Ł CQA EPA/635/R-09/005-F
Xd f » www.epa.gov/iris
TOXICOLOGICAL REVIEW
OF
1,4-Dioxane
(CAS No. 123-91-1)
In Support of Summary Information on the
Integrated Risk Information System (IRIS)
August 2010
U.S. Environmental Protection Agency
Washington, DC
-------
DISCLAIMER
This document has been reviewed in accordance with U.S. Environmental Protection
Agency policy and approved for publication. Mention of trade names or commercial products
does not constitute endorsement or recommendation for use.
-------
CONTENTS - TOXICOLOGICAL REVIEW OF 1,4-DIOXANE
(CAS No. 123-91-1)
LIST OF TABLES vii
LIST OF FIGURES xi
LIST OF ABBREVIATIONS AND ACRONYMS xiv
1. INTRODUCTION 1
2. CHEMICAL AND PHYSICAL INFORMATION 3
3. TOXICOKINETICS 6
3.1. ABSORPTION 6
3.2. DISTRIBUTION 7
3.3. METABOLISM 8
3.4. ELIMINATION 12
3.5. PHYSIOLOGICALLY BASED pharmacokinetic MODELS 12
3.5.1. Available Pharmacokinetic Data 14
3.5.2. Published PBPK Models for 1,4-Dioxane 15
3.5.2.1. Leung and Paustenbach 15
3.5.2.2. Reitzetal 16
3.5.2.3.Fisheretal 18
3.5.3. Implementation of Published PBPK Models for 1,4-Dioxane 18
3.6. RAT NASAL EXPOSURE VIA DRINKING WATER 21
4. HAZARD IDENTIFICATION 23
4.1. STUDIES IN HUMANS - EPIDEMIOLOGY, CASE REPORTS, CLINICAL
CONTROLS 23
4.1.1. Thiessetal 25
4.1.2. Buffleretal 26
4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN
ANIMALS - ORAL AND INHALATION 27
4.2.1. OralToxicity 27
4.2.1.1. Subchronic Oral Toxicity 27
4.2.1.1.1. Stoneretal 27
4.2.1.1.2. Stottetal 28
4.2.1.1.3.Kanoetal 28
4.2.1.1.4. Yamamotoetal 33
4.2.1.2. Chronic Oral Toxicity and Carcinogenicity 34
4.2.1.2.1. Argus et al 34
4.2.1.2.2. Argus et al.; Hoch-Ligeti et al 35
4.2.1.2.3. Hoch-Ligeti and Argus 36
4.2.1.2.4. Kocibaetal 37
4.2.1.2.5. National Cancer Institute (NCI) 39
4.2.1.2.6. Kano et al.; Japan Bioassay Research Center; Yamazaki et al 43
4.2.2. Inhalation Toxicity 55
in
-------
4.2.2.1. Subchronic Inhalation Toxicity 55
4.2.2. l.l.Fairleyetal 55
4.2.2.2. Chronic Inhalation Toxicity and Carcinogenicity 55
4.2.2.2.1. Torkelson et al 55
4.2.3. Initiation/Promotion Studies 56
4.2.3.1. Bull etal 56
4.2.3.2. King etal 57
4.2.3.3.Lundbergetal 58
4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND INHALATION 58
4.3.1. Giavini etal 58
4.4. OTHER DURATION OR ENDPOINT-SPECIFIC STUDIES 59
4.4.1. Acute and Short-term Toxicity 59
4.4.1.1. Oral Toxicity 59
4.4.1.2. Inhalation Toxicity 59
4.4.2. Neurotoxicity 62
4.4.2. l.Frantik etal 62
4.4.2.2. Goldberg etal 63
4.4.2.3. Kanada etal 64
4.4.2.4. Knoefel 64
4.5. MECHANISTIC DATA AND OTHER STUDIES IN SUPPORT OF THE MODE OF
ACTION 64
4.5.1. Genotoxicity 64
4.5.2. Mechanistic Studies 73
4.5.2.1. Free Radical Generation 73
4.5.2.2. Induction of Metabolism 73
4.5.2.3. Mechanisms of Tumor Induction 74
4.6. SYNTHESIS OF MAJOR NONCANCER EFFECTS 76
4.6.1. Oral 76
4.6.2. Inhalation 80
4.6.3. Mode of Action Information 80
4.7. EVALUATION OF CARCINOGENICITY 81
4.7.1. Summary of Overall Weight of Evidence 81
4.7.2. Synthesis of Human, Animal, and Other Supporting Evidence 83
4.7.3. Mode of Action Information 84
4.7.3.1. Identification of Key Events for Carcinogenicity 84
4.7.3.1.1. Liver 84
4.7.3.1.2. Nasal cavity 86
4.7.3.2. Strength, Consistency, Specificity of Association 87
4.7.3.2.1. Liver 87
4.7.3.2.2. Nasal cavity 88
4.7.3.3. Dose-Response Relationship 88
4.7.3.3.1. Liver 88
4.7.3.3.2. Nasal cavity 90
4.7.3.4. Temporal Relationship 90
4.7.3.4.1. Liver 90
4.7.3.4.2. Nasal cavity 91
4.7.3.5. Biological Plausibility and Coherence 91
4.7.3.5.1. Liver 91
4.7.3.5.2. Nasal cavity 91
IV
-------
4.7.3.6. Other Possible Modes of Action 92
4.7.3.7. Conclusions About the Hypothesized Mode of Action 92
4.7.3.7.1. Liver 92
4.7.3.7.2. Nasal cavity 93
4.7.3.8. Relevance of the Mode of Action to Humans 93
4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES 93
5. DOSE-RESPONSE ASSESSMENTS 95
5.1. ORAL REFERENCE DOSE (RfD) 95
5.1.1. Choice of Principal Studies and Critical Effect with Rationale and Justification 95
5.1.2. Methods of Analysis—Including Models (PBPK, HMD, etc.) 96
5.1.3. RfD Derivation - Including Application of Uncertainty Factors (UFs) 99
5.1.4. RfD Comparison Information 100
5.1.5. Previous RfD Assessment 104
5.2. INHALATION REFERENCE CONCENTRATION (RfC) 104
5.3. UNCERTAINTIES IN THE ORAL REFERENCE DOSE (RfD) 105
5.4. CANCER ASSESSMENT 107
5.4.1. Choice of Study/Data - with Rationale and Justification 107
5.4.2. Dose-Response Data 109
5.4.3. Dose Adjustments and Extrapolation Method(s) 110
5.4.3.1. Dose Adjustments 110
5.4.3.2. ExtrapolationMethod(s) Ill
5.4.4. Oral Slope Factor and Inhalation Unit Risk 112
5.4.5. Previous Cancer Assessment 114
5.5. UNCERTAINTIES IN CANCER RISK VALUES 115
5.5.1. Sources of Uncertainty 115
5.5.1.1. Choice of Low-Dose Extrapolation Approach 115
5.5.1.2. Dose Metric 116
5.5.1.3. Cross-Species Scaling 117
5.5.1.4. Statistical Uncertainty at the POD 117
5.5.1.5. Bioassay Selection 117
5.5.1.6. Choice of Species/Gender 117
5.5.1.7. Relevance to Humans 118
5.5.1.8. Human Population Variability 118
6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HAZARD AND DOSE
RESPONSE 120
6.1. HUMAN HAZARD POTENTIAL 120
6.2. DOSE RESPONSE 121
6.2.1. Noncancer/Oral 121
6.2.2. Noncancer/Inhalation 122
6.2.3. Cancer/Oral 122
6.2.3.1. Choice of Low-Dose Extrapolation Approach 123
6.2.3.2. Dose Metric 124
6.2.3.3. Cross-Species Scaling 124
6.2.3.4. Statistical Uncertainty at the POD 124
6.2.3.5. Bioassay Selection 124
6.2.3.6. Choice of Species/Gender 124
6.2.3.7'. Relevance to Humans 125
v
-------
6.2.3.8. Human Population Variability 125
6.2.4. Cancer/Inhalation 125
7. REFERENCES 126
APPENDIX A. SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC COMMENTS
AND DISPOSITION A-l
APPENDIX B. EVALUATION OF EXISTING PBPK MODELS FOR 1,4-DIOXANE B-l
APPENDIX C. DETAILS OF BMD ANALYSIS FOR ORAL RfD FOR 1,4-DIOXANE C-l
APPENDIX D. DETAILS OF BMD ANALYSIS FOR ORAL CSF FOR 1,4-DIOXANE D-l
APPENDIX E. COMPARISON OF SEVERAL DATA REPORTS FOR THE JBRC 2-YEAR
1,4-DIOXANE DRINKING WATER STUDY E-l
VI
-------
LIST OF TABLES
Table 2-1. Physical properties and chemical identity of 1,4-dioxane 4
Table 4-1. Incidence of histopathological lesions in F344/DuCrj rats exposed to 1,4-dioxane in
drinking water for 13 weeks 31
Table 4-2. Incidence of histopathological lesions in Crj:BDFl mice exposed to 1,4-dioxane in
drinking water for 13 weeks 33
Table 4-3. Number of incipient liver tumors and hepatomas in male Sprague- Dawley rats
exposed to 1,4-dioxane in drinking water for 13 months 36
Table 4-4. Incidence of liver and nasal tumors in male and female Sherman rats (combined)
treated with 1,4-dioxane in the drinking water for 2 years 39
Table 4-5. Incidence of nonneoplastic lesions in Osborne-Mendel rats exposed to 1,4-dioxane in
drinking water 40
Table 4-6. Incidence of nasal cavity squamous cell carcinoma and liver hepatocellular adenoma
in Osborne-Mendel rats exposed to 1,4-dioxane in drinking water 41
Table 4-7. Incidence of hepatocellular adenoma or carcinoma in B6C3Fi mice exposed to
1,4-dioxane in drinking water 43
Table 4-8. Incidence of histopathological lesions in male F344/DuCrj rats exposed to
1,4-dioxane in drinking water for 2 years 47
Table 4-9. Incidence of histopathological lesions in female F344/DuCrj rats exposed to
1,4-dioxane in drinking water for 2 years 48
Table 4-10. Incidence of nasal cavity, peritoneum, and mammary gland tumors in F344/DuCrj
rats exposed to 1,4-dioxane in drinking water for 2 years 50
Table 4-11. Incidence of liver tumors in F344/DuCrj rats exposed to 1,4-dioxane in drinking
water for 2 years 50
Table 4-12. Incidence of histopathological lesions in male Crj:BDFl mice exposed to
1,4-dioxane in drinking water for 2 years 52
Table 4-13. Incidence of histopathological lesions in female Crj:BDFl mice exposed to
1,4-dioxane in drinking water for 2 years 53
Table 4-14. Incidence of tumors in Crj:BDFl mice exposed to 1,4-dioxane in drinking water for
2 years 54
Table 4-15. Acute and short-term toxicity studies of 1,4-dioxane 60
Table 4-16. Genotoxicity studies of 1,4-dioxane; in vitro 67
Table 4-17. Genotoxicity studies of 1,4-dioxane; mammalian in vivo 71
Table 4-18. Oral toxicity studies (noncancer effects) for 1,4-dioxane 77
Table 4-19. Temporal sequence and dose-response relationship for possible key events and liver
tumors in rats and mice 89
Table 5-1. Incidence of cortical tubule degeneration in Osborne-Mendel rats exposed to
1,4-dioxane in drinking water for 2 years 98
Table 5-2. BMD and BMDL values derived from BMD modeling of cortical tubule
degeneration in male and female Osborne-Mendel rats exposed to 1,4-dioxane in
drinking water for 2 years 98
Table 5-3. Incidence of liver hyperplasia in F344/DuCrj rats exposed to 1,4-dioxane in drinking
water for 2 yearsa 98
Table 5-4. BMD and BMDL values derived from BMD modeling of liver hyperplasia in male
and female F344/DuCrj rats exposed to 1,4-dioxane in drinking water for 2 years.. 99
vn
-------
Table 5-5. Incidence of liver, nasal cavity, peritoneal, and mammary gland tumors in rats and
mice exposed to 1,4-dioxane in drinking water for 2 years (based on survival to
12 months) 108
Table 5-6. Incidence of hepatocellular adenoma or carcinoma in rats and mice exposed to
1,4-dioxane in drinking water for 2 years 110
Table 5-7. Calculated HEDs for the tumor incidence data used for dose-response modeling ..111
Table 5-8. BMD RED and BMDLHED values from models fit to tumor incidence data for rats and
mice exposed to 1,4-dioxane in drinking water for 2 years and corresponding oral
CSFs 113
Table 5-9. Summary of uncertainty in the 1,4-dioxane cancer risk estimation 119
Table B-l. Human PBPK model parameter values for 1,4-dioxane B-ll
Table B-2. PBPK metabolic and elimination parameter values resulting from re-calibration of
the human model using alternative values for physiological flow ratesa and tissue:air
partition coefficients B-13
Table B-3. PBPK metabolic and elimination parameter values resulting from recalibration of the
human model using biologically plausible values for physiological flow ratesa and
selected upper and lower boundary values for tissue:air partition coefficients B-20
Table C-l. Incidence of cortical tubule degeneration in Osborne-Mendel rats exposed to
1,4-dioxane in drinking water for 2 years C-l
Table C-2. Goodness-of-fit statistics and BMDio and BMDLio values from models fit to
incidence data for cortical tubule degeneration in male and female Osborne-Mendel
rats (NCI, 1978, 062935) exposed to 1,4-dioxane in drinking water C-2
Table C-3. Incidence of liver hyperplasia in F344/DuCrj rats exposed to 1,4-dioxane in drinking
water3 C-7
Table C-4. Benchmark dose modeling results based on the incidence of liver hyperplasias in
male and female F344 rats exposed to 1,4-dioxane in drinking water for 2 years.. C-8
Table D-l. Recommended models for rodents exposed to 1,4-dioxane in drinking water (Kano et
al., 2009, 594539) D-4
Table D-2. Data for hepatic adenomas and carcinomas in female F344 rats (Kano et al., 2009,
594539) D-5
Table D-3. BMDS dose-response modeling results for the combined incidence of hepatic
adenomas and carcinomas in female F344 rats (Kano et al., 2009, 594539) D-5
Table D-4. Data for hepatic adenomas and carcinomas in male F344 rats (Kano et al., 2009,
594539) D-8
Table D-5. BMDS dose-response modeling results for the combined incidence of adenomas and
carcinomas in livers of male F344 rats (Kano et al., 2009, 594539) D-9
Table D-6. Data for significant tumors at other sites in male and female F344 rats (Kano et al.,
2009,594539) D-14
Table D-7. BMDS dose-response modeling results for the incidence of nasal cavity tumors in
female F344 ratsa (Kano et al., 2009, 594539) D-15
Table D-8. BMDS dose-response modeling results for the incidence of nasal cavity tumors in
male F344ratsa (Kano etal., 2009, 594539) D-18
Table D-9. BMDS dose-response modeling results for the incidence of mammary gland
adenomas in female F344 rats (Kano et al., 2009, 594539) D-21
Table D-10. BMDS dose-response modeling results for the incidence of peritoneal
mesotheliomas in male F344 rats (Kano et al., 2009, 594539) D-26
Table D-l 1. Data for hepatic adenomas and carcinomas in female BDF1 mice (Kano et al., 2009,
594539) D-31
Vlll
-------
Table D-12. BMDS dose-response modeling results for the combined incidence of hepatic
adenomas and carcinomas in female BDF1 mice (Kano et al., 2009, 594539) D-32
Table D-13. BMDS LogLogistic dose-response modeling results using BMRs of 10, 30, and 50%
for the combined incidence of hepatic adenomas and carcinomas in female BDF1
mice (Kano et al., 2009, 594539) D-32
Table D-14. Data for hepatic adenomas and carcinomas in male BDF1 mice (Kano et al., 2009,
594539) D-41
Table D-15. BMDS dose-response modeling results for the combined incidence of hepatic
adenomas and carcinomas in maleBDFl mice (Kano et al., 2009, 594539) D-42
Table D-16. Summary of BMDS dose-response modeling estimates associated with liver and
nasal tumor incidence data resulting from chronic oral exposure to 1,4-dioxane in
rats and mice D-47
Table D-17. Incidence of hepatocellular carcinoma and nasal squamous cell carcinoma in male
and female Sherman rats (combined) (Kociba et al., 1974, 062929) treated with
1,4-dioxane in the drinking water for 2 years D-48
Table D-18. BMDS dose-response modeling results for the incidence of hepatocellular
carcinoma in male and female Sherman rats (combined) (Kociba et al., 1974,
062929) exposed to 1,4-dioxane in the drinking water for 2 years D-49
Table D-19. BMDS dose-response modeling results for the incidence of nasal squamous cell
carcinoma in male and female Sherman rats (combined) (Kociba et al., 1974,
062929) exposed to 1,4-dioxane in the drinking water for 2 years D-54
Table D-20. Incidence of nasal cavity squamous cell carcinoma and hepatocellular adenoma in
Osborne-Mendel rats (NCI, 1978, 062935) exposed to 1,4-dioxane in the drinking
water D-57
Table D-21. BMDS dose-response modeling results for the incidence of hepatocellular adenoma
in female Osborne-Mendel rats (NCI, 1978, 062935) exposed to 1,4-dioxane in the
drinking water for 2 years D-58
Table D-22. BMDS dose-response modeling results for the incidence of nasal cavity squamous
cell carcinoma in female Osborne-Mendel rats (NCI, 1978, 062935) exposed to
1,4-dioxane in the drinking water for 2 years D-63
Table D-23. BMDS dose-response modeling results for the incidence of nasal cavity squamous
cell carcinoma in male Osborne-Mendel rats (NCI, 1978, 062935) exposed to
1,4-dioxane in the drinking water for 2 years D-68
Table D-24. Incidence of hepatocellular adenoma or carcinoma in male and female B6C3Fi mice
(NCI, 1978, 06293 5) exposed to 1,4-dioxane in drinking water D-73
Table D-25. BMDS dose-response modeling results for the combined incidence of hepatocellular
adenoma or carcinoma in female B6C3Fi mice (NCI, 1978, 062935) exposed to
1,4-dioxane in the drinking water for 2 years D-74
Table D-26. BMDS dose-response modeling results for the combined incidence of hepatocellular
adenoma or carcinoma in male B6C3Fi mice (NCI, 1978, 062935) exposed to
1,4-dioxane in drinking water D-77
Table E-l. Nonneoplastic lesions: Comparison of histological findings reported for the 2-year
JBRC drinking water study in male F344 rats E-2
Table E-2. Nonneoplastic lesions: Comparison of histological findings reported for the 2-year
JBRC drinking water study in female F344 rats E-3
Table E-3. Neoplastic lesions: Comparison of histological findings reported for the 2-year JBRC
drinking water study in maleF344 rats E-5
Table E-4. Neoplastic lesions: Comparison of histological findings reported for the 2-year JBRC
drinking water study in female F344 rats E-6
IX
-------
Table E-5. Nonneoplastic lesions: Comparison of histological findings reported for the 2-year
JBRC drinking water study in male Crj:BDFl mice E-8
Table E-6. Nonneoplastic lesions: Comparison of histological findings reported for the 2-year
JBRC drinking water study in female Crj:BDFl mice E-10
Table E-7. Neoplastic lesions: Comparison of histological findings reported for the 2-year JBRC
drinking water study in male Crj:BDFl mice E-ll
Table E-8. Neoplastic lesions: Comparison of histological findings reported for the 2-year JBRC
drinking water study in female Crj:BDFl mice E-12
x
-------
LIST OF FIGURES
Figure 2-1. 1,4-Dioxane chemical structure 3
Figure 3-1. Suggested metabolic pathways of 1,4-dioxane in the rat 9
Figure 3-2. Plasma 1,4-dioxane levels in rats following i.v. doses of 3-5,600 mg/kg 11
Figure 3-3. General PBPK model structure consisting of blood-flow limited tissue compartments
connected via arterial and venous blood flows 13
Figure 4-1. A schematic representation of the possible key events in the delivery of 1,4-dioxane
to the liver and the hypothesized MOA(s) for liver carcinogenicity 86
Figure 4-2. A schematic representation of the possible key events in the delivery of 1,4-dioxane
to the nasal cavity and the hypothesized MOA(s) for nasal cavity carcinogenicity.. 87
Figure 5-1. Potential points of departure (POD) for liver toxicity endpoints with corresponding
applied uncertainty factors and derived RfDs following oral exposure to 1,4-dioxane.
101
Figure 5-2. Potential points of departure (POD) for kidney toxicity endpoints with
corresponding applied uncertainty factors and derived RfDs following oral exposure
to 1,4-dioxane 102
Figure 5-3. Potential points of departure (POD) for nasal inflammation with corresponding
applied uncertainty factors and derived sample RfDs following oral exposure to
1,4-dioxane 103
Figure 5-4. Potential points of departure (POD) for organ specific toxicity endpoints with
corresponding applied uncertainty factors and derived sample RfDs following oral
exposure to 1,4-dioxane 104
Figure B-l. Schematic representation of empirical model for 1,4-dioxane in rats B-3
Figure B-2. Schematic representation of empirical model for 1,4-dioxane in humans B-4
Figure B-3. Output of 1,4-dioxane blood level data from the acslXtreme implementation (left)
and published (right) empirical rat model simulations of i.v. administration
experiments B-5
Figure B-4. Output of FtEAA urine level data from acslXtreme implementation (left) and
published (right) empirical rat model simulations of i.v. administration experiments.
B-6
Figure B-5. acslXtreme predictions of blood 1,4-dioxane and urine FtEAA levels from the
empirical rat model simulations of a 6-hour, 50-ppm inhalation exposure B-7
Figure B-6. Output of 1,4-dioxane blood level data from the acslXtreme implementation (left)
and published (right) empirical human model simulations of a 6-hour, 50-ppm
inhalation exposure B-8
Figure B-7. Observations and acslXtreme predictions of cumulative FtEAA in human urine
following a 6-hour, 50-ppm inhalation exposure B-9
Figure B-8. Predicted and observed blood 1,4-dioxane concentrations (left) and urinary HEAA
levels (right) following re-calibration of the human PBPK model with tissue:air
partition coefficient values B-l3
Figure B-9. Predicted and observed blood 1,4-dioxane concentrations (left) and urinary FtEAA
levels (right) following re-calibration of the human PBPK model with tissue:air
partition coefficient values B-14
Figure B-10. Predicted and observed blood 1,4-dioxane concentrations (left) and urinary HEAA
levels (right) B-15
XI
-------
Figure B-l 1. The highest seven sensitivity coefficients (and associated parameters) for blood
1,4-dioxane concentrations (CV) at 1 (left) and 4 (right) hours of a 50-ppm
inhalation exposure B-17
Figure B-12. Comparisons of the range of PBPK model predictions from upper and lower
boundaries on partition coefficients with empirical model predictions and
experimental observations for blood 1,4-dioxane concentrations (left) and urinary
FtEAA levels (right) from a 6-hour, 50-ppm inhalation exposure B-l9
Figure B-13. Comparisons of the range of PBPK model predictions from upper and lower
boundaries on partition coefficients with empirical model predictions and
experimental observations for blood 1,4-dioxane concentrations (left) and urinary
FtEAA levels (right) from a 6-hour, 50-ppm inhalation exposure B-l9
Figure B-14. Predictions of blood 1,4-dioxane concentration following calibration of a zero-
order metabolism rate constant, kLc, to the experimental data B-21
Figure B-l 5. Predictions of blood 1,4-dioxane concentration following calibration of a zero-
order metabolism rate constant, kLc, to only the exposure phase of the experimental
data B-22
Figure B-l 6. Predictions of blood 1,4-dioxane concentration following simultaneous calibration
of a zero-order metabolism rate constant, kLC, and slowly perfused tissue:air partition
coefficient to the experimental data B-23
Figure C-l. BMD Log-probit model of cortical tubule degeneration incidence data for male rats
exposed to 1,4-dioxane in drinking water for 2 years to support the results in
Table C-2 C-3
Figure C-2. BMD Weibull model of cortical tubule degeneration incidence data for female rats
exposed to 1,4-dioxane in drinking water for 2 years to support the results in
Table C-2 C-5
Figure C-3. BMD gamma model of liver hyperplasia incidence data for F344 male rats exposed
to 1,4-dioxane in drinking water for 2 years to support results Table C-4 C-9
Figure C-4. BMD multistage (2 degree) model of liver hyperplasia incidence data for F344 male
rats exposed to 1,4-dioxane in drinking water for 2 years to support results
Table C-4 C-ll
Figure C-5. BMD Weibull model of liver hyperplasia incidence data for F344 male rats exposed
to 1,4-dioxane in drinking water for 2 years to support the results in Table C-4.. C-l3
Figure C-6. BMD quantal-linear model of liver hyperplasia incidence data for F344 male rats
exposed to 1,4-dioxane in drinking water for 2 years to support the results in
Table C-4 C-15
Figure C-7. BMD log-probit model of liver hyperplasia incidence data for F344 female rats
exposed to 1,4-dioxane in drinking water for 2 years to support the results in
Table C-4 C-17
Figure D-l. Multistage BMD model (2 degree) for the combined incidence of hepatic adenomas
and carcinomas in female F344 rats D-6
Figure D-2. Probit BMD model for the combined incidence of hepatic adenomas and
carcinomas in male F344 rats D-10
Figure D-3. Multistage BMD model (3 degree) for the combined incidence of hepatic adenomas
and carcinomas in male F344 rats D-12
Figure D-4. Multistage BMD model (3 degree) for nasal cavity tumors in female F344 rats. D-l6
Figure D-5. Multistage BMD model (3 degree) for nasal cavity tumors in male F344 rats.... D-l9
Figure D-6. LogLogistic BMD model for mammary gland adenomas in female F344 rats. .. D-22
Figure D-7. Multistage BMD model (1 degree) for mammary gland adenomas in female F344
rats D-24
xn
-------
Figure D-8.ProbitBMD model for peritoneal mesotheliomas in male F344 rats D-27
Figure D-9. Multistage BMD (2 degree) model for peritoneal mesotheliomas in male F344 rats.
D-29
Figure D-10. LogLogistic BMD model for the combined incidence of hepatic adenomas and
carcinomas in female BDF1 mice with aBMR of 10% D-33
Figure D-l 1. LogLogistic BMD model for the combined incidence of hepatic adenomas and
carcinomas in female BDF1 mice with aBMR of 30% D-35
Figure D-12. LogLogistic BMD model for the combined incidence of hepatic adenomas and
carcinomas in female BDF1 mice with aBMR of 50% D-37
Figure D-13. Multistage BMD model (1 degree) for the combined incidence of hepatic adenomas
and carcinomas in female BDF1 mice D-39
Figure D-14. LogLogistic BMD model for the combined incidence of hepatic adenomas and
carcinomas in maleBDFl mice D-43
Figure D-15. Multistage BMD model (1 degree) for the combined incidence of hepatic adenomas
and carcinomas in male BDF1 mice D-45
Figure D-l6. Probit BMD model for the incidence of hepatocellular carcinoma in male and
female Sherman rats exposed to 1,4-dioxane in drinking water D-50
Figure D-17. Multistage BMD model (1 degree) for the incidence of hepatocellular carcinoma in
male and female Sherman rats exposed to 1,4-dioxane in drinking water D-52
Figure D-18. Multistage BMD model (3 degree) for the incidence of nasal squamous cell
carcinoma in male and female Sherman rats exposed to 1,4-dioxane in drinking
water D-55
Figure D-l9. LogLogistic BMD model for the incidence of hepatocellular adenoma in female
Osborne-Mendel rats exposed to 1,4-dioxane in drinking water D-59
Figure D-20. Multistage BMD model (1 degree) for the incidence of hepatocellular adenoma in
female Osborne-Mendel rats exposed to 1,4-dioxane in drinking water D-61
Figure D-21. LogLogistic BMD model for the incidence of nasal cavity squamous cell
carcinoma in female Osborne-Mendel rats exposed to 1,4-dioxane in drinking water.
D-64
Figure D-22. Multistage BMD model (1 degree) for the incidence of nasal cavity squamous cell
carcinoma in female Osborne-Mendel rats exposed to 1,4-dioxane in drinking water.
D-66
Figure D-23. LogLogistic BMD model for the incidence of nasal cavity squamous cell
carcinoma in male Osborne-Mendel rats D-69
Figure D-24. Multistage BMD model (1 degree) for the incidence of nasal cavity squamous cell
carcinoma in male Osborne-Mendel rats D-71
Figure D-25. Multistage BMD model (2 degree) for the incidence of hepatocellular adenoma or
carcinoma in female B6C3Fi mice D-75
Figure D-26. Gamma BMD model for the incidence of hepatocellular adenoma or carcinoma in
maleB6C3Fi mice exposed to 1,4-dioxane in drinking water D-78
Figure D-27. Multistage BMD model (2 degree) for the incidence of hepatocellular adenoma or
carcinoma in male B6C3Fi mice exposed to 1,4-dioxane in drinking water D-80
Xlll
-------
LIST OF ABBREVIATIONS AND ACRONYMS
AIC
ALP
ALT
AST
ATSDR
BMD
BMD50
BMDL
BMDLio
BMDLso
BMDL50
BMDS
BMR
BrdU
BUN
BW(s)
CASE
CASRN
CHO
CI
CNS
CPK
CREST
CSF
CV
CYP450
DEN
FISH
G-6-Pase
GC
GGT
HEAA
HED(s)
HPLC
HSDB
Hz
IARC
i.p.
i.v.
IRIS
JBRC
ke
Lc
Akaike's Information Criterion
alkaline phosphatase
alanine aminotransferase
aspartate aminotransferase
Agency for Toxic Substances and Disease Registry
benchmark dose
benchmark dose at 10% extra risk
benchmark dose at 30% extra risk
benchmark dose at 50% extra risk
benchmark dose, lower 95% confidence limit
benchmark dose, lower 95% confidence limit at 10% extra risk
benchmark dose, lower 95% confidence limit at 30% extra risk
benchmark dose, lower 95% confidence limit at 50% extra risk
Benchmark Dose Software
benchmark response
5-bromo-2'-deoxyuridine
blood urea nitrogen
body weight(s)
computer automated structure evaluator
Chemical Abstracts Service Registry Number
Chinese hamster ovary (cells)
confidence interval(s)
central nervous system
creatinine phosphokinase
antikinetochore
cancer slope factor
concentration in venous blood
cytochrome P450
diethylnitrosamine
fluorescence in situ hybridization
glucose-6-phosphatase
gas chromatography
y-glutamyl transpeptidase
p-hydroxyethoxy acetic acid
human equivalent dose(s)
high-performance liquid chromatography
Hazardous Substances Data Bank
Hertz
International Agency for Research on Cancer
intraperitoneal
intravenous
Integrated Risk Information System
Japan Bioassay Research Center
1st order elimination rate of 1,4-dioxane
1 st order 1 ,4-dioxane inhalation rate constant
1st order, non-saturable metabolism rate constant for 1,4-dioxane in the liver
xiv
-------
Km
kme
k0c
LAP
LDH
LOAEL
MCV
MOA
MS
MTD
MVK
NCE
NCI
ND
NE
NOAEL
NRC
NTP
OCT
ODC
OECD
PB
PBPK
PC
PCB
PCE
PFA
PLA
POD
ppm
PRA
PSA
QCC
QPC
RBC
RfC
RfD
SCE
SDH
SMR
SRC
TPA
TWA
UF
UNEP
U.S. EPA
V
VAS
Michaelis constant for metabolism of 1,4-dioxane in the liver
1 st order elimination rate of HEAA ( 1 ,4-dioxane metabolite)
soil organic carbon-water portioning coeffecient
leucine aminopeptidase
median lethal dose
lactate dehydrogenase
lowest-observed-adverse-effect-level
mean corpuscular volume
mode of action
mass spectrometry, multi-stage
maximum tolerated dose
Moolgavkar-Venzon-Knudsen (model)
normochromatic erythrocyte
National Cancer Institute
no data, not detected
not estimated
no-observed-adverse-effect-level
National Research Council
National Toxicology Program
ornithine carbamyl transferase
ornithine decarboxylase
Organization for Economic Co-operation and Development
blood:air partition coefficient
physiologically based pharmacokinetic
partition coefficient
polychlorinated biphenyl
polychromatic erythrocyte
fat: air partition coefficient
liverair partition coefficient
point of departure
parts per million
rapidly perfused tissue: air partition coefficient
slowly perfused tissue:air partition coefficient
normalized cardiac output
normalized alveolar ventilation rate
red blood cell
inhalation reference concentration
oral reference dose
sister chromatid exchange
sorbitol dehydrogenase
standardized mortality ratio
Syracuse Research Corporation
12-O-tetradecanoylphorbol- 13 -acetate
time-weighted average
uncertainty factor
United Nations Environment Programme
U.S. Environmental Protection Agency
volts
visual analogue scale
xv
-------
Va volume of distribution
Vmax maximal rate of metabolism
Vmaxc normalized maximal rate of metabolism of 1,4-dioxane in liver
VOC(s) volatile organic compound(s)
WBC white blood cell
X2 Chi-squared
xvi
-------
FOREWORD
The purpose of this Toxicological Review is to provide scientific support and rationale
for the hazard and dose-response assessment in IRIS pertaining to chronic exposure to
1,4-dioxane. It is not intended to be a comprehensive treatise on the chemical or toxicological
nature of 1,4-dioxane.
The intent of Section 6, Major Conclusions in the Characterization of Hazard and Dose
Response, is to present the major conclusions reached in the derivation of the reference dose,
reference concentration, and cancer assessment, where applicable, and to characterize the overall
confidence in the quantitative and qualitative aspects of hazard and dose response by addressing
the quality of the data and related uncertainties. The discussion is intended to convey the
limitations of the assessment and to aid and guide the risk assessor in the ensuing steps of the
risk assessment process.
For other general information about this assessment or other questions relating to IRIS,
the reader is referred to EPA's IRIS Hotline at (202) 566-1676 (phone), (202) 566-1749 (fax), or
hotline.iris@epa.gov (email address).
xvn
-------
AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGERS/AUTHORS
EvaD. McLanahan, Ph.D.
Lieutenant, U.S. Public Health Service
National Center for Environmental Assessment
U.S. Environmental Protection Agency
Research Triangle Park, NC
Reeder Sams II, Ph.D.
National Center for Environmental Assessment
U.S. Environmental Protection Agency
Research Triangle Park, NC
AUTHORS AND CONTRIBUTORS
J. Allen Davis, MSPH
National Center for Environmental Assessment
U.S. Environmental Protection Agency
Research Triangle Park, NC
Hisham El-Masri, Ph.D.
National Health and Environmental Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC
JeffS. Gift, Ph.D.
National Center for Environmental Assessment
U.S. Environmental Protection Agency
Research Triangle Park, NC
Karen Hogan
National Center for Environmental Assessment
U.S. Environmental Protection Agency
Washington, DC
Fernando Llados
Environmental Science Center
Syracuse Research Corporation
Syracuse, NY
Michael Lumpkin, Ph.D.
Environmental Science Center
Syracuse Research Corporation
Syracuse, NY
xvin
-------
Allan Marcus, Ph.D.
National Center for Environmental Assessment
U.S. Environmental Protection Agency
Research Triangle Park, NC
Marc Odin, Ph.D.
Environmental Science Center
Syracuse Research Corporation
Syracuse, NY
Susan Rieth
National Center for Environmental Assessment
U.S. Environmental Protection Agency
Washington, DC
Andrew Rooney, Ph.D.*
National Center for Environmental Assessment
U.S. Environmental Protection Agency
Research Triangle Park, NC
* Currently at National Toxicology Program
National Institute of Environmental Health Sciences
Research Triangle Park, NC
Paul Schlosser, Ph.D.
National Center for Environmental Assessment
U.S. Environmental Protection Agency
Research Triangle Park, NC
Julie Stickney, Ph.D.
Environmental Science Center
Syracuse Research Corporation
Syracuse, NY
John Vandenberg, Ph.D.
National Center for Environmental Assessment
U.S. Environmental Protection Agency
Research Triangle Park, NC
xix
-------
REVIEWERS
This document has been provided for review to EPA scientists, interagency reviewers
from other federal agencies and White House offices, and the public, and peer reviewed by
independent scientists external to EPA. A summary and EPA's disposition of the comments
received from the independent external peer reviewers and from the public is included in
Appendix A.
INTERNAL EPA REVIEWERS
Anthony DeAngelo, Ph.D.
National Health and Environmental Effects Research Laboratory
Office of Research and Development
Nagu Keshava, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
Jason Lambert, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
Connie Meacham, M.S.
National Center for Environmental Assessment
Research Triangle Park, NC
Debra Walsh, M.S.
National Center for Environmental Assessment
Research Triangle Park, NC
Douglas Wolf, Ph.D.
National Health and Environmental Effects Research Laboratory
Office of Research and Development
xx
-------
EXTERNAL PEER REVIEWERS
George V. Alexeeff, Ph.D., DABT
Office of Environmental Health Hazard Assessment (OEHHA)
California EPA
Bruce C. Allen, M.S.
Bruce Allen Consulting
James V. Bruckner, Ph.D.
Department of Pharmaceutical and Biomedical Sciences
College of Pharmacy
The University of Georgia
Harvey J. Clewell III, Ph.D., DABT
Center for Human Health Assessment
The Hamner Institutes for Health Sciences
Lena Ernstgard, Ph.D.
Institute of Environmental Medicine
Karolinska Institutet
Frederick J. Kaskel, M.D., Ph.D.
Children's Hospital at Montefiore
Albert Einstein College of Medicine of Yeshiva University
Kannan Krishnan, Ph.D., DABT
Inter-University Toxicology Research Center (CIRTOX)
Universite de Montreal
Ragubir P. Sharma, DVM, Ph.D.
Department of Physiology and Pharmacology
College of Veterinary Medicine (retired)
The University of Georgia
xxi
-------
1. INTRODUCTION
This document presents background information and justification for the Integrated Risk
Information System (IRIS) Summary of the hazard and dose-response assessment of
1,4-dioxane. IRIS Summaries may include oral reference dose (RfD) and inhalation reference
concentration (RfC) values for chronic and subchronic exposure durations, and a carcinogenicity
assessment.
The RfD and RfC, if derived, provide quantitative information for use in risk assessments
for health effects known or assumed to be produced through a nonlinear (presumed threshold)
mode of action. The RfD (expressed in units of mg/kg-day) is defined as an estimate (with
uncertainty spanning perhaps an order of magnitude) of a daily exposure to the human
population (including sensitive subgroups) that is likely to be without an appreciable risk of
deleterious effects during a lifetime. The inhalation RfC (expressed in units of mg/m3) is
analogous to the oral RfD, but provides a continuous inhalation exposure estimate. The
inhalation RfC considers toxic effects for both the respiratory system (portal-of-entry) and for
effects peripheral to the respiratory system (extrarespiratory or systemic effects). Reference
values are generally derived for chronic exposures (up to a lifetime), but may also be derived for
acute (< 24 hours), short-term (>24 hours up to 30 days), and subchronic (>30 days up to 10% of
lifetime) exposure durations, all of which are derived based on an assumption of continuous
exposure throughout the duration specified. Unless specified otherwise, the RfD and RfC are
derived for chronic exposure duration.
The carcinogenicity assessment provides information on the carcinogenic hazard
potential of the substance in question and quantitative estimates of risk from oral and inhalation
exposure may be derived. The information includes a weight-of-evidence judgment of the
likelihood that the agent is a human carcinogen and the conditions under which the carcinogenic
effects may be expressed. Quantitative risk estimates may be derived from the application of a
low-dose extrapolation procedure. If derived, the oral slope factor is a plausible upper bound on
the estimate of risk per mg/kg-day of oral exposure. Similarly, an inhalation unit risk is a
plausible upper bound on the estimate of risk per ug/m3 air breathed.
Development of these hazard identification and dose-response assessments for
1,4-dioxane has followed the general guidelines for risk assessment as set forth by the National
Research Council (NRC, 1983, 194806). EPA guidelines and Risk Assessment Forum Technical
Panel Reports that may have been used in the development of this assessment include the
following: Guidelines for the Health Risk Assessment of Chemical Mixtures (U.S. EPA, 1986,
Note: Hyperlinks to the reference citations throughout this document will take you to the NCEA HERO database
(Health and Environmental Research Online) at http://epa. gov/hero. HERO is a database of scientific literature used
by U.S. EPA in the process of developing science assessments such as the Integrated Science Assessments (ISA)
and the Integrated Risk Information System (IRIS).
1
-------
001468). Guidelines for Mutagenicity Risk Assessment (U.S. EPA, 1986, 001466).
Recommendations for and Documentation of Biological Values for Use in Risk Assessment
(U.S. EPA, 1988, 064560), Guidelines for Developmental Toxicity Risk Assessment (U.S. EPA,
1991, 008567), Interim Policy for Particle Size and Limit Concentration Issues in Inhalation
Toxicity (U.S. EPA, 1994, 076133), Methods for Derivation of Inhalation Reference
Concentrations and Application of Inhalation Dosimetry (U.S. EPA, 1994, 006488), Use of the
Benchmark Dose Approach in Health Risk Assessment (U.S. EPA, 1995, 005992), Guidelines for
Reproductive Toxicity Risk Assessment (U.S. EPA, 1996, 030019), Guidelines for Neurotoxicity
Risk Assessment (U.S. EPA, 1998, 030021), Science Policy Council Handbook: Risk
Characterization (U.S. EPA, 2000, 052149), Benchmark Dose Technical Guidance Document
(External Review Draft) (U.S. EPA, 2000, 052150), Supplementary Guidance for Conducting
Health Risk Assessment of Chemical Mixture s (U.S. EPA, 2000, 004421: U.S. EPA, 2000,
196144), A Review of the Reference Dose and Reference Concentration Processes (U.S. EPA,
2002, 088824), Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005, 086237),
Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens
(U.S. EPA, 2005, 088823), Science Policy Council Handbook: Peer Review (U.S. EPA, 2006,
194566), and A Framework for Assessing Health Risks of Environmental Exposures to Children
(U.S. EPA, 2006, 194567).
The literature search strategy employed for this compound was based on the Chemical
Abstracts Service Registry Number (CASRN) and at least one common name. Any pertinent
scientific information submitted by the public to the IRIS Submission Desk was also considered
in the development of this document. The relevant literature was reviewed through September
2009. Note that during the development of this assessment, new data regarding the toxicity of
1,4-dioxane through the inhalation route of exposure became available. These data have not been
included in the current assessment and will be evaluated in a separate IRIS assessment.
-------
2. CHEMICAL AND PHYSICAL INFORMATION
1,4-Dioxane, a volatile organic compound (VOC), is a colorless liquid with a pleasant
odor (Hawley and Lewis, 2001, 196089; Lewis, 2000, 625540). Synonyms include diethylene
ether, 1,4-diethylene dioxide, diethylene oxide, dioxyethylene ether, and dioxane (Hawley and
Lewis, 2001, 196089). The chemical structure of 1,4-dioxane is shown in Figure 2-1. Selected
chemical and physical properties of this substance are listed in Table 2-1 below:
Figure 2-1. 1,4-Dioxane chemical structure.
Note: Hyperlinks to the reference citations throughout this document will take you to the NCEA HERO database
(Health and Environmental Research Online) at http://epa. gov/hero. HERO is a database of scientific literature used
by U.S. EPA in the process of developing science assessments such as the Integrated Science Assessments (ISA)
and the Integrated Risk Information System (IRIS).
-------
Table 2-1. Physical properties and chemical identity of 1,4-dioxane
CASRN:
Molecular weight:
Chemical formula:
Boiling point:
Melting point:
Vapor pressure:
Density:
Vapor density:
Water solubility:
Other solubilities:
Log Kow:
Henry's Law constant:
OH reaction rate constant:
Bioconcentration factor:
Conversion factors (in air):
123-91-1 (CRC, 2000, 196090)
88.10 (Merck, 2001, 595055)
C4H8O2 (Merck, 2001, 595055)
101.1 °C (Merck, 2001, 595055)
11.8°C (CRC, 2000, 196090)
40 mmHg at 25°C (Lewis, 2000, 625540)
1.0337 g/mL at 20°C (CRC, 2000, 196090)
3.03 (air = 1) (Lewis, 2000, 625540)
Miscible with water (Hawley and Lewis, 2001, 196089)
Miscible with ethanol, ether, and acetone (CRC, 2000,
196090)
-0.27 (Hansch et al., 1995, 051424)
4.80 x 10"6 atm-nrVmolecule at 25°C (Park et al., 1987,
194328)
1.09 x 10"11 cnrVmolecule sec at25°C (Atkinson, 1989,
042876)
17 (estimated using log Kow) (ACS, 1990, 004237)
0.4 (estimated using log Kow) (Meylan et al., 1999,
194377)
1 ppm = 3.6 mg/m3; 1 mg/m3 = 0.278 ppm
(25°C and 1 atm) (HSDB, 2007, 196232)
1,4-Dioxane is produced commercially through the dehydration and ring closure of
diethylene glycol (Surprenant, 2002, 196117). Concentrated sulfuric acid is used as a catalyst
(Surprenant, 2002, 196117). This is a continuous distillation process with operating
temperatures and pressures of 130-200°C and 188-825 mmHg, respectively (Surprenant, 2002,
196117). During the years 1986 and 1990, the U.S. production of 1,4-dioxane reported by
manufacturers was within the range of 10-50 million pounds (U.S. EPA, 2002, 594597). The
production volume reported during the years 1994, 1998, and 2002 was within the range of
1-10 million pounds (U.S. EPA, 2002, 594597).
Historically, 1,4-dioxane has been used as a stabilizer for the solvent 1,1,1-trichloro-
ethane (Surprenant, 2002, 196117). However, this use is no longer expected to be important due
to the 1990 Amendments to the Clean Air Act and the Montreal Protocol, which mandate the
eventual phase-out of 1,1,1-trichloroethane production in the U.S. (ATSDR, 2007, 196127:
U.S. EPA, 1990, 196139: UNEP, 2000, 196125). 1,4-Dioxane is a contaminant of some
ingredients used in the manufacture of personal care products and cosmetics. 1,4-Dioxane is also
-------
used as a solvent for cellulosics, organic products, lacquers, paints, varnishes, paint and varnish
removers, resins, oils, waxes, dyes, cements, fumigants, emulsions, and polishing compositions
(Hawley and Lewis, 2001, 196089: IARC, 1999, 196238: Merck, 2001, 595055). 1,4-Dioxane
has been used as a solvent in the formulation of inks, coatings, and adhesives and in the
extraction of animal and vegeTable oil (Surprenant, 2002, 196117). Reaction products of
1,4-dioxane are used in the manufacture of insecticides, herbicides, plasticizers, and monomers
(Surprenant, 2002, 196117).
When 1,4-dioxane enters the air, it will exist as a vapor, as indicated by its vapor pressure
(HSDB, 2007, 196232). It is expected to be degraded in the atmosphere through photooxidation
with hydroxyl radicals (HSDB, 2007, 196232: Surprenant, 2002, 196117). The estimated half-
life for this reaction is 6.7 hours (HSDB, 2007, 196232). It may also be broken down by reaction
with nitrate radicals, although this removal process is not expected to compete with hydroxyl
radical photooxidation (Grosjean, 1990, 196213). 1,4-Dioxane is not expected to undergo direct
photolysis (Wolfe and Jeffers, 2000, 196109). 1,4-Dioxane is primarily photooxidized to
2-oxodioxane and through reactions with nitrogen oxides (NOX) results in the formation of
ethylene glycol diformate (Platz et al., 1997, 196086). 1,4-Dioxane is expected to be highly
mobile in soil based on its estimated Koc and is expected to leach to lower soil horizons and
groundwater (ACS, 1990, 004237: ATSDR, 2007, 196127). This substance may volatilize from
dry soil surfaces based on its vapor pressure (HSDB, 2007, 196232). The estimated
bioconcentration factor value indicates that 1,4-dioxane will not bioconcentrate in aquatic or
marine organisms (Franke et al., 1994, 194356: Meylan et al., 1999, 194377). 1,4-Dioxane is not
expected to undergo hydrolysis or to biodegrade readily in the environment (ATSDR, 2007,
196127: HSDB, 2007, 196232). Therefore, volatilization is expected to be the dominant removal
process for moist soil and surface water. Based on a Henry's Law constant of 4.8* 10"6
atm-mVmole, the half-life for volatilization of 1,4-dioxane from a model river is 5 days and that
from a model lake is 56 days (ACS, 1990, 004237: HSDB, 2007, 196232: Park et al., 1987,
194328). 1,4-Dioxane may be more persistent in groundwater where volatilization is hindered.
Recent environmental monitoring data for 1,4-dioxane are lacking. Existing data indicate
that 1,4-dioxane may leach from hazardous waste sites into drinking water sources located
nearby (Lesage et al., 1990, 195913: Yasuhara et al., 1997, 195909: Yasuhara et al., 2003,
195095). 1,4-Dioxane has been detected in contaminated surface and groundwater samples
collected near hazardous waste sites and industrial facilities (Derosa et al., 1996, 194371).
-------
3. TOXICOKINETICS
Data for the toxicokinetics of 1,4-dioxane in humans are very limited. However,
absorption, distribution, metabolism, and elimination of 1,4-dioxane are well described in rats
exposed via the oral, inhalation, or intravenous (i.v.) routes. 1,4-Dioxane is extensively absorbed
and metabolized in humans and rats. The metabolite most often measured and reported is
p-hydroxyethoxy acetic acid (HEAA), which is predominantly excreted in the urine; however,
other metabolites have also been identified. Saturation of 1,4-dioxane metabolism has been
observed in rats and would be expected in humans; however, human exposure levels associated
with nonlinear toxicokinetics are not known.
Important data elements that have contributed to our current understanding of the
toxicokinetics of 1,4-dioxane are summarized in the following sections.
3.1. ABSORPTION
Absorption of 1,4-dioxane following inhalation exposure has been qualitatively
demonstrated in workers and volunteers. Workers exposed to a time-weighted average (TWA)
of 1.6 parts per million (ppm) of 1,4-dioxane in air for 7.5 hours showed a HEAA/l,4-dioxane
ratio of 118:1 in urine (Young et al., 1976, 062953). The authors assumed lung absorption to be
100% and calculated an average absorbed dose of 0.37 mg/kg, although no exhaled breath
measurements were taken. In a study with four healthy male volunteers, Young et al. (1977,
062956) reported 6-hour inhalation exposures of adult volunteers to 50 ppm of 1,4-dioxane in a
chamber, followed by blood and urine analysis for 1,4-dioxane and HEAA. The study protocol
was approved by a seven-member Human Research Review Committee of the Dow Chemical
Company, and written informed consent of study participants was obtained. At a concentration
of 50 ppm, uptake of 1,4-dioxane into plasma was rapid and approached steady-state conditions
by 6 hours. The authors reported a calculated absorbed dose of 5.4 mg/kg. However, the
exposure chamber atmosphere was kept at a constant concentration of 50 ppm and exhaled
breath was not analyzed. Accordingly, gas uptake could not be measured. As a result, the
absorbed fraction of inhaled 1,4-dioxane could not be accurately determined in humans. Rats
inhaling 50 ppm for 6 hours exhibited 1,4-dioxane and HEAA in urine with an HEAA to
1,4-dioxane ratio of over 3,100:1 (Young et al., 1978, 062955: Young et al., 1978, 625640).
Plasma concentrations at the end of the 6-hour exposure period averaged 7.3 ug/mL. The
authors calculated an absorbed 1,4-dioxane dose of 71.9 mg/kg; however, the lack of exhaled
Note: Hyperlinks to the reference citations throughout this document will take you to the NCEA HERO database
(Health and Environmental Research Online) at http://epa. gov/hero. HERO is a database of scientific literature used
by U.S. EPA in the process of developing science assessments such as the Integrated Science Assessments (ISA)
and the Integrated Risk Information System (IRIS).
-------
breath data and dynamic exposure chamber precluded the accurate determination of the absorbed
fraction of inhaled 1,4-dioxane.
No human data are available to evaluate the oral absorption of 1,4-dioxane.
Gastrointestinal absorption was nearly complete in male Sprague Dawley rats orally dosed with
10-1,000 mg/kg of [14C]-l,4-dioxane given as a single dose or as 17 consecutive daily doses
(Young et al., 1978, 062955: Young et al., 1978, 625640). Cumulative recovery of radiolabel in
the feces was
-------
Liver, kidney, spleen, lung, colon, and skeletal muscle tissues were collected from 1, 2, 6, and
12 hours after dosing. Distribution was generally uniform across tissues, with blood
concentrations higher than tissues at all times except for 1 hour post dosing, when kidney levels
were approximately 20% higher than blood. Since tissues were not perfused prior to analysis,
the contribution of residual blood to radiolabel measurements is unknown, though loss of
1,4-dioxane from tissues would be unknown had saline perfusion been performed. Covalent
binding reached peak percentages at 6 hours after dosing in liver (18.5%), spleen (22.6%), and
colon (19.5%). At 16 hours after dosing, peak covalent binding percentages were observed in
whole blood (3.1%), kidney (9.5%), lung (11.2%), and skeletal muscle (11.2%). Within
hepatocytes, radiolabel distribution at 6 hours after dosing was greatest in the cytosolic fraction
(43.8%) followed by the microsomal (27.9%), mitochondrial (16.6%), and nuclear (11.7%)
fractions. While little covalent binding of radiolabel was measured in the hepatic cytosol (4.6%),
greater binding was observed at 16 hours after dosing in the nuclear (64.8%), mitochondrial
(45.7%), and microsomal (33.4%) fractions. Pretreatment with inducers of mixed-function
oxidase activity did not significantly change the extent of covalent binding in subcellular
fractions.
3.3. METABOLISM
The major product of 1,4-dioxane metabolism appears to be HEAA, although there is
one report that identified l,4-dioxane-2-one as a major metabolite (Woo et al., 1977, 194355).
However, the presence of this compound in the sample was believed to result from the acidic
conditions (pH of 4.0-4.5) of the analytical procedures. The reversible conversion of HEAA and
p-l,4-dioxane-2-one is pH-dependent (Braun and Young, 1977, 194370). Braun and Young
(1977, 194370) identified HEAA (85%) as the major metabolite, with most of the remaining
dose excreted as unchanged 1,4-dioxane in the urine of Sprague Dawley rats dosed with
1,000 mg/kg of uniformly labeled l,4-[14C]dioxane. In fact, toxicokinetic studies of 1,4-dioxane
in humans and rats (Young et al., 1977, 062956: 1978, 062955: Young et al., 1978, 625640)
employed an analytical technique that converted HEAA to the more volatile l,4-dioxane-2-one
prior to gas chromatography (GC); however, it is still unclear as to whether HEAA or
l,4-dioxane-2-one is the major metabolite of 1,4-dioxane.
A proposed metabolic scheme for 1,4-dioxane metabolism (Woo et al., 1977, 194355) in
Sprague Dawley rats is shown in Figure 3-1. Oxidation of 1,4-dioxane to diethylene glycol
(pathway a), l,4-dioxane-2-ol (pathway c), or directly to l,4-dioxane-2-one (pathway b) could
result in the production of HEAA. 1,4-Dioxane oxidation appears to be cytochrome P450
(CYP450)-mediated, as CYP450 induction with phenobarbital or Aroclor 1254 (a commercial
PCB mixture) and suppression with 2,4-dichloro-6-phenylphenoxy ethylamine or cobaltous
chloride were effective in significantly increasing and decreasing, respectively, the appearance of
HEAA in the urine of male Sprague Dawley rats following 3 g/kg i.p. dose (Woo et al., 1977,
-------
062951: Woo et al., 1978, 194345). 1,4-Dioxane itself induced CYP450-mediated metabolism
of several barbiturates in Hindustan mice given i.p. injections of 25 and 50 mg/kg 1,4-dioxane
(Mungikar and Pawar, 1978, 194344). Of the three possible pathways proposed in this scheme,
oxidation to diethylene glycol and HEAA appears to be the most likely, because diethylene
glycol was found as a minor metabolite in Sprague Dawley rat urine following a single
1,000 mg/kg gavage dose of 1,4-dioxane (Braun and Young, 1977, 194370). Additionally, i.p.
injection of 100-400 mg/kg diethylene glycol in Sprague Dawley rats resulted in urinary
elimination of HEAA (Woo et al., 1977, 062950).
O. .OH ^OH JS
O
X
HOH2C CH2OH HOH2C
(b) ••"-....
+H,O
-H,O
Source: Adapted with permission from Elsevier Ltd., Woo et al. (1977, 194355: 1977,
062951).
Figure 3-1. Suggested metabolic pathways of 1,4-dioxane in the rat.
I = 1,4-dioxane; II = diethylene glycol; III = p-hydroxyethoxy acetic acid (HEAA);
IV = l,4-dioxane-2-one; V = l,4-dioxane-2-ol; VI = P-hydroxyethoxy acetaldehyde.
Note: Metabolite [V] is a likely intermediate in pathway b as well as pathway c.
The proposed pathways are based on the metabolites identified; the enzymes
responsible for each reaction have not been determined. The proposed pathways do
not account for metabolite degradation to the labeled carbon dioxide (CO2)
identified in expired air after labeled 1,4-dioxane exposure.
Metabolism of 1,4-dioxane in humans is extensive. In a survey of 1,4-dioxane plant
workers exposed to a TWA of 1.6 ppm of 1,4-dioxane for 7.5 hours, Young et al. (1976, 062953)
found HEAA and 1,4-dioxane in the worker's urine at a ratio of 118:1. Similarly, in adult male
volunteers exposed to 50 ppm for 6 hours (Young et al., 1977, 062956), over 99% of inhaled
1,4-dioxane (assuming negligible exhaled excretion) appeared in the urine as HEAA. The linear
-------
elimination of 1,4-dioxane in both plasma and urine indicated that 1,4-dioxane metabolism was a
nonsaturated, first-order process at this exposure level.
Like humans, rats extensively metabolize inhaled 1,4-dioxane, as HEAA content in urine
was over 3,000-fold higher than that of 1,4-dioxane following exposure to 50 ppm for 6 hours
(Young et al., 1978, 062955: Young et al., 1978, 625640). 1,4-Dioxane metabolism in rats was a
saturable process, as exhibited by oral and i.v. exposures to various doses of [14C]-l,4-dioxane
(Young et al., 1978, 062955: Young et al., 1978, 625640). Plasma data from Sprague Dawley
rats given single i.v. doses of 3, 10, 30, 100, 300, or 1,000 mg [14C]-l,4-dioxane/kg demonstrated
a dose-related shift from linear, first-order to nonlinear, saturable metabolism of 1,4-dioxane
between plasma 1,4-dioxane levels of 30 and 100 ug/mL (Figure 3-2). Similarly, in rats given,
via gavage in distilled water, 10, 100, or 1,000 mg [14C]-l,4-dioxane/kg singly or 10 or 1,000 mg
[14C]-l,4-dioxane/kg in 17 daily doses, the percent urinary excretion of the radiolabel decreased
significantly with dose while radiolabel in expired air increased. Specifically, with single
[14C]-l,4-dioxane/kg doses, urinary radiolabel decreased from 99 to 76% and expired
1,4-dioxane increased from <1 to 25% as dose increased from 10 to 1,000 mg/kg. Likewise,
with multiple daily doses 10 or 1,000 mg [14C]-l,4-dioxane/kg, urinary radiolabel decreased
from 99 to 82% and expired 1,4-dioxane increased from 1 to 9% as dose increased. The
differences between single and multiple doses in urinary and expired radiolabel support the
notion that 1,4-dioxane may induce its own metabolism.
10
-------
1.000 -
10 15 20 25 30 35 10 45 SO 55 60
65
Source: Used with permission from Taylor and Francis, Young et al. (1978, 062955).
Figure 3-2. Plasma 1,4-dioxane levels in rats following i.v. doses of
3-5,600 mg/kg.
1,4-Dioxane has been shown to induce several isoforms of CYP450 in various tissues
following acute oral administration by gavage or drinking water (Nannelli et al., 2005, 195067).
Male Sprague Dawley rats were exposed to either 2,000 mg/kg 1,4-dioxane via gavage for
2 consecutive days or by ingestion of a 1.5% 1,4-dioxane drinking water solution for 10 days.
Both exposures resulted in significantly increased CYP2B1/2, CYP2C11, and CYP2E1 activities
in hepatic microsomes. The gavage exposure alone resulted in increased CYP3A activity. The
increase in 2C11 activity was unexpected, as that isoform has been observed to be under
hormonal control and was typically suppressed in the presence of 2B1/2 and 2E1 induction. In
the male rat, hepatic 2C11 induction is associated with masculine pulsatile plasma profiles of
growth hormone (compared to the constant plasma levels in the female), resulting in
masculinization of hepatocyte function (Waxman et al., 1991, 196102). The authors postulated
that 1,4-dioxane may alter plasma growth hormone levels, resulting in the observed 2C11
induction. However, growth hormone induction of 2C11 is primarily dependent on the duration
between growth hormone pulses and secondarily on growth hormone plasma levels (Agrawal
11
-------
and Shapiro, 2000, 196132: Waxman et al., 1991, 196102). Thus, the induction of 2C11 by
1,4-dioxane may be mediated by changes in the time interval between growth hormone pulses
rather than changes in growth hormone levels. This may be accomplished by 1,4-dioxane
temporarily influencing the presence of growth hormone cell surface binding sites (Agrawal and
Shapiro, 2000, 196132). However, no studies are available to confirm the influence of
1,4-dioxane on either growth hormone levels or changes in growth hormone pulse interval.
In nasal and renal mucosal cell microsomes, CYP2E1 activity, but not CYP2B1/2
activity, was increased. Pulmonary mucosal CYP450 activity levels were not significantly
altered. Observed increases in 2E1 mRNA in rats exposed by gavage and i.p. injection suggest
that 2E1 induction in kidney and nasal mucosa is controlled by a transcriptional activation of
2E1 genes. The lack of increased mRNA in hepatocytes suggests that induction is regulated via
a post-transcriptional mechanism. Differences in 2E1 induction mechanisms in liver, kidney,
and nasal mucosa suggest that induction is controlled in a tissue-specific manner.
3.4. ELIMINATION
In workers exposed to a TWA of 1.6 ppm for 7.5 hours, 99% of 1,4-dioxane eliminated in
urine was in the form of HEAA (Young et al., 1976, 062953). The elimination half-life was
59 minutes in adult male volunteers exposed to 50 ppm 1,4-dioxane for 6 hours, with 90% of
urinary 1,4-dioxane and 47% of urinary HEAA excreted within 6 hours of onset of exposure
(Young et al., 1977, 062956). There are no data for 1,4-dioxane elimination in humans from oral
exposures.
Elimination of 1,4-dioxane in rats (Young et al., 1978, 062955: Young et al., 1978,
625640) was primarily via urine. Like humans, the elimination half-life in rats exposed to
50 ppm 1,4-dioxane for 6 hours was calculated to be 1.01 hours. In Sprague Dawley rats given
single daily doses of 10, 100, or 1,000 mg [14C]-l,4-dioxane/kg or multiple doses of 10 or
1,000 mg [14C]-l,4-dioxane/kg, urinary radiolabel ranged from 99% down to 76% of total
radiolabel. Fecal elimination was less than 2% for all doses. The effect of saturable metabolism
on expired 1,4-dioxane was apparent, as expired 1,4-dioxane in singly dosed rats increased with
dose from 0.4 to 25% while expired 14CC>2 changed little (between 2 and 3%) across doses. The
same relationship was seen in Sprague Dawley rats dosed i.v. with 10 or 1,000 mg
[14C]-l,4-dioxane/kg. Higher levels of 14CC>2 relative to 1,4-dioxane were measured in expired
air of the 10 mg/kg group, while higher levels of expired 1,4-dioxane relative to 14CC>2 were
measured in the 1,000 mg/kg group.
3.5. PHYSIOLOGICALLY BASED PHARMACOKINETIC MODELS
Physiologically based pharmacokinetic models (PBPK) models have been developed for
1,4-dioxane in rats and humans (Leung and Paustenbach, 1990, 062932: Reitz et al., 1990,
094806) and lactating women (Fisher et al., 1997, 194390). Each of the models simulates the
12
-------
body as a series of compartments representing tissues or tissue groups that receive blood from
the central vascular compartment (Figure 3-3). Modeling was conducted under the premise that
transfers of 1,4-dioxane between blood and tissues occur sufficiently fast to be effectively blood
flow-limited, which is consistent with the available data (Ramsey and Andersen, 1984, 063020).
Blood time course and metabolite production data in rats and humans suggest that absorption and
metabolism are accomplished through common mechanisms in both species (Young et al., 1977,
062956: 1978, 062955) (Young et al., 1978, 625640). allowing identical model structures to be
used for both species (and by extension, for mice as well). In all three models, physiologically
relevant, species-specific parameter values for tissue volume, blood flow, and metabolism and
elimination are used. The models and supporting data are reviewed below, from the perspective
of assessing their utility for predicting internal dosimetry and for cross-species extrapolation of
exposure-response relationships for critical neoplastic and nonneoplastic endpoints (also see
Appendix B).
IV
infusion inhalation
I It
o
_g
OH
Liver
.. ^
_}
•a
o
OH
"ro
OJ
t:
absorption metabolism
Figure 3-3. General PBPK model structure consisting of blood-flow
limited tissue compartments connected via arterial and venous blood flows.
Note: Orally administered chemicals are absorbed directly into the liver while
inhaled and intravenously infused chemicals enter directly into the arterial and
venous blood pools, respectively.
13
-------
3.5.1. Available Pharmacokinetic Data
Animal and human data sets available for model calibration derive from Young et al.
(1977, 062956: 1978, 062955: 1978, 6256401 Mikheev et al. (1990, 195061). and Woo et al.
(1977, 062950: 1977, 194355). Young et al. (1978, 062955: 1978, 625640) studied the
disposition of radiolabeled [14C]-l,4-dioxane in adult male Sprague Dawley rats following i.v.,
inhalation, and single and multiple oral gavage exposures. Plasma concentration-time profiles
were reported for i.v. doses of 3, 10, 30, 100, and 1,000 mg/kg. In addition, exhaled 14CC>2 and
urinary 1,4-dioxane and HEAA profiles were reported following i.v. doses of 10 and
1,000 mg/kg. The plasma 1,4-dioxane concentration-time course, cumulative urinary
1,4-dioxane and cumulative urinary HEAA concentrations were reported following a 6-hour
inhalation exposure to 50 ppm. Following oral gavage doses of 10-1,000 mg/kg, percentages of
total orally administered radiolabel were measured in urine, feces, expired air, and the whole
body.
Oral absorption of 1,4-dioxane was extensive, as only approximately 1% of the
administered dose appeared in the feces within 72 hours of dosing (Young et al., 1978, 062955)
(Young et al., 1978, 625640). Although it may be concluded that the rate of oral absorption was
high enough to ensure nearly complete absorption by 72 hours, a more quantitative estimate of
the rate of oral absorption is not possible due to the absence of plasma time course data by oral
exposure.
Saturable metabolism of 1,4-dioxane was observed in rats exposed by either the i.v. or
oral routes (Young et al., 1978, 062955: Young et al., 1978, 625640). Elimination of
1,4-dioxane from plasma appeared to be linear following i.v. doses of 3-30 mg/kg, but was
nonlinear following doses of 100-1,000 mg/kg. Accordingly, 10 mg/kg i.v. doses resulted in
higher concentrations of 14CC>2 (from metabolized 1,4-dioxane) in expired air relative to
unchanged 1,4-dioxane, while 1,000 mg/kg i.v. doses resulted in higher concentrations of
expired 1,4-dioxane relative to 14CC>2. Thus, at higher i.v. doses, a higher proportion of
unmetabolized 1,4-dioxane is available for exhalation. Taken together, the i.v. plasma and
expired air data from Young et al. (1978, 062955: 1978, 625640) corroborate previous studies
describing the saturable nature of 1,4-dioxane metabolism in rats (Woo et al., 1977, 062950:
1977, 194355) and are useful for optimizing metabolic parameters (Vmax and Km) in a PBPK
model.
Similarly, increasing single or multiple oral doses of 10-1,000 mg/kg resulted in
increasing percentage of 1,4-dioxane in exhaled air and decreasing percentage of radiolabel
(either as 1,4-dioxane or a metabolite) in the urine, with significant differences in both metrics
being observed between doses of 10 and 100 mg/kg (Young et al., 1978, 062955: Young et al.,
1978, 625640). These data identify the region (10-100 mg/kg) in which oral exposures will
result in nonlinear metabolism of 1,4-dioxane and can be used to test whether metabolic
14
-------
parameter value estimates derived from i.v. dosing data are adequate for modeling oral
exposures.
Post-exposure plasma data from a single 6-hour, 50 ppm inhalation exposure in rats were
reported (Young et al., 1978, 062955: Young et al., 1978, 625640). The observed linear
elimination of 1,4-dioxane after inhalation exposure suggests that, via this route, metabolism is
in the linear region at this exposure level.
The only human data adequate for use in PBPK model development (Young et al., 1977,
062956) come from adult male volunteers exposed to 50 ppm 1,4-dioxane for 6 hours. Plasma
1,4-dioxane and HEAA concentrations were measured both during and after the exposure period,
and urine concentrations were measured following exposure. Plasma levels of 1,4-dioxane
approached steady-state at 6 hours. HEAA data were insufficient to describe the appearance or
elimination of HEAA in plasma. Data on elimination of 1,4-dioxane and HEAA in the urine up
to 24 hours from the beginning of exposure were reported. At 6 hours from onset of exposure,
approximately 90% and 47% of the cumulative (0-24 hours) urinary 1,4-dioxane and HEAA,
respectively, were measured in the urine. The ratio of HEAA to 1,4-dioxane in urine 24 hours
after onset of exposure was 192:1 (similar to the ratio of 118:1 observed by Young et al. (1976,
062953) in workers exposed to 1.6 ppm for 7.5 hours), indicating extensive metabolism of
1,4-dioxane As with Sprague Dawley rats, the elimination of 1,4-dioxane from plasma was
linear across all observations (6 hours following end of exposure), suggesting that human
metabolism of 1,4-dioxane is linear for a 50 ppm inhalation exposure to steady-state. Thus,
estimation of human Vmax and Km from these data will introduce uncertainty into internal
dosimetry performed in the nonlinear region of metabolism.
Further data were reported for the tissue distribution of 1,4-dioxane in rats. Mikheev
et al. (1990, 195061) administered i.p. doses of [14C]-l,4-dioxane to white rats (strain not
reported) and reported time-to-peak blood, liver, kidney, and testes concentrations. They also
reported ratios of tissue to blood concentrations at various time points after dosing. Woo et al.
(1977, 062950: 1977, 194355) administered i.p. doses of [14C]-1,4-dioxane to Sprague Dawley
rats and measured radioactivity levels in urine. However, since i.p. dosing is not relevant to
human exposures, these data are of limited use for PBPK model development.
3.5.2. Published PBPK Models for 1,4-Dioxane
3.5.2.1. Leung andPaustenbach
Leung and Paustenbach (1990, 062932) developed a PBPK model for 1,4-dioxane and its
primary metabolite, HEAA, in rats and humans. The model, based on the structure of a PBPK
model for styrene (Ramsey and Andersen, 1984, 063020), consists of a central blood
compartment and four tissue compartments: liver, fat, slowly perfused tissues (mainly muscle
and skin), and richly perfused tissues (brain, kidney, and viscera other than the liver). Tissue
15
-------
volumes were calculated as percentages of total BW, and blood flow rates to each compartment
were calculated as percentages of cardiac output. Equivalent cardiac output and alveolar
ventilation rates were allometrically scaled to a power (0.74) of BW for each species. The
concentration of 1,4-dioxane in alveolar blood was assumed to be in equilibrium with alveolar
air at a ratio equal to the experimentally measured blood:air partition coefficient. Transfers of
1,4-dioxane between blood and tissues were assumed to be blood flow-limited and to achieve
rapid equilibrium between blood and tissue, governed by tissue:blood equilibrium partition
coefficients. The latter were derived from the quotient of blood:air and tissue:air partition
coefficients, which were measured in vitro (Leung and Paustenbach, 1990, 062932) for blood,
liver, fat, and skeletal muscle (slowly perfused tissue). Blood:air partition coefficients were
measured for both humans and rats. Rat tissue:air partition coefficients were used as surrogate
values for humans, with the exception of slowly perfused tissue:blood, which was estimated by
optimization to the plasma time-course data. Portals of entry included i.v. infusion (over a
period of 36 seconds) into the venous blood, inhalation by diffusion from the alveolar air into the
lung blood at the rate of alveolar ventilation, and oral administration via zero-order absorption
from the gastrointestinal tract to the liver. Elimination of 1,4-dioxane was accomplished through
pulmonary exhalation and saturable hepatic metabolism. Urinary excretion of HEAA was
assumed to be instantaneous with the generation of HEAA from the hepatic metabolism of
1,4-dioxane.
The parameter values for hepatic metabolism of 1,4-dioxane, Vmax and Km, were
optimized and validated against plasma and/or urine time course data for 1,4-dioxane and HEAA
in rats following i.v. and inhalation exposures and humans following inhalation exposure (Young
et al., 1977, 062956: 1978, 062955: 1978, 625640): the exact data (i.e., i.v., inhalation, or both)
used for the optimization and calibration were not reported. Although the liver and fat were
represented by tissue-specific compartments, no tissue-specific concentration data were available
for model development, raising uncertainty as the model's ability to adequately predict exposure
to these tissues. The human inhalation exposure of 50 ppm for 6 hours (Young et al., 1977,
062956) was reported to be in the linear range for metabolism; thus, uncertainty exists in the
ability of the allometrically-scaled value for the human metabolic Vmax to accurately describe
1,4-dioxane metabolism from exposures resulting in metabolic saturation. Nevertheless, these
values resulted in the model producing good fits to the data. For rats, the values for Vmax had to
be adjusted upwards by a factor of 1.8 to reasonably simulate exposures greater than 300 mg/kg.
The model authors attributed this to metabolic enzyme induction by high doses of 1,4-dioxane.
3.5.2.2. Reitzet al.
Reitz et al. (1990, 094806) developed a model for 1,4-dioxane and HEAA in the mouse,
rat, and human. This model, also based on the styrene model of Ramsey and Andersen (1984,
063020), included a central blood compartment and compartments for liver, fat, and rapidly and
16
-------
slowly perfused tissues. Tissue volumes and blood flow rates were defined as percentages of
total BW and cardiac output, respectively. Physiological parameter values were similar to those
used by Andersen et al. (1987, 001938), except that flow rates for cardiac output and alveolar
ventilation were doubled in order to produce a better fit of the model to human blood level data
(Young et al., 1977, 062956). Portals of entry included i.v. injection into the venous blood,
inhalation, oral bolus dosing, and oral dosing via drinking water. Oral absorption of 1,4-dioxane
was simulated, in all three species, as a first-order transfer to liver (halftime approximately
8 minutes).
Alveolar blood levels of 1,4-dioxane were assumed to be in equilibrium with alveolar air
at a ratio equal to the experimentally measured blood:air partition coefficient. Transfers of
1,4-dioxane between blood and tissues were assumed to be blood flow-limited and to achieve
rapid equilibrium between blood and tissue, governed by tissue:blood equilibrium partition
coefficients. These coefficients were derived by dividing experimentally measured (Leung and
Paustenbach, 1990, 062932) in vitro blood:air and tissue:air partition coefficients for blood, liver,
fat. Blood:air partition coefficients were measured for both humans and rats. The mouse
blood:air partition coefficient was different from rat or human values; the source of the partition
coefficient for blood in mice was not reported. Rat tissue:air partition coefficients were used as
surrogate values for humans. Rat tissue partition coefficient values were the same values as used
in the Leung and Paustenbach (1990, 062932) model (with the exception of slowly perfused
tissues) and were used in the models for all three species. The liver value was used for the
rapidly perfused tissues, as well as slowly perfused tissues. Although slowly perfused tissue:air
partition coefficients for rats were measured, the authors suggested that 1,4-dioxane in the
muscle and air may not have reached equilibrium in the highly gelatinous tissue homogenate
(Reitz et al., 1990, 094806). Substitution of the liver value provided much closer agreement to
the plasma data than when the muscle value was used. Further, doubling of the measured human
blood:air partition coefficient improved the fit of the model to the human blood level data
compared to the fit resulting from the measured value (Reitz et al., 1990, 094806). The Reitz
et al. (1990, 094806) model simulated three routes of 1,4-dioxane elimination: pulmonary
exhalation, hepatic metabolism to HEAA, and urinary excretion of HEAA. The elimination of
HEAA was modeled as a first-order transfer of 1,4-dioxane metabolite to urine.
Values for the metabolic rate constants, Vmax and Km, were optimized to achieve
agreement with various observations. Reitz et al. (1990, 094806) optimized values for human
Vmax and Km against the experimental human 1,4-dioxane inhalation data (Young et al., 1977,
062956). As noted previously, because the human exposures were below the level needed to
exhibit nonlinear kinetics, uncertainty exists in the ability of the optimized value of Vmax to
simulate human 1,4-dioxane metabolism above the concentration that would result in saturation
of metabolism. Rat metabolic rate constants were obtained by optimization to simulated data
17
-------
from a two-compartment empirical pharmacokinetic model, which was fitted to i.v. exposure
data (Young et al., 1978, 062955: Young et al., 1978, 625640). As with the Leung and
Paustenbach (1990, 062932) model, the Reitz et al. (1990, 094806) model included
compartments for the liver and fat, although no tissue-specific concentration data were available
to validate dosimetry for these organs. The derivations of human and rat HEAA elimination rate
constants were not reported. Since no pharmacokinetics data for 1,4-dioxane in mice were
available, mouse metabolic rate constants were allometrically scaled from rat and human values.
3.5.2.3. Fisher et al.
A PBPK model was developed by Fisher et al. (1997, 194390) to simulate a variety of
volatile organic compounds (VOCs, including 1,4-dioxane) in lactating humans. This model was
similar in structure to those of Leung and Paustenbach (1990, 062932) and Reitz et al. (1990,
094806) with the addition of elimination of 1,4-dioxane to breast milk. Experimental
measurements were made for blood:air and milk:air partition coefficients. Other partition
coefficient values were taken from Reitz et al. (1990, 094806). The model was not optimized,
nor was performance tested against experimental exposure data. Thus, the ability of the model to
simulate 1,4-dioxane exposure data is unknown.
3.5.3. Implementation of Published PBPK Models for 1,4-Dioxane
As previously described, several pharmacokinetic models have been developed to predict
the absorption, distribution, metabolism, and elimination of 1,4-dioxane in rats and humans.
Single compartment, empirical models for rats (Young et al., 1978, 062955; Young et al., 1978,
625640) and humans (Young et al., 1977, 062956) were developed to predict blood levels of
1,4-dioxane and urine levels of the primary metabolite, HEAA. PBPK models that describe the
kinetics of 1,4-dioxane using biologically realistic flow rates, tissue volumes, enzyme affinities,
metabolic processes, and elimination behaviors were also developed (Fisher et al., 1997, 194390;
Leung and Paustenbach, 1990, 062932; Reitz et al., 1990, 094806; Sweeney et al., 2008,
195085).
In developing updated toxicity values for 1,4-dioxane the available PBPK models were
evaluated for their ability to predict observations made in experimental studies of rat and human
exposures to 1,4-dioxane (Appendix B). The Reitz et al. (1990, 094806) and Leung and
Paustenbach (1990, 062932) PBPK models were both developed from a PBPK model of styrene
(Ramsey and Andersen, 1984, 063020), with the exception of minor differences in the use of
partition coefficients and biological parameters. The model code for Leung and Paustenbach
(1990, 062932) was unavailable in contrast to Reitz et al. (1990, 094806). The model of Reitz
et al. (1990, 094806) was identified for further consideration to assist in the derivation of toxicity
values, and the Sweeney et al. (2008, 195085) PBPK model was also evaluated.
18
-------
The biological plausibility of parameter values in the Reitz et al. (1990, 094806) human
model were examined. The model published by Reitz et al. (1990, 094806) was able to predict
the only available human inhalation data (50 ppm 1,4-dioxane for 6 hours; Young et al., (1977,
062956)) by increasing (i.e., approximately doubling) the parameter values for human alveolar
ventilation (30 L/hour/kg0'74), cardiac output (30 L/hour/kg0'74), and the blood:air partition
coefficient (3,650) above the measured values of 13 L/minute/kg0'74 (Brown et al., 1997,
020304). 14 L/hour/kg0'74 (Brown et al., 1997, 020304). and 1,825 (Leung and Paustenbach,
1990, 062932). respectively. Furthermore, Reitz et al. (1990, 094806) replaced the measured
value for the slowly perfused tissue:air partition coefficient (i.e., muscle—value not reported in
manuscript) with the measured liver value (1,557) to improve the fit. Analysis of the Young
et al. (1977, 062956) human data suggested that the apparent volume of distribution (Vd) for
1,4-dioxane was approximately 10-fold higher in rats than humans, presumably due to species
differences in tissue partitioning or other process not represented in the model. Based upon these
observations, several model parameters (e.g., metabolism/elimination parameters) were re-
calibrated using biologically plausible values for flow rates and tissue:air partition coefficients.
Appendix B describes all activities that were conducted in the evaluation of the empirical
models and the re-calibration and evaluation of the Reitz et al. (1990, 094806) PBPK model to
determine the adequacy and preference for the potential use of the models.
The evaluation consisted of implementation of the Young et al. (1977, 062956; 1978,
062955; 1978, 625640) empirical rat and human models using the acslXtreme simulation
software, re-calibration of the Reitz et al. (1990, 094806) human PBPK model, and evaluation of
the model parameters published by Sweeney et al. (2008, 195085). Using the model descriptions
and equations given in Young et al. (1977, 062956: 1978, 062955: 1978, 625640). model code
was developed for the empirical models and executed, simulating the reported experimental
conditions. The model output was then compared with the model output reported in Young et al.
(1977, 062956: 1978, 062955: 1978, 625640).
The PBPK model of Reitz et al. (1990, 094806) was re-calibrated using measured values
for cardiac and alveolar flow rates and tissue:air partition coefficients. The predictions of blood
and urine levels of 1,4-dioxane and HEAA, respectively, from the re-calibrated model were
compared with the empirical model predictions of the same dosimeters to determine whether the
re-calibrated PBPK model could perform similarly to the empirical model. As part of the PBPK
model evaluation, EPA performed a sensitivity analysis to identify the model parameters having
the greatest influence on the primary dosimeter of interest, the blood level of 1,4-dioxane.
Variability data for the experimental measurements of the tissue:air partition coefficients were
incorporated to determine a range of model outputs bounded by biologically plausible values for
these parameters. Model parameters from Sweeney et al. (2008, 195085) were also tested to
evaluate the ability of the PBPK model to predict human data following exposure to 1,4-dioxane.
19
-------
The rat and human empirical models of Young et al. (1977, 062956: 1978, 062955: 1978,
625640) were successfully implemented in acslXtreme and perform identically to the models
reported in the published papers (Figures B-3 through B-7), with the exception of the lower
predicted HEAA concentrations and early appearance of the peak HEAA levels in rat urine. The
early appearance of peak HEAA levels cannot presently be explained, but may result from
manipulations of kme or other parameters by Young et al. (1978, 062955: 1978, 625640) that
were not reported. The lower predictions of HEAA levels are likely due to reliance on a standard
urine volume production rate in the absence of measured (but unreported) urine volumes. While
the human urinary HEAA predictions were lower than observations, this is due to parameter
fitting of Young et al. (1977, 062956). No model output was published in Young et al. (1977,
062956) for comparison. The empirical models were modified to allow for user-defined
inhalation exposure levels. However, no modifications were made to model oral exposures as
adequate data to parameterize such modifications do not exist for rats or humans.
Several procedures were applied to the Reitz et al. (1990, 094806) human PBPK model to
determine if an adequate fit of the model to the empirical model output or experimental
observations could be attained using biologically plausible values for the model parameters. The
re-calibrated model predictions for blood 1,4-dioxane levels do not come within 10-fold of the
experimental values using measured tissue:air partition coefficients from Leung and Paustenbach
(1990, 062932) or Sweeney et al. (2008, 195085) (Figures B-8 and B-9). The utilization of a
slowly perfused tissue:air partition coefficient 10-fold lower than measured values produces
exposure-phase predictions that are much closer to observations, but does not replicate the
elimination kinetics (Figure B-10). Recalibration of the model with upper bounds on the
tissue:air partition coefficients results in predictions that are still six- to sevenfold lower than
empirical model prediction or observations (Figures B-12 and B-13). Exploration of the model
space using an assumption of zero-order metabolism (valid for the 50 ppm inhalation exposure)
showed that an adequate fit to the exposure and elimination data can be achieved only when
unrealistically low values are assumed for the slowly perfused tissue:air partition coefficient
(Figure B-16). Artificially low values for the other tissue:air partition coefficients are not
expected to improve the model fit, as these parameters are shown in the sensitivity analysis to
exert less influence on blood 1,4-dioxane than Vmaxc and Km. In the absence of actual
measurements for the human slowly perfused tissue:air partition coefficient, high uncertainty
exists for this model parameter value. Differences in the ability of rat and human blood to bind
1,4-dioxane may contribute to the difference in Vd. However, this is expected to be evident in
very different values for rat and human blood:air partition coefficients, which is not the case
(Table B-l). Therefore, some other, as yet unknown, modification to model structure may be
necessary.
Similarly, Sweeney et al. (2008, 195085) also evaluated the available PBPK models
(Leung and Paustenbach, 1990, 062932: Reitz et al., 1990, 094806) for 1,4-dioxane. To address
20
-------
uncertainties and deficiencies in these models, the investigators conducted studies to fill data
gaps and reduce uncertainties pertaining to the pharmacokinetics of 1,4-dioxane and HEAA in
rats, mice, and humans. The following studies were performed:
• Partition coefficients, including measurements for mouse blood and tissues (liver, kidney,
fat, and muscle) and confirmatory measurements for human blood and rat blood and
muscle.
• Blood time course measurements in mice conducted for gavage administration of
nominal single doses (20, 200, or 2,000 mg/kg) of 1,4-dioxane administered in water.
• Metabolic rate constants for rat, mouse, and human liver based on incubations of
1,4-dioxane with rat, mouse, and human hepatocytes and measurement of HEAA.
The studies conducted by Sweeney et al. (2008, 195085) resulted in partition coefficients
that were consistent with previously measured values and those used in the Leung and
Paustenbach (1990, 062932) model. Of noteworthy significance, the laboratory results of
Sweeney et al. (2008, 195085) did not confirm the human blood:air partition coefficient Reitz
et al. (1990, 094806) reported. Furthermore, Sweeney et al. (2008, 195085) estimated metabolic
rate constants (Vmaxc and Km) within the range used in the previous models (Leung and
Paustenbach, 1990, 062932: Reitz et al., 1990, 094806). Overall, the Sweeney et al. (2008,
195085) model utilized more rodent in vivo and in vitro data in model parameterization and
refinement; however, the model was still unable to adequately predict the human blood data from
Young et al. (1977, 062956).
Updated PBPK models were developed based on these new data and data from previous
kinetic studies in rats, workers, and human volunteers reported by Young et al. (1976, 062953:
1977, 062956: 1978, 062955: 1978, 625640). The optimized rate of metabolism for the mouse
was significantly higher than the value previously estimated. The optimized rat kinetic
parameters were similar to those in the 1990 models. Of the two available human studies
(Young et al., 1976, 062953: 1977, 062956), model predictions were consistent with one study,
but did not fit the second as well.
3.6. RAT NASAL EXPOSURE VIA DRINKING WATER
Sweeney et al. (2008, 195085) conducted a rat nasal exposure study to explore the
potential for direct contact of nasal tissues with 1,4-dioxane-containing drinking water under
bioassay conditions. Two groups of male Sprague Dawley rats (5/group) received drinking
water in 45-mL drinking water bottles containing a fluorescent dye mixture (Cell Tracker
Red/FluoSpheres). The drinking water for one of these two groups also contained 0.5%
1,4-dioxane, a concentration within the range used in chronic toxicity studies. A third group of
five rats received tap water alone (controls). Water was provided to the rats overnight. The next
morning, the water bottles were weighed to estimate the amounts of water consumed. Rats were
21
-------
sacrificed and heads were split along the midline for evaluation by fluorescence microscopy.
One additional rat was dosed twice by gavage with 2 mL of drinking water containing
fluorescent dye (the second dose was 30 minutes after the first dose; total of 4 mL administered)
and sacrificed 5 hours later to evaluate the potential for systemic delivery of fluorescent dye to
the nasal tissues.
The presence of the fluorescent dye mixture had no measurable impact on water
consumption; however, 0.5% 1,4-dioxane reduced water consumption by an average of 62% of
controls following a single, overnight exposure. Fluorescent dye was detected in the oral cavity
and nasal airways of each animal exposed to the Cell Tracker Red/FluoSpheres mixture in their
drinking water, including numerous areas of the anterior third of the nose along the nasal
vestibule, maxillary turbinates, and dorsal nasoturbinates. Fluorescent dye was occasionally
detected in the ethmoid turbinate region and nasopharynx. 1,4-Dioxane had no effect on the
detection of the dye. Little or no fluorescence at the wavelength associated with the dye mixture
was detected in control animals or in the single animal that received the dye mixture by oral
gavage. The investigators concluded that the findings indicate rat nasal tissues are exposed by
direct contact with drinking water under bioassay conditions.
22
-------
4. HAZARD IDENTIFICATION
4.1. STUDIES IN HUMANS - EPIDEMIOLOGY, CASE REPORTS, CLINICAL
CONTROLS
Case reports of acute occupational poisoning with 1,4-dioxane indicated that exposure to
high concentrations resulted in liver, kidney, and central nervous system (CNS) toxicity (Barber,
1934, 062913: Johnstone, 1959, 062927). Barber (1934, 062913) described four fatal cases of
hemorrhagic nephritis and centrilobular necrosis of the liver attributed to acute inhalation
exposure to high (unspecified) concentrations of 1,4-dioxane. Death occurred within 5-8 days of
the onset of illness. Autopsy findings suggested that the kidney toxicity may have been
responsible for lethality, while the liver effects may have been compatible with recovery.
Jaundice was not observed in subjects and fatty change was not apparent in the liver. Johnstone
(1959, 062927) presented the fatal case of one worker exposed to high concentrations of
1,4-dioxane through both inhalation and dermal exposure for a 1 week exposure duration.
Measured air concentrations in the work environment of this subject were 208-650 ppm, with a
mean value of 470 ppm. Clinical signs that were observed following hospital admission
included severe epigastric pain, renal failure, headache, elevation in blood pressure, agitation and
restlessness, and coma. Autopsy findings revealed significant changes in the liver, kidney, and
brain. These included centrilobular necrosis of the liver and hemorrhagic necrosis of the kidney
cortex. Perivascular widening was observed in the brain with small foci of demyelination in
several regions (e.g., cortex, basal nuclei). It was suggested that these neurological changes may
have been secondary to anoxia and cerebral edema.
Several studies examined the effects of acute inhalation exposure in volunteers. In a
study performed at the Pittsburgh Experimental Station of the U.S. Bureau of Mines, eye
irritation and a burning sensation in the nose and throat were reported in five men exposed to
5,500 ppm of 1,4-dioxane vapor for 1 minute (Yant et al., 1930, 062952). Slight vertigo was
also reported by three of these men. Exposure to 1,600 ppm of 1,4-dioxane vapor for 10 minutes
resulted in similar symptoms with a reduced intensity of effect. In a study conducted by the
Government Experimental Establishment at Proton, England (Fairley et al., 1934, 062919), four
men were exposed to 1,000 ppm of 1,4-dioxane for 5 minutes. Odor was detected immediately
and one volunteer noted a constriction in the throat. Exposure of six volunteers to 2,000 ppm for
3 minutes resulted in no symptoms of discomfort. Wirth and Klimmer (1936, 196105), of the
Institute of Pharmacology, University of Wurzburg, reported slight mucous membrane irritation
Note: Hyperlinks to the reference citations throughout this document will take you to the NCEA HERO database
(Health and Environmental Research Online) at http://epa. gov/hero. HERO is a database of scientific literature used
by U.S. EPA in the process of developing science assessments such as the Integrated Science Assessments (ISA)
and the Integrated Risk Information System (IRIS).
23
-------
in the nose and throat of several human subjects exposed to concentrations greater than 280 ppm
for several minutes. Exposure to approximately 1,400 ppm for several minutes caused a
prickling sensation in the nose and a dry and scratchy throat. Silverman et al. (1946, 063013)
exposed 12 male and 12 female subjects to varying air concentrations of 1,4-dioxane for
15 minutes. A 200 ppm concentration was reported to be tolerable, while a concentration of
300 ppm caused irritation to the eyes, nose, and throat. The study conducted by Silverman et al.
(1946, 063013) was conducted by the Department of Industrial Hygiene, Harvard School of
Public Health, and was sponsored and supported by a grant from the Shell Development
Company. These volunteer studies published in the 1930s and 1940s (Fairley et al., 1934,
062919: Silverman et al., 1946, 063013: Wirth and Klimmer, 1936, 196105: Yant et al., 1930,
062952) did not provide information on the human subjects research ethics procedures
undertaken in these studies; however, there is no evidence that the conduct of the research was
fundamentally unethical or significantly deficient relative to the ethical standards prevailing at
the time the research was conducted.
Young et al. (1977, 062956) exposed four healthy adult male volunteers to a 50-ppm
concentration of 1,4-dioxane for 6 hours. The investigators reported that the protocol of this
study was approved by a seven-member Human Research Review Committee of the Dow
Chemical Company and was followed rigorously. Perception of the odor of 1,4-dioxane
appeared to diminish over time, with two of the four subjects reporting inability to detect the
odor at the end of the exposure period. Eye irritation was the only clinical sign reported in this
study. The pharmacokinetics and metabolism of 1,4-dioxane in humans were also evaluated in
this study (see Section 3.3). Clinical findings were not reported in four workers exposed in the
workplace to a TWA concentration of 1.6 ppm for 7.5 hours (Young et al., 1976, 062953).
Ernstgard et al. (2006, 195034) examined the acute effects of 1,4-dioxane vapor in male
and female volunteers. The study protocol was approved by the Regional Ethics Review Board
in Stockholm, and performed following informed consent and according to the Helsinki
declaration. In a screening study by these investigators, no self-reported symptoms (based on a
visual analogue scale (VAS) that included ratings for discomfort in eyes, nose, and throat,
breathing difficulty, headache, fatigue, nausea, dizziness, or feeling of intoxication) were
observed at concentrations up to 20 ppm; this concentration was selected as a tentative no-
observed-adverse-effect-level (NOAEL) in the main study. In the main study, six male and six
female healthy volunteers were exposed to 0 or 20 ppm 1,4-dioxane, at rest, for 2 hours. This
exposure did not significantly affect symptom VAS ratings, blink frequency, pulmonary function
or nasal swelling (measured before and at 0 and 3 hours after exposure), or inflammatory
markers in the plasma (C-reactive protein and interleukin-6) of the volunteers. Only ratings for
"solvent smell" were significantly increased during exposure.
Only two well documented epidemiology studies were available for occupational workers
exposed to 1,4-dioxane (Buffler et al., 1978, 062914: Thiess et al., 1976, 062943). These studies
24
-------
did not provide evidence of effects in humans; however, the cohort size and number of reported
cases were small.
4.1.1. Thiess et al.
A cross-sectional survey was conducted by Thiess et al. (1976, 062943) in German
workers exposed to 1,4-dioxane. The study evaluated health effects in 74 workers, including
24 who were still actively employed in 1,4-dioxane production at the time of the investigation,
23 previously exposed workers who were still employed by the manufacturer, and 27 retired or
deceased workers. The actively employed workers were between 32 and 62 years of age and had
been employed in 1,4-dioxane production for 5-41 years. Former workers (age range not given)
had been exposed to 1,4-dioxane for 3-38 years and retirees (age range not given) had been
exposed for 12-41 years. Air concentrations in the plant at the time of the study were
0.06-0.69 ppm. A simulation of previous exposure conditions (prior to 1969) resulted in air
measurements between 0.06 and 7.2 ppm.
Active and previously employed workers underwent a thorough clinical examination and
X-ray, and hematological and serum biochemistry parameters were evaluated. The examination
did not indicate pathological findings for any of the workers and no indication of malignant
disease was noted. Hematology results were generally normal. Serum transaminase levels were
elevated in 16 of the 47 workers studied; however, this finding was consistent with chronic
consumption of more than 80 g of alcohol per day, as reported for these workers. No liver
enlargement or jaundice was found. Renal function tests and urinalysis were normal in exposed
workers. Medical records of the 27 retired workers (15 living at the time of the study) were
reviewed. No symptoms of liver or kidney disease were reported and no cancer was detected.
Medical reasons for retirement did not appear related to 1,4-dioxane exposure (e.g., emphysema,
arthritis).
Chromosome analysis was performed on six actively employed workers and six control
persons (not characterized). Lymphocyte cultures were prepared and chromosomal aberrations
were evaluated. No differences were noted in the percent of cells with gaps or other
chromosome aberrations. Mortality statistics were calculated for 74 workers of different ages
and varying exposure periods. The proportional contribution of each of the exposed workers to
the total time of observation was calculated as the sum of man-years per 10-year age group.
Each person contributed one man-year per calendar year to the specific age group in which he
was included at the time. The expected number of deaths for this population was calculated from
the age-specific mortality statistics for the German Federal Republic for the years 1970-1973.
From the total of 1,840.5 person-years, 14.5 deaths were expected; however, only 12 deaths were
observed in exposed workers between 1964 and 1974. Two cases of cancer were reported,
including one case of lamellar epithelial carcinoma and one case of myelofibrosis leukemia.
These cancers were not considered to be the cause of death in these cases and other severe
25
-------
illnesses were present. Standardized mortality ratios (SMRs) for cancer did not significantly
differ from the control population (SMR for overall population = 0.83; SMR for 65-75-year-old
men = 1.61; confidence intervals (CIs) were not provided).
4.1.2. Buffleretal.
Buffler et al. (1978, 062914) conducted a mortality study on workers exposed to
1,4-dioxane at a chemical manufacturing facility in Texas. 1,4-Dioxane exposure was known to
occur in a manufacturing area and in a processing unit located 5 miles from the manufacturing
plant. Employees who worked between April 1, 1954, and June 30, 1975, were separated into
two cohorts based on at least 1 month of exposure in either the manufacturing plant
(100 workers) or the processing area (65 workers). Company records and follow-up techniques
were used to compile information on name, date of birth, gender, ethnicity, job assignment and
duration, and employment status at the time of the study. Date and cause of death were obtained
from copies of death certificates and autopsy reports (if available). Exposure levels for each job
category were estimated using the 1974 Threshold Limit Value for 1,4-dioxane (i.e., 50 ppm)
and information from area and personal monitoring. Exposure levels were classified as low
(<25 ppm), intermediate (50-75 ppm), and high (>75 ppm). Monitoring was not conducted prior
to 1968 in the manufacturing areas or prior to 1974 in the processing area; however, the study
authors assumed that exposures would be comparable, considering that little change had been
made to the physical plant or the manufacturing process during that time. Exposure to
1,4-dioxane was estimated to be below 25 ppm for all individuals in both cohorts.
Manufacturing area workers were exposed to several other additional chemicals and processing
area workers were exposed to vinyl chloride.
Seven deaths were identified in the manufacturing cohort and five deaths were noted for
the processing cohort. The average exposure duration was not greater for those workers who
died, as compared to those still living at the time of the study. Cancer was the underlying cause
of death for two cases from the manufacturing area (carcinoma of the stomach, alveolar cell
carcinoma) and one case from the processing area (malignant mediastinal tumor). The workers
from the manufacturing area were exposed for 28 or 38 months and both had a positive smoking
history (>1 pack/day). Smoking history was not available for processing area workers. The
single case of cancer in this area occurred in a 21-year-old worker exposed to 1,4-dioxane for
1 year. The mortality data for both industrial cohorts were compared to age-race-sex specific
death rates for Texas (1960-1969). Person-years of observation contributed by workers were
determined over five age ranges with each worker contributing one person-year for each year of
observation in a specific age group. The expected number of deaths was determined by applying
the Texas 1960-1969 death rate statistics to the number of person years calculated for each
cohort. The observed and expected number of deaths for overall mortality (i.e., all causes) was
comparable for both the manufacturing area (7 observed versus 4.9 expected) and the processing
26
-------
area (5 observed versus 4.9 expected). No significant excess in cancer-related deaths was
identified for both areas of the facility combined (3 observed versus 1.7 expected). A separate
analysis was performed to evaluate mortality in manufacturing area workers exposed to
1,4-dioxane for more than 2 years. Six deaths occurred in this group as compared to
4.1 expected deaths. The use of a conditional Poisson distribution indicated no apparent excess
in mortality or death due to malignant neoplasms in this study. It is important to note that the
cohorts evaluated were limited in size. In addition, the mean exposure duration was less than
5 years (<2 years for 43% of workers) and the latency period for evaluation was less than
10 years for 59% of workers. The study authors recommended a follow-up investigation to
allow for a longer latency period; however, no follow-up study of these workers has been
published.
4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN
ANIMALS - ORAL AND INHALATION
The majority of the subchronic and chronic studies conducted for 1,4-dioxane were oral
drinking water studies. Longer-term inhalation studies consisted of only one subchronic study
(Fairley et al., 1934, 062919) and one chronic study (Torkelson et al., 1974, 094807). These
studies were not sufficient to characterize the inhalation risks of 1,4-dioxane (see Section 4.2.2.).
4.2.1. OralToxicity
4.2.1.1. Subchronic Oral Toxicity
Six rats and six mice (unspecified strains) were given drinking water containing 1.25%
1,4-dioxane for up to 67 days (Fairley et al., 1934, 062919). Using reference BWs and drinking
water ingestion rates for rats and mice (U.S. EPA, 1988, 064560), it can be estimated that these
rats and mice received doses of approximately 1,900 and 3,300 mg/kg-day, respectively. Gross
pathology and histopathology were evaluated in all animals. Five of the six rats in the study died
or were sacrificed in extremis prior to day 34 of the study. Mortality was lower in mice, with
five of six mice surviving up to 60 days. Kidney enlargement was noted in 5/6 rats and 2/5 mice.
Renal cortical degeneration was observed in all rats and 3/6 mice. Large areas of necrosis were
observed in the cortex, while cell degeneration in the medulla was slight or absent. Tubular casts
were observed and vascular congestion and hemorrhage were present throughout the kidney.
Hepatocellular degeneration with vascular congestion was also noted in five rats and three mice.
For this assessment, EPA identified the tested doses of 1,900 mg/kg-day in rats and 3,300 mg/kg-
day in mice as the lowest-observed-adverse-effect-levels (LOAELs) for liver and kidney
degeneration in this study.
4.2.1.1.1. Stoner et al. 1,4-Dioxane was evaluated by Stoner et al. (1986, 064678)for its ability
to induce lung adenoma formation in A/J mice. Six- to 8-week-old male and female A/J mice
27
-------
(16/sex/group) were given 1,4-dioxane by gavage or i.p. injection, 3 times/week for 8 weeks.
Total cumulative dose levels were given as 24,000 mg/kg (oral), and 4,800, 12,000, or
24,000 mg/kg (i.p.)- Average daily dose estimates were calculated to be 430 mg/kg-day (oral),
and 86, 210, or 430 mg/kg-day (i.p.) by assuming an exposure duration of 56 days. The authors
indicated that i.p. doses represent the maximum tolerated dose (MTD), 0.5 times the MTD, and
0.2 times the MTD. Mice were killed 24 weeks after initiation of the bioassay, and lungs, liver,
kidney, spleen, intestines, stomach, thymus, salivary, and endocrine glands were examined for
gross lesions. Histopathology examination was performed if gross lesions were detected.
1,4-Dioxane did not induce lung tumors in male or female A/J mice in this study.
4.2.1.1.2. Stott etaL In the Stott et al. (1981, 063021) study, male Sprague Dawley rats (4-
6/group) were given average doses of 0, 10, or 1,000 mg/kg-day 1,4-dioxane (>99% pure) in
their drinking water, 7 days/week for 11 weeks. It should be noted that the methods description
in this report stated that the high dose was 100 mg/kg-day, while the abstract, results, and
discussion sections indicated that the high dose was 1,000 mg/kg-day. Rats were implanted with
a [6"3H]thymidine loaded osmotic pump 7 days prior to sacrifice. Animals were sacrificed by
cervical dislocation and livers were removed, weighed, and prepared for histopathology
evaluation. [3H]-Thymidine incorporation was measured by liquid scintillation spectroscopy.
An increase in the liver to BW ratio was observed in rats from the high dose group
(assumed to be 1,000 mg/kg-day). Histopathological alterations, characterized as minimal
centrilobular swelling, were also seen in rats from this dose group (incidence values were not
reported). Hepatic DNA synthesis, measured by [3H]-thymidine incorporation, was increased
1.5-fold in high-dose rats. No changes relative to control were observed for rats exposed to
10 mg/kg-day. EPA found a NOAEL value of 10 mg/kg-day and a LOAEL value of
1,000 mg/kg-day for this study based on histopathological changes in the liver.
Stott et al. (1981, 063021) also performed several acute experiments designed to evaluate
potential mechanisms for the carcinogenicity of 1,4-dioxane. These experiments are discussed
separately in Section 4.5.2 (Mechanistic Studies).
4.2.1.1.3. Kano et al. In the Kano et al. (2008, 196245) study, groups of 6-week-old
F344/DuCrj rats (10/sex/group) and Crj:BDFl mice (10/sex/group) were administered
1,4-dioxane (>99% pure) in the drinking water for 13 weeks. The animals were observed daily
for clinical signs of toxicity. Food consumption and BWs were measured once per week and
water consumption was measured twice weekly. Food and water were available ad libitum. The
concentrations of 1,4-dioxane in the water for rats and mice were 0, 640, 1,600, 4,000, 10,000, or
25,000 ppm. The investigators used data from water consumption and BW changes to calculate
a daily intake of 1,4-dioxane by the male and female animals. Thus, male rats received doses of
approximately 0, 52, 126, 274, 657, and 1,554 mg 1,4-dioxane/kg-day and female rats received
28
-------
0, 83, 185, 427, 756, and 1,614 mg/kg-day. Male mice received 0, 86, 231, 585, 882, or
1,570 mg/kg-day and female mice received 0, 170, 387, 898, 1,620, or 2,669 mg/kg-day.
No information was provided as to when the blood and urine samples were collected.
Hematology analysis included red blood cell (RBC) count, hemoglobin, hematocrit, mean
corpuscular volume (MCV), platelet count, white blood cell (WBC) count, and differential
WBCs. Serum biochemistry included total protein, albumin, bilirubin, glucose, cholesterol,
triglyceride (rat only), alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate
dehydrogenase (LDH), leucine aminopeptidase (LAP), alkaline phosphatase (ALP), creatinine
phosphokinase (CPK) (rat only), urea nitrogen, creatinine (rat only), sodium, potassium,
chloride, calcium (rat only), and inorganic phosphorous (rat only). Urinalysis parameters were
pH, protein, glucose, ketone body, bilirubin (rat only), occult blood, and urobilinogen. Organ
weights (brain, lung, liver, spleen, heart, adrenal, testis, ovary, and thymus) were measured, and
gross necropsy and histopathologic examination of tissues and organs were performed on all
animals (skin, nasal cavity, trachea, lungs, bone marrow, lymph nodes, thymus, spleen, heart,
tongue, salivary glands, esophagus, stomach, small and large intestine, liver, pancreas, kidney,
urinary bladder, pituitary thyroid adrenal, testes, epididymis, seminal vesicle, prostate, ovary,
uterus, vagina, mammary gland, brain, spinal cord, sciatic nerve, eye, Harderian gland, muscle,
bone, and parathyroid). Dunnett's test and ^ test were used to assess the statistical significance
of changes in continuous and discrete variables, respectively.
Clinical signs of toxicity in rats were not discussed in the study report. One female rat in
the high dose group (1,614 mg/kg-day) group died, but cause and time of death were not
specified. Final BWs were reduced at the two highest dose levels in females (12 and 21%) and
males (7 and 21%), respectively. Food consumption was reduced 13% in females at
1,614 mg/kg-day and 8% in 1,554 mg/kg-day males. A dose-related decrease in water
consumption was observed in male rats starting at 52 mg/kg-day (15%) and in females starting at
185 mg/kg-day (12%). Increases in RBCs, hemoglobin, hematocrit, and neutrophils, and a
decrease in lymphocytes were observed in males at 1554 mg/kg-day. In females, MCV was
decreased at doses > 756 mg/kg and platelets were decreased at 1,614 mg/kg-day. With the
exception of the 30% increase in neutrophils in high-dose male rats, hematological changes were
within 2-15% of control values. Total serum protein and albumin were significantly decreased
in males at doses > 274 mg/kg-day and in females at doses > 427 mg/kg-day. Additional
changes in high-dose male and female rats included decreases in glucose, total cholesterol,
triglycerides, and sodium (and calcium in females), and increases in ALT (males only), AST,
ALP, and LAP. Serum biochemistry parameters in treated rats did not differ more than twofold
from control values. Urine pH was decreased in males at > 274 mg/kg-day and in females at
> 756 mg/kg-day.
Kidney weights were increased in females at >185 mg/kg-day with a maximum increase
of 15% and 44% at 1,614 mg/kg-day for absolute and relative kidney weight, respectively. No
29
-------
organ weight changes were noted in male rats. Histopathology findings in rats that were related
to exposure included nuclear enlargement of the respiratory epithelium, nuclear enlargement of
the olfactory epithelium, nuclear enlargement of the tracheal epithelium, hepatocyte swelling of
the centrilobular area of the liver, vacuolar changes in the liver, granular changes in the liver,
single cell necrosis in the liver, nuclear enlargement of the proximal tubule of the kidneys,
hydropic changes in the proximal tubule of the kidneys, and vacuolar changes in the brain. The
incidence data for histopathological lesions in rats are presented in Table 4-1. The effects that
occurred at the lowest doses were nuclear enlargement of the respiratory epithelium in the nasal
cavity and hepatocyte swelling in the central area of the liver in male rats. Based on these
histopathological findings the study authors identified the LOAEL as 126 mg/kg-day and the
NOAEL as 52 mg/kg-day.
30
-------
Table 4-1. Incidence of histopathological lesions in F344/DuCrj rats
exposed to 1,4-dioxane in drinking water for 13 weeks
Effect
Nuclear enlargement; nasal respiratory epithelium
Nuclear enlargement; nasal olfactory epithelium
Nuclear enlargement; tracheal epithelium
Hepatocyte swelling
Vacuolic change; liver
Granular change; liver
Single cell necrosis; liver
Nuclear enlargement; renal proximal tubule
Hydropic change; renal proximal tubule
Vacuolic change; brain
Nuclear enlargement; nasal respiratory epithelium
Nuclear enlargement; nasal olfactory epithelium
Nuclear enlargement; tracheal epithelium
Hepatocyte swelling
Vacuolic change; liver
Granular change; liver
Single cell necrosis; liver
Nuclear enlargement; proximal tubule
Hydropic change; proximal tubule
Vacuolic change; brain
Male dose (mg/kg-day)a
0
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
52
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
126
9/10b
0/10
0/10
9/10b
0/10
0/10
0/10
0/10
0/10
0/10
274
10/10b
10/10b
10/10b
10/10b
0/10
5/10c
5/10c
1/10
0/10
0/10
657
9/10b
9/10b
10/10b
10/10b
10/10b
2/10
2/10
5/10c
0/10
0/10
1,554
10/10b
10/10b
10/10b
10/10b
10/10b
10/10b
10/10b
9/10b
7/10b
10/10b
Female dose (mg/kg-day)a
0
0/10
0/10
0/10
0/10
0/10
2/10
2/10
0/10
0/10
0/10
83
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
185
5/10c
0/10
0/10
0/10
0/10
1/10
1/10
0/10
0/10
0/10
427
10/10b
9/10b
9/10b
0/10
0/10
5/10c
5/10
0/10
0/10
0/10
756
10/10b
10/10b
10/10b
9/10b
0/10
5/10c
5/10
8/10b
0/10
0/10
1,614
8/9b
8/9b
9/9b
9/9b
9/9b
8/9b
8/9b
9/9b
5/9c
9/9b
"Data are presented for sacrificed animals.
V < 0.01 by x2 test.
°p < 0.05.
Source: Kano et al. (2008, 196245)
Clinical signs of toxicity in mice were not discussed in the study report One male mouse
in the high-dose group (1,570 mg/kg-day) died, but no information was provided regarding cause
or time of death. Final BWs were decreased 29% in male mice at 1,570 mg/kg-day, but changed
less than 10% relative to controls in the other male dose groups and in female mice. Food
consumption was not significantly reduced in any exposure group. Water consumption was
reduced 14-18% in male mice exposed to 86, 231, or 585 mg/kg-day. Water consumption was
further decreased by 48 and 70% in male mice exposed to 882 and 1,570 mg/kg-day,
respectively. Water consumption was also decreased 31 and 57% in female mice treated with
1,620 and 2,669 mg/kg-day, respectively. An increase in MCV was observed in the two highest
dose groups in both male (882 and 1,570 mg/kg-day) and female mice (1,620 and
2,669 mg/kg-day). Increases in RBCs, hemoglobin, and hematocrit were also observed in high
31
-------
dose males (1,570 mg/kg-day). Hematological changes were within 2-15% of control values.
Serum biochemistry changes in exposed mice included decreased total protein (at
1,570 mg/kg-day in males, >1,620 mg/kg-day in females), decreased glucose (at
1,570 mg/kg-day in males, >1,620 mg/kg-day in females), decreased albumin (at
1,570 mg/kg-day in males, 2,669 mg/ kg-day in females), decreased total cholesterol
(> 585 mg/kg-day in males, >1,620 mg/kg-day in females), increased serum ALT (at
1,570 mg/kg-day in males, > 620 mg/kg-day in females), increased AST (at 1,570 mg/kg-day in
males, 2,669 mg/kg-day in females), increased ALP (> 585 mg/kg-day in males, 2,669 mg/kg-
day in females), and increased LDH (in females only at doses > 1,620 mg/kg-day). With the
exception of a threefold increase in ALT in male and female mice, serum biochemistry
parameters in treated rats did not differ more than twofold from control values. Urinary pH was
decreased in males at > 882 mg/kg-day and in females at > 1,620 mg/kg-day.
Absolute and relative lung weights were increased in males at 1,570 mg/kg-day and in
females at 1,620 and 2,669 mg/kg-day. Absolute kidney weights were also increased in females
at 1,620 and 2,669 mg/kg-day and relative kidney weight was elevated at 2,669 mg/kg-day.
Histopathology findings in mice that were related to exposure included nuclear enlargement of
the respiratory epithelium, nuclear enlargement of the olfactory epithelium, eosinophilic change
in the olfactory epithelium, vacuolic change in the olfactory nerve, nuclear enlargement of the
tracheal epithelium, accumulation of foamy cells in the lung and bronchi, nuclear enlargement
and degeneration of the bronchial epithelium, hepatocyte swelling of the centrilobular area of the
liver, and single cell necrosis in the liver. The incidence data for histopathological lesions in
mice are presented in Table 4-2. Based on the changes in the bronchial epithelium in female
mice, the authors identified the dose level of 387 mg/kg-day as the LOAEL for mice; the
NOAEL was 170 mg/kg-day (Kano et al., 2008, 196245).
32
-------
Table 4-2. Incidence of histopathological lesions in CrjrBDFl mice
exposed to 1,4-dioxane in drinking water for 13 weeks
Effect
Nuclear enlargement; nasal respiratory epithelium
Eosinophilic change; nasal respiratory epithelium
Nuclear enlargement; nasal olfactory epithelium
Eosinophilic change; nasal olfactory epithelium
Vacuolic change; olfactory nerve
Nuclear enlargement; tracheal epithelium
Accumulation of foamy cells; lung/bronchi
Nuclear enlargement; bronchial epithelium
Degeneration; bronchial epithelium
Hepatocyte swelling
Single cell necrosis; liver
Nuclear enlargement; nasal respiratory epithelium
Eosinophilic change; nasal respiratory epithelium
Nuclear enlargement; nasal olfactory epithelium
Eosinophilic change; nasal olfactory epithelium
Vacuolic change; olfactory nerve
Nuclear enlargement; tracheal epithelium
Accumulation of foamy cells; lung/bronchi
Nuclear enlargement; bronchial epithelium
Degeneration; bronchial epithelium
Hepatocyte swelling
Single cell necrosis; liver
Male dose (mg/kg-day)a
0
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
86
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
231
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
585
2/10
0/10
9/10c
0/10
0/10
7/10c
0/10
9/10c
0/10
10/10C
5/10b
882
5/10b
0/10
10/10C
0/10
0/10
9/10c
0/10
9/10c
0/10
10/10C
10/10C
1,570
0/9
5/9b
9/9c
6/9c
9/9c
9/9c
6/9c
9/9c
8/9c
9/9c
9/9c
Female dose (mg/kg-day)a
0
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
170
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
1/10
0/10
387
0/10
1/10
0/10
0/10
0/10
2/10
0/10
10/10C
0/10
1/10
0/10
898
3/10
1/10
6/10b
1/10C
0/10
9/10c
0/10
10/10C
0/10
10/10C
7/10c
1,620
3/10
5/10b
10/10C
6/10b
2/10
10/10C
10/10C
10/10C
7/10c
10/10C
10/10C
2,669
7/10c
9/10c
10/10C
6/10b
8/10c
10/10C
10/10C
10/10C
10/10C
9/10b
9/10c
"Data are presented for sacrificed animals.
V < 0.01 by x2 test.
cp < 0.05.
Source: Kano et al (2008,196245).
4.2.1.1.4. Yamamoto et al. Studies (Yamamoto et al., 1998, 196114: Yamamoto et al., 1998,
594544) in rasH2 transgenic mice carrying the human prototype c-Ha-ras gene have been
investigated as a bioassay model for rapid carcinogenicity testing. As part of validation studies
of this model, 1,4-dioxane was one of many chemicals that were evaluated. RasH2 transgenic
mice were Fl offspring of transgenic male C57BLr6J and normal female BALB/cByJ mice.
CB6Fi mice were used as a nontransgenic control. Seven- to nine-week-old mice (10-15/group)
were exposed to 0, 0.5, or 1% 1,4-dioxane in drinking water for 26 weeks. An increase in lung
33
-------
adenomas was observed in treated transgenic mice, as compared to treated nontransgenic mice.
The tumor incidence in transgenic animals, however, was not greater than that observed in
vehicle-treated transgenic mouse controls. Further study details were not provided.
4.2.1.2. Chronic Oral Toxicity and Carcinogenicity
4.2.1.2.1. Argus et al. Twenty-six adult male Wistar rats (Argus et al., 1965, 017009) weighing
between 150 and 200 g were exposed to 1,4-dioxane (purity not reported) in the drinking water
at a concentration of 1% for 64.5 weeks. A group of nine untreated rats served as control. Food
and water were available ad libitum. The drinking water intake for treated animals was reported
to be 30 mL/day, resulting in a dose/rat of 300 mg/day. Using a reference BW of 0.462 kg for
chronic exposure to male Wistar rats (U.S. EPA, 1988, 064560), it can be estimated that these
rats received daily doses of approximately 640 mg/kg-day. All animals that died or were killed
during the study underwent a complete necropsy. A list of specific tissues examined
microscopically was not provided; however, it is apparent that the liver, kidneys, lungs,
lymphatic tissue, and spleen were examined. No statistical analysis of the results was conducted.
Six of the 26 treated rats developed hepatocellular carcinomas, and these rats had been
treated for an average of 452 days (range, 448-455 days). No liver tumors were observed in
control rats. In two rats that died after 21.5 weeks of treatment, histological changes appeared to
involve the entire liver. Groups of cells were found that had enlarged hyperchromic nuclei. Rats
that died or were killed at longer intervals showed similar changes, in addition to large cells with
reduced cytoplasmic basophilia. Animals killed after 60 weeks of treatment showed small
neoplastic nodules or multifocal hepatocellular carcinomas. No cirrhosis was observed in this
study. Many rats had extensive changes in the kidneys often resembling glomerulonephritis,
however, incidence data was not reported for these findings. This effect progressed from
increased cellularity to thickening of the glomerular capsule followed by obliteration of the
glomeruli. One treated rat had an early transitional cell carcinoma in the kidney's pelvis; this rat
also had a large tumor in the liver. The lungs from many treated and control rats (incidence not
reported) showed severe bronchitis with epithelial hyperplasia and marked peribronchial
infiltration, as well as multiple abscesses. One rat treated with 1,4-dioxane developed leukemia
with infiltration of all organs, particularly the liver and spleen, with large, round, isolated
neoplastic cells. In the liver, the distribution of cells in the sinusoids was suggestive of myeloid
leukemia. The dose of 640 mg/kg-day tested in this study was a free-standing LOAEL,
identified by EPA, for glomerulonephritis in the kidney and histological changes in the liver
(hepatocytes with enlarged hyperchromic nuclei, large cells with reduced cytoplasmic
basophilia).
34
-------
4.2.1.2.2. Argus et al.; Hoch-Ligeti et al. Five groups (28-32/dose group) of male
Sprague Dawley rats (2-3 months of age) weighing 110-230 g at the beginning of the experiment
were administered 1,4-dioxane (purity not reported) in the drinking water for up to 13 months at
concentrations of 0, 0.75, 1.0, 1.4, or 1.8% (Argus et al., 1973, 062912: Hoch-Ligeti et al., 1970,
062926). The drinking water intake was determined for each group over a 3-day measurement
period conducted at the beginning of the study and twice during the study (weeks were not
specified). The rats were killed with ether at 16 months or earlier if nasal tumors were clearly
observable. Complete autopsies were apparently performed on all animals, but only data from
the nasal cavity and liver were presented and discussed. The nasal cavity was studied
histologically only from rats in which gross tumors in these locations were present; therefore,
early tumors may have been missed and pre-neoplastic changes were not studied. No statistical
analysis of the results was conducted. Assuming a BW of 0.523 kg for an adult male
Sprague Dawley rat (U.S. EPA, 1988, 064560) and a drinking water intake of 30 mL/day as
reported by the study authors, dose estimates were 0, 430, 574, 803, and 1,032 mg/kg-day. The
progression of liver tumorigenesis was evaluated by an additional group of 10 male rats
administered 1% 1,4-dioxane in the drinking water (574 mg/kg-day), 5 of which were sacrificed
after 8 months of treatment and 5 were killed after 13 months of treatment. Liver tissue from
these rats and control rats was processed for electron microscopy examination.
Nasal cavity tumors were observed upon gross examination in six rats (1/30 in the 0.75%
group, 1/30 in the 1.0% group, 2/30 in the 1.4% group, and 2/30 in the 1.8% group). Gross
observation showed the tumors visible either at the tip of the nose, bulging out of the nasal
cavity, or on the back of the nose covered by intact or later ulcerated skin. As the tumors
obstructed the nasal passages, the rats had difficulty breathing and lost weight rapidly. No
neurological signs or compression of the brain were observed. In all cases, the tumors were
squamous cell carcinomas with marked keratinization and formation of keratin pearls. Bony
structure was extensively destroyed in some animals with tumors, but there was no invasion into
the brain. In addition to the squamous carcinoma, two adenocarcinomatous areas were present.
One control rat had a small, firm, well-circumscribed tumor on the back of the nose, which
proved to be subcutaneous fibroma. The latency period for tumor onset was 329-487 days.
Evaluation of the latent periods and doses received did not suggest an inverse relationship
between these two parameters.
Argus et al. (1973, 062912) studied the progression of liver tumorigenesis by electron
microscopy of liver tissues obtained following interim sacrifice at 8 and 13 months of exposure
(5 rats/group, 574 mg/kg-day). The first change observed in the liver was an increase in the size
of the nucleus of the hepatocytes, mostly in the periportal area. Precancerous changes were
characterized by disorganization of the rough endoplasmic reticulum, an increase in smooth
endoplasmic reticulum, and a decrease in glycogen and increase in lipid droplets in hepatocytes.
35
-------
These changes increased in severity in the hepatocellular carcinomas in rats exposed to
1,4-dioxane for 13 months.
Three types of liver nodules were observed in exposed rats at 13-16 months. The first
consisted of groups of cells with reduced cytoplasmic basophilia and a slightly nodular
appearance as viewed by light microscopy. The second type of circumscribed nodule was
described consisting of large cells, apparently filled and distended with fat. The third type of
nodule was described as finger-like strands, 2-3 cells thick, of smaller hepatocytes with large
hyperchromic nuclei and dense cytoplasm. This third type of nodule was designated as an
incipient hepatoma, since it showed all the histological characteristics of a fully developed
hepatoma. All three types of nodules were generally present in the same liver. Cirrhosis of the
liver was not observed. The numbers of incipient liver tumors and hepatomas in rats from this
study (treated for 13 months and observed at 13-16 months) are presented in Table 4-3.
Table 4-3. Number of incipient liver tumors and hepatomas in male
Sprague- Dawley rats exposed to 1,4-dioxane in drinking water for
13 months
Dose (mg/kg-day)a
430
574
803
1,032
Incipient tumors
4
9
13
11
Hepatomas
0
0
3
12
Total
4
9
16
23
aPrecise incidences cannot be calculated since the number of rats per group was reported as 28-32; incidence in
control rats was not reported; no statistical analysis of the results was conducted in the study.
Source: Argus et al. (1973, 062912).
Treatment with all dose levels of 1,4-dioxane induced marked kidney alterations, but
quantitative incidence data were not provided. Qualitatively, the changes indicated
glomerulonephritis and pyelonephritis, with characteristic epithelial proliferation of Bowman's
capsule, periglomerular fibrosis, and distension of tubules. No kidney tumors were found. No
tumors were found in the lungs. One rat at the 1.4% treatment level showed early peripheral
adenomatous change of the alveolar epithelium and another rat in the same group showed
papillary hyperplasia of the bronchial epithelium. The lowest dose tested (430 mg/kg-day) was
considered a LOAEL by EPA for hepatic and renal effects in this study.
4.2.1.2.3. Hoch-Ligeti and Argus. Hoch-Ligeti and Argus (1970, 029386) provided a brief
account of the results of exposure of guinea pigs to 1,4-dioxane. A group of 22 male guinea pigs
(neither strain nor age provided) was administered 1,4-dioxane (purity not provided) in the
drinking water for at least 23 months and possibly up to 28 months. The authors stated that the
concentration of 1,4-dioxane was regulated so that normal growth of the guinea pigs was
36
-------
maintained, and varied 0.5-2% (no further information provided). The investigators further
stated that the amount of 1,4-dioxane received by the guinea pigs over a 23-month period was
588-635 g. Using a reference BW of 0.89 kg for male guinea pigs in a chronic study (U.S. EPA,
1988, 064560) and assuming an exposure period of 700 days (23 months), the guinea pigs
received doses between 944 and 1,019 mg 1,4-dioxane/kg-day. A group often untreated guinea
pigs served as controls. All animals were sacrificed within 28 months, but the scope of the
postmortem examination was not provided.
Nine treated guinea pigs showed peri- or intrabronchial epithelial hyperplasia and nodular
mononuclear infiltration in the lungs. Also, two guinea pigs had carcinoma of the gallbladder,
three had early hepatomas, and one had an adenoma of the kidney. Among the controls, four
guinea pigs had peripheral mononuclear cell accumulation in the lungs, and only one had
hyperplasia of the bronchial epithelium. One control had formation of bone in the bronchus. No
further information was presented in the brief narrative of this study. Given the limited reporting
of the results, a NOAEL or LOAEL value was not provided for this study.
4.2.1.2.4. Kociba et al. Groups of 6-8-week-old Sherman rats (60/sex/dose level) were
administered 1,4-dioxane (purity not reported) in the drinking water at levels of 0 (controls),
0.01, 0.1, or 1.0% for up to 716 days (Kociba et al., 1974, 062929). The drinking water was
prepared twice weekly during the first year of the study and weekly during the second year of the
study. Water samples were collected periodically and analyzed for 1,4-dioxane content by
routine gas liquid chromatography. Food and water were available ad libitum. Rats were
observed daily for clinical signs of toxicity, and BWs were measured twice weekly during the
first month, weekly during months 2-7, and biweekly thereafter. Water consumption was
recorded at three different time periods during the study: days 1-113, 114-198, and 446-460.
Blood samples were collected from a minimum of five male and five female control and high-
dose rats during the 4th, 6th, 12th, and 18th months of the study and at termination. Each sample
was analyzed for packed cell volume, total erythrocyte count, hemoglobin, and total and
differential WBC counts. Additional endpoints evaluated included organ weights (brain, liver,
kidney, testes, spleen, and heart) and gross and microscopic examination of major tissues and
organs (brain, bone and bone marrow, ovaries, pituitary, uterus, mesenteric lymph nodes, heart,
liver, pancreas, spleen, stomach, prostate, colon, trachea, duodenum, kidneys, esophagus,
jejunum, testes, lungs, spinal cord, adrenals, thyroid, parathyroid, nasal turbinates, and urinary
bladder). The number of rats with tumors, hepatic tumors, hepatocellular carcinomas, and nasal
carcinomas were analyzed for statistical significance with Fisher's Exact test (one-tailed),
comparing each treatment group against the respective control group. Survival rates were
compared using j^ Contingency Tables and Fisher's Exact test. Student's test was used to
compare hematological parameters, body and organ weights, and water consumption of each
treatment group with the respective control group.
37
-------
Male and female rats in the high-dose group (1% in drinking water) consumed slightly
less water than controls. BW gain was depressed in the high-dose groups relative to the other
groups almost from the beginning of the study (food consumption data were not provided).
Based on water consumption and BW data for specific exposure groups, Kociba et al. (1974,
062929) calculated mean daily doses of 9.6, 94, and 1,015 mg/kg-day for male rats and 19, 148,
and 1,599 mg/kg-day for female rats during days 114-198 for the 0.01, 0.1, and 1.0%
concentration levels, respectively. Treatment with 1,4-dioxane significantly increased mortality
among high-dose males and females beginning at about 2-4 months of treatment. These rats
showed degenerative changes in both the liver and kidneys. From the 5th month on, mortality
rates of control and treated groups were not different. There were no treatment-related
alterations in hematological parameters. At termination, the only alteration in organ weights
noted by the authors was a significant increase in absolute and relative liver weights in male and
female high-dose rats (data not shown). Histopathological lesions were restricted to the liver and
kidney from the mid- and high-dose groups and consisted of variable degrees of renal tubular
epithelial and hepatocellular degeneration and necrosis (no quantitative incidence data were
provided). Rats from these groups also showed evidence of hepatic regeneration, as indicated by
hepatocellular hyperplastic nodule formation and evidence of renal tubular epithelial
regenerative activity (observed after 2 years of exposure). These changes were not seen in
controls or in low-dose rats. The authors determined a LOAEL of 94 mg/kg-day based on the
liver and kidney effects in male rats. The corresponding NOAEL value was 9.6 mg/kg-day.
Histopathological examination of all the rats in the study revealed a total of 132 tumors in
114 rats. Treatment with 1% 1,4-dioxane in the drinking water resulted in a significant increase
in the incidence of hepatic tumors (hepatocellular carcinomas in six males and four females). In
addition, nasal carcinomas (squamous cell carcinoma of the nasal turbinates) occurred in one
high-dose male and two high-dose females. Since 128 out of 132 tumors occurred in rats from
the 12th to the 24th month, Kociba et al. (1974, 062929) assumed that the effective number of
rats was the number surviving at 12 months, which was also when the first hepatic tumor was
noticed. The incidences of liver and nasal tumors from Kociba et al. (1974, 062929) are
presented in Table 4-4. Tumors in other organs were not elevated when compared to control
incidence and did not appear to be related to 1,4-dioxane administration.
38
-------
Table 4-4. Incidence of liver and nasal tumors in male and female
Sherman rats (combined) treated with 1,4-dioxane in the drinking water for
2 years
Dose in mg/kg-day
(average of male
and female dose)
0
14
121
1307
Effective
number of
animals"
106
110
106
66
Number of tumor-
bearing animals
31
34
28
21
Number of animals
Hepatic tumors
(all types)
2
0
1
12b
Hepatocellular
carcinomas
1
0
1
10C
Nasal
carcinomas
0
0
0
od
J
"Rats surviving until 12 months on study.
bp = 0.00022 by one-tailed Fisher's Exact test.
°p = 0.00033 by one-tailed Fisher's Exact test.
dp = 0.05491 by one-tailed Fisher's Exact test.
Source: Used with permission from Elsevier, Ltd., Kociba et al. (1974, 062929).
The high-dose level was the only dose that increased the formation of liver tumors over
control (males 1,015 mg/kg-day; females 1,599 mg/kg-day) and also caused significant liver and
kidney toxicity in these animals. The mid-dose group (males 94 mg/kg-day; females 148 mg/kg-
day) experienced hepatic and renal degeneration and necrosis, as well as regenerative
hyperplasia in hepatocytes and renal tubule epithelial cells. No increase in tumor formation was
seen in the mid-dose group. No toxicity or tumor formation was observed in either sex in the
low-dose (males 9.6 mg/kg-day; females 19 mg/kg-day) group of rats.
4.2.1.2.5. National Cancer Institute (NCI). Groups of Osborne-Mendel rats (35/sex/dose) and
B6C3Fi mice (50/sex/dose) were administered 1,4-dioxane (> 99.95% pure) in the drinking
water for 110 or 90 weeks, respectively, at levels of 0 (matched controls), 0.5, or 1% (NCI, 1978,
062935). Solutions of 1,4-dioxane were prepared with tap water. The report indicated that at
105 weeks from the earliest starting date, a new necropsy protocol was instituted. This affected
the male controls and high-dose rats, which were started a year later than the original groups of
rats and mice. Food and water were available ad libitum. Endpoints monitored in this bioassay
included clinical signs (twice daily), BWs (once every 2 weeks for the first 12 weeks and every
month during the rest of the study), food and water consumption (once per month in 20% of the
animals in each group during the second year of the study), and gross and microscopic
appearance of all major organs and tissues (mammary gland, trachea, lungs and bronchi, heart,
bone marrow, liver, bile duct, spleen, thymus, lymph nodes, salivary gland, pancreas, kidney,
esophagus, thyroid, parathyroid, adrenal, gonads, brain, spinal cord, sciatic nerve, skeletal
muscle, stomach, duodenum, colon, urinary bladder, nasal septum, and skin). Based on the
measurements of water consumption and BWs, the investigators calculated average daily intakes
of 1,4-dioxane of 0, 240, and 530 mg/kg-day in male rats, 0, 350, and 640 mg/kg-day in female
39
-------
rats, 0, 720, and 830 mg/kg-day in male mice, and 0, 380, and 860 mg/kg-day in female mice.
According to the report, the doses of 1,4-dioxane in high-dose male mice were only slightly
higher than those of the low-dose group due to decreased fluid consumption in high-dose male
mice.
During the second year of the study, the BWs of high-dose rats were lower than controls,
those of low-dose males were higher than controls, and those of low-dose females were
comparable to controls. The fluctuations in the growth curves were attributed to mortality by the
investigators; quantitative analysis of BW changes was not done. Mortality was significantly
increased in treated rats, beginning at approximately 1 year of study. Analysis of Kaplan-Meier
curves (plots of the statistical estimates of the survival probability function) revealed significant
positive dose-related trends (p < 0.001, Tarone test). In male rats, 33/35 (94%) in the control
group, 26/35 (74%) in the mid-dose group, and 33/35 (94%) in the high-dose group were alive
on week 52 of the study. The corresponding numbers for females were 35/35 (100%), 30/35
(86%), and 29/35 (83%). Nonneoplastic lesions associated with treatment with 1,4-dioxane were
seen in the kidneys (males and females), liver (females only), and stomach (males only). Kidney
lesions consisted of vacuolar degeneration and/or focal tubular epithelial regeneration in the
proximal cortical tubules and occasional hyaline casts. Elevated incidence of hepatocytomegaly
also occurred in treated female rats. Gastric ulcers occurred in treated males, but none were seen
in controls. The incidence of pneumonia was increased above controls in high-dose female rats.
The incidence of nonneoplastic lesions in rats following drinking water exposure to 1,4-dioxane
is presented in Table 4-5. EPA identified the LOAEL in rats from this study as 240 mg/kg-day
for increased incidence of gastric ulcer and cortical tubular degeneration in the kidney in males;
a NOAEL was not established.
Table 4-5. Incidence of nonneoplastic lesions in Osborne-Mendel rats
exposed to 1,4-dioxane in drinking water
Cortical tubule degeneration
Hepatocytomegaly
Gastric ulcer
Pneumonia
Males (mg/kg-day)
0
0/3 r
5/31
(16%)
0/30a
8/30
(27%)
240
20/3 lb
(65%)
3/32
(9%)
5/28b
(18%)
15/31
(48%)
530
27/3 3b
(82%)
11/33
(33%)
5/30b
(17%)
14/33
(42%)
Females (mg/kg-day)
0
0/3 r
7/3 r
(23%)
0/31
6/30a
(20%)
350
0/34
11/33
(33%)
1/33
(3%)
5/34
(15%)
640
10/32b
(31%)
17/32b
(53%)
1/30
(3%)
25/32b
(78%)
aStatistically significant trend for increased incidence by Cochran-Armitage test (p < 0.05) performed for this
review.
blncidence significantly elevated compared to control by Fisher's Exact test (p < 0.05) performed for this review.
Source: NCI (1978, 062935).
40
-------
Neoplasms associated with 1,4-dioxane treatment were limited to the nasal cavity
(squamous cell carcinomas, adenocarcinomas, and one rhabdomyoma) in both sexes, liver
(hepatocellular adenomas) in females, and testis/epididymis (mesotheliomas) in males. The first
tumors were seen at week 52 in males and week 66 in females. The incidence of squamous cell
carcinomas in the nasal turbinates in male and female rats is presented in Table 4-6. Squamous
cell carcinomas were first seen on week 66 of the study. Morphologically, these tumors varied
from minimal foci of locally invasive squamous cell proliferation to advanced growths consisting
of extensive columns of epithelial cells projecting either into free spaces of the nasal cavity
and/or infiltrating into the submucosa. Adenocarcinomas of the nasal cavity were observed in
3 of 34 high-dose male rats, 1 of 35 low-dose female rats, and 1 of 35 high-dose female rats.
The single rhabdomyoma (benign skeletal muscle tumor) was observed in the nasal cavity of a
male rat from the low-dose group. A subsequent re-examination of the nasal tissue sections by
Goldsworthy et al. (1991, 062925) concluded that the location of the tumors in the nasal
apparatus was consistent with the possibility that the nasal tumors resulted from inhalation of
water droplets by the rats (see Section 4.5.2 for more discussion of Goldsworthy et al. (1991,
062925)).
Table 4-6. Incidence of nasal cavity squamous cell carcinoma and liver
hepatocellular adenoma in Osborne-Mendel rats exposed to 1,4-dioxane in
drinking water
Males (mg/kg-day)a
Nasal cavity squamous cell carcinoma
Hepatocellular adenoma
0
0/33 (0%)
2/31(6%)
240b
12/33 (36%)
2/32 (6%)
530
16/34 (47%)c
1/33 (3%)
Females (mg/kg-day)a
Nasal cavity squamous cell carcinoma
Hepatocellular adenoma
0
0/34 (0%)d
0/31 (0%)f
350
10/35 (29%)e
10/33 (30%)e
640
8/35 (23%)c
ll/32(34%)e
"Tumor incidence values were not adjusted for mortality.
bGroup not included in statistical analysis by NCI because the dose group was started a year earlier without
appropriate controls.
°p < 0.003 by Fisher's Exact test pair-wise comparison with controls.
dp = 0.008 by Cochran-Armitage test.
ep < 0.001 by Fisher's Exact test pair-wise comparison with controls.
fp = 0.001 by Cochran-Armitage test.
p = 0.001 y ocran-rmtag
Source: NCI (1978. 062935).
The incidence of hepatocellular adenomas in male and female rats is presented in Table
4-6. Hepatocellular adenomas were first observed in high-dose females in week 70 of the study.
These tumors consisted of proliferating hepatic cells oriented as concentric cords. Hepatic cell
size was variable; mitoses and necrosis were rare. Mesothelioma of the vaginal tunics of the
41
-------
testis/epididymis was seen in male rats (2/33, 4/33, and 5/34 in controls, low-, and high-dose
animals, respectively). The difference between the treated groups and controls was not
statistically significant. These tumors were characterized as rounded and papillary projections of
mesothelial cells, each supported by a core of fibrous tissue. Other reported neoplasms were
considered spontaneous lesions not related to treatment with 1,4-dioxane.
In mice, mean BWs of high-dose female mice were lower than controls during the second
year of the study, while those of low-dose females were higher than controls. In males, mean
BWs of high-dose animals were higher than controls during the second year of the study.
According to the investigators, these fluctuations could have been due to mortality; no
quantitative analysis of BWs was done. No other clinical signs were reported. Mortality was
significantly increased in female mice (p < 0.001, Tarone test), beginning at approximately
80 weeks on study. The numbers of female mice that survived to 91 weeks were 45/50 (90%) in
the control group, 39/50 (78%) in the low-dose group, and 28/50 (56%) in the high-dose group.
In males, at least 90% of the mice in each group were still alive at week 91. Nonneoplastic
lesions that increased significantly due to treatment with 1,4-dioxane were pneumonia in males
and females and rhinitis in females. The incidences of pneumonia were 1/49 (2%), 9/50 (18%),
and 17/47 (36%) in control, low-dose, and high-dose males, respectively; the corresponding
incidences in females were 2/50 (4%), 33/47 (70%), and 32/36 (89%). The incidences of rhinitis
in female mice were 0/50, 7/48 (14%), and 8/39 (21%) in control, low-dose, and high-dose
groups, respectively. Pair-wise comparisons of low-dose and high-dose incidences with controls
for incidences of pneumonia and rhinitis in females using Fisher's Exact test (done for this
review) yielded^-values < 0.001 in all cases. Incidences of other lesions were considered to be
similar to those seen in aging mice. The authors stated that hepatocytomegaly was commonly
found in dosed mice, but the incidences were not significantly different from controls and
showed no dose-response trend. EPA concluded the LOAEL for 1,4-dioxane in mice was
380 mg/kg-day based on the increased incidence of pneumonia and rhinitis in female mice; a
NOAEL was not established in this study.
As shown in Table 4-7, treatment with 1,4-dioxane significantly increased the incidence
of hepatocellular carcinomas or adenomas in male and female mice in a dose-related manner.
Tumors were first observed on week 81 in high-dose females and in week 58 in high-dose males.
Tumors were characterized by parenchymal cells of irregular size and arrangement, and were
often hypertrophic with hyperchromatic nuclei. Mitoses were seldom seen. Neoplasms were
locally invasive within the liver, but metastasis to the lungs was rarely observed.
42
-------
Table 4-7. Incidence of hepatocellular adenoma or carcinoma in B6C3Fi
mice exposed to 1,4-dioxane in drinking water
Males (mg/kg-day)a
Hepatocellular carcinoma
Hepatocellular adenoma or carcinoma
0
2/49 (4%)b
8/49 (16%)b
720
18/50 (36%)c
19/50 (38%)d
830
24/47 (51%)c
28/47 (60%)c
Females (mg/kg-day)a
Hepatocellular carcinoma
Hepatocellular adenoma or carcinoma
0
0/50 (0%)b
0/50 (0%)b
380
12/48 (25%)c
21/48 (44%)c
860
29/37 (78%)c
35/37 (95%)c
"Tumor incidence values were not adjusted for mortality.
bp < 0.001, positive dose-related trend (Cochran-Armitage test).
°p < 0.001 by Fisher's Exact test pair-wise comparison with controls.
dp = 0.014.
Source: NCI (1978, 062935).
In addition to liver tumors, a variety of other benign and malignant neoplasms occurred.
However, the report (NCI, 1978, 062935) indicated that each type had been encountered
previously as a spontaneous lesion in the B6C3Fi mouse. The report further stated that the
incidences of these neoplasms were unrelated by type, site, group, or sex of the animal, and
hence, not attributable to exposure to 1,4-dioxane. There were a few nasal adenocarcinomas
(1/48 in low-dose females and 1/49 in high-dose males) that arose from proliferating respiratory
epithelium lining of the nasal turbinates. These growths extended into the nasal cavity, but there
was minimal local tissue infiltration. Nasal mucosal polyps were rarely observed. The polyps
were derived from mucus-secreting epithelium and were otherwise unremarkable. There was a
significant negative trend for alveolar/bronchiolar adenomas or carcinomas of the lung in male
mice, such that the incidence in the matched controls was higher than in the dosed groups. The
report (NCI, 1978, 062935) indicated that the probable reason for this occurrence was that the
dosed animals did not live as long as the controls, thus diminishing the possibility of the
development of tumors in the dosed groups.
4.2.1.2.6. Kano et al.; Japan Bioassay Research Center; Yamazaki et al. The Japan
Bioassay Research Center (JBRC) conducted a 2-year drinking water study determining the
effects of 1,4-dioxane on both sexes of rats and mice. The study results have been reported
several times: once as conference proceedings (Yamazaki et al., 1994, 196120), once as a
laboratory report (JBRC, 1998, 196240), and most recently as a peer-reviewed manuscript (Kano
et al., 2009, 594539). Dr. Yamazaki also provided some detailed information (Yamazaki, 2006,
626614). Variations in the data between these three reports were noted and included: (1) the
level of detail on dose information reported; (2) categories for incidence data reported (e.g., all
animals or sacrificed animals); and (3) analysis of non- and neoplastic lesions.
43
-------
The 1,4-dioxane dose information provided in the reports varied. Specifically, Yamazaki
et al. (1994, 196120) only included drinking water concentrations for each dose group. In
contrast, JBRC (1998, 196240) included drinking water concentrations (ppm), in addition using
body weights and water consumption measurements to calculate daily chemical intake
(mg/kg-day). JBRC (1998, 196240) reported daily chemical intake for each dose group as a
range. Thus, for the External Peer Review draft of this Toxicological Review of 1,4-Dioxane
(U.S. EPA, 2009, 628630). the midpoint of the range was used. Kano et al. (2009, 594539) also
reported a calculation of daily chemical intake based on body weight and water consumption
measurements; however, for each dose group they reported a mean and standard deviation
estimate. Therefore, because the mean more accurately represents the delivered dose than the
midpoint of a range, the Kano et al. (2009, 594539) calculated mean chemical intake (mg/kg-
day) is used for quantitiative analysis of this data.
The categories for which incidence rates were described also varied among the reports.
Yamazaki et al. (1994, 196120) and Kano et al. (2009, 594539) reported histopathological results
for all animals, including dead and moribund animals; however, the detailed JBRC laboratory
findings (1998, 196240) included separate incidence reports for dead and moribund animals,
sacrificed animals, and all animals.
Finally, the criteria used to evaluate some of the data were updated when JBRC published
the most recent manuscript by Kano et al. (2009, 594539). The manuscript by Kano et al. (2009,
594539) stated that the lesions diagnosed in the earlier reports (JBRC, 1998, 196240: Yamazaki
et al., 1994, 196120) were re-examined and recategorized as appropriate according to current
pathological diagnostic criteria (see references in Kano et al. (2009, 594539)).
Groups of F344/DuCrj rats (50/sex/dose level) were exposed to 1,4-dioxane (>99% pure)
in the drinking water at levels of 0, 200, 1,000, or 5,000 ppm for 2 years. Groups of Crj:BDFl
mice (50/sex/dose level) were similarly exposed in the drinking water to 0, 500, 2,000, or
8,000 ppm of 1,4-dioxane. The high doses were selected based on results from the Kano et al.
(2008, 196245) 13-week drinking water study so as not to exceed the maximum tolerated dose
(MTD) in that study. Both rats and mice were 6 weeks old at the beginning of the study. Food
and water were available ad libitum. The animals were observed daily for clinical signs of
toxicity; and BWs were measured once per week for 14 weeks and once every 2 weeks until the
end of the study. Food consumption was measured once a week for 14 weeks and once every
4 weeks for the remainder of the study. The investigators used data from water consumption and
BW to calculate an estimate of the daily intake of 1,4-dioxane (mg/kg-day) by male and female
rats and mice. Kano et al. (2009, 594539) reported a calculated mean ± standard deviation for
the daily doses of 1,4-dioxane for the duration of the study. Male rats received doses of
approximately 0, 11±1, 55±3, or 274±18 mg/kg-day and female rats received 0, 18±3, 83±14, or
429±69 mg/kg-day. Male mice received doses of 0, 49±5, 191±21, or 677±74 mg/kg-day and
female mice received 0, 66±10, 278±40, or 964±88 mg/kg-day. For the remainder of this
44
-------
document, including the dose-response analysis, the mean calculated intake values are used to
identify dose groups. The Kano et al. (2009, 594539) study was conducted in accordance with
the Organization for Economic Co-operation and Development (OECD) Principles for Good
Laboratory Practice (GLP).
No information was provided as to when urine samples were collected. Blood samples
were collected only at the end of the 2-year study (Yamazaki, 2006, 626614). Hematology
analysis included RBCs, hemoglobin, hematocrit, MCV, platelets, WBCs and differential WBCs.
Serum biochemistry included total protein, albumin, bilirubin, glucose, cholesterol, triglyceride
(rat only), phospholipid, ALT, AST, LDH, LAP, ALP, y-glutamyl transpeptidase (GOT), CPK,
urea nitrogen, creatinine (rat only), sodium, potassium, chloride, calcium, and inorganic
phosphorous. Urinalysis parameters were pH, protein, glucose, ketone body, bilirubin (rat only),
occult blood, and urobilinogen. Organ weights (brain, lung, liver, spleen, heart, adrenal, testis,
ovary, and thymus) were measured, and gross necropsy and histopathologic examination of
tissues and organs were performed on all animals (skin, nasal cavity, trachea, lungs, bone
marrow, lymph nodes, thymus, spleen, heart, tongue, salivary glands, esophagus, stomach, small
and large intestine, liver, pancreas, kidney, urinary bladder, pituitary, thyroid, adrenal, testes,
epididymis, seminal vesicle, prostate, ovary, uterus, vagina, mammary gland, brain, spinal cord,
sciatic nerve, eye, Harderian gland, muscle, bone, and parathyroid). Dunnett's test and j^ test
were used to assess the statistical significance of changes in continuous and discrete variables,
respectively.
For rats, growth and mortality rates were reported in Kano et al. (2009, 594539) for the
duration of the study. Both male and female rats in the high dose groups (274 and 429 mg/kg-
day, respectively) exhibited slower growth rates and terminal body weights that were
significantly different (p < 0.05) compared to controls. A statistically significant reduction in
terminal BWs was observed in high-dose male rats (5%, p < 0.01) and in high-dose female rats
(18%, p < 0.01) (Kano et al., 2009, 594539). Food consumption was not significantly affected
by treatment in male or female rats; however, water consumption in female rats administered
18 mg/kg-day was significantly greater (p < 0.05) .
All control and exposed rats lived at least 12 months following study initiation
(Yamazaki, 2006, 626614): however, survival at the end of the 2-year study in the high dose
group of male and female rats (274 and 429 mg/kg-day, respectively) was approximately 50%,
which was significantly different compared to controls. The investigators attributed these early
deaths to the increased incidence in nasal tumors and peritoneal mesotheliomas in male rats and
nasal and hepatic tumors in female rats. (Yamazaki, 2006, 626614).
Several hematological changes were noted in the JBRC report (1998, 196240):
Decreases in RBC (male rats only), hemoglobin, hematocrit, and MCV; and increases in platelets
in high-dose groups were observed (JBRC, 1998, 196240). These changes (except for MCV)
also occurred in mid-dose males. With the exception of a 23% decrease in hemoglobin in high-
45
-------
dose male rats and a 27% increase in platelets in high-dose female rats, hematological changes
were within 15% of control values. Significant changes in serum chemistry parameters occurred
only in high-dose rats (males: increased phospholipids, AST, ALT, LDH, ALP, GOT, CPK,
potassium, and inorganic phosphorus and decreased total protein, albumin, and glucose; females:
increased total bilirubin, cholesterol, phospholipids, AST, ALT, LDH, GOT, ALP, CPK, and
potassium, and decreased blood glucose) (JBRC, 1998, 196240). Increases in serum enzyme
activities ranged from <2- to 17-fold above control values, with the largest increases seen for
ALT, AST, and GOT. Urine pH was significantly decreased at 274 mg/kg-day in male rats (not
tested at other dose levels) and at 83 and 429 mg/kg-day in female rats (JBRC, 1998, 196240).
Also, blood in the urine was seen in female rats at 83 and 429 mg/kg-day (JBRC, 1998, 196240).
In male rats, relative liver weights were increased at 55 and 274 mg/kg-day (Kano et al., 2009,
594539). In female rats, relative liver weight was increased at 429 mg/kg-day (Kano et al., 2009,
594539).
Microscopic examination of the tissues showed nonneoplastic alterations in the nasal
cavity, liver, and kidneys mainly in high-dose rats and, in a few cases, in mid-dose rats (Table s
4-8 and 4-9). Alterations in high-dose (274 mg/kg-day) male rats consisted of nuclear
enlargement and metaplasia of the olfactory and respiratory epithelia, atrophy of the olfactory
epithelium, hydropic changes and sclerosis of the lamina propria, adhesion, and inflammation.
In female rats, nuclear enlargement of the olfactory epithelium occurred at doses > 83 mg/kg-
day, and nuclear enlargement and metaplasia of the respiratory epithelium, squamous cell
hyperplasia, respiratory metaplasia of the olfactory epithelium, hydropic changes and sclerosis of
the lamina propria, adhesion, inflammation, and proliferation of the nasal gland occurred at
429 mg/kg-day. Alterations were seen in the liver at > 55 mg/kg-day in male rats (spongiosis
hepatis, hyperplasia, and clear and mixed cell foci) and at 429 mg/kg-day in female rats
(hyperplasia, spongiosis hepatis, cyst formation, and mixed cell foci). Nuclear enlargement of
the renal proximal tubule occurred in males at 274 mg/kg-day and in females at > 83 mg/kg-day
(JBRC, 1998, 196240).
46
-------
Table 4-8. Incidence of histopathological lesions in male F344/DuCrj rats
exposed to 1,4-dioxane in drinking water for 2 years
Nuclear enlargement; nasal respiratory epithelium0
Squamous cell metaplasia; nasal respiratory epithelium0
Squamous cell hyperplasia; nasal respiratory epithelium0
Nuclear enlargement; nasal olfactory epithelium0
Respiratory metaplasia; nasal olfactory epithelium"1
Atrophy; nasal olfactory epithelium"1
Hydropic change; lamina propriad
Sclerosis; lamina propriad
Adhesion; nasal cavityd
Inflammation; nasal cavityd
Hyperplasia; liverd
Spongiosis hepatis; liverd
Clear cell foci; liver0
Acidophilic cell foci; liver0
Basophilic cell foci; liver0
Mixed-cell foci; liver0
Nuclear enlargement; kidney proximal tubuled
Dose (mg/kg-day)a'b
0
0/50
0/50
0/50
0/50
12/50
0/50
0/50
0/50
0/50
0/50
3/50
12/50
3/50
12/50
7/50
2/50
0/50
11
0/50
0/50
0/50
0/50
11/50
0/50
0/50
0/50
0/50
0/50
2/50
20/50
3/50
8/50
11/50
8/50
0/50
55
0/50
0/50
0/50
5/50f
20/50
0/50
0/50
1/50
0/50
0/50
10/50
25/50f
9/50
7/50
8/50
14/50e
0/50
274
26/50e
31/50e
2/50
38/50e
43/50
36/50
46/50
44/50
48/50
13/50
24/50
40/50
8/50
5/50
16/50f
13/50e
50/50
"Data presented for all animals, including animals that became moribund or died before the end of the study.
bDose levels from Kano et al. (2009, 594539).
°Data from Kano et al. (2009, 594539).
dData from JBRC (1998, 196240). JBRC did not report statistical significance for the "All animals"
comparison.
ep< 0.01 by x2 test.
fp< 0.05 by x2 test.
Sources: Kano et al. (2009, 594539) and JBRC (1998, 196240).
47
-------
Table 4-9. Incidence of histopathological lesions in female F344/DuCrj
rats exposed to 1,4-dioxane in drinking water for 2 years
Nuclear enlargement; nasal respiratory epithelium0
Squamous cell metaplasia; nasal respiratory epithelium0
Squamous cell hyperplasia; nasal cavity0
Nuclear enlargement; nasal olfactory epithelium °
Respiratory metaplasia; nasal olfactory epithelium"1
Atrophy; nasal olfactory epithelium"1
Hydropic change; lamina propriad
Sclerosis; lamina propriad
Adhesion; nasal cavityd
Inflammation; nasal cavityd
Proliferation; nasal glandd
Hyperplasia; liverd
Spongiosis hepatis; liverd
Cyst formation; liverd
Acidophilic cell foci; liver0
Basophilic cell foci; liver0
Clear cell foci; liver0
Mixed-cell foci; liver0
Nuclear enlargement; kidney proximal tubuled
Dose (mg/kg-day)a'b
0
0/50
0/50
0/50
0/50
2/50
0/50
0/50
0/50
0/50
0/50
0/50
3/50
0/50
0/50
1/50
23/50
1/50
1/50
0/50
18
0/50
0/50
0/50
0/50
0/50
0/50
0/50
0/50
0/50
0/50
0/50
2/50
0/50
1/50
1/50
27/50
1/50
1/50
0/50
83
0/50
0/50
0/50
28/50e
2/50
1/50
0/50
0/50
0/50
1/50
0/50
ll/50e
1/50
1/50
1/50
31/50
5/50
3/50
6/50
429
13/50°
3 5/50 e
5/50
39/50
42/50
40/50
46/50
48/50
46/50
15/50
11/50
47/50
20/50
8/50
1/50
8/50e
4/50
ll/50f
39/50
"Data presented for all animals, including animals that became moribund or died before the end of the study.
bDose levels from Kano et al. (2009, 594539).
°Data from Kano et al. (2009, 594539).
dData from JBRC (1998, 196240). JBRC did not report statistical significance for the "All animals"
comparison.
ep< 0.01 by x2 test.
fp< 0.05 by x2 test.
Sources: Kano et al. (2009, 594539) and JBRC (1998, 196240).
NOAEL and LOAEL values for rats in this study were identified by EPA as 55 and
274 mg/kg-day, respectively, based on toxicity observed in nasal tissue of male rats (i.e., atrophy
of olfactory epithelium, adhesion, and inflammation). Metaplasia and hyperplasia of the nasal
epithelium were also observed in high-dose male and female rats. These effects are likely to be
associated with the formation of nasal cavity tumors in these dose groups. Nuclear enlargement
was observed in the nasal olfactory epithelium and the kidney proximal tubule at a dose of
83 mg/kg-day in female rats; however, it is unclear whether these alterations represent adverse
toxicological effects. Hematological effects noted in male rats given 55 and 274 mg/kg-day
(decreased RBCs, hemoglobin, hematocrit, increased platelets) were within 20% of control
values. In female rats decreases in hematological effects were observed in the high dose group
(429 mg/kg-day). A reference range database for hematological effects in laboratory animals
48
-------
(Wolford et al., 1986, 196112) indicates that a 20% change in these parameters may fall within a
normal range (10th-90th percentile values) and may not represent a treatment-related effect of
concern. Liver lesions were also seen at a dose of 55 mg/kg-day in male rats; these changes are
likely to be associated with liver tumorigenesis. Clear and mixed-cell foci are commonly
considered preneoplastic changes and would not be considered evidence of noncancer toxicity.
The nature of spongiosis hepatis as a preneoplastic change is less well understood (Bannasch,
2003, 196140: Karbe and Kerlin, 2002, 196246: Stroebel et al., 1995, 196101). Spongiosis
hepatis is a cyst-like lesion that arises from the perisinusoidal (Ito) cells (PSC) of the liver. It is
commonly seen in aging rats, but has been shown to increase in incidence following exposure to
hepatocarcinogens. Spongiosis hepatis can be seen in combination with preneoplastic foci in the
liver or with hepatocellular adenoma or carcinoma and has been considered a preneoplastic
lesion (Bannasch, 2003, 196140: Stroebel et al., 1995, 196101). This change can also be
associated with hepatocellular hypertrophy and liver toxicity and has been regarded as a
secondary effect of some liver carcinogens (Karbe and Kerlin, 2002, 196246). In the case of the
JBRC (1998, 196240) study, spongiosis hepatis was associated with other preneoplastic changes
in the liver (clear and mixed-cell foci). No other lesions indicative of liver toxicity were seen in
this study; therefore, spongiosis hepatis was not considered indicative of noncancer effects.
Serum chemistry changes (increases in total protein, albumin, and glucose; decreases in AST,
ALT, LDH, and ALP, potassium, and inorganic phosphorous) were observed in both male and
female rats (JBRC, 1998, 196240) in the high dose groups, 274 and 429 mg/kg-day, respectively.
These serum chemistry changes seen in terminal blood samples from high-dose male and female
rats are likely related to tumor formation in these dose groups.
Significantly increased incidences of liver tumors (adenomas and carcinomas) and tumors
of the nasal cavity occurred in high-dose male and female rats (Tables 4-10 and 4-11) treated
with 1,4-dioxane for 2 years (Kano et al., 2009, 594539). The first liver tumor was seen at
85 weeks in high-dose male rats and 73 weeks in high-dose female rats (vs. 101-104 weeks in
lower dose groups and controls) (Yamazaki, 2006, 626614). In addition, a significant increase
(p < 0.01, Fisher's Exact test) in mesotheliomas of the peritoneum was seen in high-dose males
(28/50 versus 2/50 in controls). Mesotheliomas were the single largest cause of death among
high-dose male rats, accounting for 12 of 28 pretermination deaths (Yamazaki, 2006, 626614).
Also, in males, there were increasing trends in mammary gland fibroadenoma and fibroma of the
subcutis, both statistically significant (p < 0.01) by the Peto test of dose-response trend. Females
showed a significant increasing trend in mammary gland adenomas (p < 0.01 by Peto's test).
The tumor incidence values presented in Tables 4-10 and 4-11 were not adjusted for survival.
49
-------
Table 4-10. Incidence of nasal cavity, peritoneum, and mammary gland
tumors in F344/DuCrj rats exposed to 1,4-dioxane in drinking water for
2 years
Dose (mg/kg-day)
Males
0
11
55
274
Females
0
18
83
429
Nasal cavity
Squamous cell carcinoma
Sarcoma
Rhabdomyosarcoma
Esthesioneuroepithelioma
0/50
0/50
0/50
0/50
0/50
0/50
0/50
0/50
0/50
0/50
0/50
0/50
3/50a
2/50
1/50
1/50
0/50
0/50
0/50
0/50
0/50
0/50
0/50
0/50
0/50
0/50
0/50
0/50
7/50a'b
0/50
0/50
1/50
Peritoneum
Mesothelioma
2/50
2/50
5/50
28/50a'b
1/50
0/50
0/50
0/50
Mammary gland
Fibroadenoma
Adenoma
Either adenoma or fibroadenoma
1/50
0/50
1/50
1/50
1/50
2/50
0/50
2/50
2/50
4/50a
2/50
6/50a
3/50
6/50
8/50
2/50
7/50
8/50
1/50
10/50
11/50
3/50
16/50^
is/so3-0
"Statistically significant trend for increased tumor incidence by Peto's test (p < 0.01).
bSignificantly different from control by Fisher's exact test (p < 0.01).
Significantly different from control by Fisher's exact test (p < 0.05).
Source: Used with permission from Elsevier, Ltd., Kano et al. (2009, 594539).
Table 4-11. Incidence of liver tumors in F344/DuCrj rats exposed to
1,4-dioxane in drinking water for 2 years
Dose (mg/kg-day)
Hepatocellular adenoma
Hepatocellular carcinoma
Either adenoma or carcinoma
Males
0
3/50
0/50
3/50
11
4/50
0/50
4/50
55
7/50
0/50
7/50
274
32/50a'b
14/50a'b
39/50a'b
Females
0
3/50
0/50
3/50
18
1/50
0/50
1/50
83
6/50
0/50
6/50
429
48/50a'b
10/50a'b
48/50a'b
"Significantly different from control by Fisher's exact test (p < 0.01).
bStatistically significant trend for increased tumor incidence by Peto's test (p < 0.01).
Source: Used with permission from Elsevier, Ltd., Kano et al. (2009, 594539).
For mice, growth and mortality rates were reported in Kano et al. (2009, 594539) for the
duration of the study. Similar to rats, the growth rates of male and female mice were slower than
controls and terminal body weights were lower for the mid (p < 0.01 for males administered
191 mg/kg-day and p < 0.05 for females administered 278 mg/kg-day) and high doses (p < 0.05
for males and females administered 677 and 964 mg/kg-day, respectively). There were no
differences in survival rates between control and treated male mice; however, survival rates were
significantly decreased compared to controls for female mice in the mid (278 mg/kg-day,
approximately 40% survival) and high (964 mg/kg-day, approximately 20% survival) dose
50
-------
groups. The study authors attributed these early female mouse deaths to the significant incidence
of hepatic tumors, and Kano et al. (2009, 594539) reported tumor incidence for all animals in the
study (N=50), including animals that became moribund or died before the end of the study.
Additional data on survival rates of mice were provided in a personal communication from
Dr. Yamazaki (2006, 626614). Dr. Yamazaki reported that the survival of mice was low in all
male groups (31/50, 33/50, 25/50 and 26/50 in control, low-, mid-, and high-dose groups,
respectively) and particularly low in high-dose females (29/50, 29/50, 17/50, and 5/50 in control,
low-, mid-, and high-dose groups, respectively). These deaths occurred primarily during the
second year of the study. Survival at 12 months in male mice was 50/50, 48/50, 50/50, and
48/50 in control, low-, mid-, and high-dose groups, respectively. Female mouse survival at
12 months was 50/50, 50/50, 48/50, and 48/50 in control, low-, mid-, and high-dose groups,
respectively (Yamazaki, 2006, 626614). Furthermore, these deaths were primarily tumor related.
Liver tumors were listed as the cause of death for 31 of the 45 pretermination deaths in high-dose
female Crj:BDFl mice (Yamazaki, 2006, 626614). For mice, growth and mortality rates were
reported in Kano et al. (2009, 594539) for the duration of the study. Similar to rats, the growth
rates of male and female mice were slower than controls and terminal body weights were lower
for the mid (p < 0.01 for males administered 191 mg/kg-day and p < 0.05 for females
administered 278 mg/kg-day) and high doses (p < 0.05 for males and females administered 677
and 964 mg/kg-day, respectively).
Food consumption was not significantly affected, but water consumption was reduced
26% in high-dose male mice and 28% in high-dose female mice. Final BWs were reduced 43%
in high-dose male mice and 15 and 45% in mid- and high-dose female mice, respectively. Male
mice showed increases in RBC counts, hemoglobin, and hematocrit, whereas in female mice,
there was a decrease in platelets in mid- and high-dose rats. With the exception of a 60%
decrease in platelets in high-dose female mice, hematological changes were within 15% of
control values. Serum AST, ALT, LDH, and ALP activities were significantly increased in mid-
and high-dose male mice, whereas LAP and CPK were increased only in high-dose male mice.
AST, ALT, LDH, and ALP activities were increased in mid- and high-dose female mice, but
CPK activity was increased only in high-dose female mice. Increases in serum enzyme activities
ranged from less than two- to sevenfold above control values. Glucose and triglycerides were
decreased in high-dose males and in mid- and high-dose females. High-dose female mice also
showed decreases in serum phospholipid and albumin concentrations (not reported in males).
Blood calcium was lower in high-dose females and was not reported in males. Urinary pH was
decreased in high-dose males, whereas urinary protein, glucose, and occult blood were increased
in mid- and high-dose female mice. Relative and absolute lung weights were increased in high-
dose males and in mid- and high-dose females (JBRC, 1998, 196240). Microscopic examination
of the tissues for nonneoplastic lesions showed significant alterations in the epithelium of the
respiratory tract, mainly in high-dose animals, although some changes occurred in mid-dose mice
51
-------
(Tables 4-12 and 4-13). Commonly seen alterations included nuclear enlargement, atrophy, and
inflammation of the epithelium. Other notable changes observed included nuclear enlargement
of the proximal tubule of the kidney and angiectasis in the liver in high-dose male mice.
Table 4-12. Incidence of histopathological lesions in male CrjrBDFl mice
exposed to 1,4-dioxane in drinking water for 2 years
Nuclear enlargement; nasal respiratory epithelium0
Nuclear enlargement; nasal olfactory epithelium0
Atrophy; nasal olfactory epithelium"1
Inflammation; nasal cavityd
Atrophy; trachea! epithelium"1
Nuclear enlargement; trachea! epithelium"1
Nuclear enlargement; bronchial epithelium"1
Atrophy; lung/bronchial epithelium"1
Accumulation of foamy cells; lungd
Angiectasis; liverd
Nuclear enlargement; kidney proximal tubuled
Dose (mg/kg-day)a'b
0
0/50
0/50
0/50
1/50
0/50
0/50
0/50
0/50
1/50
2/50
0/50
49
0/50
0/50
0/50
2/50
0/50
0/50
0/50
0/50
0/50
3/50
0/50
191
0/50
9/50e
1/50
1/50
0/50
0/50
0/50
0/50
0/50
4/50
0/50
677
31/50e
49/50e
48/50
25/50
42/50
17/50
41/50
43/50
27/50
16/50
39/50
aData presented for all animals, including animals that became moribund or died before the end of the study.
bDose levels from Kano et al. (2009, 594539).
°Data from Kano et al. (2009, 594539).
dData from JBRC (1998, 196240). JBRC did not report statistical significance for the "All animals"
comparison.
ep< 0.01 by i2 test.
Sources: Kano et al. (2009, 594539) and JBRC (1998, 196240).
52
-------
Table 4-13. Incidence of histopathological lesions in female CrjrBDFl mice
exposed to 1,4-dioxane in drinking water for 2 years
Nuclear enlargement; nasal respiratory epithelium0
Nuclear enlargement; nasal olfactory epithelium0
Atrophy; nasal olfactory epithelium"1
Inflammation; nasal cavityd
Atrophy; trachea! epithelium"1
Nuclear enlargement; bronchial epithelium"1
Atrophy; lung/bronchial epithelium"1
Accumulation of foamy cells; lungd
Dose (mg/kg-day)a'b
0
0/50
0/50
0/50
2/50
0/50
0/50
0/50
0/50
66
0/50
0/50
0/50
0/50
0/50
1/50
0/50
1/50
278
0/50
41/50e
1/50
7/50
2/50
22/50
7/50
4/50
964
41/50°
33/50e
42/50
42/50
49/50
48/50
50/50
45/50
aData presented for all animals, including animals that became moribund or died before the end of the study.
bDose levels from Kano et al. (2009, 594539).
°Data from Kano et al. (2009, 594539).
dData from JBRC (1998, 196240). JBRC did not report statistical significance for the "All animals"
comparison.
ep< 0.01 by x2 test.
Sources: Kano et al. (2009, 594539) and JBRC (1998, 196240).
NOAEL and LOAEL values for mice in this study were identified by EPA as 66 and
278 mg/kg-day, respectively, based on nasal inflammation observed in female mice. Nuclear
enlargement of the nasal olfactory epithelium and bronchial epithelium was also observed at a
dose of 278 mg/kg-day in female mice; however, it is unclear whether these alterations represent
adverse toxicological effects. The serum chemistry changes seen in terminal blood samples from
male and female mice (mid- and high-dose groups) are likely related to tumor formation in these
animals. Liver angiectasis, an abnormal dilatation and/or lengthening of a blood or lymphatic
vessel, was seen in male mice given 1,4-dioxane at a dose of 677 mg/kg-day.
Treatment with 1,4-dioxane resulted in an increase in the formation of liver tumors
(adenomas and carcinomas) in male and female mice. The incidence of hepatocellular adenoma
was statistically increased in male mice in the mid-dose group only. The incidence of male mice
with hepatocellular carcinoma or either tumor type (adenoma or carcinoma) was increased in the
low, mid, and high-dose groups. The appearance of the first liver tumor occurred in male mice at
64, 74, 63, and 59 weeks in the control, low- mid-, and high-dose groups, respectively
(Yamazaki, 2006, 626614). In female mice, increased incidence was observed for hepatocellular
carcinoma in all treatment groups, while an increase in hepatocellular adenoma incidence was
only seen in the 66 and 278 mg/kg-day dose groups (Table 4-14). The appearance of the first
liver tumor in female mice occurred at 95, 79, 71, and 56 weeks in the control, low-, mid-, and
high-dose groups, respectively (Yamazaki, 2006, 626614). The tumor incidence data presented
for male and female mice in Table 4-14 are based on reanalyzed sample data presented in Kano
53
-------
et al. (2009, 594539) that included lesions in animals that became moribund or died prior to the
completion of the 2-year study.
Katagiri et al. (1998, 193804) summarized the incidence of hepatocellular adenomas and
carcinomas in control male and female BDF1 mice from ten 2-year bioassays at the JBRC. For
female mice, out of 499 control mice, the incidence rates were 4.4% for hepatocellular adenomas
and 2.0% for hepatocellular carcinomas. Kano et al. (2009, 594539) reported a 10% incidence
rate for hepatocellular adenomas and a 0% incidence rate for hepatocellular carcinomas in
control female BDF1. The background incidence rates for male BDF1 mice were 15% and
22.8% for hepatocellular adenomas and carcinomas, respectively, out of 500 control mice in ten
2-year bioassays (Katagiri et al., 1998, 193804). Background rates for B6C3Fi mice evaluated
by the National Toxicology Program are similar (10.3% and 21.3% for hepatocellular adenomas
and carcinomas in male mice, respectively; 4.0% and 4.1% for hepatocellular adenomas and
carcinomas in female mice, respectively) to the BDF1 mice background rates observed by JBRC
(Haseman et al., 1984, 020667). Thus, the BDF1 mouse is not particularly sensitive compared to
the commonly used B6C3Fi strain and indicates that the results obtained by JBRC are
reasonable.
Table 4-14. Incidence of tumors in CrjrBDFl mice exposed to 1,4-dioxane
in drinking water for 2 years
Dose (mg/kg-day)
Males
0
49
191
677
Females
0
66
278
964
Nasal Cavity
Adenocarcinoma
Esthesioneuroepithelioma
0/50
0/50
0/50
0/50
0/50
0/50
0/50
1/50
0/50
0/50
0/50
0/50
0/50
0/50
1/50
0/50
Liver
Hepatocellular adenoma
Hepatocellular carcinoma
Either hepatocellular
adenoma or carcinoma
9/50
15/50
23/50
17/50
20/50
31/50
23/503
23/50
37/50c
11/50
36/50a'b
40/50a'b
5/50
0/50
5/50
31/503
6/50c
35/503
20/503
30/503
41/503
3/50
45/50a'b
46/50a'b
"Significantly different from control by Fisher's exact test (p < 0.01).
bStatistically significant trend for increased tumor incidence by Peto's test (p < 0.01).
Significantly different from control by Fisher's exact test (p < 0.05).
Source: Used with permission from Elsevier, Ltd., Kano et al. (2009, 594539).
A weight of evidence evaluation of the carcinogenicity studies presented in Section
4.2.1.2 is located in Section 4.7 and Table 4-19.
54
-------
4.2.2. Inhalation Toxicity
4.2.2.1. Subchronic Inhalation Toxicity
4.2.2.1.1. Fairley et al. Rabbits, guinea pigs, rats, and mice (3-6/species/group) were exposed
to 1,000, 2,000, 5,000, or 10,000 ppm of 1,4-dioxane vapor two-times a day for 1.5 hours
(3 hours/day) for 5 days/week and 1.5 hours on the 6th day (16.5 hours/week) (Fairley et al.,
1934, 062919). Animals were exposed until death occurred or were sacrificed at varying time
periods. At the 10,000 ppm concentration, only one animal (rat) survived a 7-day exposure. The
rest of the animals (six guinea pigs, three mice, and two rats) died within the first five exposures.
Severe liver and kidney damage and acute vascular congestion of the lungs were observed in
these animals. Kidney damage was described as patchy degeneration of cortical tubules with
vascular congestion and hemorrhage. Liver lesions varied from cloudy hepatocyte swelling to
large areas of necrosis. At 5,000 ppm, mortality was observed in two mice and one guinea pig
following 15-34 exposures. The remaining animals were sacrificed following 49.5 hours
(3 weeks) of exposure (three rabbits) or 94.5 hours (5 weeks) of exposure (three guinea pigs).
Liver and kidney damage in both dead and surviving animals was similar to that described for
the 10,000 ppm concentration. Animals (four rabbits, four guinea pigs, six rats, and five mice)
were exposed to 2,000 ppm for 45-102 total exposure hours (approximately 2-6 weeks). Kidney
and liver damage was still apparent in animals exposed to this concentration. Animals exposed
to 1,000 ppm were killed at intervals with the total exposure duration ranging between 78 and
202.5 hours (approximately 4-12 weeks). Cortical kidney degeneration and hepatocyte
degeneration and liver necrosis were observed in these animals (two rabbits, three guinea pigs,
three rats, and four mice). The low concentration of 1,000 ppm was identified by EPA as a
LOAEL for liver and kidney degeneration in rats, mice, rabbits, and guinea pigs in this study.
4.2.2.2. Chronic Inhalation Toxicity and Carcinogenicity
4.2.2.2.1. Torkelson et al. Whole body exposures of male and female Wistar rats (288/sex) to
1,4-dioxane vapors (99.9% pure) at a concentration of 0.4 mg/L (111 ppm), were carried out
7 hours/day, 5 days/week for 2 years (Torkelson et al., 1974, 094807). The age of the animals at
the beginning of the study was not provided. The concentration of 1,4-dioxane vapor during
exposures was determined with infrared analyzers. Food and water were available ad libitum
except during exposures. Endpoints examined included clinical signs, eye and nasal irritation,
skin condition, respiratory distress, and tumor formation. BWs were determined weekly.
Standard hematological parameters were determined on all surviving animals after 16 and
23 months of exposure. Blood collected at termination was used also for determination of
clinical chemistry parameters (serum AST and ALP activities, blood urea nitrogen [BUN], and
total protein). Liver, kidneys, and spleen were weighed and the major tissues and organs were
55
-------
processed for microscopic examination (lungs, trachea, thoracic lymph nodes, heart, liver,
pancreas, stomach, intestine, spleen, thyroid, mesenteric lymph nodes, kidneys, urinary bladder,
pituitary, adrenals, testes, ovaries, oviduct, uterus, mammary gland, lacrimal gland, lymph nodes,
brain, vagina, and bone marrow, and any abnormal growths). Nasal tissues were not obtained for
histopathological evaluation. Control and experimental groups were compared statistically using
Student's t test, Yates corrected $ test, or Fisher's Exact test.
Exposure to 1,4-dioxane vapors had no significant effect on mortality or BW gain and
induced no signs of eye or nasal irritation or respiratory distress. Slight, but statistically
significant, changes in hematological and clinical chemistry parameters were within the normal
physiological limits and were considered to be of no toxicological importance by the
investigators. Altered hematological parameters included decreases in packed cell volume, RBC
count, and hemoglobin, and an increase in WBC count in male rats. Clinical chemistry changes
consisted of a slight decrease in both BUN (control—23 ± 9.9; 111-ppm 1,4-dioxane—19.8 ±
8.8) and ALP activity (control—34.4 ±12.1; 111-ppm 1,4-dioxane—29.9 ± 9.2) and a small
increase in total protein (control—7.5 ± 0.37; 111-ppm 1,4-dioxane—7.9 ± 0.53) in male rats
(values are mean ± standard deviation). Organ weights were not significantly affected.
Microscopic examination of organs and tissues did not reveal any treatment-related effects.
Based of the lack of significant effects on several endpoints, EPA identified the exposure
concentration of 0.4 mg/L (111 ppm) as a free standing NOAEL. The trueNOAEL was likely to
be higher.
Tumors, observed in all groups including controls, were characteristic of the rat strain
used and were considered unrelated to 1,4-dioxane inhalation. The most common tumors were
reticulum cell sarcomas and mammary tumors. Using Fisher's Exact test and a significance level
ofp < 0.05, no one type of tumor occurred more frequently in treated rats than in controls. No
hepatic or nasal cavity tumors were seen in any rat.
4.2.3. Initiation/Promotion Studies
4.2.3.1. Bulletal.
Bull et al. (1986, 194336) tested 1,4-dioxane as a cancer initiator in mice using oral,
subcutaneous, and topical routes of exposure. A group of 40 female SENCAR mice (6-8 weeks
old) was administered a single dose of 1,000 mg/kg 1,4-dioxane (purity >99%) by gavage,
subcutaneous injection, or topical administration (vehicle was not specified). A group of rats
was used as a vehicle control (number of animals not specified). Food and water were provided
ad libitum. Two weeks after administration of 1,4-dioxane, 12-O-tetradecanoylphorbol-13-
acetate (TPA) (1.0 jig in 0.2 mL of acetone) was applied to the shaved back of mice
3 times/week for a period of 20 weeks. The yield of papillomas at 24 weeks was selected as a
potential predictor of carcinoma yields at 52 weeks following the start of the promotion
56
-------
schedule. Acetone was used instead of TPA in an additional group of 20 mice in order to
determine whether a single dose of 1,4-dioxane could induce tumors in the absence of TPA
promotion.
1,4-Dioxane did not increase the formation of papillomas compared to mice initiated with
vehicle and promoted with TPA, indicating lack of initiating activity under the conditions of the
study. Negative results were obtained for all three exposure routes. A single dose of
1,4-dioxane did not induce tumors in the absence of TPA promotion.
4.2.3.2. Kinget al.
1,4-Dioxane was evaluated for complete carcinogenicity and tumor promotion activity in
mouse skin (King et al., 1973, 029390). In the complete carcinogenicity study, 0.2 mL of a
solution of 1,4-dioxane (purity not specified) in acetone was applied to the shaved skin of the
back of Swiss Webster mice (30/sex) 3 times/week for 78 weeks. Acetone was applied to the
backs of control mice (30/sex) for the same time period. In the promotion study, each animal
was treated with 50 ug of dimethylbenzanthracene 1 week prior to the topical application of the
1,4-dioxane solution described above (0.2 mL, 3 times/week, 78 weeks) (30 mice/sex). Acetone
vehicle was used in negative control mice (30/sex). Croton oil was used as a positive control in
the promotion study (30/sex). Weekly counts of papillomas and suspect carcinomas were made
by gross examination. 1,4-Dioxane was also administered in the drinking water (0.5 and 1%) to
groups of Osborne-Mendel rats (35/sex/group) and B6C3Fi mice for 42 weeks (control findings
were only reported for 34 weeks).
1,4-Dioxane was negative in the complete skin carcinogenicity test using dermal
exposure. One treated female mouse had malignant lymphoma; however, no papillomas were
observed in male or female mice by 60 weeks. Neoplastic lesions of the skin, lungs, and kidney
were observed in mice given the promotional treatment with 1,4-dioxane. In addition, the
percentage of mice with skin tumors increased sharply after approximately 10 weeks of
promotion treatment. Significant mortality was observed when 1,4-dioxane was administered as
a promoter (only 4 male and 5 female mice survived for 60 weeks), but not as a complete
carcinogen (22 male and 25 female mice survived until 60 weeks). The survival of acetone-
treated control mice in the promotion study was not affected (29 male and 26 female mice
survived until 60 weeks); however, the mice treated with croton oil as a positive control
experienced significant mortality (0 male and 1 female mouse survived for 60 weeks). The
incidence of mice with papillomas was similar for croton oil and 1,4-dioxane; however, the
tumor multiplicity (i.e., number of tumors/mouse) was higher for the croton oil treatment.
Oral administration of 1,4-dioxane in drinking water caused appreciable mortality in rats,
but not mice, and increased weight gain in surviving rats and male mice. Histopathological
lesions (i.e., unspecified liver and kidney effects) were also reported in exposed male and female
rats; however, no histopathological changes were indicated for mice.
57
-------
1,4-Dioxane was demonstrated to be a tumor promoter, but not a complete carcinogen in
mouse skin, in this study. Topical administration for 78 weeks following initiation with
dimethylbenzanthracene caused an increase in the incidence and multiplicity of skin tumors in
mice. Tumors were also observed at remote sites (i.e., kidney and lung), and survival was
affected. Topical application of 1,4-dioxane for 60 weeks in the absence of the initiating
treatment produced no effects on skin tumor formation or mortality in mice.
4.2.3.3. Lundberg et al.
Lundberg et al. (1987, 062933) evaluated the tumor promoting activity of 1,4-dioxane in
rat liver. Male Sprague Dawley rats (8/dose group, 19 for control group) weighing 200 g
underwent a partial hepatectomy followed 24 hours later by an i.p. injection of 30 mg/kg
diethylnitrosamine (DEN) (initiation treatment). 1,4-Dioxane (99.5% pure with 25 ppm
butylated hydroxytoluene as a stabilizer) was then administered daily by gavage (in saline
vehicle) at doses of 0, 100, or 1,000 mg/kg-day, 5 days/week for 7 weeks. Control rats were
administered saline daily by gavage, following DEN initiation. 1,4-Dioxane was also
administered to groups of rats that were not given the DEN initiating treatment (saline used
instead of DEN). Ten days after the last dose, animals were sacrificed and liver sections were
stained for GGT. The number and total volume of GGT-positive foci were determined.
1,4-Dioxane did not increase the number or volume of GGT-foci in rats that were not
given the DEN initiation treatment. The high dose of 1,4-dioxane (1,000 mg/kg-day) given as a
promoting treatment (i.e., following DEN injection) produced an increase in the number of
GGT-positive foci and the total foci volume. Histopathological changes were noted in the livers
of high-dose rats. Enlarged, foamy hepatocytes were observed in the midzonal region of the
liver, with the foamy appearance due to the presence of numerous fat-containing cytoplasmic
vacuoles. These results suggest that cytotoxic doses of 1,4-dioxane may be associated with
tumor promotion of 1,4-dioxane in rat liver.
4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND INHALATION
4.3.1. Giavini et al.
Pregnant female Sprague Dawley rats (18-20 per dose group) were given 1,4-dioxane
(99% pure, 0.7% acetal) by gavage in water at concentrations of 0, 0.25, 0.5, or 1 mL/kg-day,
corresponding to dose estimates of 0, 250, 500, or 1,000 mg/kg-day (density of 1,4-dioxane is
approximately 1.03 g/mL) (Giavini et al., 1985, 062924). The chemical was administered at a
constant volume of 3 mL/kg on days 6-15 of gestation. Food consumption was determined daily
and BWs were measured every 3 days. The dams were sacrificed with chloroform on
gestation day 21 and the numbers of corpora lutea, implantations, resorptions, and live fetuses
were recorded. Fetuses were weighed and examined for external malformations prior to the
58
-------
evaluation of visceral and skeletal malformations (Wilson's free-hand section method and
staining with Alizarin red) and a determination of the degree of ossification.
Maternal weight gain was reduced by 10% in the high-dose group (1,000 mg/kg-day).
Food consumption for this group was 5% lower during the dosing period, but exceeded control
levels for the remainder of the study. No change from control was observed in the number of
implantations, live fetuses, or resorptions; however, fetal birth weight was 5% lower in the
highest dose group (p < 0.01). 1,4-Dioxane exposure did not increase the frequency of major
malformations or minor anomalies and variants. Ossification of the sternebrae was reduced in
the 1,000 mg/kg-day dose group (p < 0.05). The study authors suggested that the observed delay
in sternebrae ossification combined with the decrease in fetal birth weight indicated a
developmental delay related to 1,4-dioxane treatment. NOAEL and LOAEL values of 500 and
1,000 mg/kg-day were identified from this study by EPA and based on delayed ossification of
the sternebrae and reduced fetal BWs.
4.4. OTHER DURATION OR ENDPOINT-SPECIFIC STUDIES
4.4.1. Acute and Short-term Toxicity
The acute (< 24 hours) and short-term toxicity studies (<30 days) of 1,4-dioxane in
laboratory animals are summarized in Table 4-15. Several exposure routes were employed in
these studies, including dermal application, drinking water exposure, gavage, vapor inhalation,
and i.v. or i.p. injection.
4.4.1.1. Oral Toxicity
Mortality was observed in many acute high-dose studies, and LD50 values for
1,4-dioxane were calculated for rats, mice, and guinea pigs (Laug et al., 1939, 195055; see Table
4-15; Pozzani et al., 1959, 063019; Smyth et al., 1941, 060695). Clinical signs of CNS
depression were observed, including staggered gait, narcosis, paralysis, coma, and death
(de Navasquez, 1935, 196174; Laug et al., 1939, 195055; Nelson, 1951, 196087; Schrenk and
Yant, 1936, 195076). Severe liver and kidney degeneration and necrosis were often seen in
acute studies (David, 1964, 195954; de Navasquez, 1935, 196174; JBRC, 1998, 196242; Kesten
et al., 1939, 194972; Laug et al., 1939, 195055; Schrenk and Yant, 1936, 195076). JBRC (1998,
196242) additionally reported histopathological lesions in the nasal cavity and the brain of rats
following 2 weeks of exposure to 1,4-dioxane in the drinking water.
4.4.1.2. Inhalation Toxicity
Acute and short-term toxicity studies (all routes) are summarized in Table 4-15.
Mortality occurred in many high-concentration studies (Nelson, 1951, 196087; Pozzani et al.,
1959, 063019; Wirth and Klimmer, 1936, 196105). Inhalation of 1,4-dioxane caused eye and
nasal irritation, altered respiration, and pulmonary edema and congestion (Yant et al., 1930,
59
-------
062952). Clinical signs of CNS depression were observed, including staggered gait, narcosis,
paralysis, coma, and death (Nelson, 1951, 196087: Wirth and Klimmer, 1936, 196105). Liver
and kidney degeneration and necrosis were also seen in acute and short-term inhalation studies
(Drew et al., 1978, 067913: Fairley et al., 1934, 062919).
Table 4-15. Acute and short-term toxicity studies of 1,4-dioxane
Animal
Exposure route
Test conditions
Results
Dose"
Reference
Oral studies
Rat (inbred strain
and gender
unspecified)
Rat (strain and
gender unspecified)
F344/DuCrj rat
Female
Sprague Dawley rat
Female Carworth
Farms-Nelson rat
Male Wistar rat,
guinea pig
Oral via
drinking water
Oral via
drinking water
Oral via
drinking water
Gavage
Gavage
Gavage
1-10 days of
exposure
5-12 days of
exposure
14-Day exposure
0, 168, 840, 2550,
or 4,200 mg/kg by
gavage, 21 and
4 hours prior to
sacrifice
Determination of a
single dose LD50
Single dose,
LD50 determination
Ultrastructural
changes in the
kidney, degenerative
nephrosis, hyaline
droplet accumulation,
crystal formation in
mitochondria
Extensive
degeneration of the
kidney, liver damage,
mortality in
8/10 animals by
12 days
Mortality, decreased
BWs,
histopathological
lesions in the nasal
cavity, liver, kidney,
and brain
Increased ODC
activity, hepatic
CYP450 content, and
DNA single-strand
breaks
Lethality
Lethality
ll,000mg/kg-day
(5%)
ll,000mg/kg-day
(5%)
2,500 mg/kg-day
(nuclear
enlargement of
olfactory epithelial
cells),
>7,500 mg/kg-day
for all other effects
840 mg/kg (ODC
activity only)
LD50= 6,400 mg/kg
(14,200 ppm)
LD50 (mg/kg):
rat = 7, 120
guinea pig = 3,150
David (1964,
195954)
Kesten et al.
(1939,
194972)
JBRC (1998,
196242)
Kitchin and
Brown (1990,
062928)
Pozzani et al.
(1959,
063019)
Smyth et al.
(1941,
060695)
60
-------
Animal
Rat, mouse, guinea
Pig
Rabbit
Rat, rabbit
Crj:BDFl mouse
Dog
Exposure route
Gavage
Gavage
Gavage
Oral via
drinking water
Drinking water
ingestion
Test conditions
Single dose;
several dose
groups
Single gavage dose
of 0,207, 1,034, or
2,068 mg/kg-day
Single dose;
mortality after
2 weeks
14-Day exposure
3-10 days of
exposure
Results
Clinical signs of CNS
depression, stomach
hemorrhage, kidney
enlargement, and
liver and kidney
degeneration
Clinical signs of CNS
depression, mortality
at 2068 mg/kg, renal
toxicity (polyuria
followed by anuria),
histopathological
changes in liver and
kidneys
Mortality and
narcosis
Mortality, decreased
BWs,
histopathological
lesions in the nasal
cavity, liver, kidney,
and brain
Clinical signs of CNS
depression, and liver
and kidney
degeneration
Dose"
LD50 (mg/kg):
mouse = 5,900
rat = 5,400
guinea pig = 4,030
1,034 mg/kg-day
3, 160 mg/kg
10,800 mg/kg-day;
hepatocellular
swelling
11, 000 mg/kg-day
(5%)
Reference
Laug et al.
(1939,
195055)
de Navasquez
(1935,
196174)
Nelson (1951,
196087)
JBRC (1998,
196242)
Schrenk and
Yant (1936,
195076)
Inhalation studies
Male CD1 rat
Rat
Female Carworth
Farms-Nelson rat
Mouse, cat
Guinea pig
Rabbit, guinea pig,
rat, mouse
Vapor
inhalation
Vapor
inhalation
Vapor
inhalation
Vapor
inhalation
Vapor
inhalation
Vapor
inhalation
Serum enzymes
measured before
and after a single
4 hour exposure
5 hours of
exposure
Determination of a
4-hour inhalation
LC50
8 hours/day for
17 days
8-Hour exposure to
0.1-3% by volume
3 hours exposure,
for 5 days;
1.5 hour exposure
for 1 day
Increase in ALT,
AST, and OCT; no
change in G-6-Pase
Mortality and
narcosis
Lethality
Paralysis and death
Eye and nasal
irritation, retching
movements, altered
respiration, narcosis,
pulmonary edema
and congestion,
hyperemiaof the
brain
Degeneration and
necrosis in the kidney
and liver, vascular
congestion in the
lungs
1,000 ppm
6,000 ppm
LC50=51.3mg/L
8,400 ppm
0.5% by volume
10,000 ppm
Drew et al.
(1978,
067913)
Nelson (1951,
196087)
Pozzani et al.
(1959,
063019)
Wirth and
Klimmer
(1936,
196105)
Yant et al.
(1930,
062952)
Fairley et
al.(1934,
062919)
61
-------
Animal
Exposure route
Test conditions
Results
Dose"
Reference
Other routes
Male COBS/Wistar
rat
Rabbit, cat
Female
Sprague Dawley rat
CBA/J mouse
Dermal
i.v. injection
i.p. injection
i.p. injection
Nonoccluded
technique using
shaved areas of the
back and flank;
single application,
14-day observation
Single injection of
0, 207, 1,034,
1,600 mg/kg-day
Single dose;
LD50 values
determined
24 hours and
14 days after
injection
Daily injection for
7 days, 0,0.1, 1,5,
and 10%
Negative; no effects
noted
Clinical signs of CNS
depression, narcosis
at 1,034 mg/kg,
mortality at
1,600 mg/kg
Increased serum SDH
activity at l/16th of
the LD50 dose; no
change at higher or
lower doses
Slightly lower
lymphocyte response
to mitogens
8,300 mg/kg
1,034 mg/kg-day
LD50 (mg/kg):
24 hours = 4,848
14 days = 799
2,000 mg/kg-day
(10%)
Clark et al.
(1984,
194970)
de Navasquez
(1935,
196174)
Lundberg et
al. (1986,
062934)
Thurman et
al. (1978,
018767)
aLowest effective dose for positive results/ highest dose tested for negative results.
ND = no data; OCT = ornithine carbamyl transferase; ODC = ornithine decarboxylase; SDH = sorbitol
dehydrogenase
4.4.2. Neurotoxicity
Clinical signs of CNS depression have been reported in humans and laboratory animals
following high dose exposure to 1,4-dioxane (see Sections 4.1 and 4.2.1.1). Neurological
symptoms were reported in the fatal case of a worker exposed to high concentrations of
1,4-dioxane through both inhalation and dermal exposure (Johnstone, 1959, 062927). These
symptoms included headache, elevation in blood pressure, agitation and restlessness, and coma.
Autopsy findings demonstrated perivascular widening in the brain, with small foci of
demyelination in several regions (e.g., cortex, basal nuclei). It was suggested that these
neurological changes may have been secondary to anoxia and cerebral edema. In laboratory
animals, the neurological effects of acute high-dose exposure included staggered gait, narcosis,
paralysis, coma, and death (de Navasquez, 1935, 196174; Laug et al., 1939, 195055; Nelson,
1951, 196087; Schrenk and Yant, 1936, 195076; Yant et al., 1930, 062952). The neurotoxicity
of 1,4-dioxane was further investigated in several studies described below (Frantik et al., 1994,
067510; Goldberg et al., 1964, 058035; Kanada et al., 1994, 078052; Knoefel, 1935, 195914).
4.4.2.1. Frantik et al.
The acute neurotoxicity of 1,4-dioxane was evaluated following a 4-hour inhalation
exposure to male Wistar rats (four per dose group) and a 2-hour inhalation exposure to female
H-strain mice (eight per dose group) (Frantik et al., 1994, 067510). Three exposure groups and a
62
-------
control group were used in this study. Exposure concentrations were not specified, but
apparently were chosen from the linear portion of the concentration-effect curve. The
neurotoxicity endpoint measured in this study was the inhibition of the propagation and
maintenance of an electrically-evoked seizure discharge. This endpoint has been correlated with
the behavioral effects and narcosis that occur following acute exposure to higher concentrations
of organic solvents. Immediately following 1,4-dioxane exposure, a short electrical impulse was
applied through ear electrodes (0.2 seconds, 50 hertz (Hz), 180 volts (V) in rats, 90 V in mice).
Several time characteristics of the response were recorded; the most sensitive and reproducible
measures of chemically-induced effects were determined to be the duration of tonic hind limb
extension in rats and the velocity of tonic extension in mice.
Linear regression analysis of the concentration-effect data was used to calculate an
isoeffective air concentration that corresponds to the concentration producing a 30% decrease in
the maximal response to an electrically-evoked seizure. The isoeffective air concentrations for
1,4-dioxane were 1,860 ± 200 ppm in rats and 2,400 ± 420 ppm in mice. A NOAEL value was
not identified from this study.
4.4.2.2. Goldberg et al.
Goldberg et al. (1964, 058035) evaluated the effect of solvent inhalation on pole climb
performance in rats. Female rats (Carworth Farms Elias strain) (eight per dose group) were
exposed to 0, 1,500, 3,000, or 6,000 ppm of 1,4-dioxane in air for 4 hours/day, 5 days/weeks, for
10 exposure days. Conditioned avoidance and escape behaviors were evaluated using a pole
climb methodology. Prior to exposure, rats were trained to respond to a buzzer or shock stimulus
by using avoidance/escape behavior within 2 seconds. Behavioral criteria were the abolishment
or significant deferment (>6 seconds) of the avoidance response (conditioned or buzzer response)
or the escape response (buzzer plus shock response). Behavioral tests were administered on day
1, 2, 3, 4, 5, and 10 of the exposure period. Rat BWs were also measured on test days.
1,4-Dioxane exposure produced a dose-related effect on conditioned avoidance behavior
in female rats, while escape behavior was generally not affected. In the 1,500 ppm group, only
one of eight rats had a decreased avoidance response, and this only occurred on days 2 and 5 of
exposure. A larger number of rats exposed to 3,000 ppm (two or three of eight) experienced a
decrease in the avoidance response, and this response was observed on each day of the exposure
period. The maximal decrease in the avoidance response was observed in the 6,000 ppm group
during the first 2 days of exposure (75-100% of the animals were inhibited in this response). For
exposure days 3-10, the percent of rats in the 6,000 ppm group with significant inhibition of the
avoidance response ranged from 37-62%. At the end of the exposure period (day 10), the BWs
for rats in the high exposure group were lower than controls.
63
-------
4.4.2.3. Kanada et al.
Kanada et al. evaluated the effect of oral exposure to 1,4-dioxane on the regional
neurochemistry of the rat brain (Kanada et al., 1994, 078052). 1,4-Dioxane was administered by
gavage to male Sprague Dawley rats (5/group) at a dose of 1,050 mg/kg, approximately equal to
one-fourth the oral LD50. Rats were sacrificed by microwave irradiation to the head 2 hours
after dosing, and brains were dissected into small brain areas. Each brain region was analyzed
for the content of biogenic amine neurotransmitters and their metabolites using high-
performance liquid chromatography (HPLC) or GC methods. 1,4-Dioxane exposure was shown
to reduce the dopamine and serotonin content of the hypothalamus. The neurochemical profile
of all other brain regions in exposed rats was similar to control rats.
4.4.2.4. Knoefel
The narcotic potency of 1,4-dioxane was evaluated following i.p. injection in rats and
gavage administration in rabbits (Knoefel, 1935, 195914). Rats were given i.p. doses of 20, 30,
or 50 mmol/kg. No narcotic effect was seen at the lowest dose; however, rats given 30 mmol/kg
were observed to sleep approximately 8-10 minutes. Rats given the high dose of 50 mmol/kg
died during the study. Rabbits were given 1,4-dioxane at oral doses of 10, 20, 50, 75, or
100 mmol/kg. No effect on the normal erect animal posture was observed in rabbits treated with
less than 50 mmol/kg. At 50 and 75 mmol/kg, a semi-erect or staggering posture was observed;
lethality occurred at both the 75 and 100 mmol/kg doses.
4.5. MECHANISTIC DATA AND OTHER STUDIES IN SUPPORT OF THE MODE
OF ACTION
4.5.1. Genotoxicity
The genotoxicity data for 1,4-dioxane are presented in Tables 4-16 and 4-17 for in vitro
and in vivo tests, respectively. 1,4-Dioxane has been tested for genotoxic potential using in vitro
assay systems with prokaryotic organisms, non-mammalian eukaryotic organisms, and
mammalian cells, and in vivo assay systems using several strains of rats and mice. In the large
majority of in vitro systems, 1,4-dioxane was not genotoxic. Where a positive genotoxic
response was observed, it was generally observed in the presence of toxicity. Similarly,
1,4-dioxane was not genotoxic in the majority of available in vivo studies. 1,4-Dioxane did not
bind covalently to DNA in a single study with calf thymus DNA. Several investigators have
reported that 1,4-dioxane caused increased DNA synthesis indicative of cell proliferation.
Overall, the available literature indicates that 1,4-dioxane is nongenotoxic or weakly genotoxic.
Negative findings were reported for mutagenicity in in vitro assays with the prokaryotic
organisms Salmonella typhimurium, Escherichia coli, and Photobacterium phosphor sum
(Mutatox assay) (Haworth et al., 1983, 028947: Hellmer and Bolcsfoldi, 1992, 194717:
64
-------
Khudoley et al., 1987, 194949: Kwan et al., 1990, 196078: Morita and Hayashi, 1998, 195065:
Nestmann et al., 1984, 194339: Stott et al., 1981, 063021). In in vitro assays with
nonmammalian eukaryotic organisms, negative results were obtained for the induction of
aneuploidy in yeast (Saccharomyces cerevisiae) and in the sex-linked recessive lethal test in
Drosophila melanogaster (Yoon et al., 1985, 194373: Zimmermann et al., 1985, 194343). In the
presence of toxicity, positive results were reported for meiotic nondisjunction in Drosophila
(Munoz and Barnett, 2002, 195066).
The ability of 1,4-dioxane to induce genotoxic effects in mammalian cells in vitro has
been examined in model test systems with and without exogenous metabolic activation and in
hepatocytes that retain their xenobiotic-metabolizing capabilities. 1,4-Dioxane was reported as
negative in the mouse lymphoma cell forward mutation assay (McGregor et al., 1991, 194381:
Morita and Hayashi, 1998, 195065). 1,4-Dioxane did not produce chromosomal aberrations or
micronucleus formation in Chinese hamster ovary (CHO) cells (Galloway et al., 1987, 007768:
Morita and Hayashi, 1998, 195065). Results were negative in one assay for sister chromatid
exchange (SCE) in CHO (Morita and Hayashi, 1998, 195065) and were weakly positive in the
absence of metabolic activation in another (Galloway et al., 1987, 007768). In rat hepatocytes,
1,4-dioxane exposure in vitro caused single-strand breaks in DNA at concentrations also toxic to
the hepatocytes (Sina et al., 1983, 007323) and produced a positive genotoxic response in a cell
transformation assay with BALB/3T3 cells also in the presence of toxicity (Sheu et al., 1988,
195078).
1,4-Dioxane was not genotoxic in the majority of available in vivo mammalian assays.
Studies of micronucleus formation following in vivo exposure to 1,4-dioxane produced mostly
negative results, including studies of bone marrow micronucleus formation in B6C3Fi, BALB/c,
CBA, and C57BL6 mice (McFee et al., 1994, 195060: Mirkova, 1994, 195062: Tinwell and
Ashby, 1994, 195086) and micronucleus formation in peripheral blood of CD1 mice (Morita,
1994, 196085: Morita and Hayashi, 1998, 195065). Mirkova (1994, 195062) reported a dose-
related increase in the incidence of bone marrow micronuclei in male and female C57BL6 mice
24 or 48 hours after administration of 1,4-dioxane. At a sampling time of 24 hours, a dose of
450 mg/kg produced no change relative to control, while doses of 900, 1,800, and 3,600 mg/kg
increased the incidence of bone marrow micronuclei by approximately two-, three-, and fourfold,
respectively. A dose of 5,000 mg/kg also increased the incidence of micronuclei by
approximately fourfold at 48 hours. This compares with the negative results for BALB/c male
mice tested in the same study at a dose of 5,000 mg/kg and sampling time of 24 hours. Tinwell
and Ashby (1994, 195086) could not explain the difference in response in the mouse bone
marrow micronucleus assay with C57BL6 mice obtained in their laboratory (i.e., non-significant
1.6-fold increase over control) with the dose-related positive findings reported by Mirkova
(Mirkova, 1994, 195062) using the same mouse strain, 1,4-dioxane dose (3,600 mg/kg) and
sampling time (24 hours). Morita and Hayashi (1998, 195065) demonstrated an increase in
65
-------
micronucleus formation in hepatocytes following 1,4-dioxane dosing and partial hepatectomy to
induce cellular mitosis. DNA single-strand breaks were demonstrated in hepatocytes following
gavage exposure to female rats (Kitchin and Brown, 1990, 062928).
Roy et al. (2005, 196094) examined micronucleus formation in male CD1 mice exposed
to 1,4-dioxane to confirm the mixed findings from earlier mouse micronucleus studies and to
identify the origin of the induced micronuclei. Mice were administered 1,4-dioxane by gavage at
doses of 0, 1,500, 2,500, and 3,500 mg/kg-day for 5 days. The mice were also implanted with
5-bromo-2-deoxyuridine (BrdU)-releasing osmotic pumps to measure cell proliferation in the
liver and to increase the sensitivity of the hepatocyte assay. The frequency of micronuclei in the
bone marrow erythrocytes and in the proliferating BrdU-labeled hepatocytes was determined
24 hours after the final dose. Significant dose-related increases in micronuclei were seen in the
bone-marrow at all the tested doses (> 1,500 mg/kg-day). In the high-dose (3,500-mg/kg) mice,
the frequency of bone marrow erythrocyte micronuclei was about 10-fold greater than the control
frequency. Significant dose-related increases in micronuclei were also observed at the two
highest doses (> 2,500 mg/kg-day) in the liver. Antikinetochore (CREST) staining or
pancentromeric fluorescence in situ hybridization (FISH) was used to determine the origin of the
induced micronuclei. The investigators determined that 80-90% of the micronuclei in both
tissues originated from chromosomal breakage; small increase in micronuclei originating from
chromosome loss was seen in hepatocytes. Dose-related statistically significant decreases in the
ratio of bone marrow polychromatic erythrocytes (PCE):normochromatic erythrocytes (NCE), an
indirect measure of bone marrow toxicity, were observed. Decreases in hepatocyte proliferation
were also observed. Based on these results, the authors concluded that at high doses 1,4-dioxane
exerts genotoxic effects in both the mouse bone marrow and liver; the induced micronuclei are
formed primarily from chromosomal breakage; and 1,4-dioxane can interfere with cell
proliferation in both the liver and bone marrow. The authors noted that reasons for the
discrepant micronucleus assay results among various investigators was unclear, but could be
related to the inherent variability present when detecting moderate to weak responses using small
numbers of animals, as well as differences in strain, dosing regimen, or scoring criteria.
1,4-Dioxane did not affect in vitro or in vivo DNA repair in hepatocytes or in vivo DNA
repair in the nasal cavity (Goldsworthy et al., 1991, 062925: Stott et al., 1981, 063021). but
increased hepatocyte DNA synthesis indicative of cell proliferation in several in vivo studies
(Goldsworthy et al., 1991, 062925: Miyagawa et al., 1999, 195063: Stott et al., 1981, 063021:
Uno et al., 1994, 194385). 1,4-Dioxane caused a transient inhibition of RNA polymerase A and
B in the rat liver (Kurl et al., 1981, 195054), indicating a negative impact on the synthesis of
ribosomal and messenger RNA (DNA transcription). Intravenous administration of 1,4-dioxane
at doses of 10 or 100 mg/rat produced inhibition of both polymerase enzymes, with a quicker and
more complete recovery of activity for RNA polymerase A, the polymerase for ribosomal RNA
synthesis.
66
-------
1,4-Dioxane did not covalently bind to DNA under in vitro study conditions (Woo et al.,
1977, 062950). DNA alkylation was also not detected in the liver 4 hours following a single
gavage exposure (1,000 mg/kg) in male Sprague Dawley rats (Stott et al., 1981, 063021).
Rosenkranz and Klopman (1992, 004321) analyzed 1,4-dioxane using the computer
automated structure evaluator (CASE) structure activity method to predict its potential
genotoxicity and carcinogenicity. The CASE analysis is based on information contained in the
structures of approximately 3,000 chemicals tested for endpoints related to mutagenic/genotoxic
and carcinogenic potential. CASE selects descriptors (activating [biophore] or inactivating
[biophobe] structural fragments) from a learning set of active and inactive molecules. Using the
CASE methodology, Rosenkranz and Klopman (1992, 004321) predicted that 1,4-dioxane would
be inactive for mutagenicity in several in vitro systems, including Salmonella, induction of
chromosomal aberrations in CHO cells, and unscheduled DNA synthesis in rat hepatocytes.
1,4-Dioxane was predicted to induce SCE in cultured CHO cells, micronuclei formation in rat
bone marrow, and carcinogenicity in rodents.
Gene expression profiling in cultured human hepatoma HepG2 cells was performed using
DNA microarrays to discriminate between genotoxic and other carcinogens (van Delft et al.,
2004, 195087). Van Delft et al. (2004, 195087) examined this method using a training set of 16
treatments (nine genotoxins and seven nongenotoxins) and a validation set (three and three), with
discrimination models based on Pearson correlation analyses for the 20 most discriminating
genes. As reported by the authors (van Delft et al., 2004, 195087), the gene expression profile
for 1,4-dioxane indicated a classification of this chemical as a "nongenotoxic" carcinogen, and
thus, 1,4-dioxane was included in the training set as a "nongenotoxic" carcinogen. The accuracy
for carcinogen classification using this method ranged from 33 to 100%, depending on which
chemical data sets and gene expression signals were included in the analysis.
Table 4-16. Genotoxicity studies of 1,4-dioxane; in vitro
Test system
Endpoint
Test conditions
Results"
Without
activation
With
activation
Doseb
Source
Prokaryotic organisms in vitro
S. typhimurium
strains TA98, TA100,
TA1535, TA1537
S. typhimurium
strains TA98, TA100,
TA1530, TA1535,
TA1537
S. typhimurium
strains TA98, TA100,
TA1535, TA1537
Reverse
mutation
Reverse
mutation
Reverse
mutation
Plate incorporation
assay
Plate incorporation
assay
Plate incorporation
and preincubation
assays
—
—
10,000 ug/plate
ND
5,000 jig/plate
Haworth et
al. (1983,
028947)
Khudoley et
al. (1987,
194949)
Morita and
Hayashi
(1998,
195065)
67
-------
Test system
S. typhimurium
strains TA100,
TA1535
S. typhimurium
strains TA98, TA100,
TA1535, TA1537,
TA1538
E. coli K-12
uvrB/recA
E. coli
WP2/WP2uvrA
P. phosphoreum
M169
Endpoint
Reverse
mutation
Reverse
mutation
DNA repair
Reverse
mutation
Mutagenicity,
DNA damage
Test conditions
Preincubation
assay
Plate incorporation
assay
Host mediated
assay
Plate incorporation
and preincubation
assays
Mutatox assay
Results3
Without
activation
—
—
With
activation
—
ND
Doseb
103 mg
103 mg
l,150mmol/L
5,000 ug/plate
ND
Source
Nestmann et
al. (1984,
194339)
Stott et al.
(1981,
063021)
Hellmer and
Bolcsfoldi
(1992,
194717)
Morita and
Hayashi
(1998,
195065)
Kwan et al.
(1990,
196078)
Nonmammalian eukaryotic organisms in vitro
S. cerevisiae D6 1 .M
D. melanogaster
D. melanogaster
Aneuploidy
Meiotic
nondisjunction
Sex-linked
recessive lethal
test
Standard 16-hour
incubation or cold-
interruption
regimen
Oocytes were
obtained for
evaluation 24 and
48 hours after
mating
Exposure by
feeding and
injection
-T
+r
ND
NDd
NDd
4.75%
2% in sucrose
media
35,000ppmin
feed, 7 days or
50,000 ppm
(5% in water)
by injection
Zimmerman
etal. (1985,
194343)
Munoz and
Barnett
(2002,
195066)
Yoon et al.
(1985,
194373)
Mammalian cells in vitro
Rat hepatocytes
Primary hepatocyte
culture from male
F344 rats
L5178Y mouse
lymphoma cells
DNA damage;
single-strand
breaks measured
by alkaline
elution
DNA repair
Forward
mutation assay
3 -Hour exposure
to isolated primary
hepatocytes
Autoradiography
Thymidine kinase
mutagenicity assay
(trifluorothymidin
e resistance)
+Te
—
NDd
NDd
0.3 mM
ImM
5,000 ug/mL
Sina et al.
(1983,
007323)
Goldsworthy
etal. (1991,
062925)
McGregor et
al. (1991,
194381)
68
-------
Test system
L5178Y mouse
lymphoma cells
BALB/3T3 cells
CHO cells
CHO cells
CHO cells
CHO cells
CHO cells
Endpoint
Forward
mutation assay
Cell
transformation
SCE
Chromosomal
aberration
SCE
Chromosomal
aberration
Micronucleus
formation
Test conditions
Thymidine kinase
mutagenicity assay
(trifluorothymidin
e resistance)
48-Hour exposure
followed by
4 weeks
incubation; 13 day
exposure followed
by 2.5 weeks
incubation
BrdU was added
2 hours after
1,4-dioxane
addition; chemical
treatment was
2 hours with S9
and 25 hours
without S9
Cells were
harvested 8-
12 hours or 18-
26 hours after
treatment (time of
first mitosis)
3 hour pulse
treatment;
followed by
continuous
treatment of BrdU
for 23 or 26 hours
5 hour pulse
treatment, 20 hour
pulse and
continuous
treatments, or
44 hour
continuous
treatment; cells
were harvested 20
or 44 hours
following
exposure
5 hour pulse
treatment or
44 hour
continuous
treatment; cells
were harvested
42 hours following
exposure
Results3
Without
activation
+Tf
±g
With
activation
-T
NDd
Doseb
5,000 ug/mL
0.5 mg/mL
10,520 ug/mL
10,520 ug/mL
5,000 ug/mL
5,000 ug/mL
5,000 ug/mL
Source
Morita and
Hayashi
(1998,
195065)
Sheu et al.
(1988,
195078)
Galloway et
al. (1987,
007768)
Galloway et
al. (1987,
007768)
Morita and
Hayashi
(1998,
195065)
Morita and
Hayashi
(1998,
195065)
Morita and
Hayashi
(1998,
195065)
69
-------
Test system
CalfthymusDNA
Endpoint
Covalent
binding to DNA
Test conditions
Incubation with
microsomes from
3 -methy Icholanthr
ene treated rats
Results3
Without
activation
With
activation
Doseb
0.04 pmol/mg
DNA (bound)
Source
Woo et al.
(1977,
062950)
a+ = positive, ± = equivocal or weak positive, - = negative, T = toxicity. Endogenous metabolic
activation is not applicable for in vivo studies.
bLowest effective dose for positive results/highest dose tested for negative results; ND = no data.
°Rats were given doses of 0, 168, 840, 2,550, or 4,200 mg/kg at 4 and 21 hours prior to sacrifice. A 43 and
50% increase in the fraction of DNA eluted was observed for doses of 2,550 and 4,200 mg/kg,
respectively. Alkaline elution of DNA was not significantly different from control in the two lowest dose
groups (168 and 840 mg/kg).
dA dose-related increase in the incidence of bone marrow micronuclei was observed in male and female
C57BL6 mice 24 or 48 hours after administration of 1,4-dioxane. A dose of 450 mg/kg produced no
change relative to control, while doses of 900, 1,800, 3,600, and 5,000 mg/kg increased the incidence of
bone marrow micronuclei by approximately two-,three-, four- and fourfold, respectively.
eA dose-related increase in the incidence of hepatocyte micronuclei was observed in partially
hepatectomized mice 6 days after administration of 1,4-dioxane. A dose of 1,000 mg/kg produced no
change relative to control, while doses of 2,000 and 3,000 mg/kg increased the incidence of hepatocyte
micronuclei by 2.4- and 3.4-fold, respectively.
f Significant increases in the frequency of micronucleated erythrocytes were observed at each test dose of
1,4-dioxane (1,500, 2,500 and 3,500 mg/kg-day, 5 days/week).
8 A dose-related increase in the frequency of micronuclei was observed in proliferating cells with micronuclei at
2,500 and 3,500 mg/kg-day, 5 days/week. No increase in the frequency of micronuclei was seen in the non-
proliferating cells.
hNo increase in the hepatocyte labeling index was observed 24 or 48 hours following a single gavage
exposure of 1,000 mg/kg. Continuous administration of 1% 1,4-dioxane in the drinking water for up to
2 weeks produced a twofold increase in the hepatocyte labeling index.
'A similar pattern of RNA polymerase inhibition was observed at doses of 10 and 100 mg/rat. Inhibition
was more pronounced at the higher dose.
JHepatocyte viability was 86, 89, 87, 88, 78, and 86% 24 hours following exposure to 0, 1,000, 1,500,
2,000, or 4,000 mg/kg. The incidence (%) of replicative DNA synthesis was increased by 2.5-fold
(1,000 mg/kg) or 4.5-fold (1,500 and 2,000 mg/kg). No increase in replicative DNA synthesis was
observed at the highest dose (4,000 mg/kg).
kReplicative DNA synthesis was measured 24, 39, and 48 hours following a single dose of 0, 1,000, or
2,000 mg/kg. Hepatocyte viability ranged from 71 to 82%. The only increase in replicative DNA
synthesis was observed 24 hours after administration of 2,000 mg/kg (threefold increase). Cell viability
for this group was 79%.
'Replicative DNA synthesis was increased 1.5-fold in rats given 1,000 mg/kg of 1,4-dioxane for 11 weeks.
No change from control was observed in rats exposed to 10 mg/kg for 11 weeks or rats acutely exposed to
10, 100, or 1,000 mg/kg.
70
-------
Table 4-17. Genotoxicity studies of 1,4-dioxane; mammalian in vivo
Test system
Female
Sprague Dawley
Rat
Male
Sprague Dawley
Rat
Male
B6C3FJ
Mouse
Male and female
C57BL6
Mouse;
male BALB/c
Mouse
Male
CD1
Mouse
Male
CD1
Mouse
Male
CD1
Mouse
Male
CBAand
C57BL6 Mouse
Male
CD1
Mouse
Male
CD1
Mouse
Male
Sprague Dawley
Rat
Endpoint
DNA damage;
single-strand breaks
measured by alkaline
elution
DNA alkylation in
hepatocytes
Micronucleus
formation in bone
marrow
Micronucleus
formation in bone
marrow
Micronucleus
formation in
peripheral blood
Micronucleus
formation in
hepatocytes
Micronucleus
formation in
peripheral blood
Micronucleus
formation in bone
marrow
Micronuclei
formation in bone
marrow
Micronuclei
formation in
hepatocytes
DNA repair in
hepatocytes
Test Conditions
Two gavage doses given 21
and 4 hours prior to
sacrifice
Gavage; DNA isolation and
HPLC analysis 4 hours after
dosing
i.p. injection; analysis of
polychromatic erythrocytes
24 or 48 hours after dosing
Gavage; analysis of
polychromatic erythrocytes
24 or 48 hours after dosing
Two i.p. injections (I/day);
micronucleated
reticulocytes measured 24,
48, and 72 hours after the
2nd dose
Gavage, partial
hepatectomy 24 hours after
dosing, hepatocytes
analyzed 5 days after
hepatectomy
Gavage, partial
hepatectomy 24 hours after
dosing, peripheral blood
obtained from tail vein
24 hours after hepatectomy
Gavage; analysis of
polychromatic erythrocytes
from specimens prepared
24 hours after dosing
Gavage; analysis for
micronucleated erythrocytes
24 hours after dosing
Gavage; analysis for
micronuclei 24 hours after
dosing
Drinking water; thymidine
incorporation with
hydroxyurea to repress
normal DNA synthesis
Results3
+c
—
+
(C57BL6)d
- (BALB/c)
+e
+f
+g
Doseb
2,550 mg/kg
1,000 mg/kg
Single dose of
4,000 mg/kg;
3 daily doses of
2,000
900 mg/kg
(C57BL6);
5,000 mg/kg
(BALB/c)
3,200 mg/kg
2,000 mg/kg
3,000 mg/kg
3,600 mg/kg
1,500 mg/kg-day
for 5 days
2,500 mg/kg-day
for 5 days
1,000 mg/kg-day
for 1 1 weeks
Source
Kitchin and
Brown (1990,
062928)
Stott et al.
(1981,
063021)
McFee et al.
(1994,
195060)
Mirkova
(1994,
195062)
Morita (1994,
196085)
Morita and
Hayashi
(1998,
195065)
Morita and
Hayashi
(1998,
195065)
Tinwell and
Ashby (1994,
195086)
Roy et al.
(2005,
196094)
Roy et
al.(2005,
196094)
Stott et al.
(1981,
063021)
71
-------
Test system
Male
F344
Rat
Male
F344
Rat
Male
F344
Rat
Male
F344
Rat
Male
Sprague Dawley
Rat
Male
F344
Rat
Male
F344
Rat
Male
Sprague Dawley
Rat
Endpoint
DNA repair in
hepatocytes
(autoradiography)
DNA repair in nasal
epithelial cells from
the nasoturbinate or
maxilloturbinate
Replicative DNA
synthesis (i.e., cell
proliferation) in
hepatocytes
Replicative DNA
synthesis (i.e., cell
proliferation) in nasal
epithelial cells
RNA synthesis;
inhibition of RNA
polymerase A and B
DNA synthesis in
hepatocytes
DNA synthesis in
hepatocytes
DNA synthesis in
hepatocytes
Test Conditions
Gavage and drinking water
exposure; thymidine
incorporation
Gavage and drinking water
exposure; thymidine
incorporation
Gavage and drinking water
exposure; thymidine
incorporation
Drinking water exposure;
thymidine incorporation
i.v. injection; activity
measured in isolated
hepatocytes
Gavage; thymidine and
BrdU incorporation
Thymidine incorporation
Drinking water; thymidine
incorporation
Results3
+h
(1-2 -week
exposure)
+1
+J
±k
+1
Doseb
1,000 mg/kg for
2 or 12 hours;
1,500 mg/kg-day
for 2 weeks or
3,000 mg/kg-day
for 1 week
1,500 mg/kg-day
for 8 days +
1,000 mg/kg
gavage dose
12 hours prior to
sacrifice
1,000 mg/kg for
24 or 48 hours;
1,500 mg/kg-day
for 1 or 2 weeks
1,500 mg/kg-day
for 2 weeks
10 mg/rat
1,000 mg/kg
2,000 mg/kg
1,000 mg/kg-day
for 1 1 weeks
Source
Goldsworthy
etal. (1991,
062925)
Goldsworthy
etal. (1991,
062925)
Goldsworthy
etal. (1991,
062925)
Goldsworthy
etal. (1991,
062925)
Kurl et al.
(1981,
195054)
Miyagawa
(1999,
195063)
Uno et al.
(1994,
194385)
Stott et al.
(1981,
063021)
a+ = positive, ± = equivocal or weak positive, - = negative, T = toxicity. Endogenous metabolic
activation is not applicable for in vivo studies.
bLowest effective dose for positive results/highest dose tested for negative results; ND = no data.
°Rats were given doses of 0, 168, 840, 2,550, or 4,200 mg/kg at 4 and 21 hours prior to sacrifice. A 43 and
50% increase in the fraction of DNA eluted was observed for doses of 2,550 and 4,200 mg/kg,
respectively. Alkaline elution of DNA was not significantly different from control in the two lowest dose
groups (168 and 840 mg/kg).
dA dose-related increase in the incidence of bone marrow micronuclei was observed in male and female
C57BL6 mice 24 or 48 hours after administration of 1,4-dioxane. A dose of 450 mg/kg produced no
change relative to control, while doses of 900, 1,800, 3,600, and 5,000 mg/kg increased the incidence of
bone marrow micronuclei by approximately two-,three-, four- and fourfold, respectively.
eA dose-related increase in the incidence of hepatocyte micronuclei was observed in partially
hepatectomized mice 6 days after administration of 1,4-dioxane. A dose of 1,000 mg/kg produced no
change relative to control, while doses of 2,000 and 3,000 mg/kg increased the incidence of hepatocyte
micronuclei by 2.4- and 3.4-fold, respectively.
f Significant increases in the frequency of micronucleated erythrocytes were observed at each test dose of
1,4-dioxane (1,500, 2,500 and 3,500 mg/kg-day, 5 days/week).
8 A dose-related increase in the frequency of micronuclei was observed in proliferating cells with micronuclei at
2,500 and 3,500 mg/kg-day, 5 days/week. No increase in the frequency of micronuclei was seen in the non-
proliferating cells.
72
-------
hNo increase in the hepatocyte labeling index was observed 24 or 48 hours following a single gavage
exposure of 1,000 mg/kg. Continuous administration of 1% 1,4-dioxane in the drinking water for up to
2 weeks produced a twofold increase in the hepatocyte labeling index.
'A similar pattern of RNA polymerase inhibition was observed at doses of 10 and 100 mg/rat. Inhibition
was more pronounced at the higher dose.
JHepatocyte viability was 86, 89, 87, 88, 78, and 86% 24 hours following exposure to 0, 1,000, 1,500,
2,000, or 4,000 mg/kg. The incidence (%) of replicative DNA synthesis was increased by 2.5-fold
(1,000 mg/kg) or 4.5-fold (1,500 and 2,000 mg/kg). No increase in replicative DNA synthesis was
observed at the highest dose (4,000 mg/kg).
kReplicative DNA synthesis was measured 24, 39, and 48 hours following a single dose of 0, 1,000, or
2,000 mg/kg. Hepatocyte viability ranged from 71 to 82%. The only increase in replicative DNA
synthesis was observed 24 hours after administration of 2,000 mg/kg (threefold increase). Cell viability
for this group was 79%.
'Replicative DNA synthesis was increased 1.5-fold in rats given 1,000 mg/kg of 1,4-dioxane for 11 weeks.
No change from control was observed in rats exposed to 10 mg/kg for 11 weeks or rats acutely exposed to
10, 100, or 1,000 mg/kg.
4.5.2. Mechanistic Studies
4.5.2.1. Free Radical Generation
Burmistrov et al. (2001, 195972) investigated the effect of 1,4-dioxane inhalation on free
radical processes in the rat ovary and brain. Female rats (6-9/group, unspecified strain) were
exposed to 0, 10, or 100 mg/m3 of 1,4-dioxane vapor for 4 hours/day, 5 days/week, for 1 month.
Rats were sacrificed during the morning or evening following exposure and the ovaries and brain
cortex were removed and frozen. Tissue preparations were analyzed for catalase activity,
glutathione peroxidase activity, and protein peroxidation. Inhalation of 100 mg/m3 of
1,4-dioxane resulted in a significant increase (p < 0.05) in glutathione peroxidase activity, and
activation of free radical processes were apparent in both the rat ovary and brain cortex. No
change in catalase activity or protein peroxidation was observed at either concentration. A
circadian rhythm for glutathione peroxidase activity was absent in control rats, but occurred in
rat brain and ovary following 1,4-dioxane exposure.
4.5.2.2. Induction of Metabolism
The metabolism of 1,4-dioxane is discussed in detail in Section 3.3. 1,4-Dioxane has
been shown to induce its own metabolism (Young et al., 1978, 062955; Young et al., 1978,
625640). Nannelli et al. (2005, 195067) (study details provided in Section 3.3) characterized the
CYP450 isozymes that were induced by 1,4-dioxane in the liver, kidney, and nasal mucosa of the
rat. In the liver, the activities of several CYP450 isozymes were increased (i.e., CYP2B1/2,
CYP2E1, CYPC11); however, only CYP2E1 was inducible in the kidney and nasal mucosa.
CYP2E1 mRNA was increased approximately two- to threefold in the kidney and nasal mucosa,
but mRNA levels were not increased in the liver, suggesting that regulation of CYP2E1 is organ-
specific. Induction of hepatic CYPB1/2 and CYP2E1 levels by phenobarbital or fasting did not
increase the liver toxicity of 1,4-dioxane, as measured by hepatic glutathione content or serum
ALT activity. This result suggested that highly reactive and toxic intermediates did not play a
73
-------
large role in the liver toxicity of 1,4-dioxane, even under conditions where metabolism was
enhanced. This finding is similar to an earlier conclusion by Kociba et al. (1975, 062930) who
evaluated toxicity from a chronic drinking water study alongside data providing a
pharmacokinetic profile for 1,4-dioxane. Kociba et al. (1975, 062930) concluded that liver
toxicity and eventual tumor formation occurred only at doses where clearance pathways were
saturated and elimination of 1,4-dioxane from the blood was reduced. Nannelli et al. (2005,
195067) further suggested that a sustained induction of CYP2E1 may lead to generation of
reactive oxygen species contributing to target organ toxicity and regenerative cell proliferation;
however, no data were provided to support this hypothesis.
4.5.2.3. Mechanisms of Tumor Induction
Several studies have been performed to evaluate potential mechanisms for the
carcinogenicity of 1,4-dioxane (Goldsworthy et al., 1991, 062925; Kitchin and Brown, 1990,
062928: Stott et al., 1981, 063021). Stott et al. (1981, 063021) evaluated 1,4-dioxane in several
test systems, including salmonella mutagenicity in vitro, rat hepatocyte DNA repair activity in
vitro, DNA synthesis determination in male Sprague Dawley rats following acute gavage dosing
or an 11-week drinking water exposure (described in Section 4.2.1), and hepatocyte DNA
alkylation and DNA repair following a single gavage dose. This study used doses of 0, 10, 100,
or 1,000 mg/kg-day, with the highest dose considered to be a tumorigenic dose level. Liver
histopathology and liver to BW ratios were also evaluated in rats from acute gavage or repeated
dose drinking water experiments.
The histopathology evaluation indicated that liver cytotoxicity (i.e., centrilobular
hepatocyte swelling) was present in rats from the 1,000 mg/kg-day dose group that received
1,4-dioxane in the drinking water for 11 weeks (Stott et al., 1981, 063021). An increase in the
liver to BW ratio accompanied by an increase in hepatic DNA synthesis was also seen in this
group of animals. No effect on histopathology, liver weight, or DNA synthesis was observed in
acutely exposed rats or rats that were exposed to a lower dose of 10 mg/kg-day for 11 weeks.
1,4-Dioxane produced negative findings in the remaining genotoxicity assays conducted as part
of this study (i.e., Salmonella mutagenicity, in vitro and in vivo rat hepatocyte DNA repair, and
DNA alkylation in rat liver). The study authors suggested that the observed lack of genotoxicity
at tumorigenic and cytotoxic dose levels indicates an epigenetic mechanism for 1,4-dioxane
hepatocellular carcinoma in rats.
Goldsworthy et al. (1991, 062925) evaluated potential mechanisms for the nasal and liver
carcinogenicity of 1,4-dioxane in the rat. DNA repair activity was evaluated as a measure of
DNA reactivity and DNA synthesis was measured as an indicator of cell proliferation or
promotional activity. In vitro DNA repair was evaluated in primary hepatocyte cultures from
control and 1,4-dioxane-treated rats (1 or 2% in the drinking water for 1 week). DNA repair and
DNA synthesis were also measured in vivo following a single gavage dose of 1,000 mg/kg, a
74
-------
drinking water exposure of 1% (1,500 mg/kg-day) for 1 week, or a drinking water exposure of
2% (3,000 mg/kg-day) for 2 weeks. Liver to BW ratios and palmitoyl CoA oxidase activity were
measured in the rat liver to determine whether peroxisome proliferation played a role in the liver
carcinogenesis of 1,4-dioxane. In vivo DNA repair was evaluated in rat nasal epithelial cells
derived from either the nasoturbinate or the maxilloturbinate of 1,4-dioxane-treated rats. These
rats received 1% 1,4-dioxane (1,500 mg/kg-day) in the drinking water for 8 days, followed by a
single gavage dose of 10, 100, or 1,000 mg/kg 12 hours prior to sacrifice. Archived tissues from
the NCI (1978, 062935) bioassay were reexamined to determine the primary sites for tumor
formation in the nasal cavity following chronic exposure in rats. Histopathology and cell
proliferation were determined for specific sites in the nasal cavity that were related to tumor
formation. This evaluation was performed in rats that were exposed to drinking water containing
1% 1,4-dioxane (1,500 mg/kg-day) for 2 weeks.
1,4-Dioxane and its metabolite l,4-dioxane-2-one did not affect in vitro DNA repair in
primary hepatocyte cultures (Goldsworthy et al., 1991, 062925). In vivo DNA repair was also
unaffected by acute gavage exposure or ingestion of 1,4-dioxane in the drinking water for a 1- or
2-week period. Hepatocyte cell proliferation was not affected by acute gavage exposure, but was
increased approximately twofold following a 1-2-week drinking water exposure. A 5-day
drinking water exposure to 1% 1,4-dioxane (1,500 mg/kg-day) did not increase the activity of
palmitoyl coenzyme A or the liver to BW ratio, suggesting that peroxisome proliferation did not
play a role in the hepatocarcinogenesis of 1,4-dioxane. Nannelli et al. (2005, 195067) also
reported a lack of hepatic palmitoyl CoA induction following 10 days of exposure to 1.5%
1,4-dioxane in the drinking water (2,100 mg/kg-day).
Treatment of rats with 1% (1,500 mg/kg-day) 1,4-dioxane for 8 days did not alter DNA
repair in nasal epithelial cells (Goldsworthy et al., 1991, 062925). The addition of a single
gavage dose of up to 1,000 mg/kg 12 hours prior to sacrifice also did not induce DNA repair.
Reexamination of tissue sections from the NCI (1978, 062935) bioassay suggested that the
majority of nasal tumors were located in the dorsal nasal septum or the nasoturbinate of the
anterior portion of the dorsal meatus (Goldsworthy et al., 1991, 062925). No histopathological
lesions were observed in nasal section of rats exposed to drinking water containing 1%
1,4-dioxane (1,500 mg/kg-day) for 2 weeks and no increase was observed in cell proliferation at
the sites of highest tumor formation in the nasal cavity.
Female Sprague Dawley rats (three to nine per group) were given 0, 168, 840, 2,550, or
4,200 mg/kg 1,4-dioxane (99% purity) by corn oil gavage in two doses at 21 and 4 hours prior to
sacrifice (Kitchin and Brown, 1990, 062928). DNA damage (single-strand breaks measured by
alkaline elution), ODC activity, reduced glutathione content, and CYP450 content were
measured in the liver. Serum ALT activity and liver histopathology were also evaluated. No
changes were observed in hepatic reduced glutathione content or ALT activity. Light
microscopy revealed minimal to mild vacuolar degeneration in the cytoplasm of hepatocytes
75
-------
from three of five rats from the 2,550 mg/kg dose group. No histopathological lesions were seen
in any other dose group, including rats given a higher dose of 4,200 mg/kg. 1,4-Dioxane caused
43 and 50% increases in DNA single-strand breaks at dose levels of 2,550 and 4,200 mg/kg,
respectively. CYP450 content was also increased at the two highest dose levels (25 and 66%
respectively). ODC activity was increased approximately two-, five-, and eightfold above
control values at doses of 840, 2,550, and 4,200 mg/kg, respectively. The results of this study
demonstrated that hepatic DNA damage can occur in the absence of significant cytotoxicity.
Parameters associated with tumor promotion (i.e., ODC activity, CYP450 content) were also
elevated, suggesting that promotion may play a role in the carcinogenesis of 1,4-dioxane.
4.6. SYNTHESIS OF MAJOR NONCANCER EFFECTS
Liver and kidney toxicity were the primary noncancer health effects associated with
exposure to 1,4-dioxane in humans and laboratory animals. Several fatal cases of hemorrhagic
nephritis and centrilobular necrosis of the liver were related to occupational exposure (i.e.,
inhalation and dermal contact) to 1,4-dioxane (Barber, 1934, 062913: Johnstone, 1959, 062927).
Neurological changes were also reported in one case; including, headache, elevation in blood
pressure, agitation and restlessness, and coma (Johnstone, 1959, 062927). Perivascular widening
was observed in the brain of this worker, with small foci of demyelination in several regions
(e.g., cortex, basal nuclei). Liver and kidney degeneration and necrosis were observed in acute
oral and inhalation studies (David, 1964, 195954; de Navasquez, 1935, 196174; Drew et al.,
1978, 067913: Fairley et al., 1934, 062919: JBRC, 1998, 196242: Kesten et al., 1939, 194972:
Laug et al., 1939, 195055: Schrenk and Yant, 1936, 195076). The results of subchronic and
chronic studies are discussed below.
4.6.1. Oral
Table 4-18 presents a summary of the noncancer results for the subchronic and chronic
oral studies of 1,4-dioxane toxicity in experimental animals. Liver and kidney toxicity were the
primary noncancer health effects of oral exposure to 1,4-dioxane in animals. Kidney damage at
high doses was characterized by degeneration of the cortical tubule cells, necrosis with
hemorrhage, and glomerulonephritis (Argus et al., 1965, 017009; Fairley et al., 1934, 062919;
Kociba et al., 1974, 062929: NCI, 1978, 062935). Renal cell degeneration generally began with
cloudy swelling of cells in the cortex (Fairley et al., 1934, 062919). Nuclear enlargement of
proximal tubule cells was observed at doses below those producing renal necrosis (JBRC, 1998,
196240; Kano et al., 2008, 196245), but is of uncertain toxicological significance. The lowest
dose reported to produce kidney damage was 94 mg/kg-day, which produced renal degeneration
and necrosis of tubule epithelial cells in male rats in the Kociba et al. (1974, 062929) study.
Cortical tubule degeneration was seen at higher doses in the NCI (1978, 062935) bioassay
76
-------
(240 mg/kg-day, male rats), and glomerulonephritis was reported for rats given doses of
> 430 mg/kg-day (Argus et al., 1965, 017009: 1973, 062912).
Table 4-18. Oral toxicity studies (noncancer effects) for 1,4-dioxane
Species
Dose/duration
NOAEL
(mg/kg-day)
LOAEL
(mg/kg-day)
Effect
Reference
Subchronic studies
Rat and mouse
(6/species);
unknown strain
Male
Sprague Dawley
Rat
(4-6/group)
F344/DuCrj rat
(10/sex/group)
Crj:BDFl
Mouse
(10/sex/group)
Rats 0 or 1,900 mg/kg-
day; mice 0 or
3,300 mg/kg-day for
67 days
0, 10, or 1,000 mg/kg-day
for 1 1 weeks
Males 0, 52, 126, 274,
657, or 1,554 mg/kg-day;
females 0, 83, 185, 427,
756, or 1,614 mg/kg-day
for 13 weeks
Males 0, 86, 231,585,
882, or 1,570 mg/kg-day;
females 0, 170, 387, 898,
1,620, or 2,669 mg/kg-
day for 13 weeks
NA
10
52
170
1,900 rats
3,300 mice
1,000
126
387
Renal cortical degeneration
and necrosis, hemorrhage;
hepatocellular degeneration
Minimal centrilobular
hepatocyte swelling;
increased DNA synthesis
Nuclear enlargement of
nasal respiratory
epithelium; hepatocyte
swelling
Nuclear enlargement of
bronchial epithelium
Fairley et al.
(1934,
062919)
Stott et al.
(1981,
063021)
Kano et al.
(2008,
196245)
Kano et al.
(2008,
196245)
Chronic studies
Male
Wistar
Rat (26 treated,
9 controls)
Male
Sprague Dawley
rats (30/group)
Sherman rat
(60/sex/dose
group)
Osborne-Mendel
rat (35/sex/dose
level)
B6C3F! mouse
(50/sex/dose
level)
0 or 640 mg/kg-day for
63 weeks
0, 430, 574, 803, or
1,032 mg/kg-day for
13 months
Males 0, 9.6, 94, or
1,015 mg/kg-day; females
0, 19, 148, or
1,599 mg/kg-day for
2 years
Males 0, 240, or
530 mg/kg-day; females
0, 350, or 640 mg/kg-day
for 110 weeks
Males 0, 720, or
830 mg/kg-day; females
0,380, or 860 mg/kg-day
for 90 weeks
NA
NA
9.6
NA
NA
640
430
94
240
380
Hepatocytes with enlarged
hyperchromic nuclei;
glomerulonephritis
Hepatocy tomegaly ;
glomerulonephritis
Degeneration and necrosis
of renal tubular cells and
hepatocytes
Pneumonia, gastric ulcers,
and cortical tubular
degeneration in the kidney
Pneumonia and rhinitis
Argus et al.
(1965,
017009)
Argus et al.
(1973,
062912)
Kociba et al.
(1974,
062929)
NCI (1978,
062935)
NCI (1978,
062935)
77
-------
Species
F344/DuCrj rat
(50/sex/dose
level)
F344/DuCrj rat
(50/sex/dose
level)
F344/DuCrj rat
(50/sex/dose
level)
Crj:BDFl mouse
(50/sex/dose
level)
Crj:BDFl mouse
(50/sex/dose
level)
Dose/duration
Males 0, 11, 55, or
274 mg/kg-day; females
0, 18, 83, or 429 mg/kg-
day for 2 years
Males 0, 11, 55, or
274 mg/kg-day; females
0, 18, 83, or 429 mg/kg-
day for 2 years
Males 0, 11, 55, or
274 mg/kg-day; females
0, 18, 83, or 429 mg/kg-
day for 2 years
Males 0,49, 191 or
677 mg/kg-day; females
0, 66, 278, or 964 mg/kg-
day for 2 years
Males 0,49, 191 or
677 mg/kg-day; females
0, 66, 278, or 964 mg/kg-
day for 2 years
NOAEL
(mg/kg-day)
55
11
55
66
49
LOAEL
(mg/kg-day)
274
55
274
278
191
Effect
Atrophy of nasal olfactory
epithelium; nasal adhesion
and inflammation
Liver hyperplasia
Increases in serum liver
enzymes (GOT, GPT, LDH,
and ALP)
Nasal inflammation
Increases in serum liver
enzymes (GOT, GPT, LDH,
and ALP)
Reference
JBRC (1998,
196240);
Kano et al.
(2009,
594539)
JBRC (1998,
196240):
Kano et al.
(2009,
594539)
JBRC (1998,
196240):
Kano et al.
(2009,
594539)
JBRC (1998,
196240):
Kano et al.
(2009,
594539)
JBRC (1998,
196240):
Kano et al.
(2009,
594539)
Developmental studies
Sprague Dawley
rat
(18-20/group)
Pregnant dams 0, 250,
500, or 1,000 mg/kg-day
on gestation days 6-15
500
1,000
Delayed ossification of the
sternebrae and reduced fetal
BWs
Giavani et al.
(1985,
062924)
Liver effects included degeneration and necrosis, hepatocyte swelling, cells with
hyperchromic nuclei, spongiosis hepatis, hyperplasia, and clear and mixed cell foci of the liver
(Argus et al., 1965, 017009: Argus et al., 1973, 062912: Fairley et al., 1934, 062919: Kano et al.,
2008, 196245: Kociba et al., 1974, 062929: NCI, 1978, 062935). Hepatocellular degeneration
and necrosis were seen at high doses in a subchronic study (1,900 mg/kg-day in rats) (Fairley et
al., 1934, 062919) and at lower doses in a chronic study (94 mg/kg-day, male rats) (Kociba et al.,
1974, 062929). Argus et al. (1973, 062912) described a progression of preneoplastic effects in
the liver of rats exposed to a dose of 575 mg/kg-day. Early changes (8 months exposure) were
described as an increased nuclear size of hepatocytes, disorganization of the rough endoplasmic
reticulum, an increase in smooth endoplasmic reticulum, a decrease in glycogen, an increase in
lipid droplets in hepatocytes, and formation of liver nodules. Spongiosis hepatis, hyperplasia,
and clear and mixed-cell foci were also observed in the liver of rats (doses >55 mg/kg-day in
male rats) (JBRC, 1998, 196240: Kano et al., 2009, 594539). Clear and mixed-cell foci are
commonly considered preneoplastic changes and would not be considered evidence of noncancer
toxicity when observed in conjunction with tumor formation. If exposure to 1,4-dioxane had not
78
-------
resulted in tumor formation, these lesions could represent potential noncancer toxicity. The
nature of spongiosis hepatis as a preneoplastic change is less well understood (Bannasch, 2003,
196140: Karbe and Kerlin, 2002, 196246: Stroebel et al., 1995, 196101). Spongiosis hepatis is a
cyst-like lesion that arises from the peri sinusoidal Ito cells of the liver. This change is
sometimes associated with hepatocellular hypertrophy and liver toxicity (Karbe and Kerlin,
2002, 196246), but may also occur in combination with preneoplastic foci, or hepatocellular
adenoma or carcinoma (Bannasch, 2003, 196140: Stroebel et al., 1995, 196101). In the case of
the JBRC (1998, 196240) study, spongiosis hepatis was associated with other preneoplastic
changes in the liver (hyperplasia, clear and mixed-cell foci). No other lesions indicative of liver
toxicity were seen in this study; therefore, spongiosis hepatis was not considered indicative of
noncancer effects. The activity of serum enzymes (i.e., AST, ALT, LDH, and ALP) was
increased in rats and mice exposed to 1,4-dioxane, although only in groups with high incidence
of liver tumors. Blood samples were collected only at the end of the 2-year study, so altered
serum chemistry may be associated with the tumorigenic changes in the liver.
Hematological changes were reported in the JBRC (1998, 196240) study only. Mean
doses are reported based on information provided in Kano et al. (2009, 594539). Observed
increases in RBCs, hematocrit, hemoglobin in high-dose male mice (677 mg/kg-day) may be
related to lower drinking water consumption (74% of control drinking water intake).
Hematological effects noted in male rats given 55 mg/kg-day (decreased RBCs, hemoglobin,
hematocrit, increased platelets) were within 20% of control values. A reference range database
for hematological effects in laboratory animals (Wolford et al., 1986, 196112) indicates that a
20% change in these parameters may fall within a normal range (10th-90th percentile values)
and may not represent a treatment-related effect of concern.
Rhinitis and inflammation of the nasal cavity were reported in both the NCI (1978,
062935) (mice only, dose > 380 mg/kg-day) and JBRC (1998, 196240) studies (> 274 mg/kg-day
in rats, >278 mg/kg-day in mice). The JBRC (1998, 196240) study also demonstrates atrophy of
the nasal epithelium and adhesion in rats and mice. Nasal inflammation may be a response to
direct contact of the nasal mucosa with drinking water containing 1,4-dioxane (Goldsworthy et
al., 1991, 062925: Sweeney et al., 2008, 195085) or could result from systemic exposure.
Regardless, inflammation may indicate toxicity due to 1,4-dioxane exposure. A significant
increase in the incidence of pneumonia was reported in mice from the NCI (1978, 062935) study.
The significance of this effect is unclear, as it was not observed in other studies that evaluated
lung histopathology (JBRC, 1998, 196240: Kano et al., 2008, 196245: Kociba et al., 1974,
062929). No studies were available regarding the potential for 1,4-dioxane to cause
immunological effects. Metaplasia and hyperplasia of the nasal epithelium were also observed in
high-dose male and female rats (JBRC, 1998, 196240): however, these effects are likely to be
associated with the formation of nasal cavity tumors in these dose groups. Nuclear enlargement
of the nasal olfactory epithelium was observed at a dose of 83 mg/kg-day in female rats (Kano et
79
-------
al., 2009, 594539): however, it is unclear whether this alteration represents an adverse
toxicological effect. Nuclear enlargement of the tracheal and bronchial epithelium and an
accumulation of foamy cells in the lung were also seen in male and female mice give
1,4-dioxane at doses of > 278 mg/kg for 2 years (JBRC, 1998, 196240).
4.6.2. Inhalation
Only one subchronic study (Fairley et al., 1934, 062919) and one chronic inhalation study
(Torkelson et al., 1974, 094807) were identified. In the subchronic study, rabbits, guinea pigs,
rats, and mice (3-6/species/group) were exposed to 1,000, 2,000, 5,000, or 10,000 ppm of
1,4-dioxane vapor for 1.5 hours two times a day for 5 days, 1.5 hours for one day, and no
exposure on the seventh day. Animals were exposed until death occurred or were sacrificed after
various durations of exposure (3-202.5 hours). Detailed dose-response information was not
provided; however, severe liver and kidney damage and acute vascular congestion of the lungs
were noted for all exposure concentrations tested. Kidney damage was described as patchy
degeneration of cortical tubules with vascular congestion and hemorrhage. Liver lesions varied
from cloudy hepatocyte swelling to large areas of necrosis. Torkelson et al. (1974, 094807)
performed a chronic inhalation study in which male and female Wistar rats (288/sex) were
exposed to 111 ppm 1,4-dioxane vapor for 7 hours/day, 5 days/week for 2 years. Control rats
(192/sex) were exposed to filtered air. No significant effects were observed on BWs, survival,
organ weights, hematology, clinical chemistry, or histopathology. These studies were not
sufficient to characterize the inhalation risks of 1,4-dioxane, due to the nature of the available
data (i.e., free-standing LOAEL and NOAEL values).
4.6.3. Mode of Action Information
The metabolism of 1,4-dioxane in humans was extensive at low doses (<50 ppm). The
linear elimination of 1,4-dioxane in both plasma and urine indicated that 1,4-dioxane metabolism
was a nonsaturated, first-order process at this exposure level (1976, 062953; Young et al., 1977,
062956). Like humans, rats extensively metabolized inhaled 1,4-dioxane; however, plasma data
from rats given single i.v. doses of 3, 10, 30, 100, or 1,000 mg [14C]-l,4-dioxane/kg
demonstrated a dose-related shift from linear, first-order to nonlinear, saturable metabolism of
1,4-dioxane (Young et al., 1978, 062955: Young et al., 1978, 625640).
1,4-Dioxane oxidation appeared to be CYP450-mediated, as CYP450 induction with
phenobarbital or Aroclor 1254 and suppression with 2,4-dichloro-6-phenylphenoxy ethylamine
or cobaltous chloride was effective in significantly increasing and decreasing, respectively, the
appearance of HEAA in the urine of rats (Woo et al., 1977, 062951: Woo et al., 1978, 194345).
1,4-Dioxane itself induced CYP450-mediated metabolism of several barbiturates in Hindustan
mice given i.p. injections of 25 and 50 mg/kg of 1,4-dioxane (Mungikar and Pawar, 1978,
80
-------
194344). The differences between single and multiple doses in urinary and expired radiolabel
support the notion that 1,4-dioxane may induce its own metabolism. 1,4-Dioxane has been
shown to induce several isoforms of CYP450 in various tissues following acute oral
administration by gavage or drinking water (Nannelli et al., 2005, 195067). In the liver, the
activity of several CYP450 isozymes was increased (i.e., CYP2B1/2, CYP2E1, CYPC11);
however, only CYP2E1 was inducible in the kidney and nasal mucosa. CYP2E1 mRNA was
increased approximately two- to threefold in the kidney and nasal mucosa, but mRNA levels
were not increased in the liver, suggesting that regulation of CYP2E1 was organ-specific.
Nannelli et al. (2005, 195067) investigated the role of CYP450 isozymes in the liver
toxicity of 1,4-dioxane. Hepatic CYPB1/2 and CYP2E1 levels were induced by phenobarbital or
fasting and liver toxicity was measured as hepatic glutathione content or serum ALT activity.
No increase in glutathione content or ALT activity was observed, suggesting that highly reactive
and toxic intermediates did not play a large role in the liver toxicity of 1,4-dioxane, even under
conditions where metabolism was enhanced. Pretreatment with inducers of mixed-function
oxidases also did not significantly change the extent of covalent binding in subcellular fractions
(Woo et al., 1977, 062950). Covalent binding was measured in liver, kidney, spleen, lung, colon,
and skeletal muscle 1-12 hours after i.p. dosing with 1,4-dioxane. Covalent binding was highest
in liver, spleen, and colon. Within hepatocytes, 1,4-dioxane distribution was greatest in the
cytosolic fraction, followed by the microsomal, mitochondrial, and nuclear fractions.
The absence of an increase in toxicity following an increase in metabolism suggests that
accumulation of the parent compound may be related to 1,4-dioxane toxicity. This hypothesis is
supported by a comparison of the pharmacokinetic profile of 1,4-dioxane with the toxicology
data from a chronic drinking water study (Kociba et al., 1975, 062930). This analysis indicated
that liver toxicity did not occur unless clearance pathways were saturated and elimination of
1,4-dioxane from the blood was reduced. Alternative metabolic pathways (i.e., not CYP450
mediated) may be present at high doses of 1,4-dioxane; however, the available studies have not
characterized these pathways or identified any possible reactive intermediates. The mechanism
by which 1,4-dioxane induces tissue damage is not known, nor is it known whether the toxic
moiety is 1,4-dioxane or a transient or terminal metabolite.
4.7. EVALUATION OF CARCINOGENICITY
4.7.1. Summary of Overall Weight of Evidence
Under the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005, 086237).
1,4-dioxane is "likely to be carcinogenic to humans" based on evidence of liver carcinogenicity
in several 2-year bioassays conducted in three strains of rats, two strains of mice, and in guinea
pigs (Argus et al., 1965, 017009: Argus et al., 1973, 062912: Hoch-Ligeti and Argus, 1970,
029386: Hoch-Ligeti et al., 1970, 062926: JBRC, 1998, 196240: Kano et al., 2009, 594539:
81
-------
Kociba et al., 1974, 062929: NCI, 1978, 062935: Yamazaki et al., 1994, 196120). Additionally,
mesotheliomas of the peritoneum (JBRC, 1998, 196240: Kano et al., 2009, 594539: Yamazaki et
al., 1994, 1961201 mammary (JBRC, 1998, 196240: Kano et al., 2009, 594539: Yamazaki et al.,
1994,196120), and nasal tumors (Argus et al., 1973, 062912: Hoch-Ligeti et al., 1970, 062926:
JBRC, 1998, 196240: Kano et al., 2009, 594539: Kociba et al., 1974, 062929: NCI, 1978,
062935: Yamazaki et al., 1994, 196120) have been observed in rats due to exposure to
1,4-dioxane. Studies in humans are inconclusive regarding evidence for a causal link between
occupational exposure to 1,4-dioxane and increased risk for cancer; however, only two studies
were available and these were limited by small cohort size and a small number of reported cancer
cases (Buffier et al., 1978, 062914: Thiess et al., 1976, 062943).
The available evidence is inadequate to establish a mode of action (MOA) by which
1,4-dioxane induces liver tumors in rats and mice. A MOA hypothesis involving sustained
proliferation of spontaneously transformed liver cells has some support from data indicating that
1,4-dioxane acts as a tumor promoter in mouse skin and rat liver bioassays (King et al., 1973,
029390: Lundberg et al., 1987, 062933). Dose-response and temporal data support the
occurrence of cell proliferation and hyperplasia prior to the development of liver tumors (JBRC,
1998, 196240: Kociba et al., 1974, 062929) in the rat model. However, the dose-response
relationship for induction of hepatic cell proliferation has not been characterized, and it is
unknown if it would reflect the dose-response relationship for liver tumors in the 2-year rat and
mouse studies. Conflicting data from rat and mouse bioassays (JBRC, 1998, 196240: Kociba et
al., 1974, 062929) suggest that cytotoxicity may not be a required precursor event for
1,4-dioxane-induced cell proliferation. Data regarding a plausible dose response and temporal
progression (see Table 4-18) from cytotoxicity and cell proliferation to eventual liver tumor
formation are not available.
The MOA by which 1,4-dioxane produces liver, nasal, peritoneal (mesotheliomas), and
mammary gland tumors is unknown, and the available data do not support any hypothesized
carcinogenic MOA for 1,4-dioxane.
U.S. EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005, 086237)
indicate that for tumors occurring at a site other than the initial point of contact, the weight of
evidence for carcinogenic potential may apply to all routes of exposure that have not been
adequately tested at sufficient doses. An exception occurs when there is convincing information
(e.g., toxicokinetic data) that absorption does not occur by other routes. Information available on
the carcinogenic effects of 1,4-dioxane via the oral route demonstrates that tumors occur in
tissues remote from the site of absorption. Information on the carcinogenic effects of
1,4-dioxane via the inhalation and dermal routes in humans and animals is absent. (Note: Note
that during the development of this assessment, new data regarding the toxicity of 1,4-dioxane
through the inhalation route of exposure became available. These data have not been included in
the current assessment and will be evaluated in a separate IRIS assessment.) Based on the
82
-------
observance of systemic tumors following oral exposure, and in the absence of information to
indicate otherwise, it is assumed that an internal dose will be achieved regardless of the route of
exposure. Therefore, 1,4-dioxane is "likely to be carcinogenic to humans" by all routes of
exposure.
4.7.2. Synthesis of Human, Animal, and Other Supporting Evidence
Human studies of occupational exposure to 1,4-dioxane were inconclusive; in each case,
the cohort size and number of reported cases were of limited size (Buffier et al., 1978, 062914;
Thiess et al., 1976, 062943).
Several carcinogenicity bioassays have been conducted for 1,4-dioxane in mice, rats, and
guinea pigs (Argus et al., 1965, 017009: Argus et al., 1973, 062912: Hoch-Ligeti and Argus,
1970, 029386: Hoch-Ligeti et al., 1970, 062926: JBRC, 1998, 196240: Kano et al., 2009,
594539: Kociba et al., 1974, 062929: NCI, 1978, 062935: Torkelson et al., 1974, 094807:
Yamazaki et al., 1994, 196120). Liver tumors have been observed following drinking water
exposure in male Wistar rats (Argus et al., 1965, 017009), male guinea pigs (Hoch-Ligeti and
Argus, 1970, 029386). male Sprague Dawley rats (Argus et al., 1973, 062912: Hoch-Ligeti et al.,
1970, 062926). male and female Sherman rats (Kociba et al., 1974, 062929). female Osborne-
Mendel rats (NCI, 1978, 062935). male and female F344/DuCrj rats (JBRC, 1998, 196240: Kano
et al., 2009, 594539: Yamazaki et al., 1994, 196120). male and female B6C3Fi mice (NCI, 1978,
062935). and male and female Crj:BDFl mice (JBRC, 1998, 196240: Kano et al., 2009, 594539:
Yamazaki et al., 1994, 196120). In the earliest cancer bioassays, the liver tumors were described
as hepatomas (Argus et al., 1965, 017009: Argus et al., 1973, 062912: Hoch-Ligeti and Argus,
1970, 029386: Hoch-Ligeti et al., 1970, 062926): however, later studies made a distinction
between hepatocellular carcinoma and hepatocellular adenoma (JBRC, 1998, 196240; Kano et
al., 2009, 594539: Kociba et al., 1974, 062929: NCI, 1978, 062935: Yamazaki et al., 1994,
196120). Both tumor types have been seen in rats and mice exposed to 1,4-dioxane. Kociba
et al. (1974, 062929) noted evidence of liver toxicity at or below the dose levels that produced
liver tumors but did not report incidence data for these effects. Hepatocellular degeneration and
necrosis were observed in the mid- and high-dose groups of male and female Sherman rats
exposed to 1,4-dioxane, while tumors were only observed at the highest dose. Hepatic
regeneration was indicated in the mid- and high-dose groups by the formation of hepatocellular
hyperplastic nodules. Findings from JBRC (1998, 196240) also provided evidence of liver
hyperplasia in male F344/DuCrj rats at a dose level below the dose that induced a statistically
significant increase in tumor formation.
Nasal cavity tumors were also observed in Sprague Dawley rats (Argus et al., 1973,
062912: Hoch-Ligeti et al., 1970, 062926). Osborne-Mendel rats (NCI, 1978, 062935). Sherman
rats (Kociba et al., 1974, 062929). and F344/DuCrj rats (JBRC, 1998, 196240: Kano et al., 2009,
594539; Yamazaki et al., 1994, 196120). Most tumors were characterized as squamous cell
83
-------
carcinomas. Nasal tumors were not elevated in B6C3Fi or Crj :BDF1 mice. JBRC (1998,
196240) was the only study that evaluated nonneoplastic changes in nasal cavity tissue following
prolonged exposure to 1,4-dioxane in the drinking water. Histopathological lesions in female
F344/DuCrj rats were suggestive of toxicity and regeneration in this tissue (i.e., atrophy,
adhesion, inflammation, nuclear enlargement, and hyperplasia and metaplasia of respiratory and
olfactory epithelium). Some of these effects occurred at a lower dose (83 mg/kg-day) than that
shown to produce nasal cavity tumors (429 mg/kg-day) in female rats. Re-examination of tissue
sections from the NCI (1978, 062935) bioassay suggested that the majority of nasal tumors were
located in the dorsal nasal septum or the nasoturbinate of the anterior portion of the dorsal
meatus. Nasal tumors were not observed in an inhalation study in Wistar rats exposed to
111 ppm for 5 days/week for 2 years (Torkelson et al., 1974, 094807).
Tumor initiation and promotion studies in mouse skin and rat liver suggested that
1,4-dioxane does not initiate the carcinogenic process, but instead acts as a tumor promoter (Bull
et al., 1986, 194336: King et al., 1973, 029390: Lundberg et al., 1987, 062933) (see Section
4.2.3).
In addition to the liver and nasal tumors observed in several studies, a statistically
significant increase in mesotheliomas of the peritoneum was seen in male rats from the Kano et
al. (2009, 594539) study (also JBRC, 1998, 196240: Yamazaki et al., 1994, 196120). Female
rats dosed with 429 mg/kg-day in drinking water for 2 years also showed a statistically
significant increase in mammary gland adenomas (JBRC, 1998, 196240: Kano et al., 2009,
594539: Yamazaki et al., 1994, 196120). A significant increase in the incidence of these tumors
was not observed in other chronic oral bioassays of 1,4-dioxane (Kociba et al., 1974, 062929:
NCI, 1978, 062935).
4.7.3. Mode of Action Information
The MO A by which 1,4-dioxane produces liver, nasal, peritoneal (mesotheliomas), and
mammary gland tumors is unknown, and the available data do not support any hypothesized
mode of carcinogenic action for 1,4-dioxane. Available data also do not clearly identify whether
1,4-dioxane or one of its metabolites is responsible for the observed effects. The hypothesized
MO As for 1,4-dioxane carcinogenicity are discussed below within the context of the modified
Hill criteria of causality as recommended in the most recent Agency guidelines (U.S. EPA, 2005,
086237). MOA analyses were not conducted for peritoneal or mammary gland tumors due to the
absence of any chemical specific information for these tumor types.
4.7.3.1. Identification of Key Events for Carcinogenicity
4.7.3.1.1. Liver. A key event in this MOA hypothesis is sustained proliferation of
spontaneously transformed liver cells, resulting in the eventual formation of liver tumors.
Precursor events in which 1,4-dioxane may promote proliferation of transformed liver cells are
84
-------
uncertain. One study suggests that induced liver cytotoxicity may be a key precursor event to
cell proliferation leading to the formation of liver tumors (Kociba et al., 1974, 062929), however,
this study did not report incidence data for these effects. Other studies suggest that cell
proliferation can occur in the absence of liver cytotoxicity. Liver tumors were observed in female
rats and female mice in the absence of lesions indicative of cytotoxicity (JBRC, 1998, 196240;
Kano et al., 2008, 196245: NCI, 1978, 062935). Figure 4-1 presents a schematic representation
of possible key events in the MOA for 1,4-dioxane liver carcinogenicity. These include:
(1) oxidation by CYP2E1 and CYP2B1/2 (i.e., detoxification pathway for 1,4-dioxane),
(2) saturation of metabolism/clearance leading to accumulation of the parent 1,4-dioxane,
(3) liver damage followed by regenerative cell proliferation, or (4) cell proliferation in the
absence of cytotoxicity (i.e., mitogenesis), (5) hyperplasia, and (6) tumor formation. It is
suggested that liver toxicity is related to the accumulation of the parent compound following
metabolic saturation at high doses (Kociba et al., 1975, 062930): however, no in vivo or in vitro
assays have examined the toxicity of metabolites resulting from 1,4-dioxane to support this
hypothesis. Nanelli et al. (2005, 195067) demonstrated that an increase in the oxidative
metabolism of 1,4-dioxane via CYP450 induction using phenobarbital or fasting does not result
in an increase in liver toxicity. This result suggested that highly reactive and toxic intermediates
did not play a large role in the liver toxicity of 1,4-dioxane, even under conditions where
metabolism was enhanced. Alternative metabolic pathways (e.g., not CYP450 mediated) may be
present at high doses of 1,4-dioxane; although the available studies have not characterized these
pathways nor identified any possible reactive intermediates. Tumor promotion studies in mouse
skin and rat liver suggest that 1,4-dioxane may enhance the growth of previously initiated cells
(King et al., 1973, 029390: Lundberg et al., 1987, 062933). This is consistent with the increase
in hepatocyte cell proliferation observed in several studies (Goldsworthy et al., 1991, 062925:
Miyagawa et al., 1999, 195063: Stott et al., 1981, 063021: Uno et al., 1994, 194385). These
mechanistic studies provide evidence of cell proliferation, but do not indicate whether
mitogenesis or cytotoxicity is responsible for increased cell turnover.
85
-------
Toxicokinetics
Oral absorption of
1,4-dioxane
Metabolism by
CYP2E1 and
CYP2B1/2
HEAA elimination
in the urine
Metabolic
saturation and
accumulation of
1,4-dioxane in the
blood
MOA for Liver Tumors
Hepatocellular
cytotoxicity
Regenerative cell
proliferation
Hyperplasia
Tumor formation
Cell proliferation in
absence of
cytotoxicity
Hyperplasia
Tumor promotion
Figure 4-1. A schematic representation of the possible key events in the
delivery of 1,4-dioxane to the liver and the hypothesized MOA(s) for liver
carcinogenicity.
4.7.3.1.2. Nasal cavity. A possible key event in the MOA hypothesis for nasal tumors is
sustained proliferation of spontaneously transformed nasal epithelial cells, resulting in the
eventual formation of nasal cavity tumors. Precursor events in which 1,4-dioxane may promote
proliferation of transformed nasal cells are highly uncertain. Figure 4-2 presents a schematic
representation of possible key events leading to the formation of nasal cavity tumors.
Histopathological lesions in female rats were suggestive of toxicity and regeneration in this
tissue (i.e., atrophy, adhesion, inflammation, nuclear enlargement, and hyperplasia and
metaplasia of respiratory and olfactory epithelium) (JBRC, 1998, 196240: Kano et al., 2009,
594539).
86
-------
Toxicokinetics
Inhalation of
water droplets
1
Oral
absorption of
1,4-dioxane
Metabolism
by CYP2E1
and
CYP2B1/2
1
HEAA
elimination in
the urine
— »
Metabolic
saturation and
accumulation of
1,4-dioxane in the
blood, exhalation
of 1,4-dioxane in
breath
MOA for Nasal Cavity
Tumors
i
Chronic
irritation due to
direct contact
with nasal
epithelium
1
A
Regenerative
^oll
proliferation
1
Hyperplasia
1
Tumor formation
1
Cytotoxicity to
nasal cell
epithelium
Figure 4-2. A schematic representation of the possible key events in the
delivery of 1,4-dioxane to the nasal cavity and the hypothesized MOA(s)
for nasal cavity carcinogenicity.
4.7.3.2. Strength, Consistency, Specificity of Association
4.7.3.2.1. Liver. The plausibility of a MOA that would include liver cytotoxicity, with
subsequent reparative cell proliferation, as precursor events to liver tumor formation is
minimally supported by findings that nonneoplastic liver lesions occurred at exposure levels
lower than those resulting in significantly increased incidences of hepatocellular tumors (Kociba
et al., 1974, 062929) and the demonstration of nonneoplastic liver lesions in subchronic (Kano et
al., 2008, 196245) and acute and short-term oral studies (see Table 4-15). Because the incidence
of nonneoplastic lesions was not reported by Kociba et al. (1974, 062929), it is difficult to know
whether the incidence of liver lesions increased with increasing 1,4-dioxane concentration.
Contradicting the observations by Kociba et al. (1974, 062929), liver tumors were observed in
female rats and female mice in the absence of lesions indicative of cytotoxicity (JBRC, 1998,
196240: Kano et al., 2008, 196245: NCI, 1978, 062935). This suggests that cytotoxicity may not
be a requisite step in the MOA for liver cancer. Mechanistic and tumor promotion studies
suggest that enhanced cell proliferation without cytotoxicity may be a key event; however, data
showing a plausible dose response and temporal progression from cell proliferation to eventual
liver tumor formation are not available (see Sections 4.7.3.3 and 4.7.3.4). Mechanistic studies
that demonstrated cell proliferation after short-term exposure did not evaluate liver cytotoxicity
(Goldsworthy et al., 1991, 062925: Miyagawa et al., 1999, 195063: Uno et al., 1994, 194385).
Studies have not investigated possible precursor events that may lead to cell proliferation in the
absence of cytotoxicity (i.e., genetic regulation of mitogenesis).
87
-------
4.7.3.2.2. Nasal cavity. Nasal cavity tumors have been demonstrated in several rat strains
(JBRC, 1998, 196240: Kano et al., 2009, 594539: Kociba et al., 1974, 062929: NCI, 1978,
062935: Yamazaki et al., 1994, 1961201 but were not elevated in two strains of mice (JBRC,
1998, 196240: Kano et al., 2009, 594539: NCI, 1978, 062935: Yamazaki et al., 1994, 196120).
Chronic irritation was indicated by the observation of rhinitis and inflammation of the nasal
cavity in rats from the JBRC (1998, 196240) study. This study also showed atrophy of the nasal
epithelium and adhesion in rats. Regeneration of the nasal epithelium is demonstrated by
metaplasia and hyperplasia observed in rats exposed to 1,4-dioxane (JBRC, 1998, 196240: Kano
et al., 2009, 594539: Yamazaki et al., 1994, 196120).
4.7.3.3. Dose-Response Relationship
4.7.3.3.1. Liver. Table 4-19 presents the temporal sequence and dose-response relationship for
possible key events in the liver carcinogenesis of 1,4-dioxane. Dose-response information
provides some support for enhanced cell proliferation as a key event in the liver tumorigenesis of
1,4-dioxane; however, the role of cytotoxicity as a required precursor event is not supported by
data from more than one study. Kociba et al. (1974, 062929) demonstrated that liver toxicity and
hepatocellular regeneration occurred at a lower dose level than tumor formation. Hepatocellular
degeneration and necrosis were observed in the mid- and high-dose groups of Sherman rats
exposed to 1,4-dioxane, although it is not possible to discern whether this effect was observed in
both genders due to the lack of incidence data (Kociba et al., 1974, 062929). Hepatic tumors
were only observed at the highest dose (Kociba et al., 1974, 062929). Hepatic regeneration was
indicated in the mid- and high-dose group by the formation of hepatocellular hyperplastic
nodules. Liver hyperplasia was also seen in rats from the JBRC (1998, 196240) study, at or
below the dose level that resulted in tumor formation (Kano et al., 2009, 594539): however,
hepatocellular degeneration and necrosis were not observed. These results suggest that hepatic
cell proliferation and hyperplasia may occur in the absence of significant cytotoxicity. Liver
angiectasis (i.e., dilation of blood or lymphatic vessels) was observed in male mice at the same
dose that produced liver tumors; however, the relationship between this vascular abnormality and
tumor formation is unclear.
-------
Table 4-19. Temporal sequence and dose-response relationship for possible
key events and liver tumors in rats and mice
Dose (mg/kg-day)
Key event (time — >)
Metabolism
1,4-dioxane
Liver damage
Cell
proliferation
Hyperplasia
Adenomas
and/or
carcinomas
Kociba et al., (1974, 062929) — Sherman rats (male and female combined)
0
14
121
1,307
a
+b
+b
+b
a
+c
+c
a
a
a
a
+c
+c
a
a
a
+c
NCI, (1978, 062935)— female Osborne-Mendel rats
0
350
640
a
+b
+b
a
a
a
a
a
a
a
a
a
a
+c
+c
NCI, (1978, 062935)— male B6C3Fi mice
0
720
830
a
+b
+b
a
a
a
a
a
a
a
a
a
a
+c
+c
NCI, (1978, 062935)— female B6C3Fi mice
0
380
860
a
+b
+b
a
a
a
a
a
a
a
a
a
a
+c
+c
Kano et al., (2009, 594539); JBRC, (1998, 196240)— male F344/DuCrj rats
0
11
55
274
a
+b
+b
+b
a
a
a
+c,d
a
a
a
a
a
a
+o,e
+c,e
a
a
a
+c,e
Kano et al., (2009, 594539); JBRC, (1998, 196240)— female F344/DuCrj rats
0
18
83
429
a
+b
+b
+b
a
a
a
a
a
a
a
a
a
a
a
+c,e
a
a
a
+c,e
Kano et al., (2009, 594539); JBRC, (1998, 196240)— male Crj:BDFl mice
0
49
191
677
a
+b
+b
+b
a
a
a
+c,d
a
a
a
a
a
a
a
a
a
+c,e
+o,e
+c,e
89
-------
Dose (mg/kg-day)
Key event (time — >)
Metabolism
1,4-dioxane
Liver damage
Cell
proliferation
Hyperplasia
Adenomas
and/or
carcinomas
Kano et al., (2009, 594539); JBRC, (1998, 196240)— female Crj:BDFl mice
0
66
278
964
a
+b
+b
+b
a
a
a
+c,d
a
a
a
a
a
a
a
a
a
+c,e
+c,e
+c,e
3— No evidence demonstrating key event.
b+ 1,4-dioxane metabolism was not evaluated as part of the chronic bioassays. Data from pharmacokinetic studies
suggest that metabolism of 1,4-dioxane by CYP2E1 and CYP2B2 occurs immediately and continues throughout the
duration of exposure at all exposure levels.
°+ Evidence demonstrating key event.
d+ Single cell necrosis was observed in a 13 week bioassay for male rats (274 mg/kg-day), male mice (585 mg/kg-
day), and female mice (898 mg/kg-day) exposed to 1,4-dioxane in drinking water (Kano et al., 2008, 196245).
e+ Kano et al. (2009, 594539) reported incidence rates for hepatocellular adenomas and carcinomas; however,
information from JBRC (1998, 196240) on incidence of liver hyperplasia was used to create this table.
4.7.3.3.2. Nasal cavity. Toxicity and regeneration in nasal epithelium (i.e., atrophy, adhesion,
inflammation, and hyperplasia and metaplasia of respiratory and olfactory epithelium) was
evident in one study at the same dose levels that produced nasal cavity tumors (see also JBRC,
1998, 196240: Kano et al., 2009, 594539).
4.7.3.4. Temporal Relationship
4.7.3.4.1. Liver. Available information regarding temporal relationships between the key event
(sustained proliferation of spontaneously transformed liver cells) and the eventual formation of
liver tumors is limited. A comparison of 13-week and 2-year studies conducted in F344/DuCrj
rats and Crj :BDF1 mice at the same laboratory revealed that tumorigenic doses of 1,4-dioxane
produced liver toxicity by 13 weeks of exposure (JBRC, 1998, 196240: Kano et al., 2008,
196245: Kano et al., 2009, 594539). Hepatocyte swelling of the centrilobular area of the liver,
vacuolar changes in the liver, granular changes in the liver, and single cell necrosis in the liver
were observed in mice and rats given 1,4-dioxane in the drinking water for 13 weeks. Sustained
liver damage could presumably lead to regenerative hyperplasia and tumor formation following
chronic exposure. As discussed above, histopathological evidence of regenerative hyperplasia
has been seen following long-term exposure to 1,4-dioxane (JBRC, 1998, 196240: Kociba et al.,
1974, 062929). Tumors occurred earlier at high doses in both mice and rats from this study
(Yamazaki, 2006, 626614): however, temporal information regarding hyperplasia or other
possible key events was not available (i.e., interim blood samples not collected, interim sacrifices
were not performed). Argus et al. (1973, 062912) studied the progression of tumorigenesis by
electron microscopy of liver tissues obtained following interim sacrifices at 8 and 13 months of
90
-------
exposure (five rats/group, 574 mg/kg-day). The first change observed was an increase in the size
of the nuclei of the hepatocytes, mostly in the periportal area. Precancerous changes were
characterized by disorganization of the rough endoplasmic reticulum, increase in smooth
endoplasmic reticulum, and decrease in glycogen and increase in lipid droplets in hepatocytes.
These changes increased in severity in the hepatocellular carcinomas in rats exposed to
1,4-dioxane for 13 months.
Three types of liver nodules were observed in exposed rats at 13-16 months. The first
consisted of groups of these cells with reduced cytoplasmic basophilia and a slightly nodular
appearance as viewed by light microscopy. The second type of nodule was described consisting
of large cells, apparently filled and distended with fat. The third type of nodule was described as
finger-like strands, 2-3 cells thick, of smaller hepatocytes with large hyperchromic nuclei and
dense cytoplasm. This third type of nodule was designated as an incipient hepatoma, since it
showed all the histological characteristics of a fully developed hepatoma. All three types of
nodules were generally present in the same liver.
4.7.3.4.2. Nasal cavity. No information was available regarding the temporal relationship
between toxicity in the nasal epithelium and the formation of nasal cavity tumors.
4.7.3.5. Biological Plausibility and Coherence
4.7.3.5.1. Liver. The hypothesis that sustained proliferation of spontaneously transformed liver
cells is a key event within a MOA is possible based on supporting evidence indicating that
1,4-dioxane is a tumor promoter of mouse skin and rat liver tumors (Bull et al., 1986, 194336;
King et al., 1973, 029390: Lundberg et al., 1987, 062933). Further support for this hypothesis is
provided by studies demonstrating that 1,4-dioxane increased hepatocyte DNA synthesis,
indicative of cell proliferation (Goldsworthy et al., 1991, 062925; Miyagawa et al., 1999,
195063: Stott et al., 1981, 063021: Uno et al., 1994, 194385). In addition, the generally negative
results for 1,4-dioxane in a number of genotoxicity assays indicates the carcinogenicity of
1,4-dioxane may not be mediated by a mutagenic MOA. The importance of cytotoxicity as a
necessary precursor to sustained cell proliferation is biologically plausible, but is not supported
by the dose-response in the majority of studies of 1,4-dioxane carcinogenicity.
4.7.3.5.2. Nasal cavity. Sustained cell proliferation in response to cell death from toxicity may
be related to the formation of nasal cavity tumors; however, this MOA is also not established .
Nasal carcinogens are generally characterized as potent genotoxins (Ashby, 1994, 195021):
however, other MO As have been proposed for nasal carcinogens that induce effects through
other mechanisms (Green et al., 2000, 196210: Kasper et al., 2007, 195045).
The National Toxicological Program (NTP) database identified 12 chemicals from
approximately 500 bioassays as nasal carcinogens and 1,4-dioxane was the only identified nasal
91
-------
carcinogen that showed little evidence of genotoxicity (Haseman and Hailey, 1997, 195983).
Nasal tumors were not observed in an inhalation study in Wistar rats exposed to 111 ppm for
5 days/week for 2 years (Torkelson et al., 1974, 094807).
4.7.3.6. Other Possible Modes of Action
An alternate MO A could be hypothesized that 1,4-dioxane alters DNA, either directly or
indirectly, which causes mutations in critical genes for tumor initiation, such as oncogenes or
tumor suppressor genes. Following these events, tumor growth may be promoted by a number of
molecular processes leading to enhanced cell proliferation or inhibition of programmed cell
death. The results from in vitro and in vivo assays do not provide overwhelming support for the
hypothesis of a genotoxic MOA for 1,4-dioxane carcinogenicity. The genotoxicity data for
1,4-dioxane were reviewed in Section 4.5.1 and were summarized in Table 4-16. Negative
findings were reported for mutagenicity in Salmonella typhimurium, Escherichia coli, and
Photobacteriumphosphoreum (Mutatox assay) (Haworth et al., 1983, 028947; Hellmer and
Bolcsfoldi, 1992, 194717: Khudoley et al., 1987, 194949: Kwan et al., 1990, 196078: Morita and
Hayashi, 1998, 195065: Nestmann et al., 1984, 194339: Stott et al., 1981, 063021). Negative
results were also indicated for the induction of aneuploidy in yeast (Saccharomyces cerevisiae)
and the sex-linked recessive lethal test in Drosophila melanogaster (Zimmermann et al., 1985,
194343). In contrast, positive results were reported in assays for sister chromatid exchange
(Galloway et al., 1987, 007768). DNA damage (Kitchin and Brown, 1990, 062928). and in in
vivo micronucleus formation in bone marrow (Mirkova, 1994, 195062: Roy et al., 2005,
196094). and liver (Morita and Hayashi, 1998, 195065: Roy et al., 2005, 196094). Lastly, in the
presence of toxicity, positive results were reported for meiotic nondisjunction in drosophila
(Munoz and Barnett, 2002, 195066). DNA damage (Sina et al., 1983, 007323). and cell
transformation (Sheu et al., 1988, 195078).
Additionally, 1,4-dioxane metabolism did not produce reactive intermediates that
covalently bound to DNA (Stott et al., 1981, 063021: Woo et al., 1977, 062950) and DNA repair
assays were generally negative (Goldsworthy et al., 1991, 062925: Stott et al., 1981, 063021).
No studies were available to assess the ability of 1,4-dioxane or its metabolites to induce
oxidative damage to DNA.
4.7.3.7. Conclusions About the Hypothesized Mode of Action
4.7.3.7.1. Liver. The MOA by which 1,4-dioxane produces liver tumors is unknown, and
available evidence in support of any hypothetical mode of carcinogenic action for 1,4-dioxane is
inconclusive. A MOA hypothesis involving 1,4-dioxane induced cell proliferation is possible
but data are not available to support this hypothesis. Pharmacokinetic data suggest that
clearance pathways were saturable and target organ toxicity occurs after metabolic saturation.
Liver toxicity preceded tumor formation in one study (Kociba et al., 1974, 062929) and a
92
-------
regenerative response to tissue injury was demonstrated by histopathology. Liver hyperplasia
and tumor formation have also been observed in the absence of cytotoxicity (see also JBRC,
1998, 196240; Kano et al., 2009, 594539). Cell proliferation and tumor promotion have been
shown to occur after prolonged exposure to 1,4-dioxane (Bull et al., 1986, 194336; Goldsworthy
et al., 1991, 062925: King et al., 1973, 029390: Lundberg et al., 1987, 062933: Miyagawa et al.,
1999, 195063: Stott et al., 1981, 063021: Uno et al., 1994, 194385).
4.7.3.7.2. Nasal cavity. The MOA for the formation of nasal cavity tumors is unknown, and
evidence in support of any hypothetical mode of carcinogenic action for 1,4-dioxane is
inconclusive.
4.7.3.8. Relevance of the Mode of Action to Humans
Several hypothesized MO As for 1,4-dioxane induced tumors in laboratory animals have
been discussed along with the supporting evidence for each. As was stated, the MOA by which
1,4-dioxane produces liver, nasal, peritoneal, and mammary gland tumors is unknown. Some
mechanistic information is available to inform the MOA of the liver and nasal tumors but no
information exists to inform the MOA of the observed peritoneal or mammary gland tumors (see
also JBRC, 1998, 196240: Kano et al., 2009, 594539: Yamazaki et al., 1994, 196120).
4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES
There is no direct evidence to establish that certain populations and lifestages may be
potentially susceptible to 1,4-dioxane. Changes in susceptibility with lifestage as a function of
the presence of microsomal enzymes that metabolize and detoxify this compound (i.e., CYP2E1
present in liver, kidney, and nasal mucosa can be hypothesized). Vieira et al. (1996, 011956)
reported that large increases in hepatic CYP2E1 protein occur postnatally between 1 and
3 months in humans. Adult hepatic concentrations of CYP2E1 are achieved sometime between 1
and 10 years. To the extent that hepatic CYP2E1 levels are lower, children may be more
susceptible to liver toxicity from 1,4-dioxane than adults. CYP2E1 has been shown to be
inducible in the rat fetus. The level of CYP2E1 protein was increased by 1.4-fold in the maternal
liver and 2.4-fold in the fetal liver following ethanol treatment, as compared to the untreated or
pair-fed groups (Carpenter et al., 1996, 080660). Pre- and postnatal induction of microsomal
enzymes resulting from exposure to 1,4-dioxane or other drugs or chemicals may reduce overall
toxicity following sustained exposure to 1,4-dioxane.
Genetic polymorphisms have been identified for the human CYP2E1 gene (Hayashi et
al., 1991, 196219: Watanabe et al., 1994, 196099) and were considered to be possible factors in
the abnormal liver function seen in workers exposed to vinyl chloride (Huang et al., 1997,
005276). Individuals with a CYP2E1 genetic polymorphism resulting in increased expression of
this enzyme may be less susceptible to toxicity following exposure to 1,4-dioxane.
93
-------
Gender differences were noted in subchronic and chronic toxicity studies of 1,4-dioxane
in mice and rats (see Sections 4.6 and 4.7). No consistent pattern of gender sensitivity was
identified across studies.
94
-------
5. DOSE-RESPONSE ASSESSMENTS
5.1. ORAL REFERENCE DOSE (RfD)
5.1.1. Choice of Principal Studies and Critical Effect with Rationale and Justification
Liver and kidney toxicity were the primary noncancer health effects associated with
exposure to 1,4-dioxane in humans and laboratory animals. Occupational exposure to
1,4-dioxane has resulted in hemorrhagic nephritis and centrilobular necrosis of the liver (Barber,
1934, 062913; Johnstone, 1959, 062927). In animals, liver and kidney degeneration and necrosis
were observed frequently in acute oral and inhalation studies (David, 1964, 195954;
de Navasquez, 1935, 196174: Drew et al., 1978, 067913: Fairley et al., 1934, 062919: JBRC,
1998, 196242: Kesten et al., 1939, 194972: Laug et al., 1939, 195055: Schrenk and Yant, 1936,
195076). Liver and kidney effects were also observed following chronic oral exposure to
1,4-dioxane in animals (Argus et al., 1965, 017009: Argus et al., 1973, 062912: JBRC, 1998,
196240: Kano et al., 2009, 594539: Kociba et al., 1974, 062929: NCI, 1978, 062935: Yamazaki
et al., 1994, 196120) (see Table 4-18).
Liver toxicity in the available chronic studies was characterized by necrosis, spongiosis
hepatic, hyperplasia, cyst formation, clear foci, and mixed cell foci. Kociba et al. (1974, 062929)
demonstrated hepatocellular degeneration and necrosis at doses of 94 mg/kg-day (LOAEL in
male rats) or greater. The NOAEL for liver toxicity was 9.6 mg/kg-day and 19 mg/kg-day in
male and female rats, respectively. No quantitative incidence data were provided in this study.
Argus et al. (1973, 062912) described early preneoplastic changes in the liver and JBRC (1998,
196240) demonstrated liver lesions that are primarily associated with the carcinogenic process.
Clear and mixed-cell foci in the liver are commonly considered preneoplastic changes and would
not be considered evidence of noncancer toxicity. In the JBRC (1998, 196240) study, spongiosis
hepatis was associated with other preneoplastic changes in the liver (clear and mixed-cell foci)
and no other lesions indicative of liver toxicity were seen. Spongiosis hepatis was therefore not
considered indicative of noncancer effects in this study. The activity of serum enzymes (i.e.,
AST, ALT, LDH, and ALP) was increased in mice and rats chronically exposed to 1,4-dioxane
(JBRC, 1998, 196240): however, these increases were seen only at tumorigenic dose levels.
Blood samples were collected at study termination and elevated serum enzymes may reflect
changes associated with tumor formation. Histopathological evidence of liver toxicity was not
seen in rats from the JBRC (1998, 196240) study. The highest non-tumorigenic dose levels for
Note: Hyperlinks to the reference citations throughout this document will take you to the NCEA HERO database
(Health and Environmental Research Online) at http://epa. gov/hero. HERO is a database of scientific literature used
by U.S. EPA in the process of developing science assessments such as the Integrated Science Assessments (ISA)
and the Integrated Risk Information System (IRIS).
95
-------
this study approximated the LOAEL derived from the Kociba et al. (1974, 062929) study (94 and
148 mg/kg-day for male and female rats, respectively).
Kidney damage in chronic toxicity studies was characterized by degeneration of the
cortical tubule cells, necrosis with hemorrhage, and glomerulonephritis (Argus et al., 1965,
017009: Argus et al., 1973, 062912: Fairley et al., 1934, 062919: Kociba et al., 1974, 062929:
NCI, 1978, 062935). Kociba et al. (1974, 062929) described renal tubule epithelial cell
degeneration and necrosis at doses of 94 mg/kg-day (LOAEL in male rats) or greater, with a
NOAEL of 9.6 mg/kg-day. No quantitative incidence data were provided in this study (Kociba
et al., 1974, 062929). Doses of > 430 mg/kg-day 1,4-dioxane induced marked kidney alterations
(Argus et al., 1973, 062912). The observed changes included glomerulonephritis and
pyelonephritis, with characteristic epithelial proliferation of Bowman's capsule, periglomerular
fibrosis, and distension of tubules. Quantitative incidence data were not provided in this study.
In the NCI (1978, 062935) study, kidney lesions in rats consisted of vacuolar degeneration
and/or focal tubular epithelial regeneration in the proximal cortical tubules and occasional
hyaline casts. Kidney toxicity was not seen in rats from the JBRC (1998, 196240) study at any
dose level (highest dose was 274 mg/kg-day in male rats and 429 mg/kg-day in female rats).
Kociba et al. (1974, 062929) was chosen as the principal study for derivation of the RfD
because the liver and kidney effects in this study are considered adverse and represent the most
sensitive effects identified in the database (NOAEL 9.6 mg/kg-day, LOAEL 94 mg/kg-day in
male rats). Kociba et al. (1974, 062929) reported degenerative effects in the liver, while liver
lesions reported in other studies (Argus et al., 1973, 062912: JBRC, 1998, 196240) appeared to
be related to the carcinogenic process. Kociba et al. (1974, 062929) also reported degenerative
changes in the kidney. NCI (1978, 062935) and Argus et al. (1973, 062912) provided supporting
data for this endpoint; however, kidney toxicity was observed in these studies at higher doses.
JBRC (1998, 196240) reported nasal inflammation in rats (NOAEL 55 mg/kg-day, LOAEL
274 mg/kg-day) and mice (NOAEL 66 mg/kg-day, LOAEL 278 mg/kg-day).
Even though the study reported by Kociba et al. (1974, 062929) had one noteworthy
weakness, it had several noted strengths, including: (1) two-year study duration; (2) use of both
male and female rats and three dose levels, 10-fold apart, plus a control group; (3) a sufficient
number of animals per dose group (60 animals/sex/dose group; and (4) the authors conducted a
comprehensive evaluation of the animals including body weights and clinical observations, blood
samples, organ weights of all the major tissues, and a complete histopathological examination of
all rats. The authors did not report individual incidence data that would have allowed for a BMD
analysis of this robust dataset.
5.1.2. Methods of Analysis—Including Models (PBPK, BMD, etc.)
Several procedures were applied to the human PBPK model to determine if an adequate
fit of the model to the empirical model output or experimental observations could be attained
96
-------
using biologically plausible values for the model parameters. The re-calibrated model
predictions for blood 1,4-dioxane levels did not come within 10-fold of the experimental values
using measured tissue:air partition coefficients of Leung and Paustenbach (1990, 062932) or
Sweeney et al. (2008, 195085) (Figures B-8 and B-9). The utilization of a slowly perfused
tissue:air partition coefficient 10-fold lower than measured values produces exposure-phase
predictions that are much closer to observations, but does not replicate the elimination kinetics
(Figure B-10). Re-calibration of the model with upper bounds on the tissue:air partition
coefficients results in predictions that are still six- to sevenfold lower than empirical model
prediction or observations (Figures B-12 and B-13). Exploration of the model space using an
assumption of zero-order metabolism (valid for the 50 ppm inhalation exposure) showed that an
adequate fit to the exposure and elimination data can be achieved only when unrealistically low
values are assumed for the slowly perfused tissue:air partition coefficient (Figure B-16).
Artificially low values for the other tissue:air partition coefficients are not expected to improve
the model fit, as these parameters are shown in the sensitivity analysis to exert less influence on
blood 1,4-dioxane than Vmaxc and Km. This suggests that the model structure is insufficient to
capture the apparent 10-fold species difference in the blood 1,4-dioxane between rats and
humans. In the absence of actual measurements for the human slowly perfused tissue:air
partition coefficient, high uncertainty exists for this model parameter value. Differences in the
ability of rat and human blood to bind 1,4-dioxane may contribute to the difference in Vd.
However, this is expected to be evident in very different values for rat and human blood:air
partition coefficients, which is not the case (Table B-l). Therefore, some other, as yet unknown,
modification to model structure may be necessary.
Kociba et al. (1974, 062929) did not provide quantitative incidence or severity data for
liver and kidney degeneration and necrosis. Benchmark dose (BMD) modeling could not be
performed for this study and the NOAEL for liver and kidney degeneration (9.6 mg/kg-day in
male rats) was used as the point of departure (POD) in deriving the RfD for 1,4-dioxane.
Alternative PODs were calculated using incidence data reported for cortical tubule
degeneration in male and female rats (NCI, 1978, 062935) and liver hyperplasia (JBRC, 1998,
196240). The incidence data for cortical tubule cell degeneration in male and female rats
exposed to 1,4-dioxane in the drinking water for 2 years are presented in Table 5-1. Details of
the BMD analysis of these data are presented in Appendix C. Male rats were more sensitive to
the kidney effects of 1,4-dioxane than females and the male rat data provided the lowest POD for
cortical tubule degeneration in the NCI (1978, 062935) study (BMDLio of 22.3 mg/kg-day)
(Table 5-2). Incidence data (JBRC, 1998, 196240: Kano et al., 2009, 594539) for liver
hyperplasia in male and female rats exposed to 1,4-dioxane in the drinking water for 2 years are
presented in Table 5-3. Details of the BMD analysis of these data are presented in Appendix C.
Male rats were more sensitive to developing liver hyperplasia due to exposure to 1,4-dioxane
than females and the male rat data provided the lowest POD for hyperplasia in the JBRC (1998,
97
-------
196240) study (BMDLio of 23.8 mg/kg-day) (Table 5-4). The BMDLio values of 22.3 mg/kg-
day and 23.8 mg/kg-day from the NCI (1978, 062935) and JBRC (1998, 196240) studies,
respectively, are about double the NOAEL (9.6 mg/kg-day) observed by Kociba et al. (1974,
062929).
Table 5-1. Incidence of cortical tubule degeneration in Osborne-Mendel rats
exposed to 1,4-dioxane in drinking water for 2 years
Males (mg/kg-day)
0
0/3 r
240
20/3 lb
530
27/3 3b
Females (mg/kg-day)
0
0/3 la
350
0/34
640
10/32b
"Statistically significant trend for increased incidence by Cochran-Armitage test (p < 0.05) performed for this
review.
blncidence significantly elevat
Source: NCI (1978, 062935).
review.
blncidence significantly elevated compared to control by Fisher's Exact test (p < 0.001) performed for this review.
Table 5-2. BMD and BMDL values derived from BMD modeling of cortical
tubule degeneration in male and female Osborne-Mendel rats exposed to
1,4-dioxane in drinking water for 2 years
Male rats
Female rats
BMD10 (mg/kg-day)
28.8
596.4
BMDL10 (mg/kg-day)
22.3
452.4
Source: NCI (1978, 062935).
Table 5-3. Incidence of liver hyperplasia in F344/DuCrj rats exposed to
1,4-dioxane in drinking water for 2 years"
Males (mg/kg-day)
0
3/40
11
2/45
55
9/35b
274
12/22C
Females (mg/kg-day)
0
0/3 8b
18
0/37
83
1/38
429
14/24C
aDose information from Kano et al. (2009, 594539) and incidence data for sacrificed animals from JBRC (1998,
196240).
bStatistically significant compared to controls by the Dunnett's test (p < 0.05).
Incidence significantly elevated compared to control by %2 test (p < 0.01).
Sources: Kano et al. (2009, 594539): JBRC (1998, 196240).
98
-------
Table 5-4. BMD and BMDL values derived from BMD modeling of liver
hyperplasia in male and female F344/DuCrj rats exposed to 1,4-dioxane in
drinking water for 2 years
Male rats
Female rats
BMD10 (mg/kg-day)
35.9
137.3
BMDL10 (mg/kg-day)
23.8
88.5
Source: Kano et al. (2009, 594539): JBRC (1998, 196240).
5.1.3. RfD Derivation - Including Application of Uncertainty Factors (UFs)
•v-2
The RfD of 3 x 10 mg/kg-day is based on liver and kidney toxicity in rats exposed to
1,4-dioxane in the drinking water for 2 years (Kociba et al., 1974, 062929). The Kociba et al.
(1974, 062929) study was chosen as the principal study because it provides the most sensitive
measure of adverse effects by 1,4-dioxane. The incidence of liver and kidney lesions was not
reported for each dose group. Therefore, BMD modeling could not be used to derive a POD.
The RfD for 1,4-dioxane is derived by dividing the NOAEL of 9.6 mg/kg-day (Kociba et al.,
1974, 062929) by a composite UF of 300, as follows:
RfD = NOAEL / UF
9.6 mg/kg-day / 300
0.03 or 3 x 10"2 mg/kg-day
The composite UF of 300 includes factors of 10 for animal-to-human extrapolation and
for interindividual variability, and an UF of 3 for database deficiencies.
A default interspecies UF of 10 was used to account for pharmacokinetic and
pharmacodynamic differences across species. Existing PBPK models could not be used to derive
an oral RfD for 1,4-dioxane (Appendix B).
A default interindividual variability UF of 10 was used to account for variation in
sensitivity within human populations because there is limited information on the degree to which
humans of varying gender, age, health status, or genetic makeup might vary in the disposition of,
or response to, 1,4-dioxane.
An UF of 3 for database deficiencies was applied due to the lack of a multigeneration
reproductive toxicity study. A single oral prenatal developmental toxicity study in rats was
available for 1,4-dioxane (Giavini et al., 1985, 062924). This developmental study indicates that
the developing fetus may be a target of toxicity.
An UF to extrapolate from a subchronic to a chronic exposure duration was not necessary
because the RfD was derived from a study using a chronic exposure protocol.
An UF to extrapolate from a LOAEL to a NOAEL was not necessary because the RfD
was based on a NOAEL. Kociba et al. (1974, 062929) was a well-conducted, chronic drinking
water study with an adequate number of animals. Histopathological examination was performed
99
-------
for many organs and tissues, but clinical chemistry analysis was not performed. NOAEL and
LOAEL values were derived by the study authors based on liver and kidney toxicity; however
quantitative incidence data was not reported. Several additional oral studies (acute/short-term,
subchronic, and chronic durations) were available that support liver and kidney toxicity as the
critical effect (Argus et al., 1973, 062912: JBRC, 1998, 196240: Kano et al., 2008, 196245: NCI,
1978, 062935) (Tables 4-15 and 4-17). Although degenerative liver and kidney toxicity was not
observed in rats from the JBRC (1998, 196240) study at doses at or below the LOAEL in the
Kociba et al. (1974, 062929) study, other endpoints such as metaplasia and hyperplasia of the
nasal epithelium, nuclear enlargement, and hematological effects, were noted.
5.1.4. RfD Comparison Information
PODs and sample oral RfDs based on selected studies included in Table 4-18 are arrayed
in Figures 5-1 to 5-3, and provide perspective on the RfD supported by Kociba et al. (1974,
062929). These figures should be interpreted with caution because the PODs across studies are
not necessarily comparable, nor is the confidence in the data sets from which the PODs were
derived the same. PODs in these figures may be based on a NOAEL, LOAEL, or BMDL (as
indicated), and the nature, severity, and incidence of effects occurring at a LOAEL are likely to
vary. To some extent, the confidence associated with the resulting sample RfD is reflected in the
magnitude of the total UF applied to the POD (i.e., the size of the bar); however, the text of
Sections 5.1.1 and 5.1.2 should be consulted for a more complete understanding of the issues
associated with each data set and the rationale for the selection of the critical effect and principal
study used to derive the RfD.
The predominant noncancer effect of chronic oral exposure to 1,4-dioxane is
degenerative effects in the liver and kidney. Figure 5-1 provides a graphical display of effects
that were observed in the liver following chronic oral exposure to 1,4-dioxane. Information
presented includes the PODs and UFs that could be considered in deriving the oral RfD. As
discussed in Sections 5.1.1 and 5.1.2, among those studies that demonstrated liver toxicity, the
study by Kociba et al. (1974, 062929) provided the data set most appropriate for deriving the
RfD. For degenerative liver effects resulting from 1,4-dioxane exposure, the Kociba et al. (1974,
062929) study represents the most sensitive effect and dataset observed in a chronic bioassay
(Figure 5-1).
Kidney toxicity as evidenced by glomerulonephritis (Argus et al., 1965, 017009: Argus et
al., 1973, 062912) and degeneration of the cortical tubule (Kociba et al., 1974, 062929: NCI,
1978, 062935) has also been observed in response to chronic exposure to 1,4-dioxane. As was
discussed in Sections 5.1 and 5.2, degenerative effects were observed in the kidney at the same
dose level as effects in the liver (Kociba et al., 1974, 062929). A comparison of the available
datasets from which an RfD could potentially be derived is presented in Figure 5-2.
100
-------
Rhinitis and inflammation of the nasal cavity were reported in both the NCI (1978,
062935) (mice only, dose > 380 mg/kg-day) and JBRC (1998, 196240) studies (> 274 mg/kg-day
in rats, >278 mg/kg-day in mice). JBRC (1998, 196240) reported nasal inflammation in rats
(NOAEL 55 mg/kg-day, LOAEL 274 mg/kg-day) and mice (NOAEL 66 mg/kg-day, LOAEL
278 mg/kg-day). A comparison of the available datasets from which an RfD could potentially be
derived is presented in Figure 5-3.
Figure 5-4 displays PODs for the major targets of toxicity associated with oral exposure
to 1,4-dioxane. Studies in experimental animals have also found that relatively high doses of
1,4-dioxane (1,000 mg/kg-day) during gestation can produce delayed ossification of the
sternebrae and reduced fetal BWs (Giavini et al., 1985, 062924). This graphical display
(Figure 5-4) compares organ specific toxicity for 1,4-dioxane, including a single developmental
study. The most sensitive measures of degenerative liver are and kidney effects. The sample
RfDs for degenerative liver and kidney effects are identical since they were derived from the
same study and dataset (Kociba et al., 1974, 062929) and are presented for completeness.
100
Rat
Rat
Mouse
Rat
Rat
10
HI
8
Q
0.1
0.01
I
• POD
OAnimal-to-human
nHuman variation
ElLOAEL to NOAEL
dSubchronic to Chronic
BDatabase deficiencies
ORfD
Liver hyperplasia; Hepatocellular Increase in serum liver Increase in serum liver Liver hyperplasia;
NOAEL; 2 yr rat degeneration and enzymes; NOAEL; 2 yr enzymes; NOAEL; 2 yr BMDL10; 2 yr rat
drinking water study necrosis; NOAEL; 2 yr mouse drinking water rat drinking water study drinking water study
rat drinking water study study
Figure 5-1. Potential points of departure (POD) for liver toxicity endpoints
with corresponding applied uncertainty factors and derived RfDs following
oral exposure to 1,4-dioxane.
101
-------
1000
100
10
Rat
Rat
Rat
0.1 -
0.01
• POD
miAnimal-to-human
QHuman variation
E3LOAELtoNOAEL
CUSubchronic to Chronic
• Database deficiencies
ORfD
Glomerulonephritis; LOAEL; 13 month Degeneration and necrosis of tubular Cortical tubule degeneration; BMDL10;
rat drinking water study epithelium; NOAEL; 2 yr rat drinking 2 yr rat drinking water study
water study
Figure 5-2. Potential points of departure (POD) for kidney toxicity endpoints
with corresponding applied uncertainty factors and derived RfDs following
oral exposure to 1,4-dioxane.
102
-------
Mouse
100
10
o
Q
0.1
Rat
1
I
O
I
• POD
U|Animal-to-human
QHurnan variation
E]LOAELto NOAEL
CUsubchronic to Chronic
BDatabase deficiencies
ORfD
Nasal inflammation; NOAEL; 2 yr mouse drinking water study Nasal inflammation; NOAEL; 2 yr rat drinking water study
Figure 5-3. Potential points of departure (POD) for nasal inflammation with
corresponding applied uncertainty factors and derived sample RfDs
following oral exposure to 1,4-dioxane.
103
-------
1000
100 -
10 -
Rat
Rat
Rat
Mouse
m
8
Q
1 -
0.01
• POD
EDAnimal-to-human
^Human variation
gLOAELto NOAEL
^jSubchronic to Chronic
f Database deficiencies
ORfD
Degeneration and necrosis Hepatocellular Delayed ossification of Nasal inflammation;
of tubular epithelium; degeneration and necrosis; sternebrae and reduced NOAEL; 2 yr mouse
NOAEL; 2 yr rat drinking NOAEL; 2 yr rat drinking fetal body weight; NOAEL; drinking water study
water study water sduy rat study gestation days 6-
15
Figure 5-4. Potential points of departure (POD) for organ specific toxicity
endpoints with corresponding applied uncertainty factors and derived
sample RfDs following oral exposure to 1,4-dioxane.
5.1.5. Previous RfD Assessment
An assessment for 1,4-dioxane was previously posted on the IRIS database in 1988. An
oral RfD was not developed as part of the 1988 assessment.
5.2. INHALATION REFERENCE CONCENTRATION (RFC)
NOTE: During the development of this assessment, new data regarding the toxicity of
1,4-dioxane through the inhalation route of exposure became available. The IRIS Program will
evaluate the more recently published 1,4-dioxane inhalation data for the potential to derive an
RfC in a separate assessment. A description of the studies that were available at the time that this
assessment was under development are summarized below.
Inhalation studies for 1,4-dioxane evaluated in this assessment were not adequate for the
determination of an RfC value. Only one subchronic study (Fairley et al., 1934, 062919) and one
chronic inhalation study (Torkelson et al., 1974, 094807) were identified. In the subchronic
study, rabbits, guinea pigs, rats, and mice (3-6/species/group) were exposed to 1,000, 2,000,
5,000, or 10,000 ppm of 1,4-dioxane vapor for 1.5 hours two times a day for 5 days, 1.5 hours
104
-------
for one day, and no exposure on the seventh day. Animals were exposed until death occurred or
were sacrificed after various durations of exposure (3-202.5 hours). Detailed dose-response
information was not provided; however, severe liver and kidney damage and acute vascular
congestion of the lungs were observed at concentrations > 1,000 ppm. Kidney damage was
described as patchy degeneration of cortical tubules with vascular congestion and hemorrhage.
Liver lesions varied from cloudy hepatocyte swelling to large areas of necrosis.
Torkelson et al. (1974, 094807) performed a chronic inhalation study in which male and
female Wistar rats (288/sex) were exposed to 111 ppm 1,4-dioxane vapor for 7 hours/day,
5 days/week for 2 years. Control rats (192/sex) were exposed to filtered air. No significant
effects were observed on BWs, survival, organ weights, hematology, clinical chemistry, or
histopathology. Because Fairley et al. (1934, 062919) identified a free-standing LOAEL only,
and Torkelson et al. (1974, 094807) identified a free-standing NOAEL only, neither study was
sufficient to characterize the inhalation risks of 1,4-dioxane. A route extrapolation from oral
toxicity data was not performed because 1,4-dioxane inhalation causes direct effects on the
respiratory tract (i.e., respiratory irritation in humans, pulmonary congestion in animals) (Fairley
et al., 1934, 062919: Wirth and Klimmer, 1936, 196105: Yant et al., 1930, 062952). which
would not be accounted for in a cross-route extrapolation. In addition, available kinetic models
are not suitable for this purpose (Appendix B).
An assessment for 1,4-dioxane was previously posted on the IRIS database in 1988. An
inhalation RfC was not developed as part of the 1988 assessment.
5.3. UNCERTAINTIES IN THE ORAL REFERENCE DOSE (RfD)
Risk assessments need to portray associated uncertainty. The following discussion
identifies uncertainties associated with the RfD for 1,4-dioxane. As presented earlier in this
section (5.1.2 and 5.1.3), the uncertainty factor approach (U.S. EPA, 1994, 006488: U.S. EPA,
2002, 088824), was applied to a POD. Factors accounting for uncertainties associated with a
number of steps in the analyses were adopted to account for extrapolating from an animal
bioassay to human exposure, a diverse population of varying susceptibilities, and to account for
database deficiencies. These extrapolations are carried out with current approaches given the
paucity of experimental 1,4-dioxane data to inform individual steps.
An adequate range of animal toxicology data are available for the hazard assessment of
1,4-dioxane, as described throughout the previous section (Section 4). The database of oral
toxicity studies includes chronic drinking water studies in rats and mice, multiple subchronic
drinking water studies conducted in rats and mice, and a developmental study in rats. Toxicity
associated with oral exposure to 1,4-dioxane is observed predominately in the liver and kidney.
The database of inhalation toxicity studies in animals includes one subchronic bioassay in
rabbits, guinea pigs, and rats, and a chronic inhalation bioassay in rats. Although the subchronic
bioassay observed degenerative effects in the liver, kidney, and lungs of all species tested, the
105
-------
information reported from the study was insufficient to determine an exposure level below which
these effects did not occur. The only available chronic inhalation bioassay did not indicate any
treatment related effects due to exposure to 1,4-dioxane. Thus, the inhalation database lacked
sufficient information to derive toxicity values relevant to this route of exposure for 1,4-dioxane.
In addition to oral and inhalation data, there are PBPK models and genotoxicity studies of
1,4-dioxane. Critical data gaps have been identified and uncertainties associated with data
deficiencies of 1,4-dioxane are more fully discussed below.
Consideration of the available dose-response data led to the selection of the two-year
drinking water bioassay in Sherman rats (Kociba et al., 1974, 062929) as the principal study and
increased liver and kidney degeneration as the critical effects for deriving the RfD for
1,4-dioxane. The dose-response relationship for oral exposure to 1,4-dioxane and cortical tubule
degeneration in Osborne-Mendel rats (NCI, 1978, 062935) was also suitable for deriving a RfD,
but it is associated with higher a POD and potential RfD compared to Kociba et al. (1974,
062929).
The RfD was derived by applying UFs to a NOAEL for degenerative liver and kidney
effects. The incidence data for the observed effects were not reported in the principal study
(Kociba et al., 1974, 062929), precluding modeling of the dose-response. However confidence
in the LOAEL can be derived from additional studies (Argus et al., 1965, 017009; Argus et al.,
1973, 062912: JBRC, 1998, 196240: NCI, 1978, 062935) that observed effects on the same
organs at comparable dose levels and by the BMDL generated by modeling of the kidney dose-
response data from the chronic NCI (1978, 062935) study.
Extrapolating from animals to humans embodies further issues and uncertainties. The
effect and the magnitude associated with the dose at the POD in rodents are extrapolated to
human response. Pharmacokinetic models are useful to examine species differences in
pharmacokinetic processing; however, it was determined that dosimetric adjustment using
pharmacokinetic modeling was to reduce uncertainty following oral exposure to 1,4-dioxane was
not supported. Insufficient information was available to quantitatively assess toxicokinetic or
toxicodynamic differences between animals and humans, so a 10-fold UF was used to account
for uncertainty in extrapolating from laboratory animals to humans in the derivation of the RfD.
Heterogeneity among humans is another uncertainty associated with extrapolating doses
from animals to humans. Uncertainty related to human variation needs consideration. In the
absence of 1,4-dioxane-specific data on human variation, a factor of 10 was used to account for
uncertainty associated with human variation in the derivation of the RfD. Human variation may
be larger or smaller; however, 1,4-dioxane-specific data to examine the potential magnitude of
over- or under-estimation are unavailable.
Uncertainties in the assessment of the health hazards of ingested 1,4-dioxane are
associated with deficiencies in reproductive toxicity information. The oral database lacks a
multigeneration reproductive toxicity study. A single oral prenatal developmental toxicity study
106
-------
in rats was available for 1,4-dioxane (Giavini et al., 1985, 062924). This developmental study
indicates that the developing fetus may be a target of toxicity. The database of inhalation studies
is of particular concern due to the lack of a basic toxicological studies, a multigenerational
reproductive study, and developmental toxicity studies.
5.4. CANCER ASSESSMENT
5.4.1. Choice of Study/Data - with Rationale and Justification
Three chronic drinking water bioassays provided incidence data for liver tumors in rats
and mice, and nasal cavity, peritoneal, and mammary gland tumors in rats only (JBRC, 1998,
196240: Kano et al., 2009, 594539: Kociba et al., 1974, 062929: NCI, 1978, 062935: Yamazaki
et al., 1994, 196120). The dose-response data from each of these studies are summarized in
Table 5-5. With the exception of the NCI (1978, 062935) study, the incidence of nasal cavity
tumors was generally lower than the incidence of liver tumors in exposed rats. The Kano et al.
(2009, 594539) drinking water study was chosen as the principal study for derivation of an oral
cancer slope factor (CSF) for 1,4-dioxane. This study used three dose groups in addition to
controls and characterized the dose-response relationship at lower exposure levels, as compared
to the high doses employed in the NCI (1978, 062935) bioassay (Table 5-5). The Kociba et al.
(1974, 062929) study also used three dose groups and low exposures; however, the study authors
only reported the incidence of hepatocellular carcinoma, which may underestimate the combined
incidence of rats with adenoma or carcinoma. In addition to increased incidence of liver tumors,
chosen as the most sensitive target organ for tumor formation, the Kano et al. (2009, 594539)
study also noted increased incidence of peritoneal and mammary gland tumors. Nasal cavity
tumors were also seen in high-dose male and female rats; however, the incidence of nasal tumors
was much lower than the incidence of liver tumors in both rats and mice.
In a personal communication, Dr. Yamazaki (2006, 626614) provided that the survival of
mice was low in all male groups (31/50, 33/50, 25/50 and 26/50 in control, low-, mid-, and high-
dose groups, respectively) and particularly low in high-dose females (29/50, 29/50, 17/50, and
5/50 in control, low-, mid-, and high-dose groups, respectively). These deaths occurred
primarily during the second year of the study. Survival at 12 months in male mice was 50/50,
48/50, 50/50, and 48/50 in control, low-, mid-, and high-dose groups, respectively. Female
mouse survival at 12 months was 50/50, 50/50, 48/50, and 48/50 in control, low-, mid-, and high-
dose groups, respectively (Yamazaki, 2006, 626614). Furthermore, these deaths were primarily
tumor related. Liver tumors were listed as the cause of death for 31 of the 45 pretermination
deaths in high-dose female Crj :BDF1 mice (Yamazaki, 2006, 626614). Thus, the high mortality
rates in the female mice were still considered to be relevant for this analysis.
107
-------
Table 5-5. Incidence of liver, nasal cavity, peritoneal, and mammary gland
tumors in rats and mice exposed to 1,4-dioxane in drinking water for 2 years
(based on survival to 12 months)
Study
Kociba et al.
(1974, 062929)
NCI (1978,
0629351
Kano et al. (2009,
594539)
Species/strain/gender
Sherman rats, male
and female
combined3'13
Male Osborne-Mendel
ratsb
Female Osborne-
Mendel ratsb>c
Male B6C3FJ miced
Female B6C3FJ miced
Male F344/DuCrj
ratsd,e,f,g
Female F344/DuCrj
ratsd,e,f,g
MaleCrj:BDFlmiced
Female Crj:BDFl
miced
Animal dose
(mg/kg-day)
0
14
121
1,307
0
240
530
0
350
640
0
720
830
0
380
860
0
11
55
274
0
18
83
429
0
49
191
677
0
66
278
964
Tumor Incidence
Liver
l/106h
0/110
1/106
10/661
NA
NA
NA
0/3 lh
10/301
11/291
8/49h
19/501
28/471
0/50h
21/481
35/371
3/50
4/50
7/50
39/50>'k
3/50
1/50
6/50
48/50"*
23/50
31/50
37/501
40/50>'k
5/50
35/501
41/501
46/501'k
Nasal
cavity
0/106h
0/110
0/106
3/66
0/33h
12/26
16/331
0/34h
10/301
8/291
NA
NA
NA
NA
NA
NA
0/50
0/50
0/50
7/50k
0/50
0/50
0/50
s/so1*
0/50
0/50
0/50
1/50
0/50
0/50
0/50
1/50
Peritoneal
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
2/50
2/50
5/50
28/501'k
1/50
0/50
0/50
0/50
NA
NA
NA
NA
NA
NA
NA
NA
Mammary
gland
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
1/50
2/50
2/50
6/50k
8/50
8/50
11/50
18/50''k
NA
NA
NA
NA
NA
NA
NA
NA
108
-------
Incidence of hepatocellular carcinoma.
blncidence of nasal squamous cell carcinoma.
Incidence of hepatocellular adenoma.
Incidence of hepatocellular adenoma or carcinoma.
Incidence (sum) of all nasal tumors including squamous cell carcinoma, sarcoma, rhabdomyosarcoma, and
esthesioneuroepithelioma.
Incidence of peritoneal tumors (mesothelioma).
Incidence of mammary gland tumors (fibroadenoma or adenoma)
hp < 0.05; positive dose-related trend (Cochran-Armitage orPeto's test).
'Significantly different from control atp < 0.05 by Fisher's Exact test.
JSignificantly different from control atp < 0.01 by Fisher's Exact test.
kp < 0.01; positive dose-related trend (Peto's test).
NA = data were not available for modeling (no significant change from controls)
5.4.2. Dose-Response Data
Table 5-6 summarizes the incidence of hepatocellular adenoma or carcinoma in rats and
mice from the Kano et al. (2009, 594539) 2-year drinking water study. There were statistically
significant increasing trends in tumorigenic response for males and females of both species. The
dose-response curve for female mice is steep, with 70% incidence of liver tumors occurring in
the low-dose group (66 mg/kg-day). Exposure to 1,4-dioxane increased the incidence of these
tumors in a dose-related manner.
A significant increase in the incidence of peritoneal mesothelioma was observed in high-
dose male rats only (28/50 rats, Table 5-5). The incidence of peritoneal mesothelioma was lower
than the observed incidence of hepatocellular adenoma or carcinoma in male rats (Table 5-6);
therefore, hepatocellular adenoma or carcinoma data were used to derive an oral CSF for
1,4-dioxane.
109
-------
Table 5-6. Incidence of hepatocellular adenoma or carcinoma in rats and
mice exposed to 1,4-dioxane in drinking water for 2 years
Species/strain/gender
Male F344/DuCrj rats
Female F344/DuCrj rats
Male Crj:BDFl mice
Female Crj:BDFl mice
Animal dose
(mg/kg-day)
0
11
55
274
0
18
83
429
0
49
191
677
0
66
278
964
Incidence of liver tumors"
3/50
4/50
7/50
39/50b'c
3/50
1/50
6/50
48/50b'c
23/50
31/50
37/50d
40/50b'c
5/50
35/50c
41/50C
46/50b'c
""Incidence of either hepatocellular adenoma or carcinoma.
bp < 0.05; positive dose-related trend (Peto's test).
Significantly different from control atp < 0.01 by Fisher's Exact test.
dSignificantly different from control atp < 0.01 by Fisher's Exact test.
Source: Used with permission from Elsevier, Ltd., Kano et al. (2009, 594539).
5.4.3. Dose Adjustments and Extrapolation Method(s)
5.4.3.1. Dose Adjustments
Human equivalent doses (HEDs) were calculated from the administered animal doses
using a BW scaling factor (BW0'75). This was accomplished using the following equation:
TTT^ u f n \ |~ animal BW (kg)
HED = animal dose (mg/kg) x ^-^L
[ human BW (kg)
For all caculations, a human BW of 70 kg was used. HEDs for the principal study (Kano et al.,
2009, 594539) are given in Table 5-7. HEDs were also calculated for supporting studies (Kociba
et al., 1974, 062929: NCI, 1978, 062935) and are also shown in Table 5-7.
110
-------
Table 5-7. Calculated HEDs for the tumor incidence data used for dose-
response modeling
Study
Kano et al. (2009,
5945391
Kocibaetal. (1974,
062929)
NCI (1978, 062935)
Species/strain/gender
Male F344/DuCrj rats
Female F344/DuCrj rats
Male Crj:BDFl mice
Female Crj:BDFl mice
Male and female (combined)
Sherman rats
Male Osborne-Mendel rats
Female Osborne-Mendel rats
Male B6C3FJ mice
Female B6C3FJ mice
Animal BW (g)
TWA
432a
432a
432a
267a
267a
267a
47.9a
47.9a
47.9a
35. 9a
35. 9a
35. 9a
325b
325b
285C
470b
470b
310b
310b
32b
32b
30b
30b
Animal dose
(mg/kg-day)
11
81
398
18
83
429
49
191
677
66
278
964
14
121
1,307
240
530
350
640
720
830
380
860
RED
(mg/kg-day)d
3.1
23
112
4.5
21
107
7.9
31
110
10
42
145
3.7
32
330
69
152
90
165
105
121
55
124
a TWA BWs were determined from BW growth curves provided for each species and gender.
bTWA BWs were determined from BW curve provided for control animals.
°BWs of high dose male and female rats were significantly lower than controls throughout the study. TWA
represents the mean of TWA for male and females (calculated separately from growth curves).
dHEDs are calculated as HED = (animal dose) x (animal BW / human BW)°25.
Sources: Kano et al. (2009, 594539): Kociba et al. (1974, 062929): and NCI (1978, 062935).
5.4.3.2. Extrapolation Method(s)
The U.S. EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005, 086237)
recommend that the method used to characterize and quantify cancer risk from a chemical is
determined by what is known about the mode of action of the carcinogen and the shape of the
cancer dose-response curve. The linear approach is recommended if the mode of action of
carcinogenicity is not understood (U.S. EPA, 2005, 086237). In the case of 1,4-dioxane, the
mode of carcinogenic action for peritoneal, mammary, nasal, and liver tumors is unknown.
Therefore, a linear low-dose extrapolation approach was used to estimate human carcinogenic
risk associated with 1,4-dioxane exposure.
Ill
-------
However, several of the external peer review panel members (Appendix A: Summary of
External Peer Review and Public Comments and Disposition) recommended that the mode of
action data support the use of a nonlinear extrapolation approach to estimate human carcinogenic
risk associated with exposure to 1,4-dioxane and that such an approach should be presented in
the Toxicological Review. As discussed in Section 4.7.3., numerous short-term in vitro and a
few in vivo tests were nonpositive for 1,4-dioxane-induced genotoxicity. Results from two-stage
mouse skin tumor bioassays demonstrated that 1,4-dioxane does not initiate mouse skin tumors,
but it is a promoter of skin tumors initiated by DMBA (King et al., 1973, 029390). These data
suggest that a potential mode of action for 1,4-dioxane-induced tumors may involve proliferation
of cells initiated spontaneously, or by some other agent, to become tumors (Bull et al., 1986,
194336: Goldsworthy et al., 1991, 062925: King et al., 1973, 029390: Lundberg et al., 1987,
062933: Miyagawa et al., 1999, 195063: Stott et al., 1981, 063021: Uno et al., 1994, 194385).
However, key events related to the promotion of tumor formation by 1,4-dioxane are unknown.
Therefore, under the U.S. EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005,
086237), EPA concluded that the available information does not establish a plausible mode of
action for 1,4-dioxane and data are insufficient to establish significant biological support for a
nonlinear approach. EPA determined that there are no data available to inform the low-dose
region of the dose response, and thus, a nonlinear approach was not included.
Accordingly, the CSF for 1,4-dioxane was derived via a linear extrapolation from the
POD calculated by curve fitting the experimental dose-response data. The POD is the 95%
lower confidence limit on the dose associated with a benchmark response (BMR) near the lower
end of the observed data. The BMD modeling analysis used to estimate the POD is described in
detail in Appendix D and is summarized below in Section 5.4.4.
Model estimates were derived for all available bioassays and tumor endpoints (Appendix
D); however, the POD used to derive the CSF is based on the most sensitive species and target
organ in the principal study (female mice; liver tumors; Kano et al., 2009, 594539).
The oral CSF was calculated using the following equation:
BMDL
5.4.4. Oral Slope Factor and Inhalation Unit Risk
The dichotomous models available in the Benchmark Dose Software (BMDS, version
2.1.1) were fit to the incidence data for "either hepatocellular carcinoma or adenoma" in rats and
mice, as well as mammary and peritoneal tumors in rats exposed to 1,4-dioxane in the drinking
water (Kano et al., 2009, 594539: Kociba et al., 1974, 062929: NCI, 1978, 062935) (Table 5-5).
Animal doses are used for BMD modeling and HED BMD and BMDL values are calculated
using the animal TWAs (Table 5-7) and a human BW of 70kg. Doses associated with a BMR of
10% extra risk were calculated. BMDs and BMDLs from all models are reported, and the output
112
-------
and plots corresponding to the best-fitting model are shown (Appendix D). When the best-fitting
model is not a multistage model, the multistage model output and plot are also provided
(Appendix D). A summary of the BMDS model predictions for the Kano et al. (2009, 594539),
NCI (1978, 062935), and Kociba et al. (1974, 062929) studies is shown in Table 5-8.
Table 5-8. BMD HED and BMDLHED values from models fit to tumor
incidence data for rats and mice exposed to 1,4-dioxane in drinking water for
2 years and corresponding oral CSFs
Study
Kano et al.
(2009,
594539)
Kociba et al.
(1974,
062929)
NCI (1978,
062935)
Gender/strain/species
Male F344/DuCrj ratsb
Female F344/DuCrj rats'
j
Male Crj:BDFl mice
Female Crj:BDFl miced
Female Crj:BDFl miced'e
Female Crj:BDFl miced'f
Female F344/DuCrj rats8
Male F344/DuCrj rats8
Male F344/DuCrj ratsb
Female F344/DuCrj ratsd
Male and female (combined)
Sherman rats8
Male and female (combined)
Sherman ratsb
Male Osborne Mendel ratsd
Female Osborne Mendel ratsd
Female B6C3FJ micec
Male B6C3FJ miceh
Tumor type
Hepatocellular
adenoma or
carcinoma
Nasal
squamous cell
carcinoma
Peritoneal
mesothelioma
Mammary
gland
adenoma
Nasal
squamous cell
carcinomas
Hepatocellular
carcinoma
Nasal
squamous cell
carcinomas
Hepatocellular
adenoma
Hepatocellular
adenoma or
carcinoma
BMDHED3
(mg/kg-day)
17.43
19.84
5.63
0.83
3.22e
7.51f
94.84
91.97
26.09
40.01
448.24
290.78
16.10
40 07
28.75
23.12
87.98
BMDLHED3
(mg/kg-
day)
14.33
14.43
2.68
0.55
2.12e
4.95f
70.23
68.85
21.39
20.35
340.99
240.31
10.66
95 89
18.68
9.75
35.67
Oral CSF
(mg/kg-day)1
7.0 x 10'3
6.9 x 10'3
9
3.7 x 10
0.18
0.14
0.10
1.4xlO"3
1.5 x 10"3
4.7 x 1Q-3
4.9 x 10"3
2.9 x 10"4
4.2 x 10'4
9.4 x 10'3
3 9 x 1 0"3
5.4 x 10'3
l.OxlO'2
2.8 x 10'3
"Values associated with a BMR of 10% unless otherwise noted.
bProbit model, slope parameter not restricted.
'Multistage model, degree of polynomial = 2.
dLog-logistic model, slope restricted > 1.
"Values associated with a BMR of 30%.
Values associated with a BMR of 50%.
8Multistage model, degree of polynomial =3.
hGamma model.
113
-------
The multistage model did not provide an adequate fit (as determined by AIC,/>-value
< 0.1, and 'ip > |0.1|) to the data for the incidence of hepatocellular adenoma or carcinoma in
female mice (Appendix D). The high dose was dropped for the female mouse liver tumor dataset
in an attempt to achieve an adequate fit; however, an adequate fit was still not achieved.
Because the female mice were clearly the most sensitive group tested, other BMD models were
applied to the female mouse liver tumor dataset to achieve an adequate fit. The log-logistic
model was the only model that provided adequate fit for this data set due to the steep rise in the
dose-response curve (70% incidence at the low dose) followed by a plateau at near maximal
tumor incidence in the mid- and high-dose regions (82 and 92% incidence, respectively). The
predicted BMDio and BMDLio for the female mouse data are presented in Table 5-8, as well as
BMDHED and BMDLHED values associated with BMRs of 30 and 50% .
The multistage model also did not provide an adequate fit to mammary tumor incidence
data for the female rat or male rat peritoneal tumors. The predicted BMDio and BMDLio for
female rat mammary tumors and male peritoneal tumors obtained from the log-logistic and
probit models, respectively, are presented in Table 5-8.
A comparison of the model estimates derived for rats and mice from the Kano et al.
(2009, 5945391 NCI (1978, 0629351 and Kociba et al. (1974, 062929) studies (Table 5-8)
indicates that female mice are more sensitive to liver carcinogenicity induced by 1,4-dioxane
compared to other species or tumor types. The BMDL50 HED for the female mouse data was
chosen as the POD and the CSF of 0.10 (mg/kg-day)"1 was calculated as follows:
CSF = — = 0.10 (mg/kg - day)'1
4.95 mg/kg - day (BMDL50HED for female mice)
Calculation of a CSF for 1,4-dioxane is based upon the dose-response data for the most
sensitive species and gender.
Inhalation studies for 1,4-dioxane evaluated in this assessment were not adequate for the
determination of an inhalation unit risk. No treatment-related tumors were noted in a chronic
inhalation study in rats; however, only a single exposure concentration was used (111 ppm
1,4-dioxane vapor for 7 hours/day, 5 days/week for 2 years) (Torkelson et al., 1974, 094807). A
route extrapolation from oral bioassay data was not performed (Section 5.2). In addition,
available kinetic models are not suitable for this purpose (Appendix B).
During the development of this assessment, new data regarding the toxicity of
1,4-dioxane through the inhalation route of exposure became available. The IRIS Program will
evaluate the more recently published 1,4-dioxane inhalation data for the potential to derive an
inhalation unit risk in a separate assessment.
5.4.5. Previous Cancer Assessment
A previous cancer assessment was posted for 1,4-dioxane on IRIS in 1988. 1,4-Dioxane
was classified as a Group B2 Carcinogen (probable human carcinogen; sufficient evidence from
114
-------
animal studies and inadequate evidence or no data from human epidemiology studies (U.S. EPA,
1986, 199530)) based on the induction of nasal cavity and liver carcinomas in multiple strains of
rats, liver carcinomas in mice, and gall bladder carcinomas in guinea pigs. An oral CSF of 0.011
(mg/kg-day)"1 was derived from the tumor incidence data for nasal squamous cell carcinoma in
male rats exposed to 1,4-dioxane in drinking water for 2 years (NCI, 1978, 062935). The
linearized multistage extra risk procedure was used for linear low dose extrapolation.
5.5. UNCERTAINTIES IN CANCER RISK VALUES
As in most risk assessments, extrapolation of study data to estimate potential risks to
human populations from exposure to 1,4-dioxane has engendered some uncertainty in the results.
Several types of uncertainty may be considered quantitatively, but other important uncertainties
cannot be considered quantitatively. Thus an overall integrated quantitative uncertainty analysis
is not presented. Principal uncertainties are summarized below and in Table 5-9.
5.5.1. Sources of Uncertainty
5.5.1.1. Choice of Low-Dose Extrapolation Approach
The range of possibilities for the low-dose extrapolation of tumor risk for exposure to
1,4-dioxane, or any chemical, ranges from linear to nonlinear, but is dependent upon a plausible
MOA(s) for the observed tumors. The MOA is a key consideration in clarifying how risks
should be estimated for low-dose exposure. Exposure to 1,4-dioxane has been observed in
animal models to induce multiple tumor types, including liver adenomas and carcinomas, nasal
carcinomas, mammary adenomas and fibroadenomas, and mesotheliomas of the peritoneal cavity
(JBRC, 1998, 196240: Kano et al., 2009, 594539: Kociba et al., 1974, 062929: NCI, 1978,
062935). MOA information that is available for the carcinogenicity of 1,4-dioxane has largely
focused on liver adenomas and carcinomas, with little or no MOA information available for the
remaining tumor types. In Section 4.7.3, hypothesized MOAs were explored for 1,4-dioxane.
Information that would provide sufficient support for any MOA is not available. In the absence
of a MOA(s) for the observed tumor types, a linear low-dose extrapolation approach was used to
estimate human carcinogenic risk associated with 1,4-dioxane exposure.
It is not possible to predict how additional MOA information would impact the dose-
response assessment for 1,4-dioxane because of the variety of tumors observed and the lack of
data on how 1,4-dioxane or a metabolite thereof, interacts with cells starting the progression to
the observed tumors.
In general, the Agency has preferred to use the multistage model for analyses of tumor
incidence and related endpoints because they have a generic biological motivation based on
long-established mathematical models such as the Moolgavkar-Venzon-Knudsen (MVK) model.
115
-------
The MVK model does not necessarily characterize all modes of tumor formation, but it is
a starting point for most investigations and, much more often than not, has provided at least an
adequate description of tumor incidence data.
In the studies evaluated (Kano et al., 2009, 594539: Kociba et al., 1974, 062929: NCI,
1978, 062935), the multistage model provided good descriptions of the incidence of a few tumor
types in male (nasal cavity) and female (hepatocellular and nasal cavity) rats and in male mice
(hepatocellular) exposed to 1,4-dioxane (Appendix D for details). However, the multistage
model did not provide an adequate fit for the female mouse liver tumor dataset based upon the
following (U.S. EPA, 2000, 052150):
• Goodness-of-fit/>-value was not greater than 0.10;
• Akaike's Information Criterion (AIC) was larger than other acceptable models;
• Data deviated from the fitted model, as measured by their ^ residuals (values were
greater than an absolute value of one).
BMDS software typically implements the guidance in the external peer review draft
BMD technical guidance document (U.S. EPA, 2000, 052150) by imposing constraints on the
values of certain parameters of the models. When these constraints were imposed, the multistage
model and most other models did not fit the incidence data for female mouse liver adenomas or
carcinomas.
The log-logistic model was selected because it provides an adequate fit for the female
mouse data (Kano et al., 2009, 594539). A BMR of 50% was used because it is proximate to the
response at the lowest dose tested and the BMDL50 HED was derived by applying appropriate
parameter constraints, consistent with recommended use of BMDS in the BMD technical
guidance document (U.S. EPA, 2000, 052150).
The human equivalent oral CSFs estimated from tumor datasets with statistically
significant increases ranged from 4.2 x 10"4 to 0.18 per mg/kg-day (Table 5-8), a range of about
three orders of magnitude, with the extremes coming from the combined male and female rat
data for hepatocellular carcinomas (Kociba et al., 1974, 062929) and the female mouse combined
liver adenoma and carcinomas (Kano et al., 2009, 594539).
5.5.1.2. Dose Metric
1,4-Dioxane is known to be metabolized in vivo. However, it is unknown whether a
metabolite or the parent compound, or some combination of parent compound and metabolites, is
responsible for the observed toxicity. If the actual carcinogenic moiety is proportional to
administered exposure, then use of administered exposure as the dose metric is the least biased
choice. On the other hand, if this is not the correct dose metric, then the impact on the CSF is
unknown.
116
-------
5.5.1.3. Cross-Species Scaling
An adjustment for cross-species scaling (BW°75) was applied to address toxicological
equivalence of internal doses between each rodent species and humans, consistent with the 2005
Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005, 086237). It is assumed that equal
risks result from equivalent constant lifetime exposures.
5.5.1.4. Statistical Uncertainty at the POD
Parameter uncertainty can be assessed through confidence intervals. Each description of
parameter uncertainty assumes that the underlying model and associated assumptions are valid.
For the log-logistic model applied to the female mouse data, there is a reasonably small degree of
uncertainty at the 10% excess incidence level (the POD for linear low-dose extrapolation).
5.5.1.5. Bioassay Selection
The study by Kano et al. (2009, 594539) was used for development of an oral CSF. This
was a well-designed study, conducted in both sexes in two species (rats and mice) with a
sufficient number (N=50) of animals per dose group. The number of test animals allocated
among three dose levels and an untreated control group was adequate, with examination of
appropriate toxicological endpoints in both sexes of rats and mice. Alternative bioassays
(Kociba et al., 1974, 062929: NCI, 1978, 062935) were available and were fully considered for
the derivation of the oral CSF.
5.5.1.6. Choice of Species/Gender
The oral CSF for 1,4-dioxane was quantified using the tumor incidence data for the
female mouse, which was shown to be more sensitive than male mice or either sex of rats to the
carcinogenicity of 1,4-dioxane. While all data, both species and sexes reported from the Kano
et al. (2009, 594539) study, were suitable for deriving an oral CSF, the female mouse data
represented the most sensitive indicator of carcinogenicity in the rodent model. The lowest
exposure level (66 mg/kg-day or 10 mg/kg-day [HED]) resulted in a considerable and significant
increase in combined liver adenomas and carcinomas observed. Additional testing of doses
within the range of control and the lowest dose (66 mg/kg-day or 10 mg/kg-day [HED]) could
refine and reduce uncertainty for the oral CSF.
A personal communication from Dr. Yamazaki (2006, 626614) provided that the survival
of mice was particularly low in high-dose females (29/50, 29/50, 17/50, and 5/50 in control,
low-, mid-, and high-dose groups, respectively). These deaths occurred primarily during the
second year of the study. Female mouse survival at 12 months was 50/50, 50/50, 48/50, and
48/50 in control, low-, mid-, and high-dose groups, respectively (Yamazaki, 2006, 626614).
Furthermore, these deaths were primarily tumor related. Liver tumors were listed as the cause of
death for 1/21, 2/21, 8/33, and 31/45 of the pretermination deaths in control, low-, mid- and,
high-dose female Crj :BDF1 mice (Yamazaki, 2006, 626614). Therefore, because a number of
117
-------
the deaths in female mice were attributed to liver tumors, this endpoint and species was still
considered to be relevant for this analysis; however, the high mortality rate does contribute
uncertainty.
Additionally, the incidence of hepatocellular adenomas and carcinomas in historical
controls was evaluated with the data from Kano et al. (2009, 594539). Katagiri et al. (1998,
193804) summarized the incidence of hepatocellular adenomas and carcinomas in control male
and female BDF1 mice from ten 2-year bioassays at the JBRC. For female mice, out of 499
control mice, the incidence rates were 4.4% for hepatocellular adenomas and 2.0% for
hepatocellular carcinomas. Kano et al. (2009, 594539) reported a 10% incidence rate for
hepatocellular adenomas and a 0% incidence rate for hepatocellular carcinomas in control female
BDF1. These incidence rates are near the historical control values and thus are appropriate for
consideration in this assessment
5.5.1.7. Relevance to Humans
The derivation of the oral CSF is derived using the tumor incidence in the liver of female
mice. A thorough review of the available toxicological data available for 1,4-dioxane provides
no scientific justification to propose that the liver adenomas and carcinomas observed in animal
models due to exposure to 1,4-dioxane are not relevant to humans. As such, liver adenomas and
carcinomas were considered relevant to humans due to exposure to 1,4-dioxane.
5.5.1.8. Human Population Variability
The extent of inter-individual variability in 1,4-dioxane metabolism has not been
characterized. A separate issue is that the human variability in response to 1,4-dioxane is also
unknown. Data exploring whether there is differential sensitivity to 1,4-dioxane carcinogenicity
across life stages are unavailable. This lack of understanding about potential differences in
metabolism and susceptibility across exposed human populations thus represents a source of
uncertainty. Also, the lack of information linking a MO A for 1,4-dioxane to the observed
carcinogenicity is a source of uncertainty.
118
-------
Table 5-9. Summary of uncertainty in the 1,4-dioxane cancer risk estimation
Consideration/
approach
Low-dose
extrapolation
procedure
Dose metric
Cross-species
scaling
Bioassay
Species /gender
combination
Human
relevance of
mouse tumor
data
Human
population
variability in
metabolism
and response/
sensitive
subpopulations
Impact on oral slope
factor
Departure from
EPA's Guidelines for
Carcinogen Risk
Assessment POD
paradigm, if justified,
could | or t unit risk
an unknown extent
Alternatives could t
or | CSF by an
unknown extent
Alternatives could J,
orf CSF [e.g., 3.5-
fold J, (scaling by
BW) or t twofold
(scaling by BW067)]
Alternatives could t
or | CSF by an
unknown extent
Human risk could J,
or t, depending on
relative sensitivity
If rodent tumors
proved not to be
relevant to humans,
unit risk would not
apply i.e., could J,
CSF
Low-dose risk f or J,
to an unknown extent
Decision
Log-logistic
model to
determine POD,
linear low-dose
extrapolation
from POD
Used
administered
exposure
BW075 (default
approach)
JBRC (1998,
1962401
Female mouse
Liver adenomas
and carcinomas
are relevant to
humans
Considered
qualitatively
Justification
A linear low-dose extrapolation approach was used
to estimate human carcinogenic risk associated
with 1,4-dioxane exposure. Where data are
insufficient to ascertain the MO A, EPA's 2005
Guidelines for Carcinogen Risk Assessment
recommend application of a linear low-dose
extrapolation approach.
Experimental evidence supports a role for
metabolism in toxicity, but it is unclear if the
parent compound, metabolite or both contribute to
1,4-dioxane toxicity.
There are no data to support alternatives. BW0'75
scaling was used to calculate equivalent
cumulative exposures for estimating equivalent
human risks. PBPK modeling was conducted but
not deemed suitable for interspecies extrapolation.
Alternative bioassays were available and
considered for derivation of oral CSF.
There are no MOA data to guide extrapolation
approach for any choice. It was assumed that
humans are as sensitive as the most sensitive
rodent gender/species tested; true correspondence
is unknown. Calculation of the CSF for
1,4-dioxane was based on dose-response data from
the most sensitive species and gender. The
carcinogenic response occurs across species.
1,4-dioxane is a multi-site carcinogen in rodents
and the MOA(s) is unknown; carcinogenicity
observed in the rodent studies is considered
relevant to human exposure.
No data to support range of human
variability /sensitivity, including whether children
are more sensitive.
119
-------
6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HAZARD AND DOSE
RESPONSE
6.1. HