11/50 (22%)
adenocarcinoma or carcinoma 14.3% 89.5% 87.7% 100.0% 100.0%
-- p<0.001c p<0.001 p<0.001 p<0.001
Table 6-8. Incidence of primary neoplasms in male B6C3F1 mice exposed to 1,3-butadiene by
inhalation in the stop-exposure study (continued)
1/28/98
Alveolar/bronchiolar adenoma, 21/50 (42%) 36/50 (72%) 28/50 (56%) 32/50 (64%) 17/50 (34%)
adenocarcinoma, or carcinoma 54.9% 100.0% 100.0% 100.0% 100.0%
-- p<0.001c p<0.001 p<0.001 p<0.001
Liver Hepatocellular adenoma 13/50 (26%) 27/49 (55%) 19/49 (39%) 19/50 (38%) 11/50 (22%)
32.1% 91.1% 91.1% 100.0% 100.0%
-- p<0.001d p=0.042 p=0.045 p=0.284
Hepatocellular carcinoma 11/50 (22%) 14/49 (29%) 14/49 (29%) 10/50 (20%) 4/50 (8%)
26.0% 50.3% 90.9% 74.6% 50.5%
-- p=0.530d p=0.142 p=0.453 p=0.393
Hepatocellular adenoma or carcinoma 21/50 (42%) 33/49 (67%) 24/49 (49%) 24/50 (48%) 13/50 (26%)
47.9% 93.4% 94.4% 100.0% 100.0%
-- p=0.004d p=0.063 p=0.169 p=0.561
6-20
Forestomach Squamous cell papilloma 1/50 (2%) 3/50 (6%) 4/50 (8%) 4/50 (8%) 4/50 (8%)
2.5% 21.4% 28.3% 100.0% 20.1%
-- p=0.195d p=0.260 p=0.181 p=0.301
Squamous cell carcinoma 0/50 (0%) 0/50 (0%) 4/50 (8%) 5/50 (10%) 6/50 (12%)
0.0% 0.0% 51.6% 33.1% 40.9%
-- NA p<0.001d p<0.001 p<0.001
Squamous cell papilloma or carcinoma 1/50 (2%) 3/50 (6%) 7/50 (14%) 9/50 (18%) 10/50 (20%)
2.5% 21.4% 56.6% 100.0% 52.8%
-- p=0.065c p<0.001 p<0.001 p<0.001
Harderian gland Adenoma 6/50 (12%) 26/50 (52%) 20/50 (40%) 28/50 (56%) 13/50 (26%)
14.8% 87.9% 94.3% 100.0% 100.0%
-- p<0.001d p=0.001 p<0.001 p=0.046

1/28/98
Carcinoma 0/50 (0%) 2/50 (4%) 4/50 (8%) 2/50 (4%) 0/50 (0%)
0.0% 5.6% 38.8% 51.5% 0.0%
-- p=0.397d p=0.190 p=0.006 NA
Adenoma or carcinoma 6/50 (12%) 27/50 (54%) 23/50 (46%) 30/50 (60%) 13/50 (26%)
14.8% 88.3% 100.0% 100.0% 100.0%
-- p<0.001d p<0.001 p<0.001 p=0.046
Kidney Renal tubule adenoma 0/50 (0%) 4/48 (8%) 1/50 (2%) 3/49 (6%) 1/50 (2%)
0.0% 17.4% 14.3% 27.8% 6.3%
-- p=0.073d p=0.273 p=0.075 p=0.731
Braine Malignant glioma 0/50 (0%) 0/50 (0%) 2/50 (4%) 0/50 (0%) 1/50 (2%)
Malignant neuroblastomas 0/50 (0%) 0/50 (0%) 2/50 (4%) 0/50 (0%) 0/50 (0%)
6-21
Preputial gland Carcinoma 0/50 (0%) 1/50 (2%) 4/50 (8%) 4/50 (8%) 3/50 (6%)
0.0% 10.0% 16.9% 100.0% 100.0%
-- p=0.368c p=0.039 p<0.001 p=0.002
Adenoma or carcinoma 0/50 (0%) 1/50 (2%) 5/50 (10%) 4/50 (8%) 3/50 (6%)
0.0% 10.0% 22.9% 100.0% 100.0%
-- p=0.368c p=0.013 p<0.001 p=0.002
Zymbal’s gland Adenoma or carcinoma 1/50 (2%) 1/50 (2%) 2/50 (4%) 0/50 (0%) 2/50 (4%)
2.9% 4.8% 8.8% 0.0% 37.3%
-- p=0.531c p=0.178 p=0.998N p=0.009
a
b
Overall rate, number of tumor-bearing animals/number of animals examined.
c
Life table test. The p values for pairwise comparison of exposed groups with controls are beneath the exposed group incidence. N refers to negative association with control group. The life
table analysis regards neoplasms in animals dying prior to terminal kill as being (directly or indirectly) the cause of death.
d
e
No statistical analysis.
Source: NTP, 1993a.
Lymphocytic lymphomas of thymic origin occurred at a markedly increased incidence in
mice exposed to 625 ppm for 13 or 26 weeks. According to the life table test, the incidence of
lymphocytic lymphoma was also significantly increased in mice exposed to 200 ppm for 40 weeks
or 312 ppm for 52 weeks. The incidence of histiocytic sarcomas was significantly increased (life
table test) in mice in all stop-exposure groups as well.
The lower incidences of lymphocytic lymphomas at 200 ppm (40 weeks) and 312 ppm (52
weeks) compared to 625 ppm for 13 and 26 weeks, respectively, demonstrate that the concentration
of 1,3-butadiene is a greater contributing factor in the development of this lesion than the duration
of exposure, i.e., a high concentration for a short duration is more effective than a lower
concentration of longer duration. A comparison of the 200 ppm (40 weeks) versus the 625 ppm (13
weeks) and of the 312 ppm (52 weeks) versus the 625 ppm (26 weeks) lymphocytic lymphoma
results using a life table test confirms that the higher concentration/shorter duration regimen is
significantly more effective than the lower concentration/longer duration regimen within each
cumulative exposure grouping (p=0.005 for 8,000 ppm·weeks and p<0.001 for 16,000 ppm·weeks)
after survival differences are taken into account.
As observed in the 2-year study, lymphocytic lymphomas occurred very early after exposure
started: as early as 23 weeks in the group exposed to 625 ppm for 26 weeks and as early as 24
weeks in the group exposed to 625 ppm for 13 weeks. This lesion accounted for 24 and 17,
respectively, of the first 25 deaths occurring in these groups by weeks 45 and 79, respectively.
Therefore, early deaths due to lymphocytic lymphoma would have a tremendous negative effect on
the incidence of late-developing lesions.
Hemangiosarcomas of the heart, which also accounted for some of the early deaths, were
significantly increased in most stop-exposure groups compared with the controls. The highest
incidence, which was about twice as high as that of other groups, occurred in the group exposed to
312 ppm, followed by the groups exposed to 200 ppm and 625 ppm (26 weeks). The lowest
incidence occurred in the group exposed to 625 ppm for 13 weeks. Hemangiosarcomas appeared
at about 9 months in the 200, 312, and 625 ppm (26-week) stop-exposure groups. Comparison (life
table test) of groups having the same total exposures showed that the incidences of
hemangiosarcomas in mice exposed to 625 ppm were significantly lower than that of the
corresponding group exposed to 312 ppm (p=0.032) but not 200 ppm. The incidences of
hemangiosarcomas in both 625-ppm stop-exposure groups were higher than that in the 625-ppm 2-
year exposure group, probably due to longer survival of the stop-exposure groups.
The incidences of neoplastic lesions of the lung (alveolar/bronchiolar adenoma,
adenocarcinoma, or carcinoma) were significantly elevated in each exposure group. The highest
incidence occurred in the 200-ppm stop-exposure group, followed by the 312-, 625- (13 weeks),
and 625-ppm (26 weeks) groups. The adenomas developed after week 47 and the adenocarcinomas
and carcinomas developed after week 53; the late appearance of these lesions relative to lymphocytic
lymphomas probably accounted for the lowest incidence of lung neoplasms occurring in 625 ppm
(26 weeks) group. A life table analysis suggested the incidence of lung lesions in the 625 ppm (26
weeks) group was significantly greater than in the 312 ppm (52 weeks) group (p=0.013), but no
difference was detected between the 200 ppm (40 weeks) and 625 ppm (13 weeks) groups.
Mice exposed to 200 ppm 1,3-butadiene for 40 weeks had significantly increased incidences
of hepatocellular adenomas and adenomas/carcinomas combined; the incidences of hepatocellular
carcinomas analyzed alone were not significantly increased. Exposure to 1,3-butadiene at 312 ppm
or 625 ppm (13 or 26 weeks) did not increase the incidence of hepatocellular neoplasms of any type.
The earliest detection of these neoplasms was 67 weeks for the 625 ppm (13 weeks), 57 weeks for
the 200 ppm, 47 weeks for the 312 ppm, and 45 weeks for the 625 ppm (26 weeks) stop-exposure
groups. A logistic regression analysis found no differences between the 200 ppm and 625 ppm (13
weeks) or the 312 ppm and 625 ppm (26 weeks) groups.
A low incidence of squamous cell papillomas of the forestomach occurred in each of the
groups, and squamous cell carcinomas were seen in mice exposed to 312 ppm or 625 ppm for 13
and 26 weeks. The incidences of squamous cell papillomas were not significantly greater than
controls for any group, but the incidences of squamous cell carcinomas were significantly greater
by the life table test, which is considered to be the appropriate test (NTP, 1993) for these fatal
neoplasms. A life table analysis also revealed a statistically significant exposure-rate effect for the
squamous cell carcinomas in both of the total exposure groupings (p=0.019 for 8,000 ppm·weeks
and p=0.015 for 16,000 ppm·weeks), suggesting that the higher concentration/shorter duration
exposures were more potent.
The incidence of adenomas of the Harderian gland was significantly greater in each exposure
group than in the controls by a logistic regression test. A low incidence of Harderian gland
carcinomas occurred in mice exposed to 200 ppm for 40 weeks (not significant), 312 ppm for 52
weeks (p=0.006), and 625 ppm for 13 weeks (not significant). No Harderian gland carcinomas were
observed in the controls or in mice exposed to 625 ppm for 26 weeks. A logistic regression analysis
did not detect any exposure-rate effects.
Other neoplasms occurred at low incidence in the stop-exposure studies; they were
considered to be related to exposure because of their low spontaneous incidences in NTP historical
control male mice. These neoplasms occurred in the kidney, brain, Zymbal’s gland, and preputial
gland. The incidences of these neoplasms are also summarized in Table 6-8.
Renal tubule neoplasms occurred in historical male control mice; the range was 0 to 1%.
The small number of these neoplasms in each of the exposure groups are considered to be related
to administration of 1,3-butadiene because the incidences were greater than the upper range for
historical controls.
Brain neoplasms, including two neuroblastomas and two malignant gliomas, observed in
male mice exposed to 625 ppm for 13 weeks and one malignant glioma observed in male mice
exposed to 625 ppm for 26 weeks may have been related to 1,3-butadiene exposure. Brain
neoplasms are rare in untreated B6C3F1 mice; none have been reported in 574 NTP historical
control male mice. Furthermore, a low incidence of gliomas was also reported in the previous NTP
(1984) study. For these reasons, the brain neoplasms are considered exposure-related lesions.
A low incidence of preputial gland carcinomas occurred in the exposed groups in the stop-
exposure studies, and none were seen in controls. Compared with the incidence in concurrent
controls, the combined incidences of preputial gland tumors (adenoma and carcinoma) were
significant in male mice exposed to 312 ppm (52 weeks) and to 625 ppm (13 and 26 weeks) by the
life table test. Preputial gland carcinomas were not reported in a survey of NTP historical control
mice, further indicating that these neoplasms are probably related to exposure to 1,3-butadiene.
One male exposed to 200 ppm for 40 weeks, two males exposed to 625 ppm for 13 weeks,
and two males exposed to 625 ppm for 26 weeks developed Zymbal’s gland carcinomas. This lesion
did not occur in male mice exposed to 312 ppm for 52 weeks; one control male, however, developed
an adenoma. The combined incidence of Zymbal’s gland adenomas and carcinomas in animals
exposed to 625 ppm for 26 weeks was significantly increased compared with controls by the life
table test. Zymbal’s gland neoplasms are rare spontaneous neoplasms that had not been observed
in any NTP historical controls before the only occurrence of this adenoma in the control male mice
for these studies.
To summarize the results of the stop-exposure study pertaining to the relationship between
exposure level and duration of exposure: For lymphocytic lymphomas, there is strong evidence that
higher concentration/shorter duration exposures are more potent than the lower concentration/longer
duration exposures for both the 8,000 ppm-weeks and 16,000 ppm-weeks total exposure groupings.
There is also some evidence for a similar exposure-rate effect for forestomach squamous cell
carcinomas in both total exposure groupings. Any exposure-rate effects at other sites are less clear,
especially because it is difficult to distinguish a small apparent increased potency effect of higher
concentration/shorter duration exposures from an effect of longer potential postexposure follow-up
times following the shorter-duration exposures.
6.3.3. Summary of NTP (1993) Study

The 2-year inhalation study showed that 1,3-butadiene is a potent carcinogen in mice at all
concentrations evaluated. It also demonstrated that exposure to lower concentrations of 1,3-
butadiene than those used in the previous NTP (1984) study allowed expression of neoplasms at
other sites and provided clearer exposure-response relationships because of increased survival.
Statistically significant increases in the incidences of malignant tumors at one or more sites occurred
in male mice exposed to 20 ppm and in females exposed to 6.25 ppm (the lowest exposure
concentration used) 1,3-butadiene for periods up to 103 weeks. The possibility, therefore, exists that
lower exposure concentrations would also cause cancer in B6C3F1 mice. The percentage of animals
bearing malignant tumors increased from about 30% in the controls to nearly 90% in the highest
exposure group, 625 ppm. Lymphocytic lymphomas, hemangiosarcomas of the heart, lung
neoplasms, and neoplastic lesions of the forestomach, mammary gland, ovary, and liver, lesions
identified in the NTP (1984) study, were again increased in this study. In addition, the Harderian
gland and preputial gland were identified as sites of 1,3-butadiene-induced neoplasms. Tumors
observed in the kidneys, skin, Zymbal’s gland, and intestine may also have been related to 1,3-
butadiene exposure.
The stop-exposure study demonstrated that limited exposure to 1,3-butadiene also induces
neoplasms at multiple organ sites in male B6C3F1 mice. Incidences of lymphocytic lymphomas,
hemangiosarcomas of the heart, alveolar-bronchiolar neoplasms, forestomach squamous cell
neoplasms, Harderian gland neoplasms, and preputial gland neoplasms were increased compared
with controls after exposure to 625 ppm 1,3-butadiene for only 13 weeks. The stop-exposure study
also demonstrated an apparent exposure-rate effect for the induction of lymphocytic lymphomas by
1,3-butadiene. At equivalent total exposures, the induction of lymphocytic lymphomas was greater
with exposure to a higher concentration of 1,3-butadiene for a shorter time than for exposure to a
lower concentration for a longer duration.
Overall, the NTP (1993) was a very well conducted study with a precise and comprehensive
presentation of the data. Adequate numbers of animals of both sexes were exposed to multiple
concentration levels of 1,3-butadiene for a major portion of their life span. Comprehensive
histopathological evaluations were performed and mortality and tumor incidences were analyzed
statistically using multiple methods.
6.3.4. 1-Year Study (Irons et al., 1989; Irons, 1990)

To elucidate the mechanism of murine leukemogenesis, Irons and coworkers compared the
induction of thymic lymphomas and expression of murine leukemia virus in NIH Swiss male mice
and B6C3F1 male mice by exposing them to 1,250 ppm 1,3-butadiene, 6 h/day, 5 days/week for 52
weeks. Activation of an endogenous esotropic retrovirus has been associated with spontaneous
lymphomas in the B6C3F1 mouse. The NIH mouse strain was used because it does not express the
esotropic murine leukemia viruses expressed in B6C3F1 mice. The background rate for thymic
lymphoma in NIH mice is nearly zero. Although there was a marked difference between the
incidence of thymic lymphoma/leukemia in B6C3F1 mice (57%) and the incidence in similarly
exposed NIH mice (14%), the study showed that 1,3-butadiene can induce thymic lymphomas
independently of an activated retrovirus. In addition, because these studies were for only 52 weeks,
they did not necessarily allow for a full response for induction of lymphomas by 1,3-butadiene.
6.4. RELATED COMPOUNDS

The draft report on the toxicology and carcinogenicity of 4-vinyl-1-cyclohexene, a dimer
of 1,3-butadiene, was reviewed in U.S. EPA (1985). The final report (NTP, 1986) contains the
same information; therefore, the data are not summarized in this update. The basic conclusion was
that there was clear evidence of carcinogenicity of 4-vinyl-1-cyclohexene (by gavage) in female
mice based on increased ovarian neoplasms and equivocal evidence in male mice based on marginal
increases of malignant lymphomas and alveolar/bronchiolar adenomas. In rats, there was inadequate
evidence in males, at least in part because of excessive mortality, and equivocal evidence in females
based on increased neoplasms of the clitoral gland.
The 1,3-butadiene metabolites 1,2-epoxy-3-butene and 1,2:3,4-diepoxybutane have been
shown to be carcinogenic in rats when administered by skin application or subcutaneous injection
(van Duuren et al., 1963, 1966). In addition, 1,2-epoxybutane, a related compound that is used as
a stabilizer for chlorinated hydrocarbon solvents, was administered by inhalation 6 h/day, 5
days/week for 24 months at exposure concentrations of 0, 200, or 400 ppm to F344/N rats and 0,
50, or 200 ppm to B6C3F1 mice (Dunnick et al., 1988). The treatment and control groups consisted
of 50 male and 50 female animals of each species. Exposure-related inflammatory, degenerative,
and proliferative lesions occurred in the nasal cavity of both rats and mice. Neoplastic lesions were
restricted to the respiratory tract in rats. At 400 ppm, nasal papillary adenomas were observed in
seven male rats and in two female rats; none were observed in controls. In male rats exposed to 400
ppm, there was also an increased incidence of alveolar/bronchiolar adenomas or carcinomas
(combined) (5/50) compared with controls (0/50). No exposure-related neoplastic lesions were seen
in male or female mice.
6.5. DISCUSSION AND CONCLUSIONS

Previous long-term inhalation studies have shown that 1,3-butadiene is carcinogenic in rats
and mice, inducing tumors at multiple organ sites (NTP, 1984; Owen et al., 1987). Results of the
2-year inhalation study (NTP, 1993) presented in this report confirmed the carcinogenicity for 1,3-
butadiene in male and female B6C3F1 mice as demonstrated in an earlier study (NTP, 1984).
Of particular interest in this study were the large number of primary organ sites of tumor
induction by 1,3-butadiene; the early and extensive development of lymphomas; the induction of
uncommon tumors, such as hemangiosarcomas of the heart and squamous cell neoplasms of the
forestomach; and the development of malignant lung tumors at exposure concentrations as low as
6.25 ppm. Because there were no exposure levels of 1,3-butadiene at which a carcinogenic response
was not induced, it is likely that exposure to concentrations below 6.25 ppm would also cause cancer
in mice.
Exposure to 1,3-butadiene at concentrations ranging from 6.25 to 625 ppm for 2 years
caused increased incidences of neoplasms in the hematopoietic system, heart, lung, forestomach,
mammary gland, ovary, and liver, all lesions identified in the NTP (1984) study. The Harderian
gland and preputial glands were identified as additional sites, and tumors in the kidneys, skin,
Zymbal’s gland and intestine were marginally associated with 1,3-butadiene. Because of increased
survival, the study also established clearer concentration-response relationships than the 1984 study.
Competing risks of early-developing lethal lymphocytic lymphomas at high concentrations
preempted the appearance of late-developing neoplasms at some organ sites.
Separate experiments with reduced exposure durations (stop-exposure study) showed that
continued exposure is not necessary for development of neoplasms. The incidences of lymphocytic
lymphomas, hemangiosarcomas of the heart, and tumors of the lung, forestomach, Harderian gland,
and preputial gland were increased in mice exposed for only 13 weeks to 625 ppm 1,3-butadiene
and it is likely that even shorter exposure durations would have produced a carcinogenic response.
The stop-exposure study also showed that the concentration is a greater contributing factor in the
development of lymphocytic lymphomas than the duration of exposure. At comparable total
exposures, the incidence of lymphocytic lymphomas was greater with exposure to a high
concentration of 1,3-butadiene for a short time compared with a lower concentration for a longer
duration.
A morphological continuum of 1,3-butadiene-induced proliferative lesions to neoplasia or
the progression of benign to malignant neoplasms was evident for a number of sites in both the 2-
year and the stop-exposure study (NTP, 1993). Increased incidences of proliferative, nonneoplastic
lesions (hyperplasia) of the cardiac endothelium, alveolar epithelium, forestomach epithelium,
germinal epithelium and granulosa cells of the ovaries, mammary gland, and Harderian gland
probably represent treatment-related preneoplastic changes at these target sites. The distinction
between adenoma and carcinoma further reveal the biological progression of the benign lesions to
malignant neoplasia. For example, in the lungs of male mice, progression from alveolar-bronchiolar
adenoma to carcinoma was evident in the 200-ppm exposure group and in all of the stop-exposure
groups.
The mechanism of 1,3-butadiene-induced carcinogenicity is not known; however,
metabolism likely involving two reactive metabolites, 1,2-epoxy-3-butene and 1,2:3,4-
diepoxybutane, is thought to be an important factor (Chapters 3 and 4).
Results of previous carcinogenicity studies reviewed in U.S. EPA (1985) have shown
different effects of exposure to 1,3-butadiene in rats and mice, with mice being more sensitive to
the induction of carcinogenic effects than rats. The carcinogenic activity in Sprague-Dawley rats
exposed to 1000 or 8000 ppm 1,3-butadiene was largely limited to endocrine tissues or hormonal
responsive tissues, such as pancreas, Leydig cells of the testis, uterus, Zymbal gland, mammary
gland, and thyroid (Owen et al., 1987), whereas exposure of B6C3F1 mice to much lower
concentrations of 1,3-butadiene caused significantly increased incidences of mammary gland
neoplasms and granuloma cell neoplasms of the ovary as well as malignant lymphomas,
hemangiosarcomas of the heart, alveolar-bronchiolar neoplasms, squamous cell neoplasms of the
forestomach, and hepatocellular neoplasms. The reason for the species difference is not known, but
may in part be due to differences in toxicokinetics.
Toxicokinetic studies have shown species-related quantitative and qualitative differences in
the metabolism and disposition of inhaled 1,3-butadiene that may, in part, account for the observed
species variability in the toxicity (Chapter 3). For example, metabolism studies have shown that
blood concentrations of 1,3-butadiene are higher in mice than in rats, and are lower in monkeys than
in either rodent species. In vitro studies using liver microsomes have shown that the metabolism
of the reactive intermediate, 1,2-epoxy-3-butene, to the non-DNA-reactive 1,2-dihydroxybut-3-ene
is the prevalent pathway in human and rat preparations, whereas mouse liver microsomes convert
1,2-epoxy-3-butene to DNA-reactive 1,2:3,4-diepoxybutane in addition to the nonreactive 1,2-
dihydroxybut-3-ene (Csanáday and Bond, 1991).
Investigations by Irons and coworkers (Irons et al., 1989; Irons, 1990) to explain the species
differences of 1,3-butadiene-induced carcinogenicity have focused on the possibility that activation
of an endogenous leukemia retrovirus may play a critical role in 1,3-butadiene-induced lymphoma
in B6C3F1 mice. The incidence of thymic lymphomas was greater in B6C3F1 mice (57%) than in
NIH Swiss mice (14%) exposed to 1,250 ppm 1,3-butadiene for 1 year. However, the NIH Swiss
mouse does not express the endogenous leukemia retrovirus and has a very low background rate for
thymic lymphomas. Thus, the finding that exposure to 1,3-butadiene caused a 14% incidence of
thymic lymphomas in NIH Swiss mice suggests that 1,3-butadiene can induce thymic lymphomas
independently of an activated retrovirus.
Identification of activated oncogenes in chemically induced tumors also may provide
information regarding the mechanism of tumor induction by butadiene. For example, because K-ras
is the most commonly detected oncogene in human cancers, tumors from the NTP (1993) study were
evaluated for the presence of K-ras oncogenes (Goodrow et al., 1990). Activated K-ras oncogenes
were detected in 6/9 lung tumors, 3/12 hepatocellular carcinomas, and in 2/11 lymphomas obtained
from B6C3F1 mice exposed to 1,3-butadiene at concentrations ranging from 62.5 to 625 ppm. A
specific codon 13 mutation was found in most of the activated K-ras oncogenes, suggesting a
chemical-specific effect. Activated K-ras genes have not been found in spontaneously occurring
liver tumors or lymphomas (Goodrow et al., 1990) and were observed only in 1/10 of spontaneous
lung tumors in B6C3F1 mice (Goodrow et al., 1990; Reynolds et al., 1987). Furthermore, it was
shown that tumor suppressor genes are inactivated during 1,3-butadiene carcinogenesis. Soderkvist
et al. (1992) identified allelic losses in the p53 tumor suppressor gene in lung and mammary
carcinomas and lymphomas of B6C3F1 mice exposed to 1,3-butadiene, that were analogous to those
observed in a variety of human cancers.
Immune-function assays conducted by Thurmond et al. (1986) in which B6C3F1 mice were
exposed by inhalation to 1,250 ppm 1,3-butadiene for 6 or 12 weeks showed that 1,3-butadiene
exerts no significant immunosuppressive effects, suggesting that 1,3-butadiene causes neoplasia by
mechanisms other than by compromise of immune function.
In addition to the carcinogenic effects noted in the NTP (1993) study, exposure to 1,3-
butadiene caused hematological changes indicative of a partially regenerative anemia in mice
exposed to 62.5 ppm 1,3-butadiene. Mice exposed to 625 ppm exhibited bone marrow atrophy and
splenic and hepatic extramedullary hematopoiesis. Increases in mean cell volume and mean cell
hemoglobin at 625 ppm 1,3-butadiene suggested that although 1,3-butadiene caused suppression of
hematopoiesis in the bone marrow, younger larger cells may have been released into the blood from
extramedullary sites. A macrocytic-megaloblastic anemia was reported in B6C3F1 mice exposed
to 1,250 ppm 1,3-butadiene for 6 weeks (Irons et al., 1986a, b).
7. EPIDEMIOLOGIC STUDIES OF CARCINOGENICITY
This updated review presents the evaluation of studies published from 1985 through
January 1997. The follow-up proposed by Lemen et al. (1990) of the cohort studied by
Meinhardt et al. (1982) and Downs et al. (1992), an abstract submitted for the International
Symposium are not reviewed in this evaluation. Lemen et al. (1990) did not present any results,
while no details of study design and analysis were available for Downs et al. (1992). Since
1985, investigators have conducted studies of workers who produce 1,3-butadiene as a raw
material (monomer production) or who use 1,3-butadiene in styrene-butadiene rubber (SBR)
production (polymer production).
7.1. MONOMER PRODUCTION

7.1.1. Texaco Cohort
7.1.1.1. Downs et al., 1987: Mortality Among Workers at a Butadiene Facility
Investigators examined a cohort of 2,586 permanent male employees who worked a
minimum of 6 months in a Texaco butadiene manufacturing plant (monomer production) that
supplied the raw material to two adjacent SBR plants studied by Meinhardt et al. (1982) and for
which an update has been proposed by Lemen et al. (1990). Data were available for the 37-year
period from January 1, 1943, through December 31, 1979. Vital status of the cohort was
determined through the Social Security Administration (SSA). Individuals whose vital status
was unverifiable through SSA were traced through the Texas Department of Public Safety.
Death certificates were obtained from the health departments of the states where the individual
resided at the time of death. When this effort was unsuccessful, the individual’s name was
placed on a list, which was submitted to the health departments of Texas and Louisiana, to
obtain the death certificates. A trained nosologist coded the death certificates using the eighth
revision of the International Classification of Diseases (ICD).
Because quantitative exposure data had not been accumulated for individual workers, the
investigators used department codes to construct a qualitative exposure scale composed of four
groups: Group I, low exposure (included utility, office, and management workers, N = 432);
Group II, routine exposure (included process, laboratory, storage, and transport workers, N =
710); Group III, nonroutine exposure (included skilled maintenance workers, N = 993); and
Group IV, unknown exposures (N = 451). The investigators postulated that Group III workers
may have had exposure to higher concentrations with a lesser frequency than Group II workers.
Of 2,586 employees in the cohort, 175 (6.8%) were black. Scrutiny of death certificates
uncovered that 45 blacks (7.5% of total deaths) were improperly coded as whites. At this point,
investigators conducted a preliminary analysis on the total cohort, using both black and white

national death rates. The standard mortality ratios (SMRs) were higher based on black rates as
compared with white rates for four cause-specific deaths only (i.e., all lymphohematopoietic
cancers) (SMR = 169 vs. 138), lymphosarcoma (SMR = 336 vs. 220), Hodgkin’s disease (SMR
= 135 vs. 102), and leukemia (SMR = 155 vs. 119). Most of the other SMRs for both cancers
and noncancers were decreased based on black rates. Therefore, using black rates would have
underestimated the risks. Thus, the entire cohort was treated as white, and all further analyses
were conducted using white death rates.
Expected deaths were calculated using two referent populations: U.S. white males
(national comparison) and white males in a seven-county area surrounding the plants (local
comparison). The rates were standardized for age, race, sex, and calendar year. SMRs (labeled
NSMR for national comparisons and LSMR for local comparisons) were calculated in the
customary manner by dividing the observed deaths by the expected deaths and multiplying the
ratio by 100. Under the null hypothesis, the significance of the ratios of observed to expected
deaths was tested assuming that the observed (O) deaths followed a Poisson distribution using a
two-sided test and assuming a p value of <0.05 to be significant. Comparisons between Groups
I, II, and III were done by using the Mantel-Haenzel procedure for computation of relative risks
in follow-up studies with stratified data (Rothman and Boice, 1982), and power calculations
were performed using the normal approximation to the Poisson distribution (Beaumont and
Breslow, 1981). The person-years at risk were not accrued until after the sixth month of
employment.
A total of 64,800 person-years were accrued for the follow-up period. There were 603
deaths from 1943 through 1979; death certificates were obtained for 579 (96%) individuals. The
vital status was unknown for 73 individuals (2.8% of the total cohort).
Results of this investigation indicated lower than expected mortality for these workers
from all causes (NSMR = 80, p<0.05 and LSMR = 96, p>0.05, O = 603) and from all cancers
(NSMR = 84, p>0.05 and LSMR = 76, p<0.05, O = 122). However, a site-specific comparison
indicated a statistically significant increase in mortality from lymphosarcoma and
reticulosarcoma (ICD code 200, NSMR = 235, 95% confidence intervals [CI] = 101-463, O = 8)
compared with national rates and a nonsignificant excess (LSMR = 182, p>0.05) compared with
local rates.
A comparison of wartime workers (N = 1,061; 452 deaths) who had worked for at least 6
months prior to 1945 and postwar workers (N = 1,525; 151 deaths) found an increase for all
lymphohematopoietic cancers among wartime workers (NSMR = 150, 95% CI = 84-247, O =
15) and among postwar workers (NSMR = 134, p>0.05, O = 6). However, stratification reduced
sample sizes considerably. The rationale for this comparison was based on the assumption that
wartime exposures may have been higher than in postwar periods.

The analyses by duration of employment on mortality showed an increase among those
who worked <5 years for all lymphohematopoietic cancers (NSMR = 167, p>0.05, O = 11), with
most of the increase attributed to leukemia (NSMR = 187, p>0.05, O = 5) and residual
lymphohematopoietic cancers1 (i.e., non-Hodgkin’s lymphoma, multiple myeloma, and other
lymphohematopoietic cancers) (NSMR = 172, p>0.05, O = 5). Among those who worked >5
years, a nonsignificant increase was found for all lymphohematopoietic cancers (NSMR = 127,
O = 10), mainly due to an increase in residual lymphohematopoietic cancers (NSMR = 200, O = 7).
Further analyses were conducted for the four groups identified on the qualitative
exposure scale. For those with routine exposure (Group II), increases were noted for all
lymphohematopoietic cancers (NSMR = 187, p>0.05, O = 6), Hodgkin’s disease (NSMR = 197,
p>0.05, O = 1), and residual lymphohematopoietic cancers (NSMR = 282, p>0.05, O = 4). An
excess of kidney cancer (NSMR = 254, p>0.05) was also observed in this group based on one
case. Similarly, in those with nonroutine exposure (Group III), excesses were observed for all
lymphohematopoietic cancers (NSMR = 167, p>0.05, O = 10), Hodgkin’s disease (NSMR =
130, p>0.05, O = 1), leukemia (NSMR = 201, p>0.05, O = 5), and residual
lymphohematopoietic cancers (NSMR = 150, p>0.05, O = 4).
For those in the low-exposure group (Group I), excess mortality was seen for the same
cancers (excluding Hodgkin’s disease): all lymphohematopoietic cancers (NSMR = 128,
p>0.05, O = 3), leukemia (NSMR = 105, p>0.05, O = 1), and residual lymphohematopoietic
cancers (NSMR = 190, p>0.05, O = 2). In general, use of local southeast Texas coastal rates
resulted in lower SMRs for the above three groups except for Hodgkin’s disease in routine and
nonroutine exposure groups, which showed slight increases over national rates. Both of these
SMRs were based on one observed case in each group. None of the excess found in these three
groups was statistically significant.
The comparison of Groups II, III, and IV with the low-exposure group (Group I) resulted
in inconsistent findings due to a small number of cause-specific deaths and could not be reliably
interpreted.
Analyses were also done by latency and number of years worked using national rates.
Although the results for number of years worked were inconsistent for total cancers, the SMRs
increased from 80 to 93, with increasing latency for this category. Similarly, excess SMRs for
all lymphohematopoietic deaths were observed in all latency periods (0 to 9, 20 to 29, 30 to 39)
except for 10 to 19 years. The number of years of employment results showed an inverse
relationship for these cause-specific deaths. For cause-specific deaths due to lymphosarcoma
and reticulosarcoma (ICD code 200), both the latency as well as number of years employed
1
Residual lymphohematopoietic cancers include ICD codes 200, 202, 203, 208, and 209.

showed an inverse relationship. The notable finding in this analysis was for workers who had a
latency of 0 to 9 years and had worked for less than 10 years (NSMR = 1,198, p<0.01, O = 4).
This increase was statistically highly significant (tested by the author of this document using the
Poisson distribution).
This is an extensively analyzed cohort mortality study. As correctly acknowledged by
the investigators, there are a few methodological limitations to this study, the major ones being a
lack of industrial hygiene (IH) data and a lack of personal work histories. In addition, half of the
total cohort worked less than 5 years in the plant. Some of the workers from this cohort had also
worked in two neighboring SBR plants. The exposures to other chemicals in the SBR plants and
in their prior jobs are the confounders that were not adjusted for in this study. The cohort is
relatively small to start with, but stratification in several subgroups further reduced the power.
The major strength of the study is that it is conducted in a butadiene (monomer)
production facility in a cohort where confounding exposure from styrene is absent. The excesses
observed are in cancers of the lymphohematopoietic system, which are consistent with cancer
findings of the SBR plant workers. Most of the cases of malignancy are concentrated in workers
employed for less than 10 years, which may be due to the occurrence of higher exposures during
wartime years. The exposures during subsequent periods were lower. Thus, the finding of
excess cancer mortality in short-term employees is not evidence against dose-response
relationship.
7.1.1.2. Divine, 1990: An Update on Mortality Among Workers at a 1,3-Butadiene

FacilityCPreliminary Results
In 1990, Divine reported an updated analysis of the same Texaco plant (monomer
production) cohort. The follow-up on the original cohort was extended through 1985 by
updating the information on workers from company data and the SSA. Death certificates were
obtained from the health departments of Texas, Louisiana, Ohio, and Mississippi and were coded
by a trained nosologist according to the eighth revision of the ICD. The National Death Index
records were searched for workers for whom the SSA failed to provide the vital status.
Mortality analyses were performed using Monson’s computer program (Monson, 1974).
Again, the white male death rates of the U.S. population were used due to uncertainties about
race information in the company files and because there were few blacks in the cohort. Person-
years were accrued similarly to the Downs et al. (1987) study.
The qualitative exposure categories remained the same. IH sampling data at the time of
this study supported the exposure categories developed earlier. For this study, lymphosarcoma
(ICD code 200) was reported separately from the cancers of other lymphatic tissues (ICD codes
202, 203, and 208).

A total of 74,219 person-years had accrued through 1985. The number of deaths had
increased to 826, and death certificates were not available for 49 (6%) individuals. Of 2,5822
employees in the cohort, 1,708 individuals were still alive and 48 (1.9%) were lost to follow-up.
Overall, the pattern of results was unchanged from the report by Downs et al. (1987) for this
cohort. For the total cohort, the SMRs for all lymphohematopoietic cancers and Hodgkin’s
disease were increased but not significantly; however, for lymphosarcoma and reticulosarcoma,
the excess was significantly larger (SMR = 229, 95% CI = 104-435, O = 9) and accounted
almost entirely for the increase in overall lymphohematopoietic cancers. Analyses by various
subcohorts also yielded results similar to those observed in the earlier study (Downs et al.,
1987). The highest increase was observed in lymphosarcoma and reticulosarcoma among
workers who had worked more than 5 years but less than 10 years (SMR = 245, 95% CI = 79-
572, O = 5). Prewar and postwar subcohort analyses demonstrated a statistically significant
increase among the prewar subcohort for the same cause-specific deaths (SMR = 269, 95% CI =
108-555, O = 7), while an excess in the postwar subcohort was not statistically significant (SMR
= 155, 95% CI = 17-558, O = 2).
Among the subcohorts based on exposure levels, the only statistically significant excess
was observed for lymphosarcoma and reticulosarcoma among workers who were ever employed
in routine exposure category (SMR = 561, 95% CI = 181-1,310, O = 5). Among workers who
were ever employed in nonroutine exposure category, the excess was observed for all
lymphohematopoietic cancers (SMR = 141, 95% CI = 70-253, O = 11) due to an increase in
leukemia (SMR = 185, 95% CI = 68-403, O = 6). The lymphosarcoma in this group was
slightly increased (SMR = 126, 95% CI = 14-454, O = 2).
For the total cohort, no pattern with latency or duration of years worked was observed for
either all deaths or total cancer deaths. For all lymphohematopoietic cancers, excesses were
observed in the latency groups of 30+ years (SMR = 205, O = 8) and 0 to 9 years (SMR = 200,
O = 4). Both of these groups had worked less than 10 years. Deaths from lymphosarcoma were
also increased in the same duration and latency groups. For 30+ year and 0 to 9 year groups, the
SMRs were 3,333 (O = 2) and 1,333 (O = 4), respectively. No statistical test results were
presented for this analysis. Similar analyses by different exposure groups failed to show any
pattern for all lymphohematopoietic deaths and lymphosarcoma deaths among low-exposure and
unknown exposure groups. Among routinely exposed groups, the excesses were observed for
the same two latency and duration groups as for the total cohort, whereas for nonroutine
exposure the excesses were observed only for 20 to 29 and 30+ years’ latency groups who had
2
It was not explained in the paper how the cohort was reduced to 2,582 from 2,586.

worked for less than 10 years. All of these excesses were based on 3 deaths in each group,
making interpretation of these findings by exposure levels very difficult.
This also is a well-conducted study; unfortunately, the same methodological limitations
that were present in the Downs et al. (1987) study are applicable to this study. However, the
findings of this study are consistent with the earlier study, as well as with other SBR plant
studies.
7.1.1.3. Divine et al., 1993: Cancer Mortality Among Workers at a Butadiene Production
Facility
This update added another 5 years of follow-up to the earlier cohort of monomer workers
(Divine, 1990). Cohort inclusion criteria remained the same but were extended from December
31, 1979, to December 31, 1990. This yielded additional workers resulting in a total cohort of
2,749 individuals. The four exposure groups were similar to those used in earlier studies with
slight changes as follows: (1) The background exposure group (included office utility,
warehouse, and transportation workers, N = 347). This group was called the low-exposure
group in the previous two studies (Downs et al., 1987; Divine, 1990). (2) The low-exposure
group (included workers from operating units, planners and engineers, welders, carpenters, and
workers from brick masons, N = 958). This group was a combination of some of the low-
exposure and all of the unknown exposure group from the previous two studies. (3) The
nonroutine exposure group (included skilled maintenance workers such as pipefitters, tinsmiths,
instrument and electrical workers, and insulators, N = 865). (4) The routine exposure group
(included process, lab, storage, and transport workers, N = 1056). Although the last two
categories appeared to be the same as in the earlier two studies, the change in the number of
individuals in these categories was not explained in the paper. For this study, the investigators
reviewed the results of the IH data and information obtained from the plant personnel and found
that the main difference between the routine and nonroutine exposure groups was in the
frequency and not the intensity of exposure.
Monson’s computer program (Monson, 1974) was used for the analysis of this study also.
All the analytical methods included use of white male death rates of the U.S. population (since
there were very few blacks in the study, they were assumed to be white for the analysis) and
calculation of person-years. The follow-up procedures and acquisition of death certificates were
the same as in an earlier study by Divine (1990).
A total of 83,591 person-years was accrued. At the end of the follow-up period, 1,660
individuals were still alive, 38 were lost to follow-up, and 1,051 were deceased (death
certificates were obtained for 1,036 individuals).

The overall results observed in this study were similar to the earlier two studies. The
only statistically significant elevated SMR observed was for lymphosarcoma and
reticulosarcoma for workers employed for less than 5 years (SMR = 286, 95% CI = 104-622, O
= 6). Again, this increase probably came entirely from the prewar employees (SMR = 254, 95%
CI = 102-523, O = 7). The analysis by exposure group showed an increase for the same cause
in the routine exposure group (SMR = 452, 95% CI = 165-984, O = 6). The analysis by latency
and duration of employment yielded the largest increase in 0 to 9 years latency for the
individuals employed for less than 5 years (prewar individuals?). The SMR was 3,333 based on
two observed cases. No statistical test results were presented for this analysis.
7.1.1.4. Divine and Hartman, 1996: Mortality Update of Butadiene Production Workers
This recent follow-up of the same cohort added 46 more individuals to the cohort (2,795)
by extending the inclusion criteria and the follow-up period through December 31, 1994. The
person-years accrued increased to 85,581. Of 2,795 individuals, 999 were still alive, 574 were
lost to follow-up (28 known to be alive), and 1,222 were deceased (death certificates were
obtained for 1,202 individuals). The follow-up procedures and analytical techniques (for SMR
analysis) were the same as for earlier studies. The exposure categories also remained the same
for this follow-up.
Based on IH data available since 1980, each employee’s potential exposure to butadiene
was estimated by separating the employee’s work history by job categories into 1-year segments.
Two variables were used to calculate the estimated exposure (job categories and calendar time
periods). There were six exposure classes based on job categories: 0, 1, 2, 3, 4, and 5 with 0,
0.1, 0.2, 0.3, 0.4, and 0.5 weights (wt), respectively, and five calendar time periods: <1946 (wt
= 10), 1946-59 (wt = 8), 1960-76 (wt = 4), 1977-85 (wt = 2), and 1986-94 (wt = 1). The
cumulative exposure was obtained for each individual by summing up the scores for all the years
of employment. These exposure estimates were used to conduct survival analyses for: (1) total
lymphohematopoietic cancer, (2) lymphosarcoma, (3) non-Hodgkin’s lymphoma, (4) multiple
myeloma, and (5) leukemias.
Three different models were used for the survival analysis, i.e., a Cox proportional
hazard model with a time-dependent estimate of cumulative exposure, a person-time logistic
regression model with a time-dependent estimate of cumulative exposure, and a nested case-
control model using conditional logistic regression. Each case had 10 matched controls by date
of birth
(+2 years). The selection of controls without replacement was from noncases at the time of the
occurrence of each case.

The results of the SMR analyses were very similar to the earlier two follow-up studies of
this cohort (Divine, 1990; Divine et al., 1993). The survival analyses failed to show any
significant increase in the risk ratios, in any cause-specific cancer, by any of the three methods.
Although the investigators have done a good job of estimating the exposure and have
conducted various analyses, the increase observed in the prewar subcohort for lympho-
reticulosarcoma, when exposures were probably the highest, still persists. Upon completion of
this study, this cohort has 52 years of follow-up but has failed to show any increase in leukemias
which were observed in SBR production workers.
7.1.2. Shell Oil Refinery Cohort

7.1.2.1. Cowles et al., 1994: Mortality, Morbidity, and Hematological Results From a Cohort
of Long-Term Workers Involved in 1,3-Butadiene Monomer Production
Shell Oil’s Deer Park Refinery produced a butadiene monomer from 1941 to 1948 and
1970 to the present. The cohort consisted of male workers who had a minimum of 5 years
employment in the jobs with potential exposure to butadiene or at least 50% of their total
duration of employment (minimum of 3 months) in these jobs. This facility also had several
other refinery operations and chemical production units. Three different analyses were
performed on this cohort: (1) mortality, (2) morbidity, and (3) hematological.
1. Mortality Analysis:
A total of 614 employees comprised the cohort. The follow-up period was from 1948 to
December 31, 1989. Vital status was assessed from company records, SSA, master beneficiary
files, and the National Death Index (NDI). Death certificates were obtained for all the deceased
workers and coded by a trained nosologist according to the revision of the ICD in effect at the
time of death. Mortality rates of Harris County, TX, were used to compute the age-, race-, and
calendar year-adjusted SMRs, using the Occupational Cohort Mortality Analysis Program
(OCMAP) from the University of Pittsburgh.
A total of 7,232 person-years were accrued. Of 614 employees, 589 were still alive, 1
was lost to follow-up, and 24 were dead. No excess mortality, either for total deaths or total
cancers (including cause-specific cancers), was observed.
2. Morbidity Analysis:
Original cohort members who were active at some time between January 1, 1982, and
December 31, 1989, qualified for the morbidity study. Morbidity data were obtained from the
Shell Health Surveillance System. The follow-up period was from 1982 to 1991. Causes of
morbidity were coded according to the 9th revision clinical modification of the ICD. Morbidity

ratios (SMbRs) were calculated by using the internal comparison group of employees who were
active during the same time period and had no exposure to butadiene.
A total of 438 employees were included in this analysis. No excess morbidity by any
cause was observed.
3. Hematological Data Analysis:

Of 438 individuals included in the morbidity study, periodic hematological data were
available for 429 individuals. These hematological data reveal that seven hematological
outcomes were measured (between 1985 and 1991). The most recent laboratory test results were
used for the analysis. Comparisons were done with similar results from 2,600 nonexposed
employees. No differences were observed between butadiene-exposed vs. nonexposed groups.
This study has quite a few methodological limitations. The cohort is small, and deaths
are few. The number of employees selected for this study from the time period 1941-1948,
when exposure was probably higher, is unclear. Over 50% of the cohort was hired in 1970 or
later, with an average follow-up of 12 years. This means that the cohort was still young,
showing "healthy worker" effect, and enough latent period had not elapsed to show increases in
cancers, which usually have a long latent period. Thus, despite the absence of any positive
results, this study fails to provide any negative evidence towards the causal association between
butadiene and occurrence of cancer.
7.1.3. Union Carbide Cohort

7.1.3.1. Ward et al., 1995: Mortality Study of Workers in 1,3-Butadiene Production Units
Identified From a Chemical Workers Cohort
Ward et al., 1996c: Mortality Study of Workers Employed in 1,3-Butadiene
Production Units Identified From a Large Chemical Workers Cohort
The study cohort was selected from 29,139 workers at three Union Carbide Corporation
facilities in the Kanawha Valley, West Virginia. A total of 527 male workers who had worked
between 1940 and 1979 were identified from the work history records as having ever worked in
the departments where there was a potential for butadiene exposure. Only the individuals who
worked in these departments during the butadiene production period (during World War II) were
selected for the study (i.e., 364 individuals). The vital status was determined through December
31, 1990, using the National Death Index. Death certificates were obtained for decedents and
coded according to the revision of the ICD codes in effect at the time of death. Both U.S. and
Kanawha County mortality rates were used for comparison. A modified life table analysis
developed by the National Institute for Occupational Safety and Health (NIOSH) was used to
compute the SMRs.

Of 364 workers, 176 were alive, 3 were lost to follow-up, and 185 were dead at the end
of 1990. The SMR for all causes was 91, while for all cancers it was 105. Neither of them were
statistically significant. The only statistically significant increase was observed for
lymphosarcoma and reticulosarcoma, which was based on four cases (SMR = 577, 95% CI =
157-148). A county-based comparison also resulted in a similar result. By duration of
employment and latency, a statistically significant excess of the SMR was observed among
workers who were employed for more than 2 years and with more than 30 years of latency
(SMR = 1980, 95% CI = 408-5,780, O = 3).
The investigators stated that except for butadiene exposure, there were no common
exposures to other chemicals in the four individuals who had died of lymphosarcoma and
reticulosarcoma, although two of them had been assigned to an acetaldehyde unit for some time.
This study has a few methodological limitations. The cohort is very small, no
adjustments for confounding exposures to other chemicals were done, and no exposure
information is available. The qualitative exposure is assumed based on the job coded for
butadiene exposure. It is still interesting to note that the exposure in these plants was to
butadiene monomer alone either in the production process or the recovery from the olefin
cracking process and not to styrene-butadiene polymer. The only other cohort exposed to
butadiene monomer (Downs et al., 1987; Divine, 1990; Divine et al., 1993; Divine and Hartman,
1996) also found excess in lymphosarcoma and reticulosarcoma in the prewar subcohort.
Studies in monomer production workers are summarized in Table 7-1.
7.2. POLYMER PRODUCTION

7.2.1. Cohort Identified by Johns Hopkins University (JHU) Investigators
7.2.1.1. Matanoski and Schwartz, 1987: Mortality of Workers in Styrene-Butadiene Polymer
Production
This cohort mortality study of SBR polymer production workers from eight plants (seven
U.S. and one Canadian) was reviewed in a 1985 document (U.S. EPA, 1985). At that time, this
study was submitted to the U.S. Environmental Protection Agency but was not published.
1/28/98
Table 7-1. Epidemiologic studies of the health effects of exposure to 1,3-butadiene Cmonomer production
1,3-Butadiene exposure Strengths and
Authors Population studied assessment Results limitations
Downs et al. (1987) 2,586 permanent male Four exposure groups based SS NSMR = 235 and SNS Cohort of monomer production
employee cohort mortality on qualitative exposure LSMR = 182 for workers, a major strength
scale: lymphosarcoma and
Worked for at least a minimum reticulosarcoma for total Lack of IH data
of 6 months from January 1, Group I, low (N = 432) cohort
1943-December 31, 1979 ½ the cohort worked less than 5
Group II, routine (N = 710) SS NSMR R = 1,198 for years in the plant
Follow-up from 1943 through lymphosarcoma and
1979 (37 years) Group III, nonroutine (N = reticulosarcoma for latency Relatively small cohort; therefore
993) of 0-9 years and <10 years hard to interpret results after
Comparison group U.S. of employment further stratification
7-11
population (national) and 7 Group IV, unknown (N =

counties surrounding the plants 451) Lack of adjustment for
(local) confounding for people who
worked in SBR plant too
Divine (1990) Update of the cohort from Same exposure groups as the For lymphosarcoma and Same methodologic limitations as
Downs et al. (1987) earlier study reticulosarcoma: the earlier study
Cohort reduced to 2,582 SS SMR = 229 for total

cohort
Follow-up extended through
1985 SS SMR = 269 for prewar
subcohort
Comparison group U.S.
population SS SMR = 561 for routinely
exposed for less than 10
years
No pattern with latency or

duration of employment

1/28/98
Table 7-1. Epidemiologic studies of the health effects of exposure to 1,3-butadiene Cmonomer production (continued)
Divine et al. (1993) Update of the cohort from Similar exposure groups as For lymphosarcoma and Same strengths and limitations as
Divine (1990) earlier with some reticulosarcoma earlier study
redistribution of workers
Cohort increased to 2,749 as the SS SMR = 254 for prewar
inclusion period extended Group I, background (N = subcohort
through December 31, 1990 347)
Group II, low (N = 958) SS SMR = 286 for workers
Follow-up extended through Group III, nonroutine (N = employed less than 5 years
1990 865)
Group IV, routine (N =
Comparison group U.S. 1,056)
7-12
population
Divine and Hartman (1996) Update of the cohort from Based on IH data and work Results of SMR analysis 52 years follow-up
Divine et al. (1993) histories using were similar as earlier
6 exposure classes studies Exposure estimation useful
Cohort increased to 2,795 as the 5 calendar periods
inclusion period extended Survival analysis failed to Major limitation is no exposure
through December 31, 1994 Individual exposures were show any SS excess in any estimation available in prewar
estimated for each worker cause-specific cancer subcohort, which has the SS
Follow-up extended through lymphosarcoma excess
1994 Three different models used
for the survival analysis

Comparison group U.S.
population
Internal comparison
1/28/98
Table 7-1. Epidemiologic studies of the health effects of exposure to 1,3-butadiene Cmonomer production (continued)
Cowles et al. (1994) Cohort of monomer production None No excess observed in either Very small cohort
workers from 1941-1948 and mortality or morbidity study
from 1970-1994 Exposure is not certain
No hematologic differences
5 years or 50% of total duration found between the exposed Deaths are very few
worked in jobs with potential to and nonexposed employees
1,3-butadiene exposure 50% of cohort hired after 1970
when exposures were low
Mortality follow-up from 1948-
1989 (614 employees) Not enough latent period has
elapsed
7-13
Morbidity follow-up from 1982-

1989 (438 employees)
Hematologic data analyses

1985-1991 (429 employees)
Ward et al. (1995, 1996c) Cohort of 364 male employees Jobs where only 1,3- For lymphosarcoma and Small cohort
who had worked between 1940 butadiene exposure occurred reticulosarcoma
and 1979 No adjustments for confounding
SS SMR = 577 exposures to other chemicals
Employees who has worked in SS SMR = 1980 for more
monomer production during than 2 years of employment No exposure information available

World War II and 30 years of latency
Follow-up through December

31, 1990
U.S. population
Kanawha County population
SS = Statistically significant.
SNS = Statistically nonsignificant.
NSMR = National standard mortality ratios.
LSMR = Local standard mortality ratios.
IH = Industrial hygiene.
Because the findings of the published study are essentially the same, it will not be reviewed
again.
7.2.1.2. Matanoski et al., 1989: Epidemiologic Data Related to Health Effects of 1,3-Butadiene
Matanoski et al., 1990: Mortality of a Cohort of Workers in the Styrene-Butadiene
Polymer Manufacturing Industry (1943-1982)
These two publications essentially reported the same updated reanalysis of the earlier
cohort. In addition, Matanoski et al. (1989) also presented the results of the nested case-control
study in this population. Three methodological differences in the original analysis (Matanoski et
al., 1987) and the reanalysis presented in these two publications should be noted: extension of
follow-up through 1982, fewer workers whose vital status was unknown (3.4% vs.
6.6% in the earlier report), and deletion of workers from the Canadian plant who had relatively
short-term exposure (i.e., workers who had worked for less than 10 years or who had not
reached the age of 45 during employment). Analytical methods were essentially unchanged
from the earlier analysis.
In addition to information received from the SSA and the Motor Vehicle Administration,
follow-up through local plant beneficiary records and the National Death Index was done to
assess the vital status of the study cohort. Follow-up procedures for Canadian workers were
similar to the earlier study. Death certificates were obtained from the local health departments.
The total cohort was reduced from 13,920 to 13,422 in this study. Of 12,113 workers for whom
the vital status was successfully traced, 23% (2,784) were still working in the plants, 53.4%
(6,472) were alive but not working in the plants, 20.2% (2,441) had died, and vital status was
unknown for 3.4% (416). The racial distribution was 75% whites, 10% blacks, 15% unknown
(presumed to be white for the analysis), and less than 1% other.3 Death certificates were
obtained for 97.2% of the deceased individuals and were coded by a trained senior nosologist,
using the eighth revision of the ICD.
Data analyses were done by using age, race, calendar time, and cause-specific U.S.
population rates. A modified life-table program by Monson (1974) was used. The person-years
were calculated through December 31, 1982. The first-year work experience was omitted from
person-years because one of the inclusion criteria was that an individual had to have worked for
at least 1 year. A total of 251,431 person-years were accrued, of which 226,475 were
contributed by whites.
3
The percentages, which are quoted from the paper, add up to 101. This is due to the rounding of the numbers by the
authors of the paper.

Statistically significant lower SMRs for all causes of deaths (81) and for all cancers (85)
were virtually the same as in earlier studies. The SMRs for all causes of deaths by 5-year
calendar period demonstrated increasing SMRs with increasing time period, indicating a
"healthy worker" effect in earlier calendar years. Blacks showed higher SMRs than whites in
later years. A statistically significant excess for all causes of deaths was observed for blacks in
the last 3 years of follow-up (SMR = 134, 95% CI = 101-175, O = 54). Most of the cause-
specific cancer SMRs showed deficits in both races. A few cancer sites demonstrated excess
mortality in both races. Among whites, excesses were observed for esophageal cancer, kidney
cancer, Hodgkin’s disease, and other lymphohematopoietic cancers. Among blacks, excesses
were observed for stomach, liver, and prostate cancer; all lymphohematopoietic cancers;
lymphosarcoma; leukemia; and other lymphohematopoietic cancers. None of the excesses were
statistically significant.
Because the risks for kidney, digestive, and lymphohematopoietic system cancers
approached those of the reference population, which was unusual for an occupational cohort
with low overall risks, investigators further analyzed the data by work areas. For production
workers, deaths from lymphohematopoietic cancers, Hodgkin’s disease, and leukemia were
nonsignificantly increased for the total cohort and among whites (except for leukemia). The
only significant excess observed for the total cohort was for other lymphohematopoietic cancers,
which included non-Hodgkin’s lymphoma and multiple myeloma (SMR = 260, 95% CI = 119-
494, O = 9). Among blacks, however, statistically significant excesses were observed for all
lymphohematopoietic cancers (SMR = 507, 95% CI = 187-1,107, O = 6) and leukemia (SMR =
655, 95% CI = 135-1,906, O = 3). The other two excesses observed among blacks for
lymphosarcoma and other lymphohematopoietic cancers (including non-Hodgkin’s lymphoma
and multiple myeloma) were based on one and two cases, respectively, none being statistically
significant (p>0.05).
Among white maintenance workers, no excesses of lymphohematopoietic cancers were
found with the exception of Hodgkin’s disease (SMR = 170, 95% CI = 35-495), based on only
three deaths. However, rates were nonsignificantly increased for digestive tract malignancies
(i.e., esophagus, stomach, and large intestine). Among black maintenance workers,
nonsignificant excesses were observed for cancer of the rectum and stomach. For utility
workers, the numbers were reported to be too small to reach firm conclusions about risks. For
the "other" category of workers (including laboratory workers, management, and administrative
workers), excesses were observed for Hodgkin’s (SMR = 130, 95% CI = 16-472, O = 2) and
leukemia (SMR = 116, 95% CI = 43-252, O = 6) among whites and for leukemia (SMR = 246,
95% CI not given, O = 1) among blacks. Nonsignificant increased SMRs for the digestive

system among blacks were also observed for the stomach, liver, and pancreas, all of which were
based on fewer than five cases.
Analysis by duration of work or latency for the total cohort did not show an increase in
the hematopoietic cancers.
This is still the largest cohort of SBR workers. The increased follow-up, better tracing,
and exclusion of short-term workers from the Canadian plant have resulted in demonstrating the
excess mortality from malignancies of the lymphohematopoietic system, digestive system, and
kidney. However, the limitations of the earlier study of this cohort (i.e., the lack of exposure
data and inclusion of less than 50% of the population in the follow-up cohort) still exist. The
magnitude of the bias introduced by exclusion of workers (2,391) due to missing information on
total work history or crucial information such as date of birth could be substantial. Although an
attempt was made to correct the race, the race was unknown for 15% of the eligible cohort, and
this segment was assumed to be white for the analysis. This would result in an overestimation of
rates in blacks and an underestimation of rates in whites. No explanation was given as to how
the total eligible population of 13,422 was reduced to 12,113. No data were presented by
individual plants, but as indicated in the earlier study, only four plants had follow-up starting
from 1943, whereas in the other four plants the starting dates of the follow-up ranged from 1957
to 1970; thus, these latter four plants may not have had long enough follow-up for the
malignancies to develop.
7.2.1.3. Matanoski et al., 1989: Epidemiologic Data Related to Health Effects of 1,3-Butadiene
Santos-Burgoa et al., 1992: Lymphohematopoietic Cancer in Styrene-Butadiene
Polymerization Workers
To elucidate the separate contributions of 1,3-butadiene and styrene to the cancers
identified in the updated cohort study, a nested case-control study of this cohort of SBR workers
was conducted using estimates of exposure to 1,3-butadiene and to styrene for each job.
Fifty-nine cases and 193 controls (matched for duration of work) were included in the analysis.
Among the case group were 26 cases of leukemia; 18 of other lymphatic cancers, which included
10 multiple myelomas and 7 non-Hodgkin’s lymphomas; 8 Hodgkin’s lymphomas; and 6
lymphosarcomas.
Cases (workers who had lymphohematopoietic cancer as either the underlying or
contributory cause of death on death certificates) arose from the original eight plants with the
same selection criteria for the eligibility of that cohort (13,422), with the exception of the
Canadian plant. For the Canadian plant, the restriction of either 10 years of work or those who
had reached age 45 during employment was dropped from the selection of cases, which added
two more cases to lymphohematopoietic cancers. Another four cases were added in which

individuals had died of another cause of death but had a lymphohematopoietic cancer at the time
of their death. Two cases were deleted from the final analysis, one lymphosarcoma due to lack
of any controls and one non-Hodgkin’s lymphoma due to lack of job records from which
exposure could be identified.
Controls included workers from the same cohort who were alive or had died of any cause
other than malignant neoplasms. Controls were individually matched to cases by plant; age; hire
year; employment as long as or longer than the case; and, if the control was dead, then survival
to the death of the case. Based on these criteria, an average of 3.3 controls per case were
selected instead of 4 controls per case as intended by the investigators. This average of 3.3
controls per one case had more than a 90% chance of detecting the twofold risk from exposure to
1,3-butadiene. Both cases and controls had about 15 years of employment and were hired at 36
to 37 years of age, somewhat older than usually seen in occupational populations.
Exposures to 1,3-butadiene and styrene were calculated from the job records of each
subject, the number of months that each job was held, and an estimate of the 1,3-butadiene and
styrene exposure levels associated with that job. Both the job identification and exposure
estimation were done independently and without knowledge of case or control status of the
subjects. To estimate 1,3-butadiene and styrene exposures, all jobs within the rubber industry
were ranked from 0 to 10 by a group of senior engineers with many years of experience in the
industry. One-third of the jobs were determined to have no routine exposure, but almost all jobs
were thought to have intermittent exposure. Cumulative dose for both styrene and 1,3-butadiene
was calculated using the score and duration for each job in the participants’ work history.
Because the distribution of exposure scores was skewed to the right, a log transformation of the
scores was used in the analyses. As the logarithmic transformation approached normal
distribution, only the transformed exposure variables were used for the analyses.
Analyses were done by using "ever/never exposed" categories to both butadiene and
styrene and using high-exposure vs. low-exposure groups (based on mean log exposure
cumulative rank for each substance determined by combining cases and controls). Both
conditional (matched) and unconditional (unmatched) logistic regression analyses were
performed. Odds ratios (OR) for matched sets were then calculated based on maximum
likelihood estimates of the OR, and test-based confidence limits around the OR were calculated.
Unadjusted for the presence of the other chemicals and unmatched, analyses by
"ever/never exposed" to butadiene and styrene found significantly increased relative odds for
leukemia for both high and low exposures. Relative odds for butadiene were 6.82 (95% CI =
1.10-42.23) and for styrene were 4.26 (95% CI = 1.02-17.78).
Nonsignificant excesses were also observed for all lymphohematopoietic, other
lymphohematopoietic cancers for exposures to both butadiene and styrene. Other excesses were

for Hodgkin’s disease among workers exposed to butadiene and lymphosarcoma among workers
exposed to styrene.
Matched analyses demonstrated that risk for all lymphohematopoietic neoplasms was
significantly increased among workers exposed to butadiene (OR = 2.30, 95% CI = 1.13-4.71).
Separate evaluation of these neoplasms revealed that most of the association could be explained
by a significant excess risk for leukemia (OR = 9.36, 95% CI = 2.05-22.94), but other cancers in
this group were not significantly elevated. Leukemia also showed a threefold increase associated
with styrene exposure (OR = 3.13, 95% CI = 84-112).
Conditional logistic regression was used to separate the risks associated with each of
these substances. Again, there was a significant excess of leukemia associated with butadiene
(OR = 7.61, 95% CI = 1.62-35.64) and a nonsignificant excess of leukemia associated with
styrene exposures (OR = 2.92, 95% CI = 0.83-10.27). When exposures to both chemicals were
evaluated in the model as dichotomous variables, only butadiene was found to be associated with
leukemia (OR = 7.39, 95% CI = 1.32-41.33).
To determine if specific jobs within the SBR industry might explain some of the risk of
leukemia, the investigators categorized each worker according to the longest job held. A
mixed-job category that combined utilities, operation services, and laboratory jobs was
associated with a relative odds of 3.78 (95% CI = 1.2-11.9). When butadiene was added to the
model, the OR increased to 6.08 for the mixed-job category (95% CI = 1.56-23.72). The
relative odds were 13.3 (95% CI = 2.24-78.55) for association between butadiene exposure and
risk of leukemia adjusted for mixed jobs in this model. Thus, both the mixed-job category and
exposure to butadiene seem to contribute to the risk of leukemia.
The trend test for increasing risk of leukemia with increasing exposure levels of
butadiene (0 through 8) was statistically significant (trend = 3.76, p = 0.05). A similar trend was
not found for styrene. The higher risk of leukemia seen in the original cohort for black workers
could not be evaluated adequately because race was partially controlled in this nested
case-control study.
Unlike the mortality study of this cohort, the case-control study did not show other
lymphoma to be associated with production jobs, but the number of cases was small.
Interestingly, when each chemical was analyzed by stratification, there was an excess risk for
butadiene exposure when exposure to styrene was low (OR = 6.67, 95% CI = 1.06-42.7). A
similar nonsignificant increase also was observed for styrene when butadiene exposure was low.
This might have resulted from small numbers of non-Hodgkin’s lymphoma or multiple myeloma
included together with potentially different etiologies or correlated exposure data. Thus,
investigators suggest further evaluation of each cancer in this other lymphoma category should
be performed separately.

Investigators also caution that estimated exposures in this study were crude and were not
substantiated by monitoring data. As correctly pointed out by them, the original ordinal rank
does not create a perfect exposure scheme. The distribution of ranks was skewed to the right and
had to be log-transformed to differentiate between no exposure and low exposure. Matching on
duration of work may have overmatched the dose and resulted in underestimation of the risk.
Validation of diagnosis of lymphohematopoietic malignancies was not done in this study, which
is an important methodologic limitation of the study given the fact that lymphohematopoietic
cancer recording on death certificates is unreliable (Percy et al., 1981). The panel ranked 71%
of the jobs in ranks of two or less; thus misclassification of exposure based on the estimated
exposure by job as judged by the panel members is quite possible. Because the panel members
were blind concerning the status of the individual being the case or control, the distribution of
misclassification should be the same in cases and controls.
7.2.1.4. Matanoski et al., 1993: Cancer Epidemiology Among Styrene-Butadiene Rubber

Workers
This was an effort by the investigators to verify the findings of their earlier nested case-
control study among styrene-butadiene production workers (Santos-Burgoa et al., 1992). This
study had shown statistically significant elevated relative odds for leukemias. The results from
the analysis conducted with a new set of three controls per case were similar to the results from
the earlier study. The new controls were matched to all the variables except duration of work
with the case. Comparability between the previous and new controls was checked by reviewing
the information on cases and controls from the earlier study. To verify that the cause of death
was correctly coded on the death certificates, hospital records for cases were obtained. Of the 55
records reviewed, two cases had been incorrectly coded on the death certificates as
lymphohematopoietic cancers. Records were obtained for 25 out of 26 leukemia cases and were
found to be correctly coded on the death certificates.
Exposure estimation was done based on measurements provided by seven rubber plants,
the International Institute of Synthetic Rubber Producers, and NIOSH. Although there was
variability among plants, a significant correlation was observed between the log transformed
data provided by the company and the ranks of 464 job and area specific titles. Of the seven
plants that provided exposure measurements for butadiene, three had geometric means. Thus,
using the geometric means, the cohort data were reanalyzed for these three plants. The workers
who were hired before 1960 and had 10 or more years of service showed excesses for all
lymphohematopoietic cancers (SMR = 163, 95% CI = 113-227, O = 34) and leukemia and
aleukemia (SMR = 181, 95% CI = 101-299, O = 15).

This reanalysis of earlier data with new information on exposure estimation validates the
earlier results found by these investigators.
7.2.2. Cohort Identified by University of Alabama (UAB) Investigators

7.2.2.1. Delzell et al., 1996: A Follow-Up Study of Synthetic Rubber Workers
A retrospective cohort mortality study was conducted by Delzell et al. (1996) of synthetic
rubber workers employed in seven U.S. and one Canadian plant. Of the eight plants, seven
plants (including the Canadian plant) were studied by JHU (Matanoski and Schwartz, 1987;
Matanoski et al., 1989, 1990, 1993; Santos-Burgoa et al., 1992) and one (two initial plants
combined into one) by Meinhardt et al. (1982). Of seven plants studied by JHU, one located in
Texas that had a starting time of 1970 was not included in UAB study. The cohort comprised all
the male workers who had worked for at least 1 year between January 1, 1943, and January 1,
1992 (49 years), which was the end of the follow-up period. The follow-up period was shorter
for plants 1, 2, and 6 because the complete records of the employees from these plants were
available much later than 1943. The Canadian plant (plant 8) also had a shorter follow-up
period because follow-up of men who had left employment before 1950 was not feasible.
Since the inclusion criteria for this study were different, there were some additions and
deletions to the earlier study cohort. The vital status was assessed by using plant records; the
SSA’s death master file; the NDI; DMV records of Texas, Louisiana, and Kentucky for the U.S.
plants; and plant records and record linkage with the Canadian Mortality Data Base for the
Canadian plant.
Death certificates were acquired from plant and corporate offices and from state vital
records. The underlying cause of death was coded by a trained nosologist using the ninth
revision of the ICD. Any cancer was coded as a contributory cause of death. For the Canadian
decedents, the underlying cause of death was used from Canadian death registration and coded
according to the ICD revision in effect at the time of death. All ICD codes were converted to
eighth revision codes for analysis. The Ontario Cancer Registry provided the information on
incident cancer cases (including the date of diagnosis, primary site, ninth revision ICD code, and
histologic classification) for the study period.
Mortality analysis included computation of SMRs using the U.S. male general and state
population rates and Ontario male rates; SMRs by quantitative exposure (cumulative ppm-years
and peak ppm-years) to 1,3-butadiene, styrene, and benzene; and stratified internal comparisons.
Various within-cohort analyses were conducted using Poisson regression models.
This study included exposure estimation for each individual. A detailed description of
this estimation appears in Section 7.2.2.2, Macaluso et al., 1996. Complete work histories were
available for 97% of the cohort. Analysis for process group was conducted on the workers from

all the plants. Subgroup analyses were restricted to 6 plants (1,354 workers from 2 plants were
excluded from the analyses due to the lack of information on specific work areas).
Of 15,649 males who had worked in SBR and related processes, 13,586 were white and
2,063 were black. Vital status assessment indicated that 10,939 (70%) workers were alive, 3,976
(25%) were dead, and 734 (5%) were lost to follow-up. Death certificates were acquired for
3,853 (97%) individuals. A total of 386,172 person-years (336,532 for whites and 49,640 for
blacks) was accrued.
Total cohort analysis found SMRs of 87 and 93 for all causes and all cancers,
respectively. The SMR for leukemia was 131 based on 48 observed deaths (95% CI = 97-174).
The SMRs for lymphosarcoma and other lymphopoietic cancers were close to null.
Subcohorts of whites, blacks, ever hourly, and never hourly showed a similar pattern of
below null results for both all causes and all cancer deaths. Ever hourly was the only subcohort
in which statistically significant excesses were found for leukemia. The SMR was 143 ( 95% CI
= 104-191, O = 36) for this subcohort. For white ever hourly workers, the SMR was 130 (95%
CI = 91-181, O = 36), while for blacks the SMR was 227 (95% CI = 104-431, O = 9). The
lymphosarcoma SMR for this subcohort was 102 based on 4 cases, while the SMR for other
lymphopoietic cancer was 106 based on 17 cases. Neither of these excesses was statistically
significant. The further analyses of this ever hourly subcohort by year of death (<1975, 1975-
84, 1985+), year of hire (<1950, 1950-59, 1960), and age at death (<55 years, 55-64 years, 65+
years) showed statistically significant SMRs for 1985+ year of death (SMR = 187, 95% CI =
111-296, O = 18), 1950-59 year of hire (SMR = 200, 95% CI = 122-310, O = 20), and <55 years
at death (SMR = 179, 95% CI = 104-287, O = 17).
When this subcohort was further restricted to >10 years of employment and >20 years
since hire, the SMRs of 224 (95% CI = 149-323, O = 28) for all workers, 192 (95% CI = 119-
294, O = 21) for whites, and 436 (95% CI = 176-901, O = 7) for blacks were observed.
Furthermore, in this restricted subcohort, the SMRs for leukemia were 209 (95% CI = 100-385)
and 228 (95% CI = 135-160) for the workers from plants with the solution polymerization
process and workers from plants without such a process, respectively.
When analyses were done by various process groups, more than twofold increases were
observed for leukemia in polymerization process SMR = 251 (95% CI = 140-414, O = 15),
coagulation process SMR = 248 (95% CI = 100-511, O = 7), maintenance labor SMR = 265
(95% CI = 141-453, O = 13), and laboratory workers SMR = 431 (95% CI = 207-793, O = 10).
Analysis by further restricting the process groups by 5+ years of employment and 20+ years
since hire in each group showed the excesses in leukemia SMRs in the same processes as above.
Analyses by mutually exclusive process groups showed excesses for ever in
polymerization and never in maintenance labor or laboratories (O/E = 8/4.7), ever in

maintenance labor and never in polymerization or laboratories (O/E = 6/3.7), and ever in
laboratories and never in polymerization or maintenance labor (O/E = 8/1.6). Within the labor
group, leukemia increase was observed for workers ever in maintenance labor and never in
production labor (O/E = 11/3.8). On the other hand, for workers in production labor and never
in maintenance labor, the leukemia excess was negligible (O/E = 2/1.4). No excess mortality
from leukemia was observed among ever in finishing and never in polymerization process
workers (O/E = 4/4.5).
An unpublished report by the same authors (Delzell et al., 1996) submitted to the
International Institute of Synthetic Rubber Producers (IISRP) in October 1995 (Delzell et al.,
1995) included many more results of the analyses of this cohort that are relevant to this
assessment. A review of the unpublished results is presented in the following paragraphs.
Various analyses by estimated 1,3-butadiene and styrene exposures were conducted. The
RRs calculated by Poisson regression for 1,3-butadiene ppm-years adjusted for styrene ppm-
years, age, years since hire, calendar period, and race for 0, >0-19, 20-99, 100-199, and 200+
ppm-years were 1, 1.1, 1.8, 2.1, and 3.6, respectively. When analysis was restricted to leukemia
as the underlying cause of death and person-years 20+ years since hire, the results were similar.
Analysis restricted to ever hourly also showed positive results for butadiene. Various analyses
were conducted by using alternate ppm-years categories of exposure. All the analyses
consistently showed similar results, strengthening the association between 1,3-butadiene and
occurrence of leukemias. It is interesting to note that all the leukemia subjects who were
exposed to 1,3-butadiene were also exposed to styrene. There were only two leukemia cases
who had exposure to styrene but none to 1,3-butadiene.
Analysis by 1,3-butadiene peak-years and styrene peak-years demonstrated an association
with 1,3-butadiene peak-years and occurrence of leukemia when adjusted for styrene peak-years,
1,3-butadiene and styrene ppm-years, and other covariates. The association, however, was
irregular. A similar analysis for styrene peak-years was weak and imprecise.
The investigators also conducted a cancer incidence study in the Canadian plant.
Information was obtained from the Ontario Cancer Registry from 1965 to 1992. Standard
incidence ratios (SIRs) were calculated by using the male general population of Ontario. No
increased incidence was found for any cancer in this study.
This is a well-designed, -conducted, and -analyzed study. The main strengths of the
study are large cohort size; long follow-up period (49 years); availability of exposure estimations
on each individual, processes, and tasks; and in-depth analyses using both general population as
well as internal comparison groups.
There are a few limitations as correctly pointed out by the investigators. The cause of
death on death certificates was not confirmed by medical records. Histologic typing was not

available for leukemias. These limitations may have led to misclassification. Furthermore, as
pointed out in the Macaluso et al. (1996) study, there may have been misclassification of
exposure, but this was thought to be nondifferential. Two plants were eliminated from the final
analysis due to the lack of detailed work histories. Although this may have resulted in fewer
uncertainties, valuable data may have been lost due to this elimination. Nevertheless, the
association between exposure to butadiene and occurrence of leukemia was present among both
white and black workers and was fairly consistent across plants.
7.2.2.2. Macaluso et al., 1996: Leukemia and Cumulative Exposure to Butadiene, Styrene, and
Benzene Among Workers in the Synthetic Rubber Industry
A cohort mortality study conducted in synthetic rubber workers by Delzell et al. (1996)
(Section 7.2.2.1) had a component of exposure estimation. The exposures to 1,3-butadiene,
styrene, and benzene were estimated by Macaluso et al. (1996).
An exposure estimation was conducted on each and every worker based on detailed work
histories, work area/job specification, IH monitoring survey records, IH recommendations,
various records from the plants, historical aerial pictures, use of protective and safety equipment,
walk-through surveys, and interviews with plant management as well as long-term employees in
specific areas/jobs. The quantitative exposure estimation was based on process analysis, job
analysis, and exposure estimation. The job-exposure matrices (JEMs) were computed for 1,3-
butadiene, styrene, and benzene, which were linked to work histories of each employee.
Quantitative estimates of exposure to 1,3-butadiene and styrene were based on
background exposure plus task-specific exposure, using multiple exposure and point source
models, respectively. Input variables for these models were derived from several information
sources described earlier. Limited validation of exposure estimates was attempted by comparing
the available IH data from the 1970s and 1980s as well as actually measuring the air
concentrations of 1,3-butadiene and styrene under controlled conditions. The latter method
showed a good agreement among the methods of sampling, while the comparison of IH data
indicated overestimations of 1,3-butadiene exposure.
For each job, 8-h time-weighted average (TWA) intensities and the number of peak
exposures (15-min exposures over 100 ppm) were calculated. Based on job exposures, a JEM
database was developed that was linked with individual work histories to develop individual
quantitative work exposure estimates. For each individual, the exposure indices were multiplied
by the length of employment in that particular process or job and were added up for the total
employment period in various jobs to estimate the cumulative exposure.
Mortality analysis was done by calculating the SMRs and risk ratios (RR) using
estimated quantitative exposures to 1,3-butadiene, styrene, and benzene. Both cumulative ppm-

years and peak-years were calculated for each individual in the study. Person-year data were
grouped by 1,3-butadiene, styrene, and benzene ppm-years for both SMR analyses as well as RR
analyses. Comparability between cohort mortality rates and general population reference
mortality rates was assured by limiting the SMR analysis to the individuals whose underlying
cause of death was listed as leukemia (51 people). Risk ratios were computed by using the
Mantel-Haenszel method and 95% CI were computed by the Breslow method. Poisson
regression models were used for adjustment of multiple confounders and to compute within-
cohort mortality rates, and the X2 test for linear trend was used to examine the dose response.
Work histories were available for 97% of the population. Fifty-two in-depth interviews
with plant management and long-term employees identified 446 specific tasks/work areas with
potential for 1,3-butadiene, styrene, and benzene (3 plants only) exposure. Eight-hour TWAs
for 1,3-butadiene, styrene, and benzene were 0-64 ppm, 0-7.7 ppm, and <1 ppm, respectively,
the median exposures being <2 ppm for 1,3-butadiene and 0.5-1.1 ppm for styrene.
Exposure analysis found that 75% of the cohort was exposed to 1,3-butadiene, 83% was
exposed to styrene, while only 25% was exposed to benzene. The median cumulative exposure
to 1,3-butadiene, styrene, and benzene was 11.2, 7.4, and 2.9 ppm-years, respectively. The
exposure prevalence as well as median cumulative exposure was higher in individuals who had
died of leukemia. Among the leukemia decedents, 85% had exposure to 1,3-butadiene, with
their median cumulative exposure being 36.4 ppm-years. This exposure was two times higher as
compared with all decedents and three times higher as compared with all the other employees.
The exposure to styrene was present in 90% of leukemia decedents, with median cumulative
exposure in them being 22.4 ppm-years, two times and three times higher as compared with all
the decedents and all other employees, respectively. Benzene exposure was found to be less
frequent among leukemia decedents as compared with all the other employees. Analysis by
benzene exposure showed no association with the occurrence of leukemia after adjustment for
1,3-butadiene and styrene.
Leukemia SMRs increased with increasing cumulative exposure to 1,3-butadiene as well
as styrene. Mortality RRs computed for cumulative 1,3-butadiene exposure adjusted for race,
age, and cumulative styrene exposure also showed increasing RRs for increasing cumulative
exposure to 1,3-butadiene. The adjusted RRs for cumulative exposures of butadiene of 0, <1, 1-
19, 20-79, and 80+ ppm-years were 1, 2.0, 2.1, 2.4, and 4.5, respectively. The linear X2 test for
trend was statistically significant (p = 0.01). When similar RRs were computed for styrene
exposure, neither showed a consistent pattern nor a trend of increasing risk with increasing
exposure. A similar trend test was statistically not significant.
Analysis by exclusion of the nonexposed population resulted in RRs of 1, 1.5, and 1.7 for
0.1-19, 20-79, and 80+ ppm-years of the cumulative exposures of 1,3-butadiene. The linear

trend test was statistically significant (p = 0.03), substantiating the earlier finding of increasing
risk of leukemia with increasing cumulative exposure to 1,3-butadiene. Although the same
analysis suggested increasing risk of leukemia with increasing cumulative exposure to styrene
after adjustment for 1,3-butadiene and other covariates, the results were imprecise and
statistically nonsignificant.
There was neither any positive or negative interaction found between the cumulative
exposures to 1,3-butadiene and styrene.
For the last decade or so, epidemiologists have been including exposure estimation in
their studies. The methods used and efforts made to do exposure estimations are improving but
variable. This study is one of the best efforts of exposure estimations to date. The investigators
have used many available methods to come up with best estimates of exposures of 1,3-butadiene,
styrene, and benzene. They also have validated these estimates on a smaller scale. Although
this is considered as the best effort, it should be noted that these are estimates and not actual
measurements. Two plants were eliminated from the analysis because detailed work histories
were lacking. Thus it is possible that individuals may have been misclassified with respect to
process or job, resulting in either over- or underestimations of exposure. However, there is no
reason to believe that the misclassification of exposure occurred only in individuals who had
died of leukemia.
Studies in polymer production workers are summarized in Table 7-2.
7.3. SUMMARY AND DISCUSSION

1,3-Butadiene has been shown to be both mutagenic as well as carcinogenic in animals
and humans. Data in animals, particularly in mice, show that butadiene is a multisite carcinogen

1/28/98
Table 7-2. Epidemiologic studies of the health effects of exposure to 1,3-butadiene Cpolymer production
1,3-Butadiene
Authors Population studied exposure Results Strengths and limitations
assessment
Matanoski et al. Update of the cohort from Divided in four major Among production workers: Largest cohort mortality study of SBR
(1989 and 1990) Matanoski and Schwartz areas based on the workers
(1987) longest job held: SS SMR = 260 for other
lymphohematopoietic cancers Lack of exposure data
Cohort mortality of 8 SBR Production workers in whites
polymer production plant Exclusion of 50% of the population in
workers Utility workers SS SMR = 507 for all the follow-up
lymphohematopoietic cancers
Reduced cohort of 13,422 Maintenance workers and SS SMR = 655 for Four plants had follow-up ranging from
followed through 1982 leukemia in blacks 12 years to 25 years; may not be enough
7-26
All other work sites time for malignancies to develop

Worked for at least 1 year No relation observed with
latency or duration of
Comparison group U.S. employment
population
Matanoski et al. Nested case-control study Exposure to 1,3- For 1,3-butadiene: One of the strengths is attempt was
(1989) butadiene and styrene made to estimate actual exposure
Santos-Burgoa et Cases: was done by job $ Ever/never exposure

al. (1992) - of leukemia 26 identification and levels Matching may have overmatched the
- of other lymphatic associated with that job SS OR = 6.82 (high) and dose
cancers 18 4.26 (low) were found for
Estimations of job and leukemia Lack of validation of diagnosis of
Controls matched on: exposure levels were hematopoietic malignancies may have
done independently of $ Matched analyses resulted in misclassification
plant, age, hire year, the status of the case or
employment duration, control SS OR = 2.3 for all Misclassification of exposure based on
survival to the death of lymphohematopoietic cancer job categories
the case The jobs were ranked
from 0 to 10 SS OR = 9.36 for leukemia
an average 3.3 controls
(instead of intended 4) Cumulative dose was $ Conditional analyses
were selected calculated using the
score and duration for
each job
1/28/98 (continued)
1,3-Butadiene
assessment
Matanoski et A log transformation SS OR = 7.61 for leukemia

al. (1989) of the scores was
Santos-Burgoa used in the analyses $ SS OR = 6.67 for other
et al. (1992) lymphoma when styrene
(continued) Analyses were done: exposure was low
- by ever/never SS trend of 3.76 was found

exposed for increased risk of
- by high- vs. low- leukemia with increasing
exposure exposure levels of
7-27
- both matched butadiene

(conditional) and
unmatched
(unconditional)
Matanoski et Same as nested case- Exposure estimation Similar results with new Verification of cause of death
al. (1993) control study done based on controls
measurements New set of controls validates earlier
A new set of 3 controls provided by seven Reanalysis of cohort data for results
per case plants, IISRP, and three plants
NIOSH
Cause of death verified SS SMR = 163 for all LHC
by hospital records SS SMR = 181 for leukemia
and aleukemia
Cohort data reanalysis
1/28/98
(continued)
1,3-Butadiene
assessment
Macaluso et al. Cohort of seven U.S. and Exposure estimation Adjusted RRs for Methods used and efforts made for
(1996) one Canadian SBR conducted based on cumulative exposure to 1,3- exposure estimation are best efforts
workers mortality study several information butadiene of 0, <1, 1-19, 20- to date
sources including IH 79 and 80 + ppm-years
Worked for at least 1 were 1, 2.0, 2.1, 2.4, and Misclassification with respect to job
year between January 1, Quantitative exposure 4.5. may be possible but unlikely to be
1943, and January 1, estimates on Trend test was SS only in leukemia deaths
1992 background, task-
7-28
specific, multiple Exclusion of the nonexposed

Follow-up period through exposure, and point population also had similar
January 1, 1992 sources models for results with SS trend test
1,3-butadiene,
15,649 male workers styrene, and benzene
U.S. population Peak exposures

Respective State
populations where the 8-h time-weighted
plants were located intensities
Ontario male rates for Cumulative exposures

Canadian plant
Exposures estimated
Internal comparison for each individual
using Poisson regression
1/28/98 Table 7-2. Epidemiologic studies of the health effects of exposure to 1,3-butadiene Cpolymer production
(continued)
1,3-Butadiene
assessment
Delzell et al. Same as Macaluso et al. Same as Macaluso et Ever-hourly workers showed Same as Macaluso et al. (1996)
(1996) (1996) al. (1996) for leukemia
SS SMR = 143 for all ever- Cause of death not verified
Analysis by ever- hourly workers
hourly and never- SS SMR = 227 for blacks Histologic typing of leukemia not
hourly SS SMR = 187 for 1985+ available, thus leading to
year of death misclassification
Analysis by process SS SMR = 200 for 1950-59,
7-29
groups year of hire

SS SMR = 179 for <55 years
age at death
SS SMR = 224 for >10 years
of employment and >20
years since hire
(SMR = 192 for whites and
SMR = 436 for blacks both
SS)
Various process groups

showed for leukemia
SS SMR = 251 for
polymerization process
SS SMR = 265 for
maintenance labor
SS SSMR = 431 for
laboratory worker
Cancer incidence study in

Canadian plant did not show
any increased incidence for
any cancer
1/28/98
(continued)
SS = Statistically significant.
SMR = Standard mortality ratio.
IH = Industrial hygiene.
RR = Risk ratios.
OR = Odds ratio.
LHC = Lymphohematopoietic cancers.
7-30
even at the lowest dose of 6.25 ppm (NTP, 1993). Occupational populations are exposed to
butadiene in the production/recovery of butadiene monomer and production of resins and
plastics. Exposure to this colorless, odorless gas is entirely via inhalation due to its extremely
volatile nature. The general population is exposed to butadiene in ambient air, the major sources
of its release in ambient air being automotive exhaust and cigarette smoke. Its potential to cause
cancer in humans has become an important public health issue.
Butadiene becomes diluted in ambient air and is eliminated by photooxidation. Thus it is
difficult to study the health effects of exposure to butadiene in the general population. Since
exposure to butadiene is ubiquitous in the general population, "unexposed" reference populations
used in occupational cohort studies are likely to contain a substantial number of individuals who
are exposed to butadiene nonoccupationally. Furthermore, the issue of health measurement is
complicated by the fact that occupational cohorts tend to be healthier than the overall general
population and have below average mortality, which is referred to as the "healthy worker effect."
Thus the standard mortality ratios observed in occupational cohorts, computed using the general
population as the reference group, are underestimations of real risk.
7.3.1. Monomer Production

To evaluate the carcinogenicity of 1,3-butadiene, cohorts from monomer and polymer
production were studied by several investigators. The largest cohort of monomer production
workers was initially studied by Downs et al. (1987) and had three follow-ups by Divine (1990),
Divine et al. (1993), and Divine and Hartman (1996). The cohort included 2,586 workers
initially and had 2,795 individuals in the last follow-up due to an extended time period for the
inclusion criteria. The four exposure groups were identified by Downs et al. (1987) based on a
qualitative exposure scale. They remained the same in Divine’s (1990) follow-up and were
similar but slightly changed in Divine et al. (1993). In their last follow-up, based on IH data, the
investigators (Divine and Hartman, 1996) estimated the potential exposure to butadiene for each
employee by their work histories (in 1-year segments), using job categories and calender time
periods. Cumulative exposures were obtained by summing the scores of all the years of
employment.
The findings of all four investigations were essentially the same even after 52 years of
follow-up. There were deficits observed for mortality from all causes and all cancers. The only
statistically significant excess observed was for lymphosarcoma (ICD code 200). Downs et al.
(1987) observed this excess for the total cohort and for the subcohort of workers who had
worked for less than 10 years and latency of 0-9 years. This excess was seen in the prewar
subcohort in all three follow-up studies (SMR = 269, and SMR = 254 in both Divine, 1990, and
in Divine et al., 1993; Divine and Hartman, 1996). No information on exposure levels was

available for this period, but it was believed that the exposures were high during the prewar
period. When analyses were done by years of employment and latency excess for
lymphosarcoma, mortality was always found to be in individuals employed for less than 10 years
and with latency of 0-9 years. It should be noted that after 52 years of follow-up, no elevated
mortality was observed for leukemia, which was the main finding in SBR workers.
A small cohort of 364 individuals was identified from 29,139 workers at three Union
Carbide Corporation plants who had potential exposure to butadiene during World War II (Ward
et al., 1995, 1996c). The exposure to butadiene was assumed based on job categories, and no
adjustments for confounding by other chemicals were done. As observed in the Divine Studies
(1990, 1993, 1996), a statistically significant excess for lymphosarcoma (SMR = 577) also was
observed in this cohort.
A third cohort of 614 workers exposed to monomer was studied by Cowles et al. (1994)
and the study failed to show any excess mortality or morbidity. Due to several methodologic
limitations, this study failed to provide any negative evidence towards the causal association
between exposure to butadiene and occurrence of lymphosarcoma that was observed in the other
two cohorts.
7.3.2. Polymer Production

A further follow-up and reanalysis of a large SBR polymer production workers’ cohort
(Matanoski and Schwartz, 1987) was conducted by Matanoski et al. (1989, 1990). This follow-
up added 3 years to the earlier study. The findings of this follow-up were essentially the same as
the earlier study. The only statistically significant excesses were found among production
workers. Among whites the excess was for other lymphohematopoietic cancers (SMR = 260)
and among blacks the excesses were for all lymphohematopoietic cancers (SMR = 507) and
leukemia (SMR = 655). Analyses by duration of work and latency did not show any increases in
hematopoietic cancers. There were no exposure measurements or estimations done in this study.
A nested case-control study from this cohort (Matanoski et al., 1989, 1990) was
conducted by the same investigators and reported in Matanoski et al. (1989) and Santos-Burgoa
et al. (1992). Fifty-nine cases of lymphohematopoietic cancers and 193 matched controls were
identified. Exposures to 1,3-butadiene and styrene were estimated in these individuals using the
job records and levels of exposures to 1,3-butadiene and styrene associated with those jobs
independently of the case or control status. The jobs were ranked and cumulative dose was
calculated for each case and control. Analyses were conducted using log transformed scores.
The relative odds were increased for high (OR = 6.82) and low (OR = 4.26) exposures in the
ever/never exposed analysis, matched analysis (OR = 9.36), and conditional analysis (OR =
7.61) for leukemia. All the increases were statistically significant. A statistically significant

trend was also observed for increasing risk of leukemia with increasing exposure levels of
butadiene.
Because the findings of the nested case-control study were questioned by Acquavella
(1989) and Cole et al. (1993), as they were in disagreement with the base cohort study,
Matanoski et al. (1993) reevaluated the analysis of the nested case-control study by choosing a
new set of three controls per case. The investigators also verified the cause of death by
obtaining the hospital records. The findings of the new analysis were similar to the earlier
analysis.
Furthermore, they estimated the exposures to the cohort based on measurements provided
by seven rubber plants, IISRP, and NIOSH. In an analysis of the subcohort from three plants
who had the geometric means of exposure, statistically significant excesses were observed for all
lymphohematopoietic cancers (SMR = 163) as well as for leukemia and aleukemia (SMR =
181).
Delzell et al. (1996) and Macaluso et al. (1996) reported separately the two components
of the follow-up study of synthetic rubber workers. These investigators studied the seven plants
studied by Matanoski and Schwartz (1987), Matanoski et al. (1989, 1990, 1993), and Santos-
Burgoa et al. (1992) and one plant (two initial plants combined into one) by Meinhardt et al.
(1982). The follow-up period was 49 years. Investigators estimated the exposures to 1,3-
butadiene, styrene, and benzene for each worker. This was done by using various means such as
job histories, work areas, IH data, historical plant data, aerial pictures, interviews with long-term
employees and managers, walk-through surveys, etc. Quantitative exposures were calculated
and limited validation of exposure estimates were attempted using available 1970’s and 1980’s
IH data. Cumulative and peak exposures were calculated for each worker. Comparison with the
U.S. population resulted in statistically significant excesses for leukemia in ever-hourly workers
(SMR = 143) and its subcohort of blacks (SMR = 227). The excesses were also found in the
ever-hourly cohort for year of death (SMR = 187 for 1985+), year of hire (SMR = 200 for 1950-
59), age at death (SMR = 179 for <55 years), and for more than 10 years employment and more
than 20 years since hire (SMR = 192 for whites and SMR = 436 for blacks). Laboratory
workers, maintenance workers, and polymerization workers also showed increased SMRs of
431, 265, and 251, respectively. All these analyses were done adjusting for styrene and benzene.
When internal comparison was done using the estimated ppm-years exposure data, relative ratios
increased with increasing exposures. The trend test was statistically significant.
The incidence study conducted in the Canadian plant employees did not show any
increases in any cause-specific cancers.

7.3.3. Relevant Methodologic Issues and Discussion
Throughout this chapter, various methodologic issues including strengths and limitations
are discussed. The major concerns are lack of exposure information and short follow-up periods
in earlier studies, small cohort size, lack of data on confounding variables, and lack of latency
analysis in one study. Furthermore, death certificates were used by all the investigators, which
could lead to misclassification bias. Validation of diagnosis of lymphohematopoeitic cancer was
not done in any of the studies except in Matanoski et al. (1993). This is a methodologic concern
given the fact that lymphohematopoeitic cancer recording on death certificates is unreliable
(Percy et al., 1981).
Lack of exposure information is another major limitation in Cowles et al. (1994) and
Ward et al. (1995, 1996c). Cowles et al. (1994) made no attempt to even do job classification.
This cohort was very small, there were very few deaths, and more than 50% of the cohort had an
average follow-up of 12 years.
Ward et al. (1995, 1996c) also did not attempt any exposure estimation. This cohort also
was very small but was restricted to workers who had worked in the 1,3-butadiene production
period (during World War II). The high SMR for lymphosarcoma and reticulosarcoma observed
in this study was based on only four cases. They used employment of 2 years+ as surrogate for
exposure and stated that there were no other common exposures to other chemicals. Considering
that the cohort was small and only four deaths occurred from lymphosarcoma and
reticulosarcoma, it should be noted that this finding is consistent with the finding of the other
monomer facility studied by Divine (1990), Divine et al. (1993), and Divine and Hartman
(1996).
A monomer cohort study conducted by Downs et al. (1987) and followed by Divine
(1990) and Divine et al. (1993) also lacked exposure information, although the surrogate
exposure grouping was done by qualitative exposure information based on job descriptions/work
areas. The investigators attempted the exposure estimation in their last follow-up (Divine and
Hartman, 1996) and found that except for an excess observed for lymphosarcoma and
reticulosarcoma in the prewar subcohort, there were no excesses in any cause-specific cancer
mortality. However, investigators did not have any information on work histories or levels of
1,3-butadiene exposure during the prewar period, which made exposure estimation in the prewar
workers impossible. Even after 52 years of follow-up and extensive analyses, this cohort has not
observed any excess in mortality from leukemia that was observed in SBR workers.
Nonetheless, the finding of excess mortality from lymphosarcoma and reticulosarcoma is
consistent with findings of Meinhardt et al. (1982) and Ward et al. (1995, 1996c). In addition,
the excess of lymphosarcoma and reticulosarcoma in short-term workers but not in long-term
workers was consistent with the similar findings of Meinhardt et al. (1982).

Matanoski and Schwartz (1987) and Matanoski et al. (1989, 1990) did not have any
exposure information available. The cohort was distributed in four major areas based on longest
jobs held and the qualitative exposure information used as surrogate. When the nested case-
control study was undertaken by these investigators (Matanoski et al., 1989; Santos-Burgoa et
al., 1992), exposure estimation was done by using various sources only for the selected cases and
controls. They observed a statistically significant high excess from leukemia mortality, which
the authors concluded as being causally associated with exposure to 1,3-butadiene.
Matanoski et al. (1993) validated their earlier results of the nested case-control study by
using a new set of three controls per case. They also verified the cause of death noted on the
death certificates and diagnosis noted on the hospital charts. They found that the diagnosis noted
on 25 out of 26 charts agreed with the cause of death noted on the death certificates. The results
of this study were similar to the earlier nested case-control study.
This finding of a high excess of leukemia mortality in the case-control study was
questioned by Acquavella (1989) and Cole et al. (1993) because no excess leukemia mortality
was found in the base cohort study from which the cases and controls were selected. Their
argument that the results of the case-control study were statistically incompatible with the results
of the cohort study was based on the calculations of number of leukemias that should have been
seen in the cohort study, based on the relative odds observed in the case-control study. The Cole
et al. (1993) calculations resulted in approximately 104 leukemia cases if relative odds of 7.6
were applicable to 60% of the cohort that was exposed to 1,3-butadiene and an additional 9.2
expected leukemias for the remaining 40% cohort that was not exposed, resulting in an observed
113 leukemias for the cohort as against 22 leukemias actually observed in the cohort study.
Variability in both the prevalence of exposure and the relative odds were looked at by these
authors (Cole et al., 1993), and they concluded that there was no reasonable combination that
resolved the incompatibility between the findings of the cohort and case-control studies.
Matanoski and Santos-Burgoa (1994) disagreed with this criticism. They asserted that
the 60% exposure observed among the controls in the case-control study overestimated the
prevalence of exposure for the cohort population and that the matching criteria may have skewed
the control selection and produced controls who were not representative of the base cohort.
The main limitations of the cohort study were that more than 50% of the population was
excluded due to lack of work histories or start date and lack of exposure data. The follow-up for
four plants where the starting date was 1957 to 1970 may not have been long enough for
malignancies to develop. As far as the nested case-control study is concerned, as pointed out by
the authors, the estimated exposures were crude and were not substantiated by IH data. The
exposure misclassification may have occurred based on the estimated exposure by job if the jobs
were incorrectly identified for higher or lower exposure. However, the panel members were

blind towards the status of cases and controls, thus the distribution of misclassification should be
the same in cases and controls.
Although the controversy about the cohort and case-control study is still not resolved, the
nested case-control study was the first one to demonstrate a strong association between exposure
to 1,3-butadiene and occurrence of leukemias.
The Delzell et al. (1996) and Macaluso et al. (1996) cohort study is one of the best efforts
of exposure estimation to date. Some misclassification of exposure may have occurred with
respect to certain jobs, but it is unlikely to have occurred only in leukemia cases. The
investigators also did some validation of exposure estimates based on IH data. They pointed out
correctly that the excess mortality observed for leukemia was based on death certificates and was
not verified by medical records. Histologic typing of leukemia was also not available. This may
have resulted in misclassification. Two plants were eliminated from the final analysis due to the
lack of work histories, which may have resulted in the loss of valuable data.
Based on these monomer and polymer production worker cohorts, it is obvious that an
increased number of lymphohematopoietic cancers is observed in these populations. A clear
difference is becoming apparent though. Increased lymphosarcomas develop in workers exposed
to monomer (Downs et al., 1987; Divine, 1990; Divine et al., 1993; Divine and Hartman, 1996;
Ward et al., 1995, 1996c), while excess leukemias occur in workers exposed to polymer
(Matanoski et al., 1990, 1993; Santos-Burgoa et al., 1992; Delzell et al., 1996; Macaluso et al.,
1996). Furthermore, the lymphosarcomas were observed in the monomer workers, who were
probably exposed to higher levels of 1,3-butadiene for shorter periods of time (wartime workers)
and not in long-term workers with low levels of exposures. A confirmation of this observation
comes from the stop-exposure studies conducted by Melnick et al. (1990a). They observed that
at a similar total exposure, the incidence of lymphoma was greater among mice exposed to
higher concentrations of butadiene for a shorter period of time (625 ppm for 26 weeks) than
among mice exposed to a lower concentration for a longer period of time (312 ppm for 52
weeks). Consequently, this suggests that it is the concentration of 1,3-butadiene rather than the
duration of exposure that is important in the occurrence of lymphomas. There is a null
relationship between exposure to 1,3-butadiene monomer and occurrence of leukemias that is
observed in polymer workers. This may be due to very low exposures to 1,3-butadiene in
monomer production workers or exposure to a necessary co/modifying factor or a confounding
factor in SBR production workers. Data are currently lacking to confirm or refute any of these
possibilities. The findings of Delzell et al. (1996) and Macaluso et al. (1996) are inconsistent
with confounding by exposure to other chemicals. The findings of excess leukemias in SBR
production workers are consistent with a causal association with exposure to 1,3 butadiene.

7.3.4. Criteria of Causal Inference
In most situations, epidemiologic data are used to delineate the causality of certain health
effects. Several cancers have been causally associated with exposure to agents for which there is
no direct biological evidence. Insufficient knowledge about the biological bases for diseases in
humans makes it difficult to identify exposure to an agent as causal, particularly for malignant
diseases when the exposure was in the distant past. Consequently, epidemiologists and
biologists have provided a set of criteria that define a causal relationship between exposure and
health outcome. A causal interpretation is enhanced for studies that meet these criteria. None of
these criteria actually proves causality; actual proof is rarely attainable when dealing with
environmental carcinogens. None of these criteria should be considered either necessary (except
temporality of exposure) or sufficient in itself. The absence of any one or even several of these
criteria does not prevent a causal interpretation. However, if more criteria apply, it provides
credible evidence for causality.
Thus, applying the criteria of causal inference to the monomer and polymer cohort
mortality studies and one nested case-control study in which risk of lymphohematopoietic
cancers were assessed resulted in the following:
Temporality . There is temporality of exposure to 1,3-butadiene prior to the

occurrence of lymphosarcoma in monomer workers and leukemias in SBR workers.
Strength of association . Strength of association between exposure and the

occurrence of lymphosarcoma in the prewar period ranged from 154% to 477%
higher risk among workers exposed to monomer as compared with the nonexposed
general population (Divine, 1990; Divine et al., 1993; Divine and Hartman, 1996;
Ward et al., 1995, 1996c). The excess risk of leukemia ranged from 43% to 127%
higher among workers exposed to SBR in ever-hourly workers as compared with
the general population (Delzell et al., 1996). Internal comparison of SBR worker
population resulted in a 4.5-fold increased leukemia risk among the highest
exposure group in the same cohort (Macaluso et al., 1996). The nested case-control
study from the SBR cohort showed a 7.6-fold increase in the risk of leukemia
(Matanoski et al., 1989, 1993; Santos-Burgoa et al., 1992).
Consistency . Two cohort studies in monomer workers showed an increased risk of

lymphosarcoma (Divine, 1990; Divine et al., 1993; Divine and Hartman, 1996;
Ward et al., 1995, 1996c), while one cohort study (Delzell et al., 1996; Macaluso et
al., 1996) (with a cohort derived from seven U.S. plants and one Canadian plant)
and one nested case-control study (Matanoski et al., 1989, 1993; Santos-Burgoa et
al., 1995) showed an excess risk of leukemia in SBR workers. The SBR workers
cohort defined by Delzell et al. (1996) showed a fairly consistent association
between exposure to butadiene and occurrence of leukemia across plants. Excesses
for both lymphosarcoma as well as leukemia were observed by McMichael et al.
(1974, 1976) and Meinhardt et al. (1982).

Specificity . All monomer studies showed an increased risk of lymphosarcoma
while SBR studies showed an increased risk of leukemia. Overall, they show
increased risks of lymphohematopoietic system cancer among populations exposed
to 1,3-butadiene. It should be noted that exposure to a particular chemical (or drug
or radiation) may cause more than one type of leukemia or another type of
hematopoietic cancer (Linet, 1985).
Biological gradient . The biological gradient, which refers to the dose-response

relationship, was observed only in SBR workers. Both the nested case-control
study and the cohort study showed increasing risk of leukemia with increasing
exposures. Such a relationship was not observed in monomer workers. The reason
may be because a very small number of people were exposed to high levels of 1,3-
butadiene for a shorter period of time who showed the occurrence of
lymphosarcoma. They could not be further stratified to evaluate the dose response.
Biological plausibility . As described in Chapter 4, hemoglobin adducts have been

detected in humans exposed to 1,3-butadiene (Osterman-Golkar et al., 1993; Sorsa
et al., 1996). Significantly increased frequencies of hprt mutant lymphocytes were
observed in high-exposure groups by Legator et al. (1993) and Ward et al. (1994).
Mutations, chromosomal aberrations, and cell transformations, all well-established
steps in the process of carcinogenesis, were observed in human and animal studies.
This makes a convincing argument for the biological plausibility of occurrence of
leukemia in SBR workers and lymphosarcoma in monomer workers.
In conclusion, some of the causality criteria apply to monomer workers and occurrence
of lymphosarcoma while all the criteria apply well for leukemia among SBR workers. Based on
strength of association, dose-response relationship, specificity of cancer (leukemiaCspecific cell
type is not known at this time), and biological plausibility, there is sufficient evidence to
consider 1,3-butadiene a known human carcinogen.

8. PHARMACOKINETIC MODELING
8.1. INTRODUCTION
Several physiologically based pharmacokinetic (PBPK) models of 1,3-butadiene
metabolism and disposition have been developed to attempt to explain the interspecies
differences in the potency and site specificity of the carcinogenic response between mice and rats
and to provide a corresponding dosimetric basis for quantitatively extrapolating carcinogenic
potency from rodents to humans (Hattis and Wasson, 1987; Hallenbeck, 1992; Kohn and
Melnick, 1993; Johanson and Filser, 1993; Evelo et al., 1993; Medinsky et al., 1994). PBPK
models use species-specific physiological parameters such as alveolar ventilation rates and blood
flow rates, chemical-specific distribution parameters such as blood:air and tissue:blood partition
coefficients, and species- and chemical-specific metabolic rates to elucidate the
pharmacokinetics (i.e., the uptake, distribution, metabolism, and excretion) of a chemical.
Ideally, such models provide species-specific target tissue doses of the toxicologically
active form(s) of the chemical. Carcinogenic risks from bioassay data can then be extrapolated
to humans on the basis of equivalent effective doses, reducing some of the uncertainties that
occur when interspecies extrapolation is based simply on exposure to the parent compound,
especially when nonlinear physiological processes are involved. Assumptions must still be made
to the effect that the mechanisms of action of the active form(s) of the compound at the target
tissue(s) are the same across species and that the tissues of different species are equally sensitive.
If these assumptions are not valid, pharmacodynamic data and modeling are required for more
precise risk assessment.
PBPK models that fall short of describing target tissue doses of the active form(s) of a
chemical may still be useful for improving the dosimetric basis of interspecies extrapolation for
quantitative risk assessment. For example, it is well established that metabolic activation of
1,3-butadiene is probably necessary for its carcinogenic action (Chapter 4). Therefore, a PBPK
model describing the production and disposition of 1,2-epoxy-3-butene (EB), the first product of
metabolic activation of 1,3-butadiene, may be able to provide a better dose metric than the
default methodology of using exposure to 1,3-butadiene itself.
This chapter reviews and analyzes the six PBPK models for 1,3-butadiene that are
currently available and assesses their usefulness for quantitative risk assessment of 1,3-butadiene
based on interspecies extrapolation. Each of these PBPK models assumes, for simplicity, that
the transfer of 1,3-butadiene to tissues is blood flow-limited, that each tissue compartment is
"well mixed," and that tissue concentrations are in equilibrium with the venous blood
concentration leaving the tissue.

8.2. PBPK MODELS FOR 1,3-BUTADIENE
8.2.1. Hattis and Wasson (1987)
The first PBPK model for 1,3-butadiene was that of Hattis and Wasson (1987). They
defined the effective dose of 1,3-butadiene as the amount that is metabolically converted to EB
and used this dose as a basis for a risk assessment of occupational 1,3-butadiene exposure. Their
model consists of three compartments: a fat compartment; a muscle compartment; and a liver
and vessel-rich compartment, which includes the brain, heart, kidneys, and other small visceral
organs. The transfer of 1,3-butadiene between blood and tissues is assumed to be blood flow-
limited. Metabolism to the monoepoxide is ascribed to the entire liver and vessel-rich
compartment and is assumed to follow simple Michaelis-Menten kinetics. No further
metabolism of EB is considered.
The only chemical-specific parameter values then available were whole-body maximal
metabolic rates for mice and rats inferred from the chamber study data of Kreiling et al. (1986b).
These data provided the KM and preliminary Vmax estimates for the liver and vessel-rich
compartment. Tissue:blood and blood:air partition coefficients were estimated from chemical
structure and solubility data using empirical relationships (e.g., Fiserova-Bergerova and Diaz,
1986). Model simulations were then run, adjusting KM and the partition coefficients to fit the
blood 1,3-butadiene concentration data of Bond et al. (1986), to derive "best estimates" for these
parameters. Human metabolic rates were estimated by allometric scaling of the mouse and rat
rates because no PBPK data were available for human metabolism of 1,3-butadiene. The
parameter values used by Hattis and Wasson (1987) are summarized in Table 8-1.
No additional data were available at that time for an independent validation of this
model. A minimal sensitivity analysis was conducted by varying KM and the blood:air partition
coefficient among a few values and observing the effect on the ultimate risk estimates. Hattis
and Wasson (1987) claimed that their model is not very sensitive to reasonable differences in
partition coefficients. Similarly, the model is insensitive to the precise value of the metabolic
parameters because, given the blood:air partition coefficient values that were used, metabolic
conversion in their model is limited by blood flow to the liver and vessel-rich compartment.
Hattis and Wasson concluded that differences in pharmacokinetics fail to account for differences
in carcinogenesis between mice and rats and that, with respect to risk assessment, uncertainties
in the PBPK modeling are trivial compared with the differences in apparent sensitivities between
these species.

Table 8-1. Parameter values used in the Hattis and Wasson (1987) PBPK model
Parameter Rat Mouse Human
Alveolar ventilation 0.15 0.0233 11.38a

(L/min) 4.8
Weight (kg) 0.40 0.028 70
Qf (L/min) 0.0136 0.00192 0.69a

0.35b
Qm (L/min) 0.0226 0.00319 2.61a

1.1b
Qlvr (L/min) 0.1042 0.01617 5.09a

4.35b
Vf (L) 0.028 0.0028 14.024
Vm (L) 0.300 0.0196 34.756
Vlvr (L) 0.036 0.00308 8.513
Blood:air partition --------------------------------- 0.35 ---------------------------------

coefficientc
--------------------------------- 118.2 --------------------------------
Pf
--------------------------------- 5.26 ---------------------------------
Pm
--------------------------------- 5.4 ----------------------------------
Plvr
Vmax (mol/min) 1.47E-6d 1.87E-7d 8.0E-5e
KM (mol/L) --------------------------------- 5E-6 f ---------------------------------
a
Awake.
b
Asleep.
c
The blood:air partition coefficient of 0.35 is the "best estimate" value from "fitting" the model. The
tissue:blood partition coefficients (P) are from functions of the blood:air partition coefficient for which the
"best estimate" value of 0.35 was used. Partition coefficients are assumed to be the same across species.
d
From Kreiling et al. (1986b).
e
From allometric scaling of the rodent values.
f
"Best estimate" from "fitting" the model.
Subscripts f, m, and lvr designate the fat, muscle, and liver and vessel-rich compartments (tissues), respectively.
Q: tissue blood flow rate.
V: tissue volume.
P: tissue:blood partition coefficient.

The Hattis and Wasson (1987) model is not discussed further here because it has been
superseded by new data and other modeling efforts.
8.2.2. Hallenbeck (1992)

Hallenbeck (1992) reported having done a PBPK-based cancer risk assessment for
1,3-butadiene; however, he provided no details of the PBPK model that he used. Furthermore,
he used the area under the 1,3-butadiene concentration-versus-time curve for the lung as his
tissue-dose surrogate, taking no account of metabolic activation. As presented, this model
contributes nothing to the current state of knowledge regarding the pharmacokinetic modeling of
1,3-butadiene.
8.2.3. Kohn and Melnick (1993)

The PBPK model of Kohn and Melnick (1993) focuses on the disposition of EB in the
mouse, rat, and human. This model incorporates additional tissues (compartments) and
metabolic reactions based on experimental data that were not available at the time of the Hattis
and Wasson (1987) model; however, it also relies on theoretically derived partition coefficients.
The Kohn and Melnick model is blood flow-limited and consists of six compartments: lung,
blood, fat, liver, other rapidly perfused tissues (viscera), and slowly perfused tissues (muscle).
Metabolism occurs in the liver, lung, and viscera compartments. The metabolic reactions
include conversion of 1,3-butadiene to EB, the conversion of EB to 1,2:3,4-diepoxybutane
(DEB), the enzymatic hydrolysis of EB, and the enzymatic conjugation of EB with glutathione.
With the exception of the partition coefficients, which were derived in advance from
published methodologies, all of the mouse, rat, and human parameter estimates were from the
literature; none of them were adjusted to obtain a fit to experimental data. The parameter values
used by Kohn and Melnick (1993) are summarized in Table 8-2. Blood:tissue partition
coefficients for 1,3-butadiene were from Hattis and Wasson (1987). The blood:air partition
coefficients reported by Csanády et al. (1992) for 1,3-butadiene and EB were used as lung:air
partition coefficients. The fat:blood partition coefficient for EB was calculated using an
empirical relationship from Lyman et al. (1990), whereas the tissue:blood partition coefficients
of EB for the other tissues were derived using the method of Fiserova-Bergerova and Diaz
(1986). These are essentially the same procedures used by Hattis and Wasson (1987).
Michaelis-Menten kinetics were used to describe the oxidation of 1,3-butadiene and EB
by the cytochrome P-450 isozyme CYP2E1, the hydrolysis of EB by epoxide hydrolase, and the
glutathione S-transferase-catalyzed conjugation of EB with glutathione. KM and Vmax values for
each of these reactions in the liver and lung of the mouse, rat, and human were taken from the in

Table 8-2. Parameter values used in the Kohn and Melnick (1993)
PBPK model
Parameter Mouse Rat Human

a
Physiological parameters
Body weight (kg) 0.028 0.4 70
Cardiac output (L/h) 1.044 7.32 660b
Ventilation rate (L/h) 2.64 15.6 1,200b
Fraction blood 0.05 0.054 0.077
Fraction fat 0.04 0.08 0.144
Fraction liver 0.062 0.05 0.025
Fraction viscera 0.05 0.083 0.037
Fraction muscle 0.78 0.59 0.547
Fat flow fraction 0.05 0.07 0.036
Liver flow fraction 0.16 0.16 0.16
Viscera flow fraction 0.52 0.40 0.446
Muscle flow fraction 0.19 0.36 0.361
c
Partition coefficients
Air partition BD 1.5
Fat partition BD 118.2
Liver partition BD 5.49
Viscera partition BD 5.34
Muscle partition BD 5.26
Air partition EB 60
Fat partition EB 1.8083
Liver partition EB 0.6545
Viscera partition EB 0.6348
Muscle partition EB 0.6533

Table 8-2. Parameter values used in the Kohn and Melnick (1993)
PBPK model (continued)
Parameter Mouse Rat Human
Biochemical parametersd
Liver V cyt1 (nmol/h/mg) 155.4 35.4 70.8
Liver Km cyt1 (mM) 0.002 0.00375 0.00514
Liver V cyt2 (nmol/h/mg) 12
Liver Km cyt2 (mM) 0.0156
Liver V EH (nmol/h/mg) 347.4 148.8 1,110
Liver Km EH (mM) 1.59 0.26 0.58
Liver V GST (nmol/h/mg) 30,000 14,460 2,706
Liver Km GST (mM) 35.3 13.8 10.4
Liver micro prot (mg/L) 11,600 16,800 14,500
Liver cyto prot (mg/L) 82,800 108,000 58,000
Lung V cyt1 (nmol/h/mg) 138.6 9.6 9
Lung Km cyt1 (mM) 0.00501 0.00775 0.002
Lung k hydr (h-1/mg) 0.1116 0.0792 0.1914
Lung V GST (nmol/h/mg) 6,380 2,652
Lung Km GST (mM) 36.5 17.4
Lung k GST (h-1/mg) 0.1536
Lung micro prot (mg/L) 3,000 3,000 3,000
Lung cyto prot (mg/L) 82,800 108,000 58,000
a
Compartment volumes are given as fractions of body weight; compartment blood flow rates are given as
fractions of cardiac output.
b
Human cardiac output at rest: 336 L/h; human ventilation rate at rest: 240 L/h.
c
Lung:air and tissue:blood; assumed same for all species.
d
Data from Csanády et al. (1992).
BD: 1,3-butadiene; EB: 1,2-epoxy-3-butene.

V: Vmax; Km: KM.
cyt1 denotes oxidative metabolism of butadiene to EB; cyt2 denotes oxidative metabolism of EB.
EH: epoxide hydrolase.
GST: glutathione S-transferase.
micro prot: microsomal protein; cyto prot: cytoplasmic protein.
k hydr: apparent first-order rate constant for EB hydrolysis; k gst: apparent first-order rate constant
for glutathione conjugation.

vitro data of Csanády et al. (1992). The lung values were also assumed to apply to the viscera
compartment. Csanády et al. detected DEB formation only in mouse liver preparations.
Therefore, Kohn and Melnick (1993) included this reaction only in the mouse liver compartment
and only as a disappearance route for EB; the distribution of DEB was not further modeled. 1,3-
butadiene and EB were treated as competitive inhibitors of each other in the rate equations for
mouse liver CYP2E1. Finally, although glutathione was treated as saturating for glutathione S-
transferase in the mouse, rat, and human liver, glutathione conjugation with EB in human lung
and viscera was assumed to be first order.
To validate their model, Kohn and Melnick (1993) compared predicted 1,3-butadiene
absorption and blood concentrations for mice and rats with the measurements of Bond et al.
(1986). They also modified the model to include a chamber compartment and compared
predicted EB concentrations in the chamber and maximum metabolic elimination rates with the
Laib et al. (1990) results for mice and rats. Kohn and Melnick claimed that their model
predictions are comparable to the experimental results except for overestimates in the blood 1,3-
butadiene concentrations, which they ascribed to inadequacies in the model or experimental
sources of error in the blood concentration measurements.
To assess the sensitivity of the model to the values of various parameters, relative
sensitivity coefficients for different model variables were estimated by finite differences, as
given by Frank (1978). The physiological parameters to which the model was the most sensitive
were the lung:air partition coefficient and the cardiac output. Because the ventilation rate is
greater than the rate of 1,3-butadiene absorption, the lung:air partition coefficient and the cardiac
output are the major parameters governing 1,3-butadiene uptake. Predicted 1,3-butadiene
concentrations were not very sensitive to variations in the biochemical parameters; however,
monoepoxide levels were somewhat more sensitive to the parameters describing hepatic
glutathione S-transferase and epoxide hydrolase kinetics.
Based on their model simulations, Kohn and Melnick (1993) reported that 1,3-butadiene
uptake and the disposition of EB are controlled to a greater extent by physiological parameters
than by biochemical parameters. The model further suggests that storage in fat is a significant
fraction of retained 1,3-butadiene, especially in rats and humans. Kohn and Melnick also found
that predicted EB tissue concentrations do not correlate with tumor incidences in mice and rats,
and they concluded that other factors are crucial in 1,3-butadiene-induced carcinogenesis. These
other factors may include pharmacokinetic variables that were not part of the model, such as
accumulation of the diepoxide or formation of other metabolites or mechanistic
(pharmacodynamic) phenomena, such as formation of DNA adducts or efficiency of DNA
repair.

The Kohn and Melnick (1993) model appears to have a reasonable basic structure, in
terms of the compartments and metabolic reactions included, given the biochemical parameters
that are currently available. A major strength of their model is that none of the parameter
estimates is adjusted to fit experimental data. Two important drawbacks of the model are the use
of empirically derived partition coefficients and the lumping of various tissues with different
metabolic capabilities (Chapter 3) into a viscera compartment, which is assumed to have the
same metabolic activity as the lung. Partition coefficients for 1,3-butadiene and EB have
recently been measured by Johanson and Filser (1993) and Medinsky et al. (1994), and
experimental values for the 1,3-butadiene partition coefficients are substantially less than the
empirically derived estimates, which suggests that the specific results reported by Kohn and
Melnick may not be relevant. For example, the role of physiological parameters in controlling
1,3-butadiene uptake and the amount of 1,3-butadiene storage in fat may not, in fact, be as great
as the Kohn and Melnick model predicts (Medinsky et al., 1994).
8.2.4. Johanson and Filser (1993)

Johanson and Filser (1993) developed a PBPK model for 1,3-butadiene and EB
disposition in rats and mice. Their model is blood flow-limited and consists of four main
physiological compartmentsClungs and arterial blood, muscle and vessel-rich tissues, fat, and
liverCas well as a chamber compartment and an intrahepatic subcompartment. Metabolism is
assumed to take place exclusively in the liver. The metabolic reactions include oxidation of 1,3-
butadiene to EB; hydrolysis of EB; intrahepatic first-pass hydrolysis of EB; conjugation of EB
with glutathione, which is described by a "ping-pong" mechanism; and the turnover and
depletion of hepatic glutathione.
In contrast with the previous PBPK modeling efforts for 1,3-butadiene, Johanson and
Filser (1993) conducted in vitro studies of rat homogenates to obtain empirical values for the
tissue:air partition coefficients for 1,3-butadiene and EB. All physiological parameters were
taken from Arms and Travis (1988), except the alveolar ventilation rates, which were reduced to
60% of those suggested by Arms and Travis on the basis of generalized observations of uptake
rates of various gases in closed-chamber experiments (Johanson and Filser, 1992). For the
oxidative metabolism of 1,3-butadiene, the model uses the Vmax values from the in vitro studies
of Filser et al. (1992). A KM value was derived by fitting the model to the in vivo data of Lieser
(1983) for the rat and Kreiling (1986b) for the mouse because the model could not reproduce the
results observed in these closed-chamber studies the KM values of either Filser et al. (1992) or
Csanády et al. (1992). Values for the metabolic parameters pertaining to the conjugation of EB
with glutathione and to the hydrolysis of EB were taken from the in vitro data of Kreuzer et al.
(1991). The value of the "intrinsic KM" for the intrahepatic hydrolysis of EB (see below) was set

to 20% of the "apparent KM" value of Kreuzer et al. because the model then fit various in vivo
data. The flow rate between the hepatic and intrahepatic compartments was estimated from the
kinetic parameters. The physiological and biochemical parameter values used by Johanson and
Filser (1993) are summarized in Table 8-3.
In terms of the metabolic reactions involved, the Johanson and Filser (1993) model
differs from the Kohn and Melnick (1993) model in that further oxidation of EB to DEB is not
included, conjugation of EB with glutathione is described by the two-substrate ordered
sequential ping-pong mechanism (reviewed by Mannervik, 1985) rather than by Michaelis-
Menten kinetics, and glutathione turnover and the intrahepatic first-pass hydrolysis of EB are
incorporated. Given the KM values for glutathione conjugation used in the model, the
conjugation of EB becomes rate-limited by glutathione only when glutathione is almost
completely depleted. Cytosolic glutathione turnover is depicted by zero-order production and
first-order elimination. Intrahepatic first-pass hydrolysis of EB is hypothesized to occur, based
on the observations of Filser and Bolt (1984), because of proximity of the monooxygenase to the
epoxide hydrolase in the endoplasmic reticulum. Newly formed EB within this intrahepatic
compartment will be more readily hydrolyzed than EB that must diffuse in from outside the
compartment, as reflected by a lower KM in the intrahepatic compartment.
To attempt to validate the model, Johanson and Filser (1993) compared simulated results
with the data from various in vivo experiments. In addition to the 1,3-butadiene kinetics data
used to fit the KM for 1,3-butadiene oxidation and the EB kinetics data of Filser and Bolt (1984)
for the rat and Kreiling (1987) for the mouse that were used to fit the intrinsic KM for
intrahepatic first-pass hydrolysis, the model apparently reproduces the EB concentrations
appearing in chamber air as a result of 1,3-butadiene exposure in the experiments of Rolzhäuser
(1985) for the rat and Kreiling (1987) for the mouse. However, it is not clear from the text
whether these experimental data were also used to fit the intrinsic KM. The model also
reproduces the glutathione concentrations observed by Deutschmann (1988) in rat and mouse
liver after 1,3-butadiene exposure, and Johanson and Filser claimed that no model parameters
were fitted to these data. Finally, simulated blood concentrations of EB approximate those
observed by Bond et al. (1986) in the mouse but are slightly higher than those observed in the
rat.
No sensitivity analysis for the model parameters was reported.
The results of Johanson and Filser’s (1993) model simulations suggest that the internal
dose of EB, expressed as the concentration of EB or the area under the concentration-time curve

Table 8-3. Parameter values used in the Johanson and Filser (1993) PBPK model
1/28/98
Parameter Mouse Rat
Physiological data
Body weight (g) Standard animal 25 250
Simulations 27.5 157.5-217.5a
Alveolar ventilation (mL/min) Standard animal 15 70.2

Simulations proportional to bw2/3
Cardiac output (mL/min) Standard animal 17 83

Simulations proportional to bw2/3
Blood flows Muscle and VRG 66 66

(% of cardiac output) Fat 9 9
Liver 25 25
Compartment volumes Lung and arterial 1 1

8-10
(% of body weight) Muscle and VRG 75 80

Fat 10 7
Liver 5.5 4
b
1,3-Butadiene Lung and arterial, muscle and VRG, liver 0.25

Fat 7.23
Blood 3.03
1,2-Epoxy-3-butene Lung and arterial, muscle and VRG, liver 0.706
Fat 1.89
Blood 83.4
Table 8-3. Parameter values used in the Johanson and Filser (1993) PBPK model (continued)
1/28/98
Parameter Mouse Rat

Metabolic constants
1,3-Butadiene oxidation Microsomal protein (mg/g liver) 30 30
Vmax (nmol·min-1·mg-1)c 3.22 2.17
KM (µmol/L air)d 5 5
EB hydrolysis Microsomal protein (mg/g liver) 30 30
Vmax (nmol·min-1·mg-1)e 19 17
Apparent KM (mmol/L)e 1.5 0.7
Intrinsic KM (% of apparent KM)d 20% 20%
EB conjugation Cytosolic protein (mg/g liver) 95 95
Vmax/KM of EB (µL·min-1·mg-1)e 15 11
KM toward EB (mmol/L)e 100 100
KM toward glutathione (mmol/L)f 0.1 0.1
Glutathione kinetics Initial steady-state concentration (mmol/L) 8.31g 5.56g
5.5h 4.2h
8-11
Elimination rate constant (h-1)f

0.15 0.15
a
Depending on experiment simulated.
b
Tissue:blood and blood:air; assumed same for all species.
c
From Filser (1992).
d
Obtained by best fit.
e
Kreuzer et al. (1991).
f
Average of literature data.
g
Deutschmann and Laib (1989).
h
Kreiling et al. (1988).
VRG: vessel-rich tissue group.

EB: 1,2-epoxy-3-butene.
bwb = (body weight)b.
in the venous blood, the other compartments, or the whole body, is at most about three times
greater in the mouse than in the rat for a given exposure concentration. The greatest differences
in internal dose of EB between the two species result from 1,3-butadiene exposure
concentrations of above 1,000 ppm, when glutathione depletion occurs in the mouse but not in
the rat after 6 to 9 h of exposure. Once again, the relatively small interspecies differences in
body burden of EB indicated by PBPK modeling cannot explain the striking differences in
cancer response between mice and rats exposed to 1,3-butadiene. Johanson and Filser suggested
that differences in the kinetics of DEB or nonmetabolic factors, such as differences in immune
response or in the expression of oncogenes, may be responsible for the interspecies differences
in cancer response.
A major advancement found in the PBPK model of Johanson and Filser (1993) is the use of
experimentally derived partition coefficients, especially because these values differ substantially
from the theoretically estimated values. A further strength of their analysis is that they
compared the simulation results with data from several different experiments. The Johanson and
Filser model also incorporates hepatic glutathione turnover and depletion as well as intrahepatic
first-pass hydrolysis of EB, although the significance of these refinements is unknown. Some of
the limitations of the model include the exclusion of extrahepatic metabolism and of further
metabolism of EB to DEB. In addition, the values of the KM for 1,3-butadiene oxidation and of
the intrinsic KM for intrahepatic first-pass hydrolysis of EB were obtained by fitting in vivo data.
Finally, no sensitivity analysis was reported, although, for example, it was acknowledged that
wide ranges of glutathione concentrations and turnover rates have been observed. Therefore, it
is unknown how sensitive the model is to changes in these and other parameters. Johanson and
Filser are reportedly working on a corresponding PBPK model for humans, but it has not yet
been published.
8.2.5. Evelo et al. (1993)

Evelo et al. (1993) present a PBPK model for the uptake, distribution, and metabolic
clearance of 1,3-butadiene in mice and rats. Their stated objective was to investigate the relative
importance of liver and lung metabolism at different 1,3-butadiene exposure concentrations.
The Evelo et al. model has six physiological compartments: liver, fat, muscle, a vessel-rich
group, the bronchial area of the lung, and the alveolar area of the lung. A chamber compartment
is also included for validation against the data from closed-chamber experiments. 1,3-Butadiene
metabolism is assigned to both the alveolar and bronchial areas of the lung and to the liver. Gas
exchange occurs in the alveolar area of the lung.
Values for the standard physiological parameters were allometrically scaled from the data
of Travis (1988). Volumes and blood flows for the two separate lung compartments were taken

from Greep and Weis (1977). Tissue:blood and blood:air partition coefficients were
theoretically estimated using the regression analysis method of Fiserova-Bergerova and Diaz
(1986), as was done previously by Hattis and Wasson (1987).
To describe the oxidation of 1,3-butadiene to EB, Evelo et al. (1993) calculated the ratios
of the maximum metabolic activity between the liver and the lung from the in vitro data of
Schmidt and Loesser (1985) for the mouse and for the rat. Then, the total (whole-body)
maximum metabolic activities, the KMs, and "the most probable distribution" of metabolic
activity between the alveolar and bronchial areas of the lung were derived by optimizing the
model against the closed-chamber data of Kreiling et al. (1986b) for the mouse and Bolt et al.
(1984) for the rat. The only options considered for the distribution of the metabolic activity of
the lung were that all the metabolism took place in either one of the two areas, that it was equal
in each area, or that it was distributed relative to the volumes of each area; the best fit was found
using the latter distribution. The values of the physiological and metabolic parameters used in
the Evelo et al. model are summarized in Table 8-4.
The only independent validation of the model was against the whole-body extraction
ratios reported by Dahl et al. (1990). Evelo et al. (1993) calculated extraction ratios of 8.4% for
the mouse and 5.2% for the rat, whereas Dahl et al. found ratios of 12.8% for the mouse and
4.3% for the rat. Evelo et al. also noted that the whole-body Vmax value obtained for the rat by
fitting the model to the data of Bolt et al. (1984) does not fall within the range of values allowed
by experimental error based on the gas-uptake studies of Laib et al. (1992).
Evelo et al. (1993) stated that sensitivity analyses found the model optimization to be
relatively insensitive to variability in the value of KM. No other sensitivity analysis results are
reported.
The model simulations of Evelo et al. (1993) suggest that the relative importance of 1,3-
butadiene metabolism in the mouse lung is greater than the distribution of metabolic activity
would imply, especially at exposure concentrations of less than 200 ppm and for KM values of
less than the "best fit" value. Evelo et al. concluded that there is a strong first-pass effect in the
mouse lung. At higher concentrations, alveolar metabolism is saturated, and liver metabolism
becomes relatively more important. The relative importance of lung metabolism also increases
with decreasing exposure concentration for the rat and human, especially with lower values of
KM; however, unlike for the mouse, the lung metabolism never exceeds the liver metabolism.
Evelo et al. suggested that the higher rate of metabolic activation in the mouse lung could be
responsible for the mouse’s greater sensitivity to developing lung carcinomas and heart
hemangiosarcomas from exposure to 1,3-butadiene.

Table 8-4. Parameter values used in the Evelo et al. (1993) PBPK model
Parameter Mice Rats
Physiological parameters
Body mass (kg) 0.0275 0.215
Cardiac output (mL/min) 24.83 75.93

Alveolar ventilation (mL/min) 24.5 118.7
Blood flows (mL/min):
Liver
Fat 6.14 19.17
Muscle 2.34 6.52
Vessel-rich tissue 3.81 11.13
Bronchial lung area 10.75 33.60
Alveolar lung area 1.79 5.514
23.04 70.42
Volumes (mL):
Liver
Fat 1.65 8.63
Muscle 2.94 14.0
Vessel-rich tissue 19.09 162.7
Bronchial lung area 1.17 9.49
Alveolar lung area 0.2 1.29
0.18 1.63
a
Blood:air 0.894
Fat:blood 32.362
Liver:blood 2.675
Muscle:blood 1.871
Kidney:blood 1.690
Lung:blood 1.272
Brain:blood 2.355
Vessel rich:bloodb 2.02
Metabolic parameters
Vmax,total (µmol·hr-1·kg-1) 465 200

Vmax,liver (µmol·hr-1·kg-1) 318 171
Vmax,bronchial (µmol·hr-1·kg-1) 77 13
Vmax,alveolar (µmol·hr-1·kg-1) 70 16
KM (µM) 8 5
a
Same for all species.
b
Mean value of kidney:blood and brain:blood.

The Evelo et al. (1993) model suffers from a number of serious weaknesses. Several
important parameters are not empirically derived. The partition coefficients are estimated
theoretically, and the whole-body Vmax and KM are optimized. For the rat, this exercise
generated a Vmax value that was inconsistent with other in vivo data. Furthermore, sensitivity
analyses revealed that the optimization was insensitive to variability in the value of KM, so there
is considerable uncertainty in the actual value of this parameter. The results pertaining to the
relative importance of lung metabolism, however, are highly sensitive to the value of KM. The
separation of the lung into alveolar and bronchial areas and the "optimized" distribution of lung
metabolism between the two areas also appear tenuous. Other limitations of the model are that
metabolism is limited to the lung and the liver and that further metabolism of EB is not
incorporated. In addition, the model was not adequately validated, and only limited sensitivity
analyses are described. Finally, results for humans are discussed; however, the parameters used
for the human model are not fully reported.
8.2.6. Medinsky et al. (1994)

The most recent PBPK model published for butadiene is the model of Medinsky et al.
(1994) for 1,3-butadiene and EB uptake and metabolism in mice and rats. The Medinsky et al.
model is a venous equilibration, flow-limited model with six physiological compartmentsCliver,
lung, fat, slowly perfused tissue group, rapidly perfused tissue group, and bloodCand a
compartment representing the air in closed-chamber experiments. The model describes the
oxidative metabolism of 1,3-butadiene in the liver and lung, as well as hydrolysis and
glutathione conjugation of EB in the liver. In the mouse, hepatic oxidation of EB is also
included. In addition to measuring actual partition coefficients, Medinsky et al. conducted
closed-chamber experiments of 1,3-butadiene uptake with both mice and rats to test the
predictions of their model.
Medinsky et al. (1994) measured partition coefficients for 1,3-butadiene and EB
experimentally in vitro for both mouse and rat tissues. They found no significant differences
between the two species, except for the muscle:air partition coefficient for 1,3-butadiene and the
fat:air coefficient for EB (although the ultimate fat:blood coefficient was not significantly
different). Organ and body weights were taken from specific experiments on 1,3-butadiene.
The remaining physiological parameters were based on average literature values, with the
exception of alveolar ventilation rate. Alveolar ventilation rates, conventionally defined as 70%
of measured total ventilation rates, yielded overestimates of 1,3-butadiene uptake at low
concentrations, consistent with observations by Johanson and Filser (1992) for other volatile
organic chemicals. Therefore, "apparent" alveolar ventilation rates were obtained by

optimization to provide rates that yielded the best fit of the model to the EB uptake data. The
optimized rates represented 63% of alveolar ventilation for both rats and mice.
Oxidation of 1,3-butadiene and EB (the latter in mouse liver only) and hydrolysis of EB
were described using Michaelis-Menten kinetics. Glutathione conjugation of EB was assumed to
be first order, based on the large KM value reported by Csanády et al. (1992).
Rate constants for the metabolism of 1,3-butadiene and EB were taken from the in vitro
data of Csanády et al. (1992). Apparent enzyme affinities (KM) measured in vitro were used
directly, whereas maximum metabolic rates (Vmax) were scaled to the whole organs. However,
when the organ microsomal concentrations reported by Csanády et al. are used to scale the
metabolic rates similarly reported by Csanády et al., "[1,3-butadiene] uptake from the closed
chamber is underestimated." Therefore, Medinsky et al. (1994) used literature values that were
two to six times greater for microsomal concentrations in the liver and lung in order to
successfully simulate the chamber study results. The parameter values used in the Medinsky et
al. model are summarized in Table 8-5.
For validation of the model components pertaining to EB uptake and metabolism, model
predictions were compared with the EB uptake data from the closed-chamber experiments of
Filser and Bolt (1984) for rats and Kreiling et al. (1987) for mice, although these were the same
data used to optimize the alveolar ventilation rates. The model predictions were deemed
"adequate," although EB uptake was overestimated at the highest exposure concentration,
especially for the rats (3,000 ppm). Medinsky et al. (1994) then compared model simulations of
1,3-butadiene uptake to their own closed-chamber data for mice and rats exposed to 1,3-
butadiene and to data from the closed-chamber experiments of Bolt et al. (1984) for rats and
Kreiling et al. (1986b) for mice and concluded that the model adequately predicted the in vivo
uptake results. Medinsky et al. also compared model predictions with the 1,3-butadiene
retention data of Bond et al. (1986) and found the results similar for exposure concentrations up
to about 100 ppm. At higher concentrations, the model overestimated butadiene retention
observed in mice. Furthermore, the blood concentrations of EB following 1,3-butadiene
exposure, as reported by Bond et al. were overestimated by the model for both mice (except at
the lowest exposure) and rats by about two- to fourfold, although Medinsky et al. suggested that
the discrepancy might be attributable to EB loss from the blood during sampling.
No comprehensive sensitivity analysis for the model parameters was reported. Medinsky
et al. (1994) did note that use of the microsomal concentrations reported by Csanády et al.
(1992) resulted in underestimation of the 1,3-butadiene uptake from chamber studies. In
addition, they investigated whether the model was sensitive to the different values obtained for
the muscle:air

Table 8-5. Parameter values used in the Medinsky et al. (1993) PBPK model
Parameter Rat Mouse
Physiological parameters:
Alveolar ventilation (L/hr/kg)a 17 41
Cardiac output (L/hr/kg)b 17 41
Body weight (kg)c 0.215-0.475 0.028-0.035
Blood flows (fraction of cardiac output):

Liver 0.25 0.25
Fat 0.09 0.09
Lung 1.0 1.0
Slowly perfused tissues 0.15 0.15
Rapidly perfused tissues 0.51 0.51
Organ volumes (fraction of body weight):

Liver 0.05 0.0624
Fat 0.09 0.10
Lung 0.0053 0.005
Slowly perfused tissues 0.71 0.70
Rapidly perfused tissues 0.0347 0.0226
Partition coefficients for 1,3-butadiene:

Blood:air 1.49 1.34
Liver:blood 0.799 1.01
Lung:blood 0.617 1.10
Muscle:blood 0.987 2.99
Fat:blood 14.9 14.3
Partition coefficients for EB:

Blood:air 50.4 36.6
Liver:blood 1.43 1.15
Lung:blood 1.09 1.54
Muscle:blood 0.393 0.645
Fat:blood 2.74 2.49
Tissue concentrations
Liver microsomal concentration (mg/g liver) 35 35
Lung microsomal concentration (mg/g lung) 20 20
Liver cytosolic concentration (mg/g liver)d 108 82.8

Table 8-5. Parameter values used in the Medinsky et al. (1993) PBPK model
(continued)
Parameter Rat Mouse
Rate constants for oxidative metabolism of

1,3-butadiene d
Liver Vmax (µmol/kg/hr) 62 338
KM (µmol/L) 3.75 2.00
Lung Vmax (µmol/kg/hr)
KM (µmol/L) 1.01 21.6
7.75 5.01
d
Rate constants for EB metabolism in the liver
Oxidation Vmax (µmol/kg/hr) 26
KM (µmol/L) 15.6
Hydrolysis Vmax (µmol/kg/hr) 260 754

KM (µmol/L) 260 1590
glutathione conjugation K (L/kg/hr) 5.66 4.36
a
Obtained by optimization.
b
Ventilation/perfusion = 1.
c
Depending on experiment simulated.
d
From Csanády et al. (1992), with Vmax values scaled to whole organ using above microsomal concentrations.
EB: 1,2-epoxy-3-butene.

partition coefficients for the mouse and rat and determined that the species-specific coefficients
provided the best fits to their 1,3-butadiene uptake results for the two species. Medinsky et al.
also determined that the inclusion of lung metabolism improves the model fit for the mouse,
especially at lower exposure concentrations, but has little affect for the rat.
Based on their model simulations, Medinsky et al. (1994) suggested that lung metabolism
may play an important role in 1,3-butadiene uptake and carcinogenesis. Their model predicts
locally generated concentrations of EB that are 15 times greater in the mouse lung than in the rat
lung, for a 6-h exposure to 10 ppm. Medinsky et al. recommended that more research be done to
characterize 1,3-butadiene metabolism and target cells in the mouse lung and to understand the
pharmacokinetics of DEB in different species. They further claimed that "quantitation of the
concentrations of [1,3-butadiene], [EB], and [DEB] in target and non-target tissues of rats and
mice after exposure to [1,3-butadiene] is essential for validation of existing models before these
models can be applied to predict behavior in humans."
One of the major strengths of the Medinsky et al. (1994) model is that they
experimentally measured partition coefficients and confirmed the results of Johanson and Filser
(1993), suggesting that the empirical values for the partition coefficients for 1,3-butadiene differ
significantly from the theoretical values used in previous models. Medinsky et al. also
conducted closed-chamber experiments to obtain validation data for their model and investigated
the role of lung metabolism in 1,3-butadiene uptake. Some limitations of the model include the
fact that metabolism was restricted to the liver and lung, although other tissues are known to
metabolize 1,3-butadiene as well (Chapter 3). In addition, the alveolar ventilation rates were
determined by fitting experimental closed-chamber data, and there are uncertainties about the
actual values for organ microsomal contents. Finally, only 1,3-butadiene oxidation was
described in the lung, although rate constants for further metabolism of EB are also available
from Csanády et al. (1992).
8.3. SUMMARY
Pharmacokinetic modeling of 1,3-butadiene has not yet elucidated the reasons for the
interspecies differences in carcinogenic response between mice and rats. It appears that either
the PBPK models are not sufficiently sophisticated to adequately model the relevant
pharmacokinetics (e.g., the models may need to incorporate the production and disposition of
DEB) or a pharmacodynamic component(s) (e.g., DNA susceptibility or repair) is required to
accurately correlate dose to response.
Furthermore, uncertainties in the existing PBPK models and data make them unreliable
for use in risk assessment. Serious uncertainties exist pertaining to the model structures,
parameter values, and validation. For example, there are discrepancies among the models and

data as to the importance of extrahepatic and extrapulmonary metabolism, competitive
interaction between 1,3-butadiene and EB for oxidative metabolism, and glutathione depletion,
and none of the models fully describe the kinetics for DEB.
With respect to the parameter values, there are disagreements about the ventilation rate,
which is a key parameter for determining 1,3-butadiene delivery, and about metabolic
parameters. For example, measurements of Vmax and KM for the oxidation of 1,3-butadiene to
EB in mouse, rat, and human liver microsomes by Csanády et al. (1992) and by Duescher and
Elfarra (1994) differ by up to 80-fold, and Seaton et al. (1995) measured reaction rates for the
oxidation of EB to DEB by rat and human liver microsomes that Csanády et al. were unable to
detect (Chapter 3). Use of the in vitro metabolic data of Csanády et al. (1992) in the 1,3-
butadiene PBPK models appears to result in an underprediction of total metabolism. Such
underprediction could result from (1) an inability of the in vitro data to reflect the in vivo
metabolic potency, (2) inaccuracies in the measurement of metabolic reaction rates or
microsomal protein content in the tissues, or (3) a deficiency in the models such that they do not
fully characterize 1,3-butadiene metabolism (e.g., by not including metabolism in other tissues).
This is a critical issue for any PBPK-based extrapolation of carcinogenic risk from rodents to
humans because there are no appropriate human in vivo PBPK data for 1,3-butadiene and thus
interspecies extrapolation must rely on in vitro data or allometric scaling. There is also a paucity
of human in vitro data for extension of the PBPK models to humans. The few measurements
that have been made on a few metabolic parameters show a high amount of variability.
Another area of uncertainty is that of model validation. The existing models have been
subjected to a very limited validation, mostly by comparison of simulation results with chamber
uptake data. Virtually all of the model reports claim that the existing models adequately fit the
validation data, despite important differences among the models. In some cases, this is not
surprising because some of the model parameters have been determined by optimization against
data similar to those being used for validation. In other cases, it suggests that the chamber data
are relatively insensitive to various features of the models and might be of limited use for model
validation. For the PBPK models to be more reliable, they should be validated against tissue
concentration data for various metabolites in various tissues. More recently, these data have
become available (Chapter 3), although they must be interpreted with caution because it appears
that metabolites in some of the tissues are subject to further metabolism during the lag time
between the termination of exposure and the measurement of tissue concentrations. The results
of simulations using the Medinsky et al. (1994) model suggest that the model does not conform
adequately to the tissue concentration data. Any PBPK model for 1,3-butadiene would require
more rigorous validation before it could be considered reliable for use in risk assessment.

8.4. CONCLUSIONS
As discussed above, the existing PBPK models and data cannot explain the interspecies
differences in 1,3-butadiene carcinogenicity. Uncertainties in the model structures and
parameter values also prohibit their use in refining risk assessment dosimetry at this time. Some
areas in which more research is needed include (1) evaluation of the kinetics of DEB in rodents
as well as in humans, (2) investigation of the validity of the in vitro metabolic data for
extrapolating to in vivo exposure, (3) clarification of the values of various physiological
parameters such as the ventilation rate, (4) better characterization of the distribution of values for
the human metabolic rates, and (5) more measurement of tissue concentrations of metabolites for
model validation. It is possible that more information on the specific mechanisms of action is
required to explain interspecies differences in the various target tissues.
In any event, the existing PBPK models and data are inadequate for developing a reliable
alternative to the default methodology of using exposure to the parent compound as a dose
surrogate for extrapolation of the carcinogenic risk from animals to humans. Any attempt to
extrapolate the risk in rodents to humans, given the dramatic and unresolved interspecies
differences between the mouse and rat, would involve far greater uncertainties than basing a risk
assessment on the occupational data of Delzell et al. (Chapter 7). Ideally, a reliable, well-
validated PBPK model with parameter values for humans could also be applied to analyzing
different human exposure scenarios (e.g., extrapolating from occupational to environmental
exposures). However, there are too many uncertainties in the PBPK modeling for that to be
practicable at this time.

9. QUANTITATIVE RISK ASSESSMENT FOR 1,3-BUTADIENE
9.1. EPIDEMIOLOGICALLY BASED CANCER RISK ASSESSMENT

9.1.1. Exposure-Response Modeling
In general, it is preferable to use high-quality epidemiologic data when they are available
over toxicologic data for quantitative risk assessment purposes. In the past, available
epidemiologic data on 1,3-butadiene have been inadequate for quantitative risk assessment, and
previous risk assessments relied primarily on models based on the NTP mouse bioassay studies
(reviewed in Chapter 1).
The recently reported findings by Delzell et al. (1995) from a retrospective cohort
mortality study of synthetic production workers exposed to 1,3-butadiene (reviewed in Chapter
7) present an opportunity to perform a quantitative risk assessment based on human data. The
investigators developed a job exposure matrix (JEM) for 1,3-butadiene, styrene, and benzene
based on industrial hygiene data, which contained estimates of the average daily exposure (in
ppm based on the 8-h TWA) and the number of annual peaks (defined as > 100 ppm for 1,3-
butadiene and 50 ppm for styrene) for each area and job code for each study year. The
investigators were then able to estimate cumulative exposures (ppm*years and peak*years) by
linking the JEM with the study subject’s work histories.
Delzell et al. (1995) investigated the relationship between cumulative exposure to 1,3-
butadiene and leukemia mortality using Poisson regression analysis (Frome and Checkoway,
1985). The models controlled for the potentially confounding effects of age (40-49, 50-59, 60-
69, 70-79, 80+), years since hire (10-19, 20-29, 30+), calendar period (1950-59, 1960-69, 1970-
79, 1980-89, 1990-91), and race (black, other). Plant was considered as a possible confounder
but was dropped from the final models because it did not affect the estimated parameters for 1,3-
butadiene or styrene. Few subjects were exposed to benzene, and benzene did not appear to
confound the relationship between 1,3-butadiene or styrene exposure and leukemia mortality.
Hence, the model results presented in the report did not control for benzene exposure.
Different functional forms of the relationship between the relative rate (RR) and
measures of exposure were evaluated by Delzell et al. (1995) including the following:
X
(1) Multiplicative: RR = e
(2) Power: RR = e [ln(1+X)]
(3) Linear Excess: RR = 1 + X
(4) Polynomial Excess: RR = 1 + 1 Xp + 2 Xq +....

where X represents the 1,3-butadiene or styrene exposure categories using the midpoints of the
intervals, represents the estimated model parameters, and the powers "p" and "q" are fixed real
numbers. Although many polynomial functions (model 4) were considered, only the results
from a square root model were presented because this was considered to provide the best fit.
This model may be represented as:
(5) Square Root: RR = 1 + 1 X½
The Poisson regression analyses revealed a positive exposure-response relationship between

cumulative exposure to 1,3-Butadiene or styrene and leukemia mortality. This relationship was
evident both in models that represented these exposures as categorical variables (see Table 59 in
Delzell et al., 1995) and in models where exposure was represented using continuous variables
as described above. 1,3-Butadiene and styrene exposures among exposed study subjects were
found to be moderately correlated (Spearman’s rank correlation, r=0.53). The relationship
between 1,3-butadiene cumulative exposure and leukemia mortality appeared to be independent
of the styrene exposure and was not appreciably altered by inclusion of styrene cumulative
exposure in the model. On the other hand, the relationship between styrene cumulative exposure
and leukemia mortality was weakened and irregular when 1,3-butadiene cumulative exposure
was controlled for. These findings suggest that 1,3-butadiene cumulative exposure is a more
likely explanation for the leukemia excess observed in this cohort than styrene cumulative
exposure.
Analyses of peak years indicated an association between this variable and leukemia
mortality even after controlling for cumulative exposure, but this relationship was irregular in
the categorical regression analyses. Excluding exposures that occurred within 5 or 10 years of
death (i.e., lagging exposures) only slightly increased the exposure-response relationship for 1,3-
butadiene cumulative exposure; whereas excluding exposures within 20 years of death weakened
and almost eliminated the relationship (i.e., see Table 63 in Delzell et al., 1995).
The results that were obtained by the investigators from fitting the alternative relative
rate models described above are summarized in Table 9-1. These results are from models that
simultaneously evaluated the effects of 1,3-butadiene and styrene exposure. The regression
parameter for 1,3-butadiene cumulative exposure was found to be statistically significantly
greater than 0 (p<0.05) in all of the models evaluated, whereas a nonsignificant and weaker
relationship was observed for styrene.
The power and square root models were found to provide the best fit to the data based on
comparison of the model deviances. However, the differences in deviances between the various

Table 9-1. Results from exposure-response models of continuous cumulative
exposure to 1,3-butadiene and styrene using alternative structural forms
reported by Delzell et al. a
1,3-Butadiene (ppm-years) Styrene (ppm-years)
Structural
model form Model estimate LRT Model Estimate LRT
deviance (S.E.) b p-value c deviance (S.E.) b p-value c
Multiplicative: 486.0 0.0041 0.04 485.9 0.0052 0.34
RR = e X (0.0019) (0.0053)
Linear: 486.0 0.0068 0.04 485.7 0.0079 0.30
RR = 1 + X (0.0050) (0.0088)
Power: 485.6 0.2028 0.03 485.2 0.1494 0.21
[ln(1 + X)]
RR = e (0.0972) (0.1183)
Square root: 485.6 0.1293 0.03 485.4 0.0968 0.23
RR = 1 + 1X1/2 (0.1024) (0.1090)
a
Adapted from Table 67 in Delzell et al. (1995). Results presented are adjusted for age, calendar year, years since
hire, race, and exposure to 1,3-butadiene or styrene.

b
S.E. is the standard error for the exposure parameter estimates.
c
LRT, likelihood ratio test for the exposure effect (1,3-butadiene or styrene).
models are slight. The authors expressed a preference for the square root model as the best
model based on its goodness of fit and its simplicity. This model was refined into a "final
model" by omitting styrene and race because the effect of these variables on the estimated
parameter for 1,3-butadiene exposure was considered to have been minimal. In addition, certain
age, calendar year, and years since hire categories were collapsed for the final model for similar
reasons. The final model is summarized in Table 9-2. The relationship between cumulative 1,3-
butadiene exposure and leukemia mortality was highly statistically significant in this model
(p=0.002).
9.1.2. Prediction of Lifetime Excess Risk of Leukemia

The relative rate models presented in the report by Delzell et al., which are summarized
in Tables 9-1 and 9-2, were used as a basis for predicting the lifetime excess risk of leukemia
mortality for varying levels of continuous environmental exposures to 1,3-butadiene. These
lifetime risk estimates were made using the relative rate estimates and an actuarial program that

Table 9-2. Results from "final" square root exposure-response model of
a
continuous cumulative exposure to 1,3-butadiene reported by Delzell et al.
eta Likelihood ratio test
Variable Estimate S.E. b 2
(d.f.) c p-value
Loglinear terms
Constant -10.02 0.47
Age: 13.2 (2) 0.001
40-69 0
70-79 0.89 0.33
80+ 1.71 0.48
Calendar year: 3.85(1) 0.050
1950-89 0
1990-91 0.72 0.34
Years since hire: 7.64 (1) 0.006
10-19 0
20+ 1.09 0.44
Linear term
(1,3-butadiene ppm- 0.17 0.10 9.41 (1) 0.002
years)0.5
a
This table is an adaptation of Table 68 in Delzell et al. (1995).
b
S.E. is the standard error of the parameter estimate.
c
Chi-square ( 2) and degrees of freedom (d.f.) based on the likelihood ratio statistic.
takes into account the effects of competing causes of death.1 U.S. age-specific mortality rates
for all race and gender groups combined (NCHS, 1993) were used to specify the leukemia and
all-cause background rates in the actuarial program. Exposures to 1,3-butadiene were assumed
to be continuous for the entire lifetime, and the risks were computed up to age 85. The
occupational 1,3-butadiene exposures in the epidemiologic study were converted to continuous
environmental exposures by multiplying the occupational exposure estimates by a factor to
account for differences in the number of days exposed per year (365/240 days) and another
1
This program is an adaptation of the approach that was previously used in BEIR IV.
Health Risks of Radon and Other Internally Deposited Alpha Emitters. National Academy Press,
Washington, DC, 1988, pp. 131-134.

factor to account for differences in the amount of air inhaled per day (20/10 m3). The reported
standard errors for the 1,3-butadiene regression coefficients were used to compute the upper
95% confidence limits for the relative rates based on a normal approximation.
Point estimates and one-sided upper 95% confidence limits for lifetime risk of leukemia
associated with varying levels of environmental exposure to 1,3-butadiene based on the
alternative model forms are illustrated in Figures 9-1 to 9-5. Estimates of risks and exposure
levels corresponding to levels of risk of potential regulatory interest are presented in Tables 9-3
and 9-4. These estimates appear to vary by several orders of magnitude depending on the model
used. For example, at the 1 in a million risk level, the 95% upper confidence intervals for 1,3-
butadiene exposure range from 0.1 ppb (parts per billion) (based on the multiplicative model) to
1 e-6 ppb (based on the final square root model).
Consistent with the proposed EPA cancer guidelines, these results were also used to
estimate the exposure level (ECp; "effective concentration") and 95% lower confidence intervals
(LECp) associated with varying levels of risk (p) ranging from 0.1 to 10%, which are
summarized in Table 9-5. Although the new EPA guidelines emphasize the derivation of
exposure levels associated with a 10% risk level, this does not seem reasonable in this instance.
The 10% level of risk is associated with exposure levels that are higher than most of the
exposures experienced by the workers in this epidemiologic study. Furthermore, based on the
actuarial program described above, a relative rate of 19 would be required for adults over the age
of 20 to increase the lifetime risk of leukemia death by 10%, but the leukemia standardized
mortality ratios (SMRs) reported by Delzell et al. (1995) were considerably lower.2 Hence,
these considerations suggest that using a 10% risk level would be an upward extrapolation in this
case. A 1% or even a lower (e.g., 0.1%) risk level would seem to be a more reasonable choice in
this circumstance. The analogous relative rates for increased risks of 1% or 0.1% are 2.7 and
1.17, respectively, which better correspond with the set of SMRs reported by Delzell et al.
(1995). The exposure levels corresponding to a 1% risk level are illustrated in Figures 9-1 to 9-
5. When a 1% risk level is used, the LEC1 from these analyses ranges from 0.07 to 0.6 ppm
based on the different relative rate models. Using the final model presented by Delzell et al.
(1995) would yield an LEC1 of 0.12 ppm.
2
The maximum reported SMR was 13.33. This SMR was based on two leukemia deaths
among black men from plant #2 with at least 10 years of work (not all of which was salaried) and
at least 20 years of elapsed time since hired. (See Table 29 of Delzell et al., 1995.)

Figure 9-1. Excess risk and 95% upper confidence limit excess risk estimates based on the
multiplicative model reported by Delzell et al., 1995.*
* Multiplicative model: RR= e
Figur
e 9-2. Excess risk and 95% upper confidence limit excess risk estimates based on the
power model reported by Delzell et al., 1995.*
* Power model: RR= e [1v(1+X)]

linear excess relative rate model reported by Delzell et al., 1995.*
* Linear excess model: RR=1 +
final square root model reported by Delzell et al., 1995.*
* Final square root model: RR=1 + x½

square root model reported by Delzell et al., 1995.*
* Square root model: RR=1 + x½

Table 9-3. Maximum likelihood estimates (MLEs) of excess risk with one-sided
95% upper confidence limits (95% UCL) from several models reported by
Delzell et al. (1995) for continuous lifetime exposures to varying concentrations
of 1,3-butadiene
Concentration MLE 95% UCL
Model (ppm) excess risk excess risk
Multiplicative: 1.0E-04 5.2E-07 9.2E-07
X
RR = e 1.0E-03 5.2E-06 9.2E-06
1.0E-02 5.3E-05 9.3E-05
Power: 1.0E-04 2.6E-05 4.6E-05
[ln(1+X)]
RR = e 1.0E-03 2.4E-04 4.4E-04
1.0E-02 1.6E-03 3.1E-03
Linear: 1.0E-04 8.7E-07 1.9E-06
RR = 1 + X 1.0E-03 8.7E-06 1.9E-05
1.0E-02 8.7E-05 1.9E-04
Initial square root: 1.0E-04 1.1E-04 2.6E-04
RR = 1 + 1 X1/2 1.0E-03 3.6E-04 8.4E-04
1.0E-02 1.1E-03 2.6E-03
Final square root: 1.0E-04 1.5E-04 3.0E-04
RR = 1 + 1 X1/2 1.0E-03 4.8E-04 9.4E-04
1.0E-02 1.5E-03 3.0E-03
Ratios are also presented in Table 9-5 that were calculated by dividing the excess risk (p)
by the corresponding LECp for each model. Each ratio is the slope of the line segment
connecting the point (LECp, p) with the origin. Based on the LEC1, these ratios vary by
approximately one order of magnitude from 0.016 to 0.15. If these LEC1-based ratios were used
to calculate the concentration corresponding to a 1 in a million excess lifetime risk by linear
interpolation3, the values would range from 7 to 64 parts per trillion. The final model presented
by Delzell et al. (1995) would yield a corresponding exposure level of 12 parts per trillion.
Table 9-4. MLEs of parts per million continuous exposure concentrations
associated with varying excess risk levels with one-sided 95% lower confidence
3
Linear interpolation between the origin and the point (LECp, p) is also referred to as
“linear extrapolation.”

limits (95% LCL) based on relative rate results of several models reported by
Delzell et al. (1995) and U.S. population rates
Excess MLE 95% LCL
Model risk (ppm) (ppm)
Multiplicative: 1E-6 1.9E-4 1.1E-4
X
RR = e 1E-5 1.9E-3 1.1E-3
1E-4 1.9E-2 1.1E-2
Power: 1E-6 3.9E-6 2.2E-6
[ln(1+X)]
RR = e 1E-5 3.9E-5 2.2E-5
1E-4 4.0E-4 2.2E-4

Linear: 1E-6 1.1E-4 0.52E-4
RR = 1 + X 1E-5 1.1E-3 0.52E-3
1E-4 1.1E-2 0.52E-2
Initial square root: 1E-6 7.6E-9 1.4E-9
RR = 1 + 1 X1/2 1E-5 7.6E-7 1.4E-7
1E-4 7.6E-5 1.4E-5
Final square root: 1E-6 4.4E-9 1.1E-9
RR = 1 + 1 X1/2 1E-5 4.4E-7 1.1E-7
1E-4 4.4E-5 1.1E-5
9.1.3. Sources of Uncertainty

It is apparent from the results presented in Table 9-5 that one major source of uncertainty
is the choice of the model for the prediction of risk. The range of values of the LEC at either of
the 1% and 10% excess risk levels spanned approximately one order of magnitude, whereas the
range for the 0.1% level spanned nearly two orders. In this instance, it seems more reasonable to
utilize the results at the 1% risk level because this corresponds to exposures that are within the
range of this epidemiologic study. However, it is not possible to clearly choose one of the
relative rate models as the best for risk assessment purposes because none of the models has a
biologic basis. Furthermore, all the models summarized in Table 9-1 fit the observed data nearly

Table 9-5. Maximum likelihood (EC p) and 95% lower-bound (LEC p)
estimates of the continuous exposure concentrations associated with varying
levels of excess risk (p)
1,3-Butadiene exposure
levels (ppm)
Percentage Maximum Lower 95%
Structural excess risk likelihood bound
model form (p) (EC p) (LEC p) Ratio a
Multiplicative model: 10 3.3 1.87 5.3 E-2
X
RR = e 1 1.12 0.64 1.6 E-2
0.1 0.18 0.10 1.0 E-2
Power: 10 1000 15 6.7 E-3
[ln(1+X)]
RR = e 1 0.57 0.066 1.5 E-1
0.1 0.0054 0.0025 4.0 E-1
Linear model: 10 12.5 5.65 1.8 E-2
RR = 1 + X 1 1.16 0.525 1.9 E-2
0.1 0.116 0.0525 1.9 E-2
Initial square root: 10 88 16.8 5.9 E-3
1 0.77 0.145 6.9 E-2
0.1 0.0076 0.00144 6.9 E-1
Final square root: 10 51 13.5 7.4 E-3
RR = 1 + 1 X1/2 1 0.45 0.12 8.3 E-2
0.1 0.0044 0.0012 8.3 E-1
a
The ratio is the excess risk (p/100%) divided by the one-sided lower 95% confidence limit on the exposure
estimate (LECp).
as well. Moreover, for a given linear extrapolation, the ratios in Table 9-5 show that the
sensitivity of the result to the choice of excess risk level varies considerably for these models,
with the linear model being least sensitive and the two square root models being most sensitive.
Of the two square root models, however, the final relative rate model could be advantageous to
the other model if the omitted parameters for the effects of race and styrene exposure are
unnecessary.

A major source of uncertainty in this analysis is the potential for misclassification of
exposures in the study by Delzell et al. (1995). This is a frequent limitation of nearly all
epidemiologic studies of this type for quantitative risk assessment purposes. The exposures of
this study were based on modeling a relatively extensive set of data. However, questions have
been raised concerning the accuracy of exposure estimates, particularly for some ill-defined
tasks (letter from Elizabeth Moran, CMA, March 25, 1996). For example, the work histories of
maintenance laborers do not indicate whether they were vessel cleaners (a high-exposure
category) or building cleaners (a low-exposure category). The full impact of this potential for
exposure misclassification is unknown, but preliminary analyses suggest that it may have
dampened and possibly distorted the observed dose-response relationship (letter from Delzell
and Macaluso to Aparna Koppikar, April 2, 1996).
Another concern about the study has been expressed regarding the assignment of peak
exposures in the analysis, which was defined as average exposures equal to or greater than 100
ppm over 15 min. It has been suggested that there were tasks with extremely high peak
exposures (thousands of ppm) over very short time periods (seconds to a few minutes) (letter
from Delzell and Macaluso to Aparna Koppikar, April 2, 1996). The models used in this risk
assessment assume a constant dose-rate effect and do not consider the potential for the effects of
peak exposures. The potential impact of work area assignments and butadiene peaks on leukemia
mortality in this study population is an active area of research among the investigators at the
University of Alabama who conducted the study by Delzell et al. (1995).
9.1.4. Summary and Conclusions

Risk estimates for environmental exposures are derived from an analysis by Delzell et al.
(1995) of an occupational retrospective cohort mortality study of approximately 16,000 workers
in six North American styrene-butadiene rubber manufacturing plants. The analysis of this study
is based on follow-up during 1943-1991, with an average follow-up of 25 years and about 25%
of the cohort deceased. While overall mortality and all cancer mortality were below expected
values based on general population regional rates, the increase in leukemias was statistically
significant (SMR = 1.43, 95% C.I. = 1.04-1.91) for all ever-hourly men (Delzell et al., 1996).
The consistency of this leukemia result with other findings from previous epidemiology studies
with 1,3-butadiene plus other data led to the conclusion that this increase was due to 1,3-
butadiene and to the decision to perform a quantitative risk assessment with this database.
While this cohort had been previously studied (Matanoski et al., 1987, 1988, 1989, 1990,
1994), the Delzell et al. update and analyses are especially noteworthy for their extensive work
on exposure estimation based on detailed reviews of individual job histories and a job exposure
matrix (Delzell et al., 1995; Macaluso et al., 1996). The careful work on exposure allowed

better estimates of risk and dose response. Exposure metrics included cumulative ppm-years and
number of years with peak exposures of at least 100 ppm for at least 15 min. Additional
individual worker exposure information on both styrene and benzene allowed analyses to adjust
for these potential confounding exposures. The Delzell et al. (1995) report includes these
analyses.
The Delzell et al. analysis used Poisson regression analysis with nine categories for
cumulative exposure of 1,3-butadiene and nine categories of exposure for styrene. The analysis
also included, as covariates, adjustments for age, race, calendar year, and years since hire.
Relative rate models run within the Poisson analysis included the (1) multiplicative, (2) power,
(3) linear, and (4) square root models. The parameter representing cumulative 1,3-butadiene
exposure was found to be statistically significant in all the models evaluated, and all models fit
the data adequately in the observable range. The cumulative styrene exposure parameter was
positive for all the models, but not statistically significant. While Delzell et al. selected the
square root model as their final choice because of a slightly better likelihood fit, none of the
models fit the data significantly better or worse than the others.
The quantitative risk analysis presented here uses the results of the Delzell et al. analyses,
which include the styrene exposure variable as a covariate, to extrapolate risk from occupational
work-time exposure to lifetime environmental continuous exposure. This is done by adjusting
the 1,3-butadiene parameter estimates calculated by Delzell et al. to reflect continuous rather
than work-time exposures and by using life table modeling techniques to convert the relative rate
exposure-response relationship to a lifetime additional risk dose-response relationship. These
techniques have been used before by EPA as well as other governmental agencies.
After calculation of the exposure-response relationship, the low-exposure extrapolation is
done in two ways reflecting the different approaches used in EPA’s 1986 Guidelines for
Carcinogen Risk Assessment (U.S. EPA, 1986) and those currently proposed for revision (U.S.
EPA, 1996). For the 1986 Guidelines, the risk estimates are calculated as a potency or slope
factor derived from fitting a linear model (default case) to the observed data and applying the
same model to lower exposure concentrations. For the proposed guidelines revisions, the risk
estimates are obtained by first calculating a "point of departure" within the range of observation
using any of the appropriate models and then extrapolating to 0 by means of a straight line. The
LED10 (i.e., lower confidence limit on a dose associated with 10% extra risk) is proposed in the
guidelines revisions as the standard point of departure; however, the LEC01 and EC01 are used
here because 1% is within the observable range of increased leukemia deaths for the different
1,3-butadiene exposure groups in the Delzell et al. study, because exposure levels are expressed
as exposure concentrations rather than doses, and because the issue of whether to use LEDs or
EDs in the final guidelines has not yet been resolved.

The results of the extrapolations using the four relative rate models are shown in the
quantitative risk analysis and presented in Figures 9-1 to 9-4. They show that although in the
observable risk range of 1%, the MLEs of required continuous exposure (EC01) are close,
varying 2.6-fold from 0.45 to 1.16 ppm, the LEC01 estimates range from 0.066 to 0.64, or about
10-fold. Furthermore, as the risk extrapolation decreases 10-fold to a 0.001 risk level, the ML
exposure estimates for the various models diverge much more rapidly, a 45-fold range from
0.004 ppm to 0.18 ppm. At the 10-5 risk level, the exposure estimates diverge by nearly four
orders of magnitude. Clearly, the final risk estimates based on the 1986 guidelines extrapolation
procedures are highly dependent on the choice of model, but those of the proposed guidelines
revisions, which extrapolate from the LEC01, are less affected.
For the 1986 guidelines approach, the model of choice is the linear default. This choice
is based more on historical precedence and biological plausibility arguments than on statistical
fit or conservatism. In fact, for a 10-6 risk level the linear model is much less protective of
public health, by nearly five orders of magnitude, than is the Delzell et al. square root model
choice. For this approach, the maximum likelihood potency (slope) estimate is:
B = 8.7 × 10-3 (ppm)-1.
For the suggested default approach under the proposed guidelines revisions, the EC01
level is chosen because that is within the observable response range of leukemia deaths. At the
EC01 level, the different models provide dose estimates ranging from 0.45 ppm to 1.16 ppm and
the 95% LCLs on dose ranging from 0.066 to 0.64. Without specific directions for choice from
the proposed guidelines, potency estimates based on each of the models examined by Delzell et
al. are presented in Table 9-6.
The cancer potency estimates using EC01s as the point of departure range from 8.7 ×
-3
10 /ppm (linear model) to 0.022/ppm (final square root model). The square root model was the
model preferred by Delzell et al. based on goodness of fit and simplicity; thus they chose that
model for various refinements, resulting in the final square root model. The cancer potency
estimates based on LEC01s range from 0.016/ppm to 0.15/ppm, with the final square root model
yielding 0.083/ppm while the linear model yields 0.019/ppm. Although the proposed Guidelines
do not offer explicit guidance on choice of model, it may be appropriate in this particular case to
use the final square root model to obtain the point of departure because this model benefits from
the refinements performed by Delzell et al.

Table 9-6. Cancer potency (unit risk) estimates based on linear extrapolation
from the LEC 01 or EC 01 calculated from the models presented by Delzell et al.
Potency estimate Potency estimate
(ppm -1 ) (ppm -1 )
Model EC 01 (ppm) (i.e., 0.01/EC 01 ) LEC 01 (ppm) (i.e., 0.01/LEC 01 )
Multiplicative 1.12 8.9 × 10-3 0.64 0.016
Power 0.57 0.018 0.066 0.15
Linear 1.16 8.7 × 10-3 0.525 0.019
Initial square root 0.77 0.013 0.145 0.069
Final square root 0.45 0.022 0.12 0.083
As the estimates of choice, the MLEs of both the potency and EC01 are chosen. The main
reason for this choice is that these estimates are based on human data from a large, well-
conducted study. Although EPA has historically used upper-limit potency estimates for animal-
to-human extrapolations, these upper limits derive their use more from computational
instabilities of the MLEs in the quantitative risk models used. Human-to-human extrapolations
typically use a simpler linear model form that does not have these instabilities. Furthermore, the
human data inherently engender far less uncertainty in the risk estimates, so one may have more
confidence in the use of MLEs from human data than from animal data.
9.2. CANCER RISK ESTIMATES BASED ON RODENT BIOASSAYS

9.2.1. Rat-Based Estimates
Cancer risk estimates based on the 1981 Hazelton rat inhalation study of 1,3-butadiene
were presented in EPA’s 1985 1,3-butadiene risk assessment (U.S. EPA, 1985). 95% upper-
limit incremental lifetime unit cancer risk estimates for humans were calculated using the
linearized multistage (LMS) model, after estimating the equivalent human dose assuming 1,3-
butadiene retention based on results of a 1985 NTP absorption study (NTP, 1985; see EPA’s
1985 report for further details). The upper limit based on the male rat tumor incidence data for
Leydig cell tumors, pancreatic exocrine tumors, and/or Zymbal gland carcinomas was 4.2 × 10-3
per ppm 1,3-butadiene exposure. The upper limit based on the female rat tumor incidence data
for mammary gland carcinomas, thyroid follicular tumors, and/or Zymbal gland carcinomas was
5.6 × 10-2 per ppm 1,3-butadiene exposure.

These rat-based estimates are not considered the most appropriate estimates of human
risk; they are merely presented for comparison purposes. EPA believes that the mouse is likely
to represent a better rodent model for human cancer risk from 1,3-butadiene (see below) and that
the cancer risk estimates derived from the epidemiologic data are the best available estimates for
human risk.
9.2.2. Mouse-Based Estimates

Cancer risk estimates based on the 1984 NTP mouse inhalation study were presented in
EPA’s 1985 1,3-butadiene risk assessment; however, revisions to these estimates are warranted
because of the new data provided by the 1993 NTP mouse inhalation bioassay, which examined
cancer response from exposure to lower 1,3-butadiene concentrations than those used in the
1984 study (NTP 1984, 1993; see Chapter 6). Groups of male and female B6C3F1 mice were
exposed to 1,3-butadiene concentrations of 0, 6.25, 20, 62.5, 200, or 625 ppm 1,3-butadiene for
6 hours/day, 5 days/week, for up to 104 weeks. Significant increases in tumor incidence were
observed at multiple sites: the hematopoietic system (lymphomas; histiocytic sarcomas [males]),
heart (hemangiosarcomas), lung, forestomach, Harderian gland, liver, preputial gland (males),
ovary (females), and mammary gland (females), when adjusted for intercurrent mortality
(Melnick and Huff, 1993). Significant increases in lung cancer incidence were observed in
female mice at 1,3-butadiene exposure levels down to 6.25 ppm, the lowest level tested.
9.2.2.1. Quantal
When EPA estimates cancer risks for humans from rodent bioassay data, the risk
estimates are generally calculated from the incidence of rodents of the most sensitive species,
strain, and sex bearing tumors at any of the sites displaying treatment-attributable increases. In
the case of 1,3-butadiene, so many sites demonstrated significant tumor increases attributable to
1,3-butadiene that background levels of tumor-bearing animals obfuscate the effects of 1,3-
butadiene when all these tumor sites are combined. Therefore, risk estimates were derived from
the incidence of female (most sensitive sex) mice with malignant lymphomas, heart
hemangiosarcomas, lung tumors (alveolar/bronchiolar adenomas or carcinomas), mammary
gland tumors (carcinomas, adenocanthomas, or malignant mixed tumors), or benign or
malignant ovary granulosa cell tumors (Table 9-7). These sites were considered to be the most
relevant sites with low background tumor incidence. Most of the impact on the low-dose linear
extrapolation is from the lung tumor response, because the lung tumor incidences show the

Table 9-7. Dose-response data for linearized multistage model
Administered exposure
(ppm) Control 6.25 20 62.5 200
Human equivalent
exposure (ppm) 0 1.1 3.6 11 36
Number of mice with
tumorsa 6/50 19/49 26/50 31/50 46/49
Number of mice at riskb
a
Lymphocytic lymphomas, heart hemangiosarcomas, alveolar/bronchiolar adenomas or carcinomas, mammary
gland
tumors (carcinomas, adenocanthonomas, malignant mixed tumors), or benign or malignant ovary granulosa cell
tumors.
b
Female mice surviving to the time of the first significant tumor, which was a lymphocytic lymphoma at day 203.
largest increases at the lowest exposures. The 625 ppm exposure group was not included in the
dose-response analysis because all of the mice were dead by week 65, and the tumor response
was already virtually saturated in the 200 ppm exposure group. Note also that mice that died
before the time of observation of the first tumor were considered to be not at risk and were
excluded from the incidence denominators.
Human equivalent exposures were based on ppm 1,3-butadiene exposure, adjusted for
continuous daily exposure (e.g., 6.25 ppm × 6/24 × 5/7 = 1.12 ppm). No attempt was made to
adjust for internal doses of reactive 1,3-butadiene metabolites because the PBPK data were
inadequate to develop reliable PBPK models (Chapter 8). No adjustments were made for 1,3-
butadiene absorption because there are no adequate human data. Furthermore, there is no reason
to expect nonlinearities in absorption at the lowest exposures (at least < 625 ppm).
A 95% upper-limit incremental lifetime unit cancer risk (extra risk) for humans was
calculated from the incidence data in Table 9-7 using the LMS model. The multistage model has
the form:
P(d) = 1 - exp [-(q0 + q1d + q2d2 + ... + qkdk)]
where P(d) represents the lifetime risk (probability) of cancer at dose d, and parameters qi 0,
for I=0, 1, ..., k. Extra risk over the background tumor rate is defined as
[P(d) - P(0)] / [1 - P(0)].

Point estimates of the dose coefficients (qis), and consequently the extra risk function, at
any dose d, are calculated by maximizing the likelihood function with respect to the tumor
incidence data. The incremental lifetime unit cancer risk for humans (q1*) is defined as the 95%
UCL on the parameter q1, which is the linear dose coefficient, for extra risk. This 95% UCL
represents a plausible upper bound for the true risk. The 95% UCL was calculated using the
computer program GLOBAL86 (Howe and Van Landingham, 1986). Both the model and the
curve-fitting methodology used are described in detail by Anderson et al. (1983).
The tumor incidence data in Table 9-7 generated the following results using the LMS
model (GLOBAL86):
MLEs of dose coefficients:
q0 = 0.2629
q1 = 0.07643
q2 = 0.0
q3 = 0.0
q4 = 0.0
p-value for chi-square goodness of fit > 0.01
q1* = 0.10
Thus, the incremental unit cancer risk estimate (95% UCL) for humans calculated from the
mouse 1993 NTP inhalation bioassay results is 0.10 per ppm for continuous lifetime inhalation
exposure to 1,3-butadiene. The MLE of risk appears to be nearly linear between 1 ppm and 1
ppb and is about 0.075 per ppm 1,3-butadiene exposure.
Under EPA’s proposed new cancer risk assessment guidelines (U.S. EPA, 1996), unit
cancer risk estimates for genotoxic chemicals, such as 1,3-butadiene, would be derived by
straight linear extrapolation to 0 from the LED10 (estimated 95% UCL on the dose corresponding
to a 10% cancer risk). Using the LEC10 generated for the LMS model by GLOBAL86 yields a
unit cancer risk of 0.10/1.0 ppm = 0.10 per ppm, the same as the q1*. Using the EC10 yields
0.10/1.4 7.1 × 10-2 per ppm.
MLE of risk for a dose of 1 ppm = 7.4 × 10-2
MLE of risk for a dose of 1 ppb = 7.6 × 10-5
MLE of dose for a risk of 0.10 (EC10) = 1.4 ppm
95% UCL on dose for a risk of 0.10 (LEC10) = 1.0 ppm
The unit cancer risk estimate (95% UCL) derived above is intended to depict a plausible
upper limit on the risk of developing any 1,3-butadiene-attributable tumor over a full (70-year)
lifetime. However, using the quantal incidence data for total tumor-bearing mice in each
exposure group does not fully characterize the cancer potency reflected by the mouse bioassay

results. First, the methodology does not take into account the fact that many of the mice in the
higher exposure groups had tumors at multiple significant sites. Second, the methodology
ignores the fact that survival was significantly decreased in female mice exposed to 20 ppm or
more 1,3-butadiene as a result of fatal 1,3-butadiene-attributable tumors. Time-to-tumor
analyses conducted for specific tumor sites are presented below and can be used to evaluate the
time component of the cancer risk.
9.2.2.2. Time-to-Tumor
The mouse inhalation bioassay results demonstrate different dose-response relationships
for different tumor sites. To assess the characteristics of the dose-response relationships for
different tumor sites, time-to-tumor analyses were performed to adjust for competing mortality
from cancer at other sites.
Time-to-tumor analyses were conducted from the individual mice data, including the 9-
month and 15-month interim sacrifice data, for sites demonstrating an increased cancer
incidence. Benign and malignant tumors were combined for sites where appropriate. Thus
time-to-tumor analyses were performed for lung alveolar/bronchiolar adenomas or carcinomas;
lymphocytic lymphomas; heart hemangiosarcomas; hepatocellular adenomas or carcinomas;
Harderian gland adenomas or carcinomas; forestomach squamous cell papillomas or carcinomas;
malignant or benign ovary granulosa cell tumors (female); and mammary gland
adenocanthomas, carcinomas, or malignant mixed tumors (female). Preputial gland carcinomas
in male mice were not analyzed because not all the tissues were examined microscopically.
Data from the 625 ppm exposure groups were excluded from analysis because of
excessive early mortality, as in the quantal analysis discussed above. In addition, data from
interim sacrifices for specific sites were excluded for dose groups for which it appeared that
complete histopathological examination for that site was not performed on the entire interim
sacrifice group.
Human equivalent exposures were based on ppm 1,3-butadiene exposure, adjusted for
continuous daily exposure, as described above.
The general model used for the time-to-tumor (or time-to-response) analyses was the
multistage Weibull model, which has the form
P(d,t) 1 - exp[-(q0 + q1d + q2d2 + ... + qkdk)*(t - t0)z]
where P(d,t) represents the probability of a tumor (or other response) by age t (in bioassay
weeks) for dose d (human equivalent exposure), and parameters z 1, t0 0, and qi 0 for i=0, 1, ...,
k, where k = the number of dose groups - 1. The parameter t0 represents the time between when

a potentially fatal tumor becomes observable and when it causes death (see below). The
analyses were conducted using the computer software TOX_RISK version 3.5 (Crump et al.,
ICF Kaiser International, Ruston, LA), which is based on Weibull models taken from Krewski et
al. (1983). Parameters are estimated using the method of maximum likelihood.
Specific n-stage Weibull models were selected for the individual tumor types for each
sex based on the values of the log likelihoods according to the strategy used by NIOSH (1991a).
If twice the difference in log likelihoods was less than a chi-square with degrees of freedom
equal to the difference in the number of stages included in the models being compared, then the
models were considered comparable and the most parsimonious model (i.e., the lowest-stage
model) was selected.
Tumor types were categorized by tumor context as either fatal or incidental tumors.
Incidental tumors are those tumors thought not to have caused the death of an animal, while fatal
tumors are thought to have resulted in animal death. Lymphocytic lymphomas, histiocytic
sarcomas, and heart hemangiosarcomas were treated as fatal tumors, unless observed at an
interim or terminal sacrifice, in which case they were considered incidental. Furthermore, these
fatal tumors were deemed rapidly fatal, and t0 was set equal to 0 (it was felt that there were
insufficient data to reliably estimate t0 in any event). Tumors at all other sites were treated as
incidental. This is basically the same determination as that made by NIOSH (1991a), except the
NIOSH report dealt with preliminary data that did not distinguish histiocytic sarcomas from
lymphomas. NIOSH further cited the work of Portier et al. (1986) analyzing tumor types in
NTP historical controls to lend support to these tumor context assumptions.
Parameter estimates for the time-to-tumor analyses for each tumor type are presented in
Tables 9-8 (based on female mouse data) and 9-9 (male mice). For all tumor types except the
heart hemangiosarcomas (both sexes) and the forestomach (male mice), the one-stage Weibull
was the preferred model. For male mice, the heart hemangiosarcomas and forestomach tumors
were best described by the two-stage model, while for female mouse heart hemangiosarcomas, a
three-stage model was preferred.
Human unit cancer risk (or potency) estimate results (extra risk) are presented in Tables
9-10 (based on female mouse data) and 9-11 (male mice). Mouse lung tumors convey the
greatest amount of extrapolated risk to humans from both the female mouse data (q1* =
0.14/ppm 1,3-butadiene exposure) and the male mouse data (q1* = 0.10/ppm). Note that the unit
risk estimate of 0.14/ppm generated from the female mouse lung tumor data using a time-to-
tumor

Table 9-8. Parameter estimates for multistage Weibull time-to-tumor
model based on female mouse tumor incidence, w/o 625 ppm group
Tissue Q0 Q1 Q2 Q3 Z
Lymphocytic 6.23 × 10-10 1.67 × 10-10 - - 3.92

lymphoma
Heart 0 0 0 2.88 × 10-
17
6.10
hemangiosarcoma
Lung 5.83 × 10-9 3.40 × 10-9 - - 3.69
Mammary 2.47 × 10-6 5.42 × 10-5 - - 1.27
Liver 2.12 × 10-8 2.11 × 10-9 - - 3.58
Forestomach 0 1.29 × 10-9 - - 3.43
Harderian gland 1.50 × 10-5 2.06 × 10-6 - - 2.03
Ovary 7.83 × 10-9 1.48 × 10-8 - - 3.05
Histiocytic sarcoma 3.68 × 10-14 1.23 × 10-14 - - 6.03
Table 9-9. Parameter estimates for multistage Weibull time-to-tumor

model based on male mouse tumor incidence, w/o 625 ppm group
Tissue Q0 Q1 Q2 Z
Lymphocytic 1.84 × 10-8 1.28 × 10-9 - 3.08

lymphoma
Heart 0.0 0.0 1.14 × 10-23 10.0
hemangiosarcoma
Lung 1.38 × 10-7 9.53 × 10-8 - 3.27
Liver 1.40 × 10-4 5.57 × 10-6 - 1.83
Forestomach 9.68 × 10-10 0.0 3.83 × 10-11 3.39
Harderian gland 1.65 × 10-7 7.45 × 10-8 - 2.90
Histiocytic sarcoma 0.0 1.04 × 10-13 - 5.50

Table 9-10. Human unit cancer risk estimates (extra risk, computed for risks of
10 -6 ) based on female mouse tumor incidences, w/o 625 ppm group using multistage
Weibull time-to-tumor model
Q1* MLE EC 10 LEC 10 0.1/LEC 10

Tissue (ppm -1 ) (ppm -1 ) (ppm) (ppm) (ppm -1 )
Lymphocytic lymphoma 0.0239 0.0128 8.08 4.33 0.0231

Heart hemangiosarcoma 4.27 × 10-3 3.99 × 10-6 11.6 9.24 0.0108
Lung 0.1404 0.0980 1.06 0.737 0.1357
Mammary 0.0321 0.0203 5.09 3.23 0.0310
Liver 0.0631 0.0366 2.82 1.64 0.0610
Forestomach 0.0215 0.0112 9.22 4.80 0.0208
Harderian gland 0.0443 0.0258 4.00 2.33 0.0429
Ovary 0.0358 0.0218 4.74 2.89 0.0346
Histiocytic sarcoma 0.1283 3.36 × 10-3 30.8 0.806 0.1241
Table 9-11. Human unit cancer risk estimates (extra risk, computed for risks of
10 -6 ) based on male mouse tumor incidences, w/o 625 ppm group using multistage
Weibull time-to-tumor model
Q1* MLE EC 10 LEC 10 0.1/LEC 10

Tissue (ppm -1 ) (ppm -1 ) (ppm) (ppm) (ppm -1 )
Lymphocytic lymphoma 6.437 × 10-3 2.220 × 10-3 46.6 16.1 6.224 × 10-3
Heart hemangiosarcoma 0.01266 4.040 × 10-3 12.0 7.59 0.01318
Lung 0.1023 0.06998 1.48 1.01 0.09890
Liver 0.04447 0.02720 3.80 2.33 0.04300
Forestomach 4.258 × 10-3 1.660 × 10-5 19.2 13.3 7.517 × 10-3
Harderian gland 0.07402 0.05398 1.92 1.40 0.07157
Histiocytic sarcoma 0.02162 0.01394 7.42 4.78 0.02090

model is greater than the unit risk estimate of 0.10/ppm generated above from multiple female
mouse tumor sites when only the quantal data were used and decreased survival time was not
taken into account.
Although the time-to-tumor modeling does help account for decreased survival times in
the mice, considering the tumor sites individually does not convey the total amount of risk
potentially arising from the sensitivity of multiple sites. To get some indication of the total unit
risk from multiple tumor sites, assuming the multiple sites are mechanistically independent, the
MLEs of the unit potency from the Weibull time-to-tumor models were summed across tumor
sites and estimates of the 95% upper bound on the summed unit potency were calculated. The
TOX_RISK software provides MLEs and 95% UCL’s for human risk at various exposure levels,
allowing for the calculation of unit potency estimates at those exposure levels.
When the MLEs of unit potency calculated at 1 ppb from the female mouse data were
summed across the female mouse tumor sites, the MLE of the total unit risk was 0.23/ppm
continuous lifetime 1,3-butadiene exposure. A 95% upper bound for the total potency was
calculated by assuming a normal distribution for the risk estimates, deriving the variance of the
risk estimate for each tumor site from its 95% UCL according to the formula
95% UCL = MLE + 1.645 ,
where the standard deviation is the square root of the variance, summing the variances across
tumor sites to obtain the variance of the sum of the MLEs, and calculating the 95% UCL on the
sum from the variance of the sum using the same formula. The resulting 95% UCL on the unit
potency for the total unit risk was 0.38/ppm. In comparison, summing the q1*s across the female
mouse tumor sites yielded 0.50/ppm.
The unit potencies were also summed using a Monte Carlo analysis and the software
Crystal Ball version 4.0 (Decisioneering, Denver, CO). Normal distributions were assumed for
the unit potency for each tumor site, with the mean equal to the MLE and as calculated from
the above formula. A distribution around the sum of the MLEs was then generated by
simulating the sum of unit potencies picked from the distributions for each tumor site (according
to probabilities determined by those distributions) 10,000 times. The mean for the sum and the
95th percentile on the distribution were the same as the sum of MLEs and 95% UCL calculated
above, as they should be. However, a sensitivity analysis based on the contribution to variance
revealed that variability associated with the unit potency estimate for the histiocytic sarcomas
was contributing more than 83% of the variance on the sum, and some of the simulated sums
were negative (the distributions for the unit potency estimates were not constrained for the
summation analyses). Excluding the histiocytic sarcomas yielded the same MLE of total risk of

0.23/ppm; however, the 95% UCL decreased to 0.29/ppm. The lung, which then contributes the
most to the sum, contributed about 55% of the variance, followed by the liver with 20%, and no
simulated sums were negative.
The same analyses were performed summing the estimates of unit potency derived from
the male mouse data for the different tumor sites. The resulting MLE for the total unit risk was
0.18/ppm lifetime 1,3-butadiene exposure with a 95% UCL of 0.22/ppm. The lung contributed
about 56% to the variance, followed by the Harderian gland with about 20%. Histiocytic
sarcomas contributed only 3% in this case, and all simulated sums were positive.
Finally, the summation analyses were repeated for unit potency estimates calculated at 1
ppm exposure for comparison with the estimates calculated at 1 ppb. For the female mouse-
based risks (excluding histiocytic sarcomas), the sum of the MLEs was 0.22/ppm (2% lower than
at 1 ppb) and the 95% UCL on the sum was 0.28/ppm (4% lower than at 1 ppb). Thus, the total
unit potency estimates are reasonably linear up to 1 ppm continuous lifetime exposure. Recall
from Table 9-8 that the selected model for the heart hemangiosarcomas in the female mouse was
nonlinear; however, the unit risk estimates based on the heart hemangiosarcomas at these
extrapolated doses are lower than for the other sites and do not affect the total risk summed
across tumor sites. Similarly, the male mouse based- results (both the sum of the MLEs and the
95% UCL on the sum) calculated at 1 ppm were 2% lower than those calculated at 1 ppb. For
the male mice, the selected models for both the heart hemangiosarcomas and the forestomach
tumors were nonlinear (Table 9-9); however, as with the female heart hemangiosarcomas, the
risks from these sites have little impact on the total risk.
The results of these summation analyses are summarized in Table 9-12.
9.2.3. Discussion
Based on the analyses discussed above, the best estimate for an upper bound on human
extra cancer risk from continuous lifetime exposure to 1,3-butadiene derived from animal data is
about 0.3/ppm. This estimate reflects the time-to-tumor response as well as the exposure-
response relationships for the multiple tumor sites (excluding histiocytic sarcomas) in the most
sensitive species and sex (the female mouse). Histiocytic sarcomas were excluded because they
introduced excessive variance into the upper bound while contributing only negligibly to the
MLE of total unit risk.
The greatest source of uncertainty in this estimate is from the interspecies extrapolation
of risk from the mouse to humans. The two rodent species for which bioassay data were

Table 9-12. Unit potency estimates (extra risk) summed across tumor sites
Sum of MLEs 95% UCL on Sum of q 1*s

(ppm -1 ) sum (ppm -1 ) (ppm -1 )
Female mouse tumor sites
(calculated at 1 ppb) 0.23 0.38 0.50
Female sites excluding
histiocytic sarcomas (at 1 ppb) 0.23 0.29 0.37
Female sites excluding
histiocytic sarcomas (at 1 ppm) 0.22 0.28 0.36
Male mouse tumor sites
(at 1 ppb) 0.18 0.22 0.27
Male mouse tumor sites
(at 1 ppm) 0.17 0.21 0.26
availableCthe mouse and the ratCvaried significantly in their carcinogenic responses to 1,3-
butadiene, in terms of both site specificity and degree of response (Chapter 6). The mouse and
rat also exhibit substantial quantitative differences in their metabolism of 1,3-butadiene to
potentially reactive metabolites (Chapter 3). Unfortunately, existing pharmacokinetic models
have been unable to explain the species differences in carcinogenic response (Chapter 8), and it
is likely that there are pharmacodynamic as well as pharmacokinetic differences between the
mouse and rat with respect to their sensitivities to 1,3-butadiene.
The mouse was the more sensitive species to the carcinogenic effects of 1,3-butadiene
exposure and, hence, the more conservative (public health protective) for the extrapolation of
risk to humans. In addition, the mouse appears to be the more relevant species for extrapolation
to humans in terms of site specificity, as 1,3-butadiene induces tumors of the
lymphohematopoietic system in both mice and humans. Melnick and Kohn (1995) further
suggest that the genetic mutations observed in 1,3-butadiene-induced mouse tumors are
analogous to genetic alterations frequently observed in human tumors.
In addition to uncertainties pertaining to the relevance of the rodent models to human
risk, there is uncertainty in quantitatively scaling the animal risks to humans. Ideally, a PBPK
model for the internal dose of the reactive metabolite(s) would decrease some of the quantitative
uncertainty in interspecies extrapolation; however, current PBPK models are inadequate for this
purpose (Chapter 8). In vitro metabolism data for humans suggest that interhuman variability in
the capacity to metabolically activate 1,3-butadiene nearly spans the range between rats and mice
(Chapter 3).

Another major source of uncertainty in the unit potency estimate of 0.3/ppm is the
extrapolation of high-dose risks observed in the mouse bioassay to lower doses that would be of
concern from human environmental exposures. A multistage Weibull time-to-tumor model was
the preferred model because it can take into account the differences in mortality between the
exposure groups in the mouse bioassay; however, it is unknown how well this model is
predicting the low-dose extrapolated risks for 1,3-butadiene.
There are also uncertainties pertaining to the specific assumptions used in conducting
these multistage Weibull time-to-tumor analyses. Some alternative analyses were performed to
consider the sensitivity of the results to some of these assumptions. For example, for each of the
tumor types assumed to be fatal, alternative analyses were conducted in which the modeling
software estimated t0. In each case, the resulting q1*s, EC10s, and LEC10s were identical to those
generated when t0 was set equal to 0 a priori.
In addition, analyses were performed on the lymphocytic lymphoma data including the
625 ppm group, as this was the exposure group most affected by lymphocytic lymphomas and
relatively few animals in this group survived to develop tumors at other sites. From the female
mouse data, the resulting q1* was 0.515/ppm, or roughly twice that calculated when the 625 ppm
group was excluded. From male mice, the q1* was 0.0215/ppm, or roughly 3 times higher than
that obtained when the 625 ppm group was excluded.
NIOSH (1991a) examined the sensitivity of its results for each tumor type to (1) model
selection (i.e., stage of Weibull model) from among models deemed to be comparable, (2) tumor
context assumptions, and (3) exclusion/inclusion of the 625 ppm exposure group, and generally
found only small discrepancies in the results. Moreover, uncertainties in some of the model
assumptions are trivial compared with the major uncertainties introduced by the interspecies and
high-to-low dose extrapolations.
In conclusion, because of the high uncertainty in extrapolating 1,3-butadiene cancer risks
from rodents to humans and the existence of good-quality occupational epidemiology data with
exposure measures, the epidemiology-based risk estimates presented at the beginning of this
chapter are the preferred human risk estimates. The rodent-based estimates are presented
primarily for comparison purposes. Realizing that different quantitative methodologies and
assumptions were used to calculate the various risk estimates, recall that the estimated upper
bound (95% UCL) on human incremental lifetime unit cancer risk from continuous 1,3-
butadiene exposure was 6 × 10-2/ppm based on the female rat tumors, 3 × 10-1/ppm based on the
female mouse tumors, and 2 × 10-2/ppm and 6 × 10-3/ppm based on lymphocytic lymphomas in
female and male mice, respectively (lymphocytic lymphomas being the tumor site that most
closely resembles the lymphohematopoietic cancers observed in male workers exposed to 1,3-
butadiene). The best estimate (MLE) of human incremental lifetime unit cancer risk

extrapolated from the leukemias observed in occupational epidemiology studies was 9 × 10-
3
/ppm.
9.3. REPRODUCTIVE AND DEVELOPMENTAL TOXICITY

9.3.1. Introduction
The reproductive and developmental effects of 1,3-butadiene are among the effects (both
cancer and noncancer) observed at the lowest exposure levels following short-term or chronic
inhalation exposure. Data on reproductive and developmental effects were available from three
types of studies for modeling and calculation of a benchmark concentration (BMC). In the first
type of study, developmental toxicity of 1,3-butadiene was evaluated in studies in mice and rats
that included 10-day exposures via inhalation at 0, 40, 200, and 1,000 ppm on gestation days
(gd) 6-15 for 6 h/day (Hackett et al., 1987a, b). In rats, no effects were detected at any exposure
level for developmental toxicity (200 ppm was the NOAEL for maternal toxicity), while reduced
fetal weights were seen in mice at all exposure levels (Table 9-13). Thus, 40 ppm was
considered a LOAEL for mice.
In the second type of study, male-mediated effects of 1,3-butadiene were evaluated in a
dominant lethal study in which CD-1 mice were exposed to 0, 12.5, or 1,250 ppm for 6 h/day, 5
days/week, 10 weeks (Anderson et al., 1993, 1995). One group of females at each exposure
level was killed on gd 17, while another was allowed to litter. At 12.5 ppm, the frequency of
late deaths and congenital abnormalities on gd 17 were increased, while in litters allowed to
deliver their pups, changes in implantation numbers, postimplantation loss, litter size, and weight
at birth and at weaning were significantly different only at 1,250 ppm. In addition, body weights
of F1 males at all time points and of F1 females at several time points between 8 and 71 weeks of
age were significantly increased above controls at both 12.5 ppm and 1,250 ppm. Based on the
data from animals killed on gd 17, there was no NOAEL for dominant lethal effects in the study
(Table 9-14).
In the third type of study, reproductive effects of 1,3-butadiene were seen in lifetime
studies in mice after chronic inhalation exposure to 6.25, 20, 62.5, 200, and 625 ppm for 6
h/day, 5 days/week (NTP, 1993). The lowest exposure level studied in mice (6.25 ppm) showed
increased ovarian atrophy and was considered a LOAEL (Table 9-15). Minimal data from
studies on rats suggested their lesser sensitivity to chronic exposure than for mice in that effects
on fertility were noted only at high exposure levels (600 ppm and above).

Table 9-13. Prenatal (developmental) toxicity study (Hackett et al., 1987b)
Species/strain: Pregnant CD-1 mice

Exposure time: Gestational day (GD) 6-15
Exposure regimen: 6 h/day
Exposure levels: 0, 40, 200, or 1,000 ppm
Fetal Weight Data

Mean fetal
Exposure level No. litters weight/litter
0 18 1.341
40 ppm 19 1.282
200 ppm 21 1.126
1000 ppm 20 1.038
Table 9-14. Male-mediated developmental toxicity (Anderson et al., 1993, 1995)
Species/strain: CD-1 mice, adult males

Exposure time: 10 weeks
Exposure regimen: 6 h/day, 5 days/week
Exposure levels: 0, 12.5 ppm, 1250 ppm
No. implants % Early and late

Exposure level Number exposed (no. preg. females) deaths % Live implants
0 25 12.09 (23) 4.68 94.6
12.5 ppm 25 12.75 (24) 7.52 92.2
1250 ppm 50 10.68 (38) 22.91 76.8
Mean litter size at Mean litter size at

birth Mean no. implants % Post- weaning
Exposure level (no. litters) (no. litters) implantation loss (no. litters)
0 12.22 (18) 12.81 (16) 4.88 12.17 (18)
12.5 ppm 11.14 (21) 12.35 (17) 9.05 10.95 (20)
1250 ppm 9.06 (33) 10.47 (32) 23.88 9.03 (33)

Table 9-15. NTP chronic study (1993)
1/28/98
Species/strain: Male and female B6C3F1 mice

Exposure regimen: 6 h/day, 5 days/week for 2 years
Exposure levels: 0, 6.25, 20, 62.5, 200, or 625 ppm
Incidence Data - Ovarian Atrophy

Ovarian atrophy-9 mo Ovarian atrophy-15 mo Ovarian atrophy-2 years
Exposure level
No. examined % Affected No. examined % Affected No. examined % Affected
0 10 0 10 0 49 8.16
6.25 ppm -- -- 10 0 49 38.78
20.00 ppm -- -- 10 10 48 66.67
62.50 ppm 10 0 10 90 50 84.00
200.00 ppm 10 90 10 70 50 86.00
625.00 ppm 8 100 2 100 79 87.34
9-29
Ovarian Atrophy - Lesion Distribution

Number (%)
Ovarian atrophy-9 mo Ovarian atrophy-15 mo Ovarian atrophy-2 years
Exposure level
Minimal Mild Moderate Minimal Mild Moderate Minimal Mild Moderate Marked
0.00 0 0 0 0 0 0 1 (2) 2 (4) 1 (2) 0
6.25 ppm -- -- -- 0 0 0 0 15 (31) 4 (8) 0
20.00 ppm -- -- -- 1 (10) 0 0 1 (2) 23 (48) 8 (17) 0
62.50 ppm 0 0 0 1 (10) 7 (70) 1 (10) 3 (6) 18 (36) 21 (42) 0
200.00 ppm 0 0 9 (90) 0 1 (10) 6 (60) 0 9 (18) 34 (18) 0
625.00 ppm 0 0 8 (100) 0 0 2 (100) 0 19 (24) 47 (59) 3 (4)
Table 9-15. NTP chronic study (1993) (continued)
1/28/98
Incidence Data - Uterine and Testicular Atrophy

Uterine atrophy-9 mo Uterine atrophy-15 mo Uterine atrophy-2 years
Exposure level
No. examined No. (%) No. examined No. (%) No. examined No. (%) Affected
Affected Affected
0 10 0 10 0 50 1 (2)
6.25 ppm -- -- 1 0 49 0
20 ppm -- -- 10 0 50 1 (2)
62.5 ppm 10 0 10 0 49 1 (2)
200 ppm 10 3 (30) 10 0 50 8 (16)
625 ppm 8 6 (75) 2 2 (100) 78 41 (53)

9-30
Testicular atrophy-9 mo Testicular atrophy-15 mo Testicular atrophy-2 years

Exposure level
No. examined No. (%) No. examined No. (%) No. examined No. (%) Affected
Affected Affected
0 10 0 10 0 50 1 (2)
6.25 ppm -- -- -- -- 50 3 (6)
20 ppm -- -- 1 0 50 4(8)
62.5 ppm -- -- -- -- 48 2 (4)
200 ppm 10 0 10 0 49 6 (12)
625 ppm 10 6 (60) 7 4 (57) 72 53 (74)

In conclusion, each of these three types of studies indicated the potential for 1,3-
butadiene to affect reproduction and development in mice at low levels of exposure.
9.3.2. Fetal Weight Modeling

Fetal weight data (Table 9-13) were fit using a log-logistic model for developmental
toxicity, as described by Allen et al. (1994a). The TERALOG model software (ICF Kaiser
International, KS Crump Group) was used for this purpose. This model allows for nesting of
fetal data within litters and takes into account intralitter correlations and litter size. To apply this
model, the individual fetal weights were converted to dichotomous data using two different
values as the cutoff for defining an adverse level of response:
(1) a decrease below the 5th percentile of the control distribution, or
(2) a decrease below the 10th percentile.
The model was used to estimate: (a) the EC05* and the LEC05** associated with a 5% additional
risk of obtaining a fetal weight below the 5th percentile of the controls, or (b) the EC10 and
LEC10 associated with a 10% additional risk of obtaining a fetal weight below the 10th percentile
of controls, based on Kavlock et al. (1995). The model can be expressed as:
P(d, s) = + 1 s + [1 - - 1 s]/{1 + exp[ + 2 s- log (d-d0)]}
where P(d, s) is the probability of a low-weight fetus at dose d and litter size s, and the
parameters , , , 1 , and 2 are estimated by methods of maximum likelihood. In order to get
an acceptable fit, an intercept parameter (d0) was included in the model (sometimes referred to as
a threshold parameter, i.e., the point at which the model can no longer distinguish from
background). The parameter constraints were: d0 0; 1; 0 - 1 s 1.
Fetal weight also was modeled as the average of mean fetal weights per litter using the
continuous power model (Allen et al., 1994b). The THWC model software (ICF Kaiser
International, KS Crump Group) was used for this purpose. Several cutoff values were used,
based on Kavlock et al. (1995):
(1) a 5% reduction in mean fetus weight/litter from the control mean,
*
The EC is the effective (exposure) concentration associated with a given level of risk,
5% in this case.
**
The LEC is the lower confidence limit on the effective concentration associated with a
given level of risk. The LEC is also known as the benchmark concentration.

(2) a reduction in mean fetus weight/litter to the 25th percentile of the control
distribution, and
(3) a reduction in mean fetus weight/litter to 0.5 SD below the control mean.
The continuous power model can be expressed as:
m(d) = + d,
where m(d) is the mean of the mean fetus weight/litter for dose d, and , , and are parameters
estimated by maximum likelihood methods. The parameter constraints were: 0; 1.
2
Goodness of fit was determined by a test for the log-logistic model, and by an F test
for the continuous power model (U.S. EPA, 1995, Appendix A). The model was considered to
provide an acceptable fit if the p value was greater than 0.05 and a graphical display of the data
showed a good fit of the model.
A third approach used to model fetal weight data was the hybrid approach proposed by
Gaylor and Slikker (1990) and further developed by Crump (1995). The BENCH_C model
software (ICF Kaiser International, KS Crump Group) was used for this purpose. This approach
uses all of the information contained in the original observations by modeling changes in mean
response as a function of exposure concentration, but defines ECs and LECs in terms of
probability of response. The continuous data are fit using a model that incorporates parameters
from the quantal model. Several models are possible within the software for both continuous
data and quantal risk estimates. For this study, the log-logistic model (not including litter size)
was used for the quantal risk estimates and the following model for the continuous portion of the
hybrid model:
m(d) = m(0) + [N-1(1-P0) - N-1{(1-P0)[-1/[1+( dk)]]}]
where N is the standard normal distribution function, m(d) is the mean response at dose d, is
the standard deviation of the response fixed for all dose groups, and and k are the log-logistic
model parameters estimated by the maximum likelihood method. The parameter constraints
were: k 1; 0.
Crump (1995) indicated that a background rate (P0) of 5% and an EC corresponding to
10% additional risk corresponds to a change from the control mean of 0.61 SD. Since a change
in mean fetal weight/litter of 0.5 SD corresponded on average to a NOAEL in studies by
Kavlock et al. (1995), a P0 of 0.05 and an EC10 (10% additional risk) were used here.

Results of the modeling approaches for fetal weight are shown in Table 9-16 and Figures
9-6 to 9-8. The log-logistic model resulted in an adequate fit of the data. Since the log-logistic
model requires converting continuous data to quantal responses, the continuous power model
was also applied, but did not give an adequate fit with all four exposure levels. When fit to the
first three exposure levels, an adequate fit was obtained. The continuous power model gave
similar ECs and LECs but these were somewhat larger than those obtained with the log-logistic
model except for the one based on a cutoff using the 25th percentile. The hybrid approach
resulted in a quantal estimate of dose at the LEC10 that was lower than that for either the log-
logistic or continuous power model.
All three models have strengths and limitations that must be considered. The log-logistic
model accounts for intralitter correlation and litter size, but requires conversion of continuous
data to quantal responses. Neither the continuous power nor the hybrid model are currently
structured to account for intralitter correlation or litter size. The version of the hybrid model
used here does not allow use of the standard deviation ( ) for individual dose groups, so the at
dose d0 was used for all dose groups. The continuous power and hybrid models take advantage
of the power of modeling the continuous data, but the hybrid model expresses the EC and LEC
as a quantal estimate of risk, allowing direct comparison with ECs and LECs for quantal
endpoints. Given the various advantages and limitations of these models, the hybrid model is
considered the preferred approach for modeling continuous data.
Table 9-16. Fetal weight modeling (LOAEL = 40 ppm)

Model Response Cutoff EC LEC p-Value
Log-logistic (1-4)a Individual fetal 5th percentile EC05 = 46.85 LEC05 = 0.079
weight 27.02
10th percentile EC10 = 49.69 LEC10 = 0.067

38.89
Continuous power Mean fetal 5% relative 65.08 53.51 0.77

(1-3)a weight/litter reduction
25th percentile 45.10 36.66
0.5 SD absolute 50.99 41.44

reduction
Hybrid modela Mean fetal P0 = 0.05 EC10 = 28.19 LEC10 = 0.08

(1-4) weight/litter 13.67
a
Exposure levels modeled in each case are shown in parentheses.

Figure 9-6. Observed versus predicted dose (exposure) probability P(d) of fetal weight
reduction below the 10th percentile of controls using log-logistic model.
Figure 9-7. Observed versus predicted mean fetal weight per litter using continuous
model.

Figure 9-8. Observed versus predicted percent of mean fetal weights per litter less than the
5th percentile of controls (P 0 = 0.05) using hybrid model.
9.3.3. Male-Mediated Developmental Toxicity Modeling

Several endpoints from animals killed at gd 17 and after birth were modeled using a log-
linear model:
y(d) = + x [ln(1 + d)]
This model was used because of the wide spacing of doses and the lack of linearity in the dose-
response relationship. The data were limited in that only two exposure levels in addition to
controls were used, and the exposure levels differed by two orders of magnitude.
Although a statistically significant effect was noted at 12.5 ppm and 1,250 ppm for the
incidence of late deaths in the original paper (Anderson et al., 1993), the response in late deaths
at the higher exposure level was lower than at 12.5 ppm, probably because there were so many
early deaths at the higher level. For the same reason, the incidence of congenital abnormalities
was higher at 12.5 ppm than at 1,250 ppm. When early and late deaths were combined, a
consistently increasing response with increasing exposure level was seen. When combined, the
incidence in the controls was increased from 0 to 13 (4.68% of total implants), while in the 12.5
ppm group the incidence increased from 7 (2.29%) to 23 (7.52%).

Unfortunately, fetal weights were not reported in the prenatal portion of the dominant
lethal study, and only total litter weights (which are confounded by the number of live pups)
were reported in the postnatal portion of the study. When mean pup weight per litter was
calculated, there was no difference among F1 controls and treated offspring, and in some cases, a
slight increase was seen (data not shown). This is interesting in light of the fact that treated F1
male and female weights were increased above controls at 8 through 71 weeks of age. No
modeling of these data was conducted.
Table 9-17 shows the results of modeling the dominant lethal data. The ECs and LECs
for both 5% and 10% responses are shown. The log-linear model gave a good fit for all the data
except for the number of implants in the prenatal study (see Figures 9-9 to 9-15; note that "dose"
refers to 24-h adjusted exposure). This apparently was due to the fact that the number of
implants was somewhat higher in the 12.5 ppm group than in controls or the 1,250 ppm group.
Given that these data are from fetuses or pups within litters, it is likely that an EC05 and LEC05
can be estimated from the data with some degree of reliability. Also, based on the studies of
Allen et al. (1994a and b), the LEC05 (BMC05) for such endpoints was similar to the NOAEL on
average. Although certain endpoints not modeled here (late fetal deaths and congenital
malformations) were statistically increased in both the 12.5 ppm and 1,250 ppm exposure
a
Table 9-17. ECs and LECs for male-mediated developmental toxicity
Prenatal data Postnatal data
Post- Litter
No. Early and Live No. implantation size at Litter size
Estimate implants late deaths implants implants loss birth at weaning
EC05 0.21 3.4 3.5 0.12 3.2 0.1 0.1
LEC05 0.12 2.4 2.4 0.08 2.2 0.07 0.07
EC10 0.47 18 19 0.26 16 0.20 0.20
LEC10 0.26 10 11 0.17 9.0 0.15 0.15

p-Value 0.12 0.66 0.99 0.95 0.99 0.54 0.45
NOAEL 220 2.2 2.2 220 2.2 2.2 2.2
ppm ppm ppm ppm ppm ppm ppm
a
Exposures were adjusted to 24-h daily exposures (e.g., 12.5 6 5 2.2 ppm).
24 7

Figure 9-9. Observed versus predicted mean number of implants (prenatal) using log-linear model.
Figure 9-10. Observed versus predicted proportion of early and late deaths per implantation (prenatal)
using log-linear model .

Figure 9-11. Observed versus predicted proportion of live implants (prenatal) using log-linear model.
Figure 9-12. Observed versus predicted mean number of implants (postnatal) using log-linear model.

Figure 9-13. Observed versus predicted proportion of post-implantation losses (postnatal) using log-linear
model.
Figure 9-14. Observed versus predicted mean litter size at birth using log-linear model.

Figure 9-15. Observed versus predicted mean litter size at weaning using log-linear model.
groups, no other endpoints showed a statistically significant increase at 12.5 ppm by pairwise comparison.
However, there was a trend toward an increase in the incidence of early and late fetal deaths and percent
postimplantation loss, and a decrease in percent live implants and litter size at birth and at weaning in the 12.5
ppm exposure group. Given the overall effect seen on development in this study, the NOAEL for most endpoints
was considered to be much closer to 12.5 ppm than to 1,250 ppm. Since litter size at birth and at weaning showed
the lowest ECs and LECs, these endpoints will be used for calculation of an RfC.
9.3.4. Ovarian, Uterine, and Testicular Atrophy Modeling

The quantal Wiebull model was used initially to model all data. In cases where this model did not
provide a good fit of the data, a log-logistic model was used. The 15-month and chronic ovarian atrophy data
could not be fit adequately using the quantal Weibull model. A log-logistic model similar to that used for fetal
weight (setting 2 S and 2 S to zero) was found to fit the data well. The model was run to determine the
2
probability of additional risk and extra risk. Goodness of fit was determined by a test. The model was
considered to give a good fit if the p value was greater than 0.05 and a graphical display of the data showed a
good fit of the model.
An attempt was made to model various levels of severity in the lesions seen, based on the data shown in
Table 9-15. The data for moderate lesions were fit using the quantal Weibull model (Allen et al., 1994b) for
dichotomous data. This model can be expressed as:

P(d) = 1 - exp[-( + d )],
where P(d) is the probability of response at exposure level d and , , and are parameters that are estimated
from the observed dose-response data. Parameter constraints were 0; 0; > 0. The model was run to
2
determine the probability of additional risk. Goodness of fit was determined by a test. The model was
considered to provide an acceptable fit if the p value was greater than 0.05 and a graphical display of the data
showed a good fit of the model.
Table 9-18 gives the results of fitting the log-logistic model to the 2-year ovarian atrophy data for
exposure groups 1-5 and 1-4. The model gave a poor fit for all six exposure groups, because of leveling off of the
response at exposures above 62.5 ppm (36 ppm adjusted for continuous exposure). The best fit of the model was
for exposure groups 1-4, although the model also fit exposure groups 1-5 well (Figure 9-16; exposures adjusted for
continuous exposure), and the EC10s and LEC10s obtained for groups 1-4 and 1-5 were similar. As expected,
LEC10s were
lowest for ovarian atrophy at 2 years. Moderate ovarian atrophy at 2 years also was modeled using the quantal
Weibull model with exposure groups 1-5 or 1-4. The EC10 and LEC10 were higher than those for all lesions.
Ovarian atrophy data for all six exposure groups at 9 and 15 months were fit using the quantal Weibull or log-
logistic model.
Table 9-18. ECs and LECs for ovarian, uterine, and testicular atrophy using the quantal Weibull
and log-logistic models a
Endpoint Model NOAEL/LOAEL EC 10 LEC 10 p-Value
Ovarian atrophy - 2 years Log-logistic (1-5)b 1.1 ppm (LOAEL) 0.32 0.22 0.11
0.29c 0.21c
Log-logistic (1-4) 0.27 0.18 0.96
0.24c 0.17c
Ovarian atrophy - 2 year Quantal Weibull (1-5) 1.1 ppm 3.02 2.35 0.55
Moderate lesions only
Quantal Weibull (1-4) 2.31 1.67 0.96
Ovarian atrophy - 15 mos Log-logistic (1-6) 1.1 ppm 2.10 0.72 0.66
Ovarian atrophy - 9 mos Quantal Weibull (1-6) 11 ppm 20.04 9.95 0.83
Uterine atrophy Quantal Weibull (1-6) 11 ppm 29.37 18.43 0.66
Testicular atrophy Quantal Weibull (1-6) 36 ppm 40.59 25.64 0.55

a
Exposures were adjusted for continuous exposure (e.g., 6.25 6 5 1.1 ppm)
b
Exposure levels included in the model. 24 7
c
Extra risk. All other values are estimates of additional risk.

Figure 9-16. Ovarian atrophy (groups 1-5) using log-logistic model.
Uterine and testicular atrophy data also were modeled using the quantal Weibull model. The quantal
Weibull model resulted in an acceptable fit of the 2-year uterine atrophy and
testicular atrophy data (Table 9-18 and Figures 9-17 and 9-18; exposures adjusted for continuous exposure).
However, the EC10s and LEC10s were much higher for these endpoints than for 9-month, 15-month or 2-year
ovarian atrophy data. LEC10s were estimated because it has been shown (Allen et al., 1994b) that, for quantal
responses, the LEC10 is near or below the range of detectable responses. Also, the Proposed Guidelines for
Carcinogen Risk Assessment (EPA, 1996) propose use of an LED10 as the default point of departure for low-dose
extrapolation, and use of an LEC10 as a default for noncancer estimation of an RfC would be consistent with this
approach.
Although some 9- and 12-month interim sacrifice data were available for ovarian, uterine, and testicular
atrophy (Table 9-15), these were less than ideal for modeling because smaller numbers of animals were killed and
not all dose groups were represented. In addition, some animals died or became moribund and were killed before
the 2-year death time point. To account for the variability in time of death, time-to-response analyses were done
using the multistage Weibull model as discussed in Section 9.2.2.2. Exposures were adjusted to the equivalent

Figure 9-17. Uterine atrophy (groups 1-6) using quantal Weibull model.
Figure 9-18. Testicular atrophy (groups 1-6) using quantal Weibull model .

continuous lifetime exposures. An EC10 and an LEC10 were calculated in each case. All the reproductive
responses were treated as incidental, not fatal. Parameter estimates for each reproductive endpoint are presented
in Table 9-19.
Results of the Weibull time-to-response model are shown in Table 9-20. The ECs and LECs were similar
to those from other models used for ovarian atrophy, uterine atrophy, and testicular atrophy, with the exception of
those from the modeling of testicular atrophy including the highest exposure group, for which the Weibull time-
to-response model yields results roughly five times lower than the quantal Weibull model. The quantal Weibull
model results for uterine and testicular atrophy were for additional risk, while the Weibull time-to-response results
were for extra risk; however, because of the low background rates of both uterine and testicular atrophy,
additional risk and extra risk should be nearly the same. The results of the time-to-response model are used in the
calculation of an RfC.
The time-to-response model also allows for the calculation of risks at ages less than full lifetime. Thus, if
one is concerned about ovarian or uterine atrophy primarily during a woman’s reproductive years, one can
calculate corresponding EC10s and LEC10s. Assuming reproductive capabilities until 45 years of age yields EC10
1.3 ppm and LEC10 1.1 ppm for ovarian atrophy (625 ppm dose group excluded) and EC10 31 ppm and LEC10
22 ppm for uterine atrophy (625 ppm group included).
Table 9-19. Parameters for Weibull time-to-response model used to model reproductive effects
observed in mice based on ppm butadiene exposure 1
625 ppm
group
Response included Q0 Q1 Q2 Z
Ovarian atrophy no 4.86 × 10-6 7.06 × 10-6 - 2.21
-7 -6
yes 9.01 × 10 1.32 × 10 - 2.58
-5 -5
Uterine atrophy no 6.73 × 10 5.28 × 10 - 1.0
yes 9.08 × 10-5 9.74 × 10-6 1.31 × 10-6 1.0
-4 -5
Testicular no 4.28 × 10 2.24 × 10 - 1.0
atrophy -4 -4
yes 1.60 × 10 1.52 × 10 - 1.0
1
Each response was considered to be incidental with induction time, T0=0. See Section 9.2.2.2 on time-to-tumor
modeling of the mouse carcinogenicity data for a discussion of the Weibull model structure and selection.

Table 9-20. Human benchmark 1,3-butadiene exposure concentrations
calculated for reproductive effects observed in mice using a Weibull
time-to-response model (extra risk)
625 ppm group Based on ppm butadiene exposure
included
Response EC 10 LEC 10
Ovarian no 0.497 0.382
atrophy
yes 0.473 0.369
Uterine no 18.8 12.0
atrophy
yes 24.0 15.6
Testicular no 44.3 15.9
atrophy
yes 6.54 5.39
9.3.5. Summary and Conclusions

ECs and LECs were estimated for three types of exposure scenarios to 1,3-butadiene based on different
endpoints:
1. Short-term exposure (10 days)Cfetal weight reduction
2. Subchronic exposure (10 weeks)Cmale-mediated developmental toxicity
3. Chronic exposureCovarian, uterine and testicular atrophy
These analyses demonstrate approaches for estimation of ECs and LECs based on continuous and quantal data.
Results of the fetal weight analysis illustrate how both continuous and quantal modeling approaches can
be used for continuous data. All of the LECs calculated were below the LOAEL of 40 ppm, except for two LECs
calculated using the continuous power model, which were near this value. Since the hybrid modeling approach is
considered the preferred method for modeling continuous data, the LEC10 of 13.7 ppm from this model will be
used for calculating the reference concentration for developmental toxicity for short-term exposure (RfC DT).
Results of the analysis for male-mediated developmental toxicity following 10 weeks of exposure gave
ECs and LECs much lower than those from the 10-day exposures based on fetal weight. Therefore, the LEC 10 for
the dominant lethal study will be used to calculate an RfC for a subchronic exposure scenario.
Modeling of the 2-year ovarian atrophy data, the effect occurring at the lowest chronic exposure level,
gave a good fit with the log-logistic model, but only when the highest exposure level was dropped. This approach
was justified because the responses leveled off for the top three exposure groups. The LECs derived for a 10%
increase in additional risk or extra risk were 5- to 6-fold below the LOAEL of 6.25 ppm. When the time-to-
response model was applied to account for interim sacrifice data and early mortality, an LEC10 of 0.38 ppm (extra
risk) was calculated, a value similar to that using the log-logistic model.
Ovarian atrophy has been shown to be related to the amount of the diepoxide metabolite in the tissue
(Doerr et al., 1996). Uterine atrophy may be secondary to ovarian atrophy, and thus may also be related to the
amount of diepoxide metabolite formation. Modeling of the ovarian atrophy and uterine atrophy data was
considered based on internal dose of the diepoxide metabolite. However, an adequate pharmacokinetic model was
not available to estimate levels of the diepoxide metabolite (Chapter 8).

RfC calculations will be made for both ovarian atrophy, the reproductive effect occurring at the lowest
chronic exposure level, and testicular atrophy, the reproductive effect observed in male mice following chronic
exposure.
9.4. REFERENCE CONCENTRATIONS FOR REPRODUCTIVE AND DEVELOPMENTAL EFFECTS

9.4.1. Introduction
As discussed in Chapter 5 and Section 9.3, a variety of reproductive and developmental effects have been
observed in mice and rats exposed to 1,3-butadiene by inhalation. (There are no human reproductive or
developmental data available for 1,3-butadiene.) In this section, sample reference concentrations (RfCs) are
calculated for the most sensitive reproductive and developmental endpoints, i.e., those effects exhibiting responses
at the lowest exposure concentrations from various exposure scenarios, using both the traditional NOAEL/LOAEL
approach and the "benchmark dose" approach (Crump, 1984). A reference concentration (or dose) is an estimate
of a daily exposure to humans that is "likely to be without an appreciable risk of deleterious [noncancer] effects
during a lifetime" (Barnes et al., 1988). The final reported RfC will be based on the endpoint resulting in the
lowest calculated RfC level. This RfC will be solely an RfC for reproductive and developmental effects (R/D
RfC) and not a true RfC because other noncancer endpoints were not considered.
9.4.2. Calculation of RfCs

The most sensitive developmental effect was decreased fetal weight in the mouse. The most sensitive
reproductive effects observed in subchronic exposure studies were decreased litter size at birth and at weaning in
dominant lethal studies of mice (i.e., male mice are exposed to 1,3-butadiene and effects on litters are measured
after mating to unexposed females). These effects are highly correlated and both yielded the same modeled
effective dose results (Table 9-17). Litter size at birth reflects both decreased implants and increased fetal deaths,
while litter size at weaning also reflects neonatal deaths. Dominant lethal effects in humans would likely be
manifested as spontaneous abortions, miscarriages, stillbirths, or very early deaths. From chronic exposure studies
(2-year bioassays), the most sensitive reproductive endpoints were ovarian atrophy in female mice and testicular
atrophy in male mice.
Table 9-21 summarizes the EC10 (i.e., the exposure concentration resulting in a 10% increase in risk
based on modeling the exposure-response data in the observable range), the LEC10 (i.e., the 95% lower
confidence limit on the exposure concentration estimated to result in a 10% increase in risk), and the NOAEL
(i.e., no observed adverse effect level) or LOAEL (i.e., lowest observed adverse effect level; reported when no
NOAEL was observed) for these 1,3-butadiene-induced effects. Table 9-21 also provides sample calculations of
RfCs using the NOAEL (or LOAEL) as well as the LEC10 as "points of departure." Uncertainty factors are then
applied to the "point of departure" to calculate the RfC.
Typically, a factor of 10 is used for interspecies uncertainty when the "point of departure" is based on
nonhuman data; however, when ppm equivalence across species is assumed as was done here, a factor of 3 is used
instead. Thus, in Table 9-21, an interspecies uncertainty factor of 3 was used for all endpoints except ovarian
atrophy. For ovarian atrophy, there is convincing evidence that the diepoxide metabolite (1,2:3,4-diepoxybutane,
DEB) is required to elicit the effect and, while the differences cannot be quantified without an adequate
physiologically based pharmacokinetic (PBPK) model, it is expected that humans produce lower concentrations of
DEB than mice, based on differences in metabolic rates. Thus, an uncertainty factor of 1.5 was used for ovarian

atrophy to account for differences between mice and humans in the amount of DEB produced, yet allow that
humans may be more sensitive to DEB.
A large degree of human variability has been observed in metabolic activities that could affect 1,3-
butadiene toxicity. For example, Seaton et al. (1995) measured a 60-fold variation in the initial rate of oxidation
of 1,2-epoxy-3-butene (EB) to DEB in microsomes from 10 different human livers. However, overall variability
in total metabolism and susceptibility is unknown, thus the conventional intraspecies uncertainty factor of 10 for
human variability was used for each endpoint in Table 9-21.
With respect to the acute/subchronic-to-chronic uncertainty factor, none was needed for ovarian or
testicular atrophy because these effects were based on chronic studies. No acute-to-chronic uncertainty factor was
used for fetal weight either, because only exposures during

Table 9-21. Points of departure and RfC calculations for reproductive and developmental effects of 1,3-butadiene
1/28/98
Acute/
subchronic- LOAEL-to-
NOAEL Interspecies Intraspecies to-chronic NOAEL Risk RfC based on
(or LOAEL) EC 10 LEC 10 uncertainty uncertainty uncertainty uncertainty reduction NOAEL RfC based on
Effect (ppm) (ppm) (ppm) factor factor factor factor factor a (ppm) LEC 10 (ppm)
Decreased 28 14 3 10 1b 3 0.14
fetal weight
40 (LOAEL)
(10 d, 6h/d, 3 10 1b 10 0.13
GD 6-15)
Decreased 0.20c 0.15c 3 10 3 3 0.0005

litter size at
birth (or at
weaning)
(dominant
lethal effect)
2.2 (LOAEL)
(10 week, 3 10 3 10 0.002
adjusted to 24
9-48
h/d)
Ovarian 0.50d 0.38d 1.5e 10 1 3 0.008

atrophy
1.1 (LOAEL)
(2 year, 1.5e 10 1 10 0.007
adjusted to 24
h/d)
Testicular 6.5d 5.4d 3 10 1 3 0.05

atrophy
36
(2 year, 3 10 1 1 1.2
adjusted to 24
h/d)
a
To decrease risk to below what would be a detectable level, analogous to the LOAEL-to-NOAEL uncertainty factor.
b
Although from acute study, only exposure during gestation is assumed to be relevant to fetal weight.
cAdjusted to 24-h daily exposure.
d
Adjusted to chronic continuous exposure.
e
There is strong evidence that ovarian atrophy is caused specifically by the metabolite 1,2:3,4-diepoxybutane, and humans are thought to produce less of this metabolite than mice, although their relative
sensitivity to the metabolite is unknown (see text).
gestation are relevant. Although dominant lethal effects appear to occur with exposure during a
specific time period of spermatogenesis (i.e., only certain stages of developing sperm appear
susceptible), chronic exposure might result in continuous induction of these effects, so a factor
of 3 was used.
Under the NOAEL/LOAEL approach, the NOAEL is defined as the exposure level for
which there is no observed adverse effect, although it is circumscribed by the detection limit of
the study. For endpoints for which there is no NOAEL, an uncertainty factor of 10 is typically
used to attempt to extrapolate from the LOAEL to a level at which there are presumed to be no
detectable effects. In the benchmark dose approach, the typical "point of departure" corresponds
to a 10% increased response level, which is explicitly not a no-effect level. In this risk
assessment, a risk reduction factor of 3 was used to extrapolate to a level below which no
detectable effects would be expected, analogous to the LOAEL-to-NOAEL uncertainty factor.
Final guidance on this methodology is still being developed by EPA.
In addition to the sample RfCs presented in Table 9-21 for lifetime 1,3-butadiene
exposure, two RfCs were calculated for subchronic exposure. An RfCDT of 0.14 ppm for
developmental toxicity from short-term exposures was calculated for decreased fetal weight,
using the same factors depicted in Table 9-21. This RfCDT is identical to the sample RfC
calculated for decreased fetal weight because no subchronic-to-chronic uncertainty factor was
used in that calculation. Finally, an RfC for subchronic exposure was calculated for the
decreased litter size endpoints from the subchronic dominant lethal study. Using the LEC10 of
0.15 ppm and uncertainty factors of 3 for interspecies extrapolation, 10 for intraspecies
variability, and 3 for risk reduction (analogous to the LOAEL-to-NOAEL uncertainty factor), as
described above, yields an R/D RfC for subchronic exposure of 0.0015 ppm.
9.4.3. Discussion
The EC10s in Table 9-21 suggest that the dominant lethal (male-mediated) effect is the
most sensitive reproductive/developmental endpoint (i.e., the "critical" endpoint), and thus
should be the basis for the final R/D RfC. The dominant lethal effect also yields the lowest
sample RfC of 0.5 ppb. To arrive at the final R/D RfC, a further uncertainty factor of 3 is used
to account for the lack of comprehensive reproductive testing, especially the absence of a
multigenerational study. This final calculation yields an R/D RfC of 0.15 ppb.
There are substantial uncertainties in estimating low-exposure human risks for
reproductive and developmental effects observed in animals exposed to high concentrations of
an agent. It is generally believed that there is a nonlinear low-dose exposure-response
relationship for noncancer effects, and perhaps a threshold, although this is difficult to
demonstrate empirically. The shape of this low-dose exposure-response relationship is unclear,

however, so RfCs are calculated for noncancer effects rather than exposure-based risk estimates.
The major uncertainties considered in deriving an RfC include the extrapolation of effects
observed in animals to humans (interspecies extrapolation), the potential existence of sensitive
human subpopulations resulting from human (intraspecies) variability, and various deficiencies
in the database. These areas of uncertainty are addressed to some extent by the uncertainty
factors. Other methodological uncertainties arise in the determination of the "point of departure"
and in the selection of the relevant exposure metric for equating animal exposure-response
relationships to humans.
There are a number of limitations in using the NOAEL/LOAEL approach for obtaining a
"point of departure"; these have inspired the development of an alternative "benchmark dose" (or
concentration) methodology. First, the NOAEL/LOAEL approach relies on one exposure level
and ignores the rest of the exposure-response data. Second, the NOAEL/LOAELs depend
explicitly on the specific exposure levels selected for the study. They are also a function of
study power because a LOAEL is the lowest exposure level with a statistically significant
increase in an adverse effect, whereas a NOAEL could represent an increase that failed to attain
statistical significance. Finally, NOAEL/LOAELs are not readily comparable across endpoints
or studies because they can refer to different response levels.
The alternative benchmark concentration approach involves modeling the full exposure-
response curve in the observable range and calculating an effective concentration (EC)
corresponding to some level of response (e.g., 10%) that can be used as a point of comparison
across endpoints and studies (the 10% effect level is typically at the low end of the observable
range, although sometimes a lower level of response can be estimated). The LEC10 is being
considered as the default "point of departure" to take into account statistical variability around
the EC10 estimate. While the benchmark concentration approach alleviates some of the
limitations of the NOAEL/LOAEL approach, there are still uncertainties regarding the
appropriate exposure-response model to use. It is generally expected that models that provide a
good fit to the data in the observable range should yield reasonably similar EC10s, as shown for
quantal models by Allen et al. (1994b).
As shown in Table 9-21, these two approaches yielded nearly identical RfCs for
decreased fetal weight and for ovarian atrophy. For the dominant lethal effect of decreased litter
size, the RfCs were similar, with that from the NOAEL/LOAEL approach four-fold higher than
that from the benchmark concentration approach. For testicular atrophy, on the other hand, the
NOAEL-based RfC is over 20 times higher than the LEC10-based RfC. At least part of this
discrepancy is likely attributable to the fact that the time-to-response modeling conducted to
derive the LEC10 took into account the decreased survival times in the higher exposure groups in
the chronic study. This had the effect of increasing the effective percent affected in the

midrange of the exposure-response curve, which otherwise is fairly flat. This assessment
advances the use of the benchmark dose/concentration approach.
Uncertainties also exist in the choice of exposure metric. Ideally, NOAEL or LOAELs
and LEC10s (or EC10s) should be converted to appropriate human equivalent exposures before
using these exposure levels as "points of departure." Theoretically, this is best accomplished by
using a PBPK model to convert animal exposures to biologically effective doses to the target
organ and then to convert these tissue concentrations back to human exposures to the parent
compound. Unfortunately, the current PBPK data and models are inadequate for use in risk
assessment; therefore, exposure concentrations of 1,3-butadiene are used as the default exposure
metric (this risk assessment assumes equivalence of effects from equivalent ppm exposures
across species). For the lifetime chronic exposure study, demonstrating ovarian and testicular
atrophy, mouse exposure concentrations were adjusted to human equivalent continuous chronic
exposures.
For the subchronic and acute studies, however, the appropriate time frame for exposure
averaging is unclear. Typically, daily exposures resulting in nondevelopmental effects have
been adjusted to an equivalent 24-h exposure, while exposures resulting in developmental effects
have not been adjusted (U.S. EPA IRIS online database, 1997). Consistent with this approach,
1,3-butadiene exposures resulting in dominant lethal effects have been adjusted to a 24-h
exposure, whereas exposure levels from fetal weight studies have not been adjusted. The
exposure concentrations for these subchronic and acute effects have not been adjusted to reflect
total duration of exposure because the critical time frames are unknown. Thus, for example, a
1-day exposure is treated equivalently to a 10-week exposure to the same daily level. Also, for
developmental effects, a 4-h exposure to 50 ppm would be treated equivalently to an 8-h
exposure to 50 ppm.
Finally, there are uncertainties in the uncertainty factors used to derive the RfC from the
"point of departure." These factors are largely arbitrary. In particular, the shape of the
exposure-response curve below the observable range is unknown, and it is uncertain that the
NOAEL or the LOAEL/10 or the LEC10/3 actually represent no-effect levels, independent of the
application of the interspecies and intraspecies uncertainty factors.
9.4.4. Conclusions
In conclusion, an R/D RfC of 0.15 ppb was calculated for the critical endpoint of the
dominant lethal effect of decreased litter size at birth (or at weaning), based on mouse data. This
reference concentration, the uncertainties discussed above notwithstanding, is presumed to
represent a daily exposure to humans that is likely to be without an appreciable risk of
reproductive or developmental effects during a lifetime.

In addition, an RfCDT of 0.14 ppm for developmental toxicity from short-term exposures
was calculated based on fetal weight data for mice, and an R/D RfC for subchronic exposure of
0.0015 ppm was obtained based on the dominant lethal results in mice. Each of these RfCs was
calculated using benchmark concentration methodology.
9.5. CONCLUSIONS ON QUANTITATIVE RISK ESTIMATES

In this chapter, a lifetime extra unit cancer risk (MLE) of 9 × 10-3 per ppm of continuous
1,3-butadiene exposure was calculated based on linear modeling and extrapolation of the excess
leukemia mortality reported in a high-quality occupational epidemiology study. Using this
cancer potency estimate, the chronic exposure level resulting in an increased cancer risk of 10-6
can be estimated as follows: (10-6)/(9 × 10-3/ppm) = 1 × 10-4 ppm = 0.1 ppb. The 95% UCL on
the unit cancer risk was 2 × 10-2 per ppm.
A range of human cancer potency estimates from 4 × 10-3/ppm to 0.29/ppm was also
calculated based on a variety of tumors observed in mice and rats exposed to 1,3-butadiene.
These risk estimates are considered inferior to those based on the epidemiological data, primarily
because of the large uncertainties in extrapolating 1,3-butadiene cancer risks across species in
light of the large unexplained differences in responses of rats and mice.
In addition, benchmark doses and reference concentrations were calculated for an
assortment of reproductive and developmental effects observed in mice exposed to 1,3-
butadiene. An R/D RfC of 0.15 ppb was obtained for the critical effect of decreased litter size at
birth (or at weaning) observed in dominant lethal studies of mice, using a benchmark
concentration approach to obtain the "point of departure." This R/D RfC is presumed to be a
chronic exposure level without "appreciable risk" of reproductive or developmental effects.
Although other noncancer effects were not examined, the reproductive endpoints were quite
sensitive, and it is likely that the R/D RfC is protective against other noncancer effects as well.
Finally, an RfCDT of 0.1 ppm for developmental toxicity from short-term exposures was
calculated from mouse fetal weight data, and an R/D RfC for subchronic exposures of 0.0015
ppm was derived from the dominant lethal results in mice, each using benchmark concentration
methodology.

10. WEIGHT OF EVIDENCE
10.1. EVALUATION
1,3-Butadiene is a colorless, odorless chemical that exists in ambient air in gaseous form.
This extremely volatile chemical is very slightly soluble in water and is not found in soil and food.
Thus, exposure to 1,3-butadiene is mainly via inhalation. Increased mortality from leukemias and
lymphomas was observed among male workers occupationally exposed to 1,3-butadiene in
polymer and monomer production, respectively. No information is available in females. The data
from one Canadian and seven U.S. polymer production plants show that exposure to 1,3-
butadiene is causally associated with occurrence of leukemias (cell type is not known at this time).
Two lifetime inhalation studies in mice and one lifetime inhalation study in rats found
occurrence of malignant tumors in multiple sites in both mice and rats. Increased occurrence of
lymphomas in a 1-year inhalation study in Swiss mice indicated that the presence of retrovirus was
not an essential factor for the development of 1,3-butadiene-induced lymphomas.
Once inhaled, 1,3-butadiene is distributed throughout the body. The relative distribution
of 1,3-butadiene in different organs is unknown at this time. 1,3-butadiene is metabolized by
oxidation to a monoepoxide, diepoxide, and epoxy diol. Which metabolite(s) is responsible for
the causation of cancer is still uncertain. Differences in measured concentration levels of these
metabolites in mice and rats do not provide an explanation for the differences observed in
malignancies in these two species. All three of these metabolites have been shown to be
mutagenic in vivo and in vitro.
10.2. CONCLUSION
Based on the overall evidence from human, animal, and mutagenicity studies, 1,3-
butadiene is concluded to be a known human carcinogen.

11. RISK CHARACTERIZATION
11.1. INTRODUCTION
The U.S. Environmental Protection Agency (EPA) first published a health assessment of
1,3-butadiene in 1985. The 1985 assessment concluded that 1,3-butadiene was a possible human
carcinogen and calculated an upper bound cancer potency estimate of 0.25/ppm based on mouse
data. Since then, a number of new studies on 1,3-butadiene have been completed in various
disciplines such as epidemiology, toxicology, and pharmacokinetics. The purpose of this effort
was to review the new information and determine if any changes were needed to the earlier
conclusions.
This reassessment is intended to serve as a source document for risk assessors inside and
outside the Agency. Its development, however, was prompted primarily by a request from EPA’s
Office of Mobile Sources (OMS) to support decision making regarding the Air Toxic Rule’s
Section 202 (l) (2) of the Clean Air Act Amendment. The scope of the document has been limited
to address only the health effects specifically requested by OMS: carcinogenicity, mutagenicity,
and reproductive/developmental toxicity. Similarly, a detailed exposure assessment was not
requested and not conducted. For background purposes, however, some exposure information
has been included.
The major findings of this report are as follows. First, sufficient evidence exists to
consider 1,3-butadiene a known human carcinogen. The evidence for this includes findings in
epidemiologic studies as well as clear evidence that 1,3-butadiene is an animal carcinogen and is
metabolized into genotoxic metabolites by experimental animals and humans.
Second, based on linear modeling of human data, the best estimate of lifetime extra cancer
risk from continuous 1,3-butadiene exposure is about 9 × 10-3/ppm, or 9 × 10-6/ppb. In other
words, it is estimated that 9 persons in 1 million exposed to 1 ppb 1,3-butadiene continuously for
their lifetimes would develop cancer as a result of their exposure. Lower cumulative exposures
are expected to result in risks that are proportionately lower.
Third, although there are no human data on reproductive or developmental effects, a
variety of such effects have been observed in mice and rats exposed to 1,3-butadiene. A
reproductive/developmental reference concentration (RfC) of 0.05 ppb was calculated based on
the critical reproductive effect of reduced litter sizes, reflecting increased prenatal mortality,
observed among the offspring of male mice exposed to 1,3-butadiene.
Fourth, there are insufficient data to determine if children or other special subpopulations
are differentially affected by exposure to 1,3-butadiene. Heavy smokers are likely to be more
heavily exposed than the general population.

This chapter will briefly summarize and integrate the critical data and analyses on which
these findings are based and discuss the strengths and weaknesses of those data and the resulting
confidence in the findings. With the exception of the section on special subpopulations, all of the
sections in this chapter discuss material presented in the earlier chapters of this assessment.
11.2. EXPOSURE OVERVIEW

Approximately 3 billion pounds of 1,3-butadiene are produced annually in the United
States. 1,3-Butadiene is used primarily in the manufacture of styrene-butadiene rubber, plastics,
and thermoplastic resins. Environmental releases occur from process vents during these
operations. 1,3-Butadiene is not a component of gasoline or diesel fuel, but is formed as a by-
product of incomplete combustion. Mobile sources, including both on-road and nonroad engines,
are estimated to account for 79% of all 1,3-butadiene emissions (EPA, 1992). 1,3-Butadiene
emissions from vehicles are reduced by catalytic converters; total emissions may decline as older
cars without converters are removed from service.
The compound is highly volatile and slightly soluble in water. Thus, environmental
releases result primarily in emissions to the atmosphere. In the atmosphere, 1,3-butadiene
undergoes rapid destruction by photoinitiated reactions, and 50% of it is removed in
approximately 6 hours (U.S. DHHS, 1992). Although it is degraded rapidly in the atmosphere,
1,3-butadiene is almost always present at low concentrations in urban and suburban areas.
Because of this, the general population is exposed to some levels via inhalation. 1,3-Butadiene is
not found in significant amounts in food, soil, water, plants, fish, or sediment. Therefore, the
predominant pathway of exposure is via inhalation.
Monitoring done from 1987 to 1994 by Aerometric Information Retrieval System at more
than 20 different urban and suburban locations detected ambient air levels of 1,3-butadiene
ranging from 0.22 to 1.02 µg/m3 (0.10 to 0.46 ppb). Indoor air levels are likely to be higher than
ambient levels when smoking occurs. 1,3-Butadiene emissions from cigarettes have been
measured to be 200 to 400 µg/cigarette, and levels in smoke-filled bars have been found to range
from 2.7 to 19 µg/m3 (1.2 to 8.4 ppb) (Lofroth et al., 1989; Brunnemann et al., 1990).
11.3. CANCER HAZARD ASSESSMENT

11.3.1. Human Evidence
Sufficient evidence exists to consider 1,3-butadiene a known human carcinogen.
In most situations, epidemiologic data are used to delineate the causality of certain health
effects. Several cancers have been causally associated with exposure to agents for which there is
no direct biological evidence. Insufficient knowledge about the biological bases for diseases in
humans makes it difficult to identify exposure to an agent as causal, particularly for malignant

diseases when the exposure was in the distant past. Consequently, epidemiologists and biologists
have provided a set of criteria supportive of a causal relationship between an exposure and a
health outcome. A causal interpretation is enhanced for studies that meet these criteria. None of
these criteria actually proves causality; actual proof is rarely attainable when dealing with
environmental carcinogens. None of these criteria should be considered either necessary (except
temporality of exposure) or sufficient in itself. The absence of any one or even several of these
criteria does not prevent a causal interpretation. However, if more criteria apply, it provides
credible evidence for causality. The following discussion addresses the strengths and limitations
of the epidemiologic studies of workers occupationally exposed to 1,3-butadiene, from which the
human evidence is derived, and then summarizes how adequately the causality criteria apply.
The conclusion of “sufficient evidence” of human carcinogenicity is based on more than 10
epidemiologic studies examining five different groups of workers. These studies are summarized
in Table 11-1.
The strongest evidence comes from the follow-up study of a cohort of 15,000 synthetic
rubber workers (UAB cohort) conducted by Delzell et al. (1996) and Macaluso et al. (1996) and
reported in two components. The cohort was derived from seven U.S. plants and one Canadian
plant. The follow-up was from 1943 to 1994. Investigators estimated the exposures to 1,3-
butadiene, styrene, and benzene for each worker (Macaluso et al., 1996). Quantitative exposures
were calculated and limited validation of exposure estimates were attempted by various means.
Cumulative and peak exposures were calculated for each worker. Comparison with the U.S.
population resulted in significant excesses for leukemia in ever-hourly workers (43% higher than
general population) and its subcohort of blacks (127%) (Delzell et al., 1996). Significant excesses
were also found in the ever-hourly subcohort for year of death (87% for 1985+), year of hire
(100% for 1950-59), age at death (79% for <55 years), and for more than 10 years employment
and more than 20 years since hire (92% for whites and 336% for blacks). Laboratory workers,
maintenance workers, and polymerization workers also showed higher risks of 331%, 165%, and
151%, respectively. All these analyses were conducted adjusting for styrene and benzene. When
internal comparison was carried out using the estimated ppm-years
exposure data, risk ratios increased with increasing exposures. These findings demonstrate
specificity and strength of association. A fairly consistent association between exposure to
butadiene and occurrence of leukemia across the plants was also found. Furthermore, the trend
test for increasing risk of leukemia with increasing exposure to 1,3-butadiene was statistically
significant (dose response).
The major strengths of this study are as follows. First, the study had detailed and
comprehensive quantitative exposure estimations for 1,3-butadiene, styrene, and benzene for

Table 11-1. Summary of epidemiologic studies
Number of
workers,
Plants dates studied Authors Approach Significant findings
7 U.S. and 1 15,000, Delzell et al., Cohort study Excess mortality due to
Canadian 1943-1994 1996 using leukemia;
polymer Macaluso et al., quantitative leukemia risk increased
production plants 1996 exposure with increasing
(UAB cohort)a estimates for exposure level
each worker
7 U.S. and 1 13,500, Matanoski and Cohort studies Excess mortality due to
Canadian 1943 - 1985 Schwartz, 1987 using qualitative lumpho- hematopoietic
polymer Matanoski et al., exposures; cancers;
production plants 1989, 1990, and case-control leukemia risk increased
(JHU cohort)a 1993 study using with increasing
Santos-Burgoa et estimated exposure level in case-
al., 1992 quantitative control study
exposures for
each case and
control
1 U.S. monomer 2,800, Downs et al., Cohort studies Excess mortality due
production plant 1943-1994 1987 using qualitative to lymphosarcoma
(Texaco cohort) Divine, 1990 exposures, last in prewar workers
Divine et al., 1993 study made
Divine and quantitative
Hartman, 1996 exposure
estimates
3 U.S. monomer 364, Ward et al., 1995 Cohort study Excess mortality due to
production plants 1940-1990 and 1996a using qualitative lymphosarcoma in
(Union Carbide exposures World War II workers
cohort)
1 U.S. monomer 614, Cowles et al., Cohort study No increase in mortality

production plant 1948-1989 1994 using qualitative or morbidity
(Shell Oil Deer exposures
Park cohort)
a
Six U.S. plants and one Canadian plant were common in Johns Hopkins University (JHU) and University of
Alabama, Birmingham (UAB) studies.

each individual. Second, the cohort was large, with a long follow-up period of 49 years. Third,
both external and internal comparison showed similar results. Fourth, adjustments for potential
confounding factors were carried out. Fifth, analyses by duration of employment and for latency
were conducted.
The study had some limitations. First, some misclassification of exposure may have
occurred with respect to certain jobs, but it is unlikely to have occurred only in leukemia cases,
because the exposures were calculated a priori to health effects evaluation. Second, the excess
mortality observed for leukemia was based on death certificates and was not verified by medical
records. This may have resulted in some misclassification of leukemias. Third, histologic typing
of leukemia was also not available. Thus, currently it is not known whether a single cell type or
more than one cell type is associated with the exposure to 1,3-butadiene.
A large cohort of synthetic rubber workers (JHU cohort)1, assembled from one Canadian
and seven U.S. plants, was also studied by Matanoski and Schwartz (1987) and then followed up
by Matanoski et al. (1989, 1990). The follow-up included a nested case-control study (Santos-
Burgoa et al., 1992). Approximately 13,500 individuals were followed from 1943 to 1985. A
significant excess of lymphohematopoietic cancer was observed in the cohort study. The nested
case-control study from this cohort, comprising 59 cases of lymphohematopoietic cancers and 193
matched controls, found significantly increased relative odds for leukemia. Increases of 7 times in
the high-exposure group and of 4 times in the low-exposure group were observed in the
ever/never exposed analysis, of 9 times in the matched analysis, and of 8 times in the conditional
analysis (specificity and strength of association). Exposures to 1,3-butadiene and styrene were
estimated for each case and control using job records and levels of exposures to 1,3-butadiene and
styrene associated with those jobs, independently of the case or control status. A significant trend
of increasing risk of leukemia with increasing exposure level of 1,3-butadiene was also observed
(dose response).
The findings of excess leukemia risk in the nested case-control study were questioned by
Acquavella (1989) and Cole et al. (1993), as these findings were inconsistent with the absence of
excess leukemia risk in the base cohort study. Thus, Matanoski et al. (1993) reevaluated the
original nested case-control study by choosing a new set of three controls per case. The
investigators also verified the cause of death by obtaining the hospital records (25 out of 26 were
correctly recorded on the death certificates). The findings of the new analysis were similar to
those of the earlier analysis. Although the controversy about the cohort and case-control study is
still not resolved, the nested case-control study demonstrates a strong association between
exposure to 1,3-butadiene and occurrence of leukemias.
1
One Canadian plant and six U.S. plants were common in the JHU and UAB studies.

The main strengths of the JHU cohort study are as follows. First, this was the first large
cohort study of polymer production workers. Second, adjustments for confounding exposures
were conducted. Third, analyses by duration of employment and for latency were carried out.
Fourth, the nested case-control study was well conducted and well analyzed, with quantitative
estimation of exposures for each case and control as well as verification of leukemia.
Limitations of the JHU cohort study included the exclusion of more than 50% of the
population because of the lack of work histories, work start date, and exposure data. In addition,
the follow-up for four plants, where the starting date was 1957 to 1970, may not have been long
enough for malignancies to develop. As far as the nested case-control study is concerned, the
estimated exposures were crude and not substantiated by air monitoring data. Exposure
misclassification may have occurred based on the estimated exposures by job if the jobs were
incorrectly identified for higher or lower exposure. However, the panel members were blind
toward the status of cases and controls; thus, the distribution of misclassification should be the
same in cases and controls.
Three different cohorts of monomer production workers were studied. The largest cohort
of approximately 2,800 workers in a Texaco plant followed from 1943 to 1994 by several
investigators (Downs et al., 1987; Divine, 1990; Divine et al., 1993; Divine and Hartman, 1996).
All the investigations essentially found lower than expected mortality from all causes and total
cancers as compared to the general population. The only significant excess mortality observed
was for lymphosarcoma in the prewar subcohort of workers who had worked for less than 10
years and had a latency of 0-9 years; 154% to 169% higher than the general population. Even
though exposures were estimated in the last follow-up, no information about exposure levels was
available for the prewar period; however, it is believed that exposures were high.
The major strengths of this study are, first, it is the largest cohort of monomer workers.
Second, it had a long follow-up period of 52 years. Third, analyses by duration of employment,
and for latency, as well as adjustment for potential confounding factors were conducted. Fourth,
the exposures in each individual were estimated in the last follow-up.
The main limitation was lack of exposure information in the earlier follow-ups.
Furthermore, although the investigators estimated the exposures for each individual in their last
follow-up, no information was available on work histories or levels of 1,3-butadiene exposure
during the prewar period, which made exposure estimation in the prewar workers impossible.
A small cohort of 364 individuals who had potential exposure to 1,3-butadiene at three
Union Carbide plants during World War II was studied by Ward et al. (1995, 1996a). This
investigation also found a statistically significant excess for lymphosarcoma by 477%, which was
based on four cases (specificity and strength of association). The observation of excess
lymphosarcoma was consistent with the finding in the Texaco cohort study. The main limitations

of this study are that the cohort was small and that exposures were assumed based on job
categories. In addition, there was no analysis for latency or adjustments for potential confounding
by exposure to other chemicals.
Cowles et al. (1994) studied the third cohort of 614 workers. This study failed to show
any increased mortality or morbidity. Due to several methodologic limitations such as lack of
exposure information, short follow-up, and lack of information on confounders, this study failed
to provide any negative evidence toward the causal association between exposure to 1,3-
butadiene monomer and occurrence of lymphosarcoma that was observed in the other two
studies.
All the epidemiologic studies, cohort and nested case-control, evaluated for this
assessment are observational studies in occupationally exposed populations. As such, they have
various methodologic strengths and limitations as discussed above. A common limitation to all
the studies is the use of death certificates, which could lead to misclassification bias. Validation of
diagnosis of lymphohematopoietic cancer was not done in any of the studies except in Matanoski
et al. (1993). This is a methodologic concern, given the fact that lymphohematopoietic cancer
recording on death certificates is unreliable (Percy et al., 1981).
Based on these monomer and polymer production workers’ cohorts, it is obvious that an
increased number of lymphohematopoietic cancers is observed in these populations. A clear
difference is becoming apparent, though. Increased lymphosarcomas develop in monomer
workers, whereas excess leukemias occur in polymer workers. Furthermore, the lymphosarcomas
observed in the monomer workers were among wartime workers, who were probably exposed to
higher levels of 1,3-butadiene for shorter periods of time and not in long-term workers with low
levels of exposure. A similar observation comes from the stop-exposure studies conducted by
Melnick et al. (1990c). They observed that for a given total exposure, the incidence of lymphoma
was greater among mice exposed to higher concentrations of butadiene for a shorter period of
time (625 ppm for 26 weeks) than among mice exposed to a lower concentration for a longer
period of time (312 ppm for 52 weeks). Consequently, this suggests that it may be the
concentration of 1,3-butadiene rather than the duration of exposure that is important in the
occurrence of lymphomas. There is a null relationship between exposure to 1,3-butadiene
monomer and occurrence of leukemias, which are observed in polymer workers. This may be due
to the exposure patterns for 1,3-butadiene in monomer production workers or to the absence of
exposure to a necessary co/modifying factor or a confounding factor that occurs in polymer
production workers. Data are currently lacking to confirm or refute any of these possibilities.
The findings of the UAB study, which investigated styrene and benzene exposures as well,
suggest that the observed associations of leukemia with 1,3-butadiene exposure are not due to

confounding by exposure to other chemicals. The findings of excess leukemias in polymer
production workers are consistent with a causal association with exposure to 1,3-butadiene.
Table 11-2 shows the application of the causality criteria to the studies discussed above.
As these criteria are well satisfied, it is concluded that there is sufficient evidence to
consider 1,3-butadiene a known human carcinogen.
11.3.2. Animal Data

1,3-Butadiene is an animal carcinogen.
Chronic bioassay studies provide unequivocal evidence that 1,3-butadiene is a multisite
carcinogen in both rats and mice. These studies also demonstrate that the mouse is more sensitive
than the rat to 1,3-butadiene-induced carcinogenicity and develops tumors at different sites,
although the reasons for these interspecies differences are not understood at this time. The most
sensitive site was the female mouse lung, which exhibited significantly increased tumor incidence
at the lowest exposure concentration tested (6.25 ppm).
Table 11-2. Epidemiologic causality criteria
Criteria Monomer plant workers Polymer plant workers

Temporality: exposure Yes Yes
occurred prior to effect
Specificity of cancer Lymphosarcoma Leukemia (specific cell type[s] not
known at this time)
Strength of association 154% to 477% higher mortality 7 to 9 times higher relative odds
from lymphosarcoma than general for leukemia (nested case-control
population study)a;
151% to 331% higher mortality
from leukemia than general
population
Consistency 2 of 3 studies agree Fairly consistent across the plants
Dose-response relationship Cannot be demonstrated due to Yes
lack of quantitative exposure data
Biological plausibility Yes Yes
a
Relative odds is the ratio of the frequency of exposure to 1,3-butadiene in cases to the frequency of exposure to
1,3-butadiene in controls, where both the cases and controls are from the same occupational cohort.

11.3.3. Other Supportive Data
1,3-Butadiene is metabolized into genotoxic metabolites by experimental animals and
humans.
Metabolic activation is required for 1,3-butadiene carcinogenicity, and there is evidence
that 1,3-butadiene is metabolized to at least three genotoxic metabolites: a monoepoxide (1,2-
epoxy-3-butene, EB), a diepoxide (1,2:3,4-diepoxybutane, DEB), and an epoxydiol (3,4-epoxy-
1,2-butanediol). The enzymes responsible for the metabolic activation of 1,3-butadiene to these
epoxide metabolites exist in humans as well as mice and rats. EB and DEB have been measured
in the blood of rats, mice, and monkeys after 1,3-butadiene exposure, and their production by
human tissues has been observed in vitro. Formation of 3,4-epoxy-1,2-butanediol has been
observed in vitro using tissues from mice, rats, and humans. Activation rates for 1,3-butadiene
are typically higher in the mouse than in the rat, reflected by higher tissue concentrations of EB
and DEB in the mouse versus the rat. Activation rates in humans exhibit a high degree of
variability and appear to span the range between mice and rats.
Among the genotoxic effects of 1,3-butadiene is an N7-alkylguanine adduct that has been
observed in the liver DNA of exposed mice and in the urine of an exposed worker. Similarly,
increased frequencies of hprt mutations have been observed in the lymphocytes of mice and rats
exposed to 1,3-butadiene and in lymphocytes of occupationally exposed workers. Even though
these mutations may not be directly related to tumor development, they provide in vivo evidence
of similarities in the disposition and genotoxic action of 1,3-butadiene between mice and humans.
11.3.4. Cancer Characterization

1,3-Butadiene is a known human carcinogen.
This characterization is supported by the three findings discussed above: (1)
epidemiologic studies showing increased leukemias in workers occupationally exposed to
1,3-butadiene (by inhalation), (2) laboratory studies showing that 1,3-butadiene causes a variety
of tumors in mice and rats by inhalation, and (3) studies demonstrating that 1,3-butadiene is
metabolized into genotoxic metabolites by experimental animals and humans. The specific
mechanisms of 1,3-butadiene-induced carcinogenesis are unknown; however, it is virtually certain
that the carcinogenic effects are mediated by genotoxic metabolites of 1,3-butadiene. Under
EPA’s 1986 Guidelines for Carcinogen Risk Assessment (U.S. EPA, 1986), 1,3-butadiene would
be classified as a “Group A”—Human Carcinogen. It is characterized as a “Known Human
Carcinogen” according to EPA’s 1996 Proposed Guidelines for Carcinogen Risk Assessment
(U.S. EPA, 1996).

11.4. QUANTITATIVE RISK ESTIMATION FOR CANCER
Lifetime extra cancer risk is estimated to be about 9 × 10-3 per ppm continuous
1,3-butadiene exposure, based on human data.
The Delzell et al. (1995) retrospective cohort study of more than 15,000 male
styrene-butadiene rubber production workers provides high-quality epidemiologic data for
estimating the human cancer risk from 1,3-butadiene exposure. In the Delzell et al. study,
1,3-butadiene exposure was estimated for each job and work area for each study year, and these
estimates were linked to workers’ work histories to derive cumulative exposure estimates for each
individual worker. Consistent with EPA’s 1986 Guidelines for Carcinogen Risk Assessment
(U.S. EPA, 1986) and evidence of the genotoxicity of 1,3-butadiene, the linear relative rate
exposure-response model reported by Delzell et al. was used to calculate a maximum likelihood
estimate (MLE) of 8.7 × 10-3/ppm (or 9 × 10-3/ppm, rounded to one significant figure) for lifetime
extra risk of leukemia mortality from continuous environmental 1,3-butadiene exposure. The
corresponding 95% upper limit on unit risk is 0.02/ppm. There were insufficient exposure-
response data to calculate a lymphoma risk estimate from the monomer cohorts.
Alternatively, interpreting the proposed new carcinogen risk assessment guidelines (U.S.
EPA, 1996), linear extrapolation from the LEC01 or the EC01 (i.e., the 95% lower confidence limit
or MLE, respectively, of the exposure concentration associated with a 1% increased risk) is
warranted given the clear genotoxicity of 1,3-butadiene and the fact that a 1% increase in risk is
within the range of the epidemiology data. The models presented by Delzell et al. yield LEC01 and
EC01 values ranging from 0.066 to 0.64 ppm and from 0.45 to 1.16 ppm, respectively. The
corresponding cancer potency estimates range from 0.016/ppm to 0.15/ppm (based on the LEC01)
and from 8.7 × 10-3/ppm to 0.022/ppm (based on the EC01). The square root model provided the
best fit to the data and was chosen by Delzell et al. for further refinements. Thus, their final
square root model might be the appropriate model to select for determination of the ultimate
“point of departure” for linear extrapolation. Based on this model, a cancer potency estimate of
0.08/ppm is obtained from the LEC01 of 0.12 ppm, and a potency estimate of 0.02/ppm is
obtained from the EC01 of 0.45 ppm. These unit risk estimates are roughly two- and fourfold
higher, respectively, than the MLE and upper bound estimates calculated using the linear model
described above.
For comparison purposes, human unit cancer risk estimates based on extrapolation from
the results of lifetime animal inhalation studies are summarized in Table 11-3. These potency
estimates are 95% upper confidence limits on unit cancer risk calculated from incidence data on
all significantly elevated tumor sites using a linearized low-dose extrapolation model. Such
estimates are generally considered by EPA to represent plausible upper bounds on the extra unit
cancer risk to humans. Table 11-3 also includes unit risk estimates based only on the

Table 11-3. Estimates of upper bounds on human extra unit cancer risk
(potency) from continuous lifetime exposure to 1,3-butadiene based on animal
inhalation bioassays
Upper bound on
Species Sex Tumor sites/types potency (ppm -1)
M Leydig cell, pancreatic exocrine cell, Zymbal 4.2 × 10-3
a
Rat gland
F Mammary gland, thyroid follicular cell, Zymbal 5.6 × 10-2
gland
M Lymphocytic lymphomas, histiocytic sarcomas,
b
Mouse heart hemangiosarcomas, lung, forestomach, 0.22
Harderian gland, liver, preputial gland
F Lymphocytic lymphomas, heart
hemangiosarcomas, lung, forestomach, Harderian 0.29
gland, liver, ovary, mammary gland
M Lymphocytic lymphomas 6.4 × 10-3
F Lymphocytic lymphomas 2.4 × 10-2
a
From U.S. EPA’s 1985 assessment; linearized multistage model.
b
Based on 1993 NTP study; Weibull multistage time-to-tumor model.
lymphocytic lymphomas in mice, because this was the tumor type in rodents most analogous to
the lymphohematopoietic cancers observed in workers exposed to 1,3-butadiene.
In both rodent species, females are apparently more sensitive than males, as evidenced by
the higher risk estimates. The “best estimate” (i.e., MLE from the linear model) of 8.7 × 10-3/ppm
for extra cancer risk from the human (male) leukemia data exceeds the upper bound estimates
based on the male rat data and on the male mouse data for lymphocytic lymphomas, and is 25
times lower than the upper bound estimate based on all male mouse tumors.
Human health risk estimates based on extrapolation from high-quality epidemiologic
results are preferable to those based on rodent data because they avoid the uncertainties inherent
in extrapolating across species and, typically, the human exposures in epidemiologic studies are
closer to anticipated environmental exposures than the high exposures used in animal studies, thus
reducing the extent of low-dose extrapolation. In the case of 1,3-butadiene, while the rat
exposures were far in excess of human exposures, the lowest EXPOSURE in the 1993 NTP
mouse study (4.7 ppm, 8 h TWA) is within the range of occupational exposures (0.7-1.7 ppm
median and 39-64 ppm max 8 h TWAs for work-area groups). However, interspecies differences

in tumor sites and susceptibilities between rats and mice are especially pronounced, and the
biological bases for these differences are unresolved. A review of available pharmacokinetic data
and models revealed that the state of the science is currently inadequate for either explaining
interspecies differences or improving on default dosimetry assumptions. Therefore, the
quantitative extrapolation of rodent risks to humans is highly uncertain for 1,3-butadiene.
Even though high-quality human data were used for the quantitative cancer risk estimation
for 1,3-butadiene, there are inevitable uncertainties in the calculated risk estimate. First, there are
uncertainties inherent in the epidemiologic study itself. In particular, there are uncertainties in
the retrospective estimation of 1,3-butadiene exposures, which could have resulted in exposure
misclassification. Nondifferential exposure misclassification would tend to bias estimates of effect
toward the null, resulting in an underestimate of risk. Differential misclassification could bias
results in either direction.
Second, there are uncertainties regarding the appropriate dose metric for dose-response
analysis. Although the dose surrogate of cumulative exposure (i.e., ppm × years) yielded highly
statistically significant exposure-response relationships, cumulative exposure is strongly correlated
with other possible exposure measures, and there may be a dose-rate effect (e.g., risk at high
exposures may be more than proportionately greater than at lower exposures) obscured in the
analysis, or operative at exposures below the observable range but relevant to low-dose
extrapolation.
Third, there are uncertainties pertaining to the model for low-dose extrapolation.
Although Delzell et al. expressed preference for the square root model based on its goodness of
fit, the four exposure-response models that they investigated were virtually indistinguishable on
statistical grounds, and because the specific mechanisms of 1,3-butadiene carcinogenesis are
unknown, there is no biological basis for choosing one model over another. Even though the
models give similar results in the observable range, they deviate substantially at lower exposures.
For example, at a lifetime continuous exposure of 1 ppb, the preferred model of Delzell et al.
yields a cancer potency estimate almost two orders of magnitude higher than that obtained by the
linear model. However, there was no apparent biological reason to depart from a default
assumption of linearity, so the linear model was used in this risk assessment.
Fourth, it is uncertain which potential modifying or confounding factors should be
included in the model. The linear model of Delzell et al., which was used in this risk assessment,
adjusted for age, calendar year, years since hire, race, and exposure to styrene. However, these
investigators dropped styrene and race from their preferred square root model to obtain their final
model. Furthermore, there may be other relevant factors that weren’t included in the models at
all.

Fifth, there are uncertainties in the parameter estimates used in the models. The study of
Delzell et al. is large, providing some degree of reliability in the parameter estimates; however,
especially given the large human variability that has been observed in metabolic activities that
could affect cancer risk from 1,3-butadiene exposure, the generalizability of the occupational
results is unclear.
In addition, there are important concerns raised by comparison with the rodent data.
First, the rodent studies suggest that 1,3-butadiene is a multi-site carcinogen. It is possible that
humans may also be at risk of 1,3-butadiene-induced carcinogenicity at other sites and that the
epidemiologic study had insufficient power to detect the other excess risks. In the mouse, for
example, the lung is the most sensitive tumor site. Significant excesses of lung cancer may not
have been detectable in the epidemiologic study because of the high background rates of lung
cancer in humans. Delzell et al. did observe a slight increase in lung cancer among maintenance
workers. The reported excess cancer risk estimate, which is based only on leukemias, may be an
underestimate if other sites are also at risk.
Second, both the rat and mouse studies suggest that females are more sensitive to
1,3-butadiene-induced carcinogenicity than males, and the mammary gland in females was the
only tumor site common to both species. If female humans are also more sensitive than males,
then the male-based risk estimates calculated from the epidemiology study would underestimate
risks to females.
Despite these uncertainties, confidence in the excess cancer risk estimate of 9 ×
10-3/ppm is relatively high. First, the estimate is based on human data. Furthermore, these data
are from a large, high-quality epidemiologic study in which 1,3-butadiene exposures were
estimated for each individual a priori to conducting the exposure-response analysis. Although
there are uncertainties in the exposure estimation, a serious attempt was made to reconstruct
historical exposures for specific tasks and work areas. It is virtually unprecedented to have such a
comprehensive exposure assessment for individual workers in such a large occupational
epidemiologic study. In addition, the assumption of linearity for low-dose extrapolation is
reasonable given the clear evidence of genotoxicity by 1,3-butadiene metabolites.
Using the cancer potency estimate of 9 × 10-3/ppm, the chronic (70 year) exposure level
resulting in an increased cancer risk of 10-6 (i.e., one in a million) can be estimated as follows:
(10-6)/(9 × 10-3/ppm) = 1 × 10-4ppm = 0.1 ppb.
11.5. SUMMARY OF REPRODUCTIVE/DEVELOPMENTAL EFFECTS

A variety of reproductive and developmental effects have been observed in mice and
rats exposed to 1,3-butadiene by inhalation. There are no human data on reproductive or
developmental effects.

The most sensitive developmental endpoint was decreased fetal weight in the mouse.
Decreases were observed at the lowest exposure concentration (40 ppm, 6 h/day, gestation days
6-15); thus there was no NOAEL for this effect. Generally, however, it is thought that there is an
exposure threshold, and while effects on fetal growth in humans cannot be ruled out, they are not
expected to occur from low environmental exposures to 1,3-butadiene. No developmental
toxicity was observed in rats.
The most sensitive reproductive endpoints observed in subchronic exposure studies were
litter size at birth and at weaning in dominant lethal studies of mice (i.e., male mice are exposed to
1,3-butadiene and effects on litters are measured after mating to unexposed females). Litter size
at birth reflects both decreased implants and increased fetal deaths, while litter size at weaning
also reflects neonatal deaths. Dominant lethal effects in humans would likely be manifested as
spontaneous abortions, miscarriages, stillbirths, or very early deaths. The dominant lethal
responses are believed to represent a genotoxic effect; however, a large number of sperm would
have to be affected to result in any meaningful increase in risk, because the chances of any single
sperm both having a critical mutation and fertilizing an egg are minuscule. Thus, dominant lethal
effects are not expected in humans exposed to low environmental exposures, although the
possibility of such effects or of transmissible genetic mutations cannot be ruled out.
From chronic exposure studies (2-year bioassays), the most sensitive reproductive effects
were ovarian atrophy in female mice and testicular atrophy in male mice. Testicular atrophy was
primarily a high-exposure effect and likely has an exposure threshold. Ovarian atrophy, on the
other hand, was observed at the lowest exposure level (6.25 ppm, 6 h/day, 5 days/week, for 2
years), although an exposure threshold is assumed for this endpoint as well. Uterine atrophy was
also observed in the highest exposure groups: however, this is thought to be a secondary effect of
the ovarian atrophy. The mechanisms of ovarian atrophy are unknown, although there is strong
evidence that the effect is mediated by the diepoxide metabolite. It is further expected, based on
metabolic data, that humans would produce lower concentrations of this metabolite than do mice.
Thus, it is likely that humans are less sensitive to 1,3-butadiene-induced ovarian atrophy than are
mice. No reproductive effects were reported in the 2-year rat study. In conclusion, ovarian
atrophy is not expected in humans from environmental exposures to 1,3-butadiene; although, the
effect cannot be ruled out.
11.6. QUANTITATIVE ESTIMATION (RfC) FOR REPRODUCTIVE/

DEVELOPMENTAL EFFECTS
An RfC for reproductive and developmental effects of 0.15 ppb was obtained for the
critical effect of decreased litter size at birth (or at weaning), based on subchronic dominant
lethal studies in the mouse.

A reference concentration (RfC) is an estimate of the daily exposure to humans that is
“likely to be without appreciable risk of deleterious [noncancer] effects during a lifetime.” The
RfC is calculated for the “critical [noncancer] effect,” i.e., the effect for which an increased
response is observed at the lowest concentration used in the study, or for which benchmark
concentration modeling yields the lowest EC10. In this assessment, the RfC is only for
reproductive and developmental effects (R/D RfC), because other noncancer effects were not
considered. Of the 1,3-butadiene reproductive/developmental effects, the critical effect was
decreased litter size at birth or at weaning (both of these effects yielded the same EC10), as
observed in dominant lethal studies of male mice. An R/D RfC was calculated based on the
LEC10, which was calculated using benchmark concentration methodology, and uncertainty
factors for interspecies extrapolation (3), intraspecies variability (10), extrapolation from
subchronic study to chronic exposure (3), the absence of multigenerational studies (3), and “risk
reduction” to extrapolate to a level at which no detectable effects are expected (analogous to the
LOAEL-to-NOAEL uncertainty factor) (3). The resulting R/D RfC is 0.15 ppb [0.15
ppm/(3×10×3×3×3)]. The actual risks at low exposure levels are unknown; the R/D RfC merely
provides a bound on chronic exposure below which no “appreciable risk” of reproductive or
developmental effects is expected.
Although other noncancer effects were not examined, the reproductive endpoints were
quite sensitive, and it is likely that the R/D RfC is protective against other noncancer effects as
well.
In addition, a RfCDT of 0.1 ppm for developmental toxicity from short-term exposures was
calculated from the mouse fetal weight data, and a R/D RfC for subchronic exposures of 0.0015
ppm was derived from the dominant lethal results in mice, each using benchmark concentration
methodology to obtain the “point of departure” for applying uncertainty factors.
11.7. SPECIAL SUBPOPULATIONS

11.7.1. Sensitive Subpopulations
It is uncertain whether children or other subpopulations have greater susceptibility to
exposure to 1,3-butadiene than the general population.
There is no information available on health effects in children from exposure to 1,3-
butadiene at this time. Occurrence of leukemia is causally associated with exposure to 1,3-
butadiene in adults, and leukemia is one of the most common cancers in children. Furthermore,
leukemia risk in children has been shown to increase with simultaneous exposure to multiple risk
factors (Gibson et al., 1968). Thus, exposure to 1,3-butadiene may be an additional risk factor
increasing the leukemia risk further in children.

Tobacco smoke contains 1,3-butadiene as well as other carcinogens, and there are a few
studies suggesting that parental smoking increases the risk of leukemia or lymphoma in children
(John et al., 1991; Stjernfeldt et al., 1986). The overall evidence, however, is inconclusive
because other studies observed no increased risk. Furthermore, if there is an effect in children
from parental smoking, it is unclear whether it is attributable to preconception effects on fathers’
sperm, in utero exposure of the fetus, and/or postnatal exposure to environmental tobacco smoke.
Because metabolic activation of 1,3-butadiene to epoxide metabolites is believed to be
necessary for carcinogenicity, it is possible that genetic differences in metabolic or detoxification
enzymes could result in different risks to different human subpopulations. For example,
investigators have observed that polymorphism in glutathione-S-transferase genes confers
differential susceptibility to the induction of sister chromatid exchanges by butadiene metabolites
in cultured human lymphocytes. However, the critical/rate-limiting mechanistic steps are
unknown at present; thus, it is unknown whether or not there are actual human subpopulations
that may have notably different susceptibility to 1,3-butadiene.
11.7.2. Highly Exposed Subpopulations

Some subpopulations may be at greater risk than the general population as a result of
higher exposure to 1,3-butadiene.
Heavy smokers may be highly exposed to 1,3-butadiene due to its formation in tobacco
smoke. Cigarette smoke has been shown to be a risk factor for various types of leukemias. It
should be noted, however, that known and suspected leukemogenic constituents of tobacco
smoke include benzene, polonium-210, nitrosamines, and hydrocarbons in addition to 1,3-
butadiene (Schottenfeld and Fraumeni, 1996).
11.8. FUTURE RESEARCH NEEDS

Although 1,3-butadiene is classified as a known human carcinogen in this assessment,
there are some data gaps in various areas which, if filled, will refine the assessment. The specific
research needs are as follows:
Epidemiology
The medical records for the leukemia cases in the studies by Delzell et al. and
Macaluso et al. should be reviewed to verify the cell types of leukemias.
Further follow-up of these studies is recommended because it will give an opportunity
to observe whether any noncancer effects, such as cardiovascular, or any cancers with
a longer latency period are associated with exposure to 1,3-butadiene.

Studies in other polymer facilities around the world could also add to the human
evidence of carcinogenicity.
All epidemiologic studies to date have examined male cohorts. Some butadiene
production facilities around the world (e.g., China) employ women in their
laboratories. If the number of women in these facilities is large enough, a
reproductive/developmental study would help determine if female workers are at risk
of reproductive effects or if exposed fetuses are at risk of developmental effects.
A reproductive study of exposed males is also needed to examine potential dominant

lethal effects in humans.
Toxicology
Elucidation of the mechanisms responsible for the interspecies differences in sensitivity
to 1,3-butadiene could assist in resolving questions about the human risk for
reproductive effects and for cancer at sites for which the Delzell et al. study may have
had insufficient power to detect an effect.
Molecular biology
Once the mechanisms of 1,3-butadiene-induced health effects are better understood,
information on polymorphisms in human metabolic enzymes (or DNA repair enzymes,
etc.) could help define sensitive subpopulations.
11.9. SUMMARY AND CONCLUSIONS

The purpose of this effort was to review the new information that has become available
since EPA’s 1985 health assessment of 1,3-butadiene and to determine if any changes were
needed to the earlier conclusions.
1,3-Butadiene is a gas used commercially in the production of styrene-butadiene rubber,
plastics, and thermoplastic resins. The major environmental source of 1,3-butadiene is the
incomplete combustion of fuels from mobile sources (e.g., automobile exhaust). Tobacco smoke
can be a significant source of 1,3-butadiene in indoor air.
This assessment concludes that 1,3-butadiene is a known human carcinogen, based on
three types of evidence: (1) epidemiologic studies showing increased leukemias in workers
occupationally exposed to 1,3-butadiene (by inhalation), (2) studies showing that 1,3-butadiene
causes a variety of tumors in mice and rats by inhalation, and (3) studies demonstrating that 1,3-
butadiene is metabolized into genotoxic metabolites by experimental animals and humans.
The specific mechanisms of 1,3-butadiene-induced carcinogenesis are unknown; however, it is
virtually certain that the carcinogenic effects are mediated by genotoxic metabolites of
1,3-butadiene.
The best estimate of human lifetime extra cancer risk from chronic exposure to
1,3-butadiene is 9 × 10-3 per ppm based on linear modeling and extrapolation of the increased

leukemia risks observed in occupationally exposed workers. Although there is uncertainty in
extrapolating from occupational exposures to lower environmental exposures, this risk estimate
has the advantage of being based on a large, high-quality human study, and linear extrapolation is
warranted by the known genotoxicity of 1,3-butadiene metabolites. The corresponding estimate
of the chronic exposure level of 1,3-butadiene resulting in an extra cancer risk of 10-6 (i.e., one in
a million) is 0.1 ppb. The 95% upper bound on unit risk from the linear model is 0.02/ppm.
1,3-Butadiene also causes a variety of reproductive and developmental effects in mice and
rats; no human data on these effects are available. The most sensitive effect was reduced litter
size at birth and at weaning observed in studies in which exposed male mice were mated with
unexposed females. In humans, such an effect might be manifested as an increased risk of
spontaneous abortions, miscarriages, stillbirths, or very early deaths. Based on this critical effect
of reduced litter size, a reference concentration (i.e., a chronic exposure level presumed to be
“without appreciable risk”) of 0.15 ppb for reproductive and developmental effects was calculated
from the modeled benchmark concentration (LED10) of 0.15 ppm. The actual risks at low
exposure levels are unknown; this RfC merely provides a bound on chronic exposure below which
no “appreciable risk” of reproductive or developmental effects is expected.
There are insufficient data from which to draw any conclusions on potentially sensitive
subpopulations.
In summary, the primary changes in EPA’s conclusions about the health effects of 1,3-
butadiene from the 1985 document to this one are:
The cancer classification has been changed from probable to known human
carcinogen.
The unit cancer risk estimate has been changed from 0.25/ppm (upper bound based on
mouse data) to 0.009/ppm (best estimate based on linear modeling and extrapolation
of human data).
For the first time, an RFC (0.15 ppb) is calculated for reproductive/developmental
effects.

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DRAFT NCEA-W-0267

DO NOT CITE OR QUOTE January 1998

Health Risk Assessment

National Center for Environmental Assessment

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

LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xii

AUTHORS, CONTRIBUTORS, AND REVIEWERS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiv

2. OVERVIEW OF EXPOSURE TO 1,3-BUTADIENE . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1

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2.4.1. Air . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-8

3. METABOLISM AND PHARMACOKINETICS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1

5. REPRODUCTIVE AND DEVELOPMENTAL EFFECTS . . . . . . . . . . . . . . . . . . . . . . . . 5-1

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5.4. SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-27

6. TOXICITY IN ANIMALS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-1

7. EPIDEMIOLOGIC STUDIES OF CARCINOGENICITY . . . . . . . . . . . . . . . . . . . . . . . . 7-1

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Santos-Burgoa et al., 1992: Lymphohematopoietic Cancer in

8. PHARMACOKINETIC MODELING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-1

9. QUANTITATIVE RISK ASSESSMENT FOR 1,3-BUTADIENE . . . . . . . . . . . . . . . . . . 9-1

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9.3.4. Ovarian, Uterine, and Testicular Atrophy Modeling . . . . . . . . . . . . . . . . . . . 9-40

10. WEIGHT OF EVIDENCE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10-1

11. RISK CHARACTERIZATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11-1

12. REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12-1

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1-1 Carcinogenicity assessments of 1,3-butadiene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-12

3-1 Metabolic pathways of 1,3-butadiene metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4

5-1 Reproductive tract lesions in female B6C3F1 mice exposed to 1,3-butadiene

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5-4 Developmental toxicity in Sprague-Dawley CD rats exposed to

6-1 Survival of male and female B6C3F1 mice exposed to 1,3-butadiene by

7-1 Epidemiologic studies of the health effects of exposure to 1,3-butadieneC

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9-1 Results from exposure-response models of continuous cumulative

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developmental effects of 1,3-butadiene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-48

11-1 Summary of epidemiologic studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11-4

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3-1 Some pathways in the metabolism of butadiene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-3

6-1 Kaplan-Meier survival curves for male B6C3F1 mice exposed to

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1.2. SUMMARY OF PAST CARCINOGEN RISK ASSESSMENTS OF 1,3-BUTADIENE

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1.2.2. Summary of IARC’s Evaluation of 1,3-Butadiene (IARC, 1986, 1992)

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1.2.4. California Air Resources Board (CARB, 1991)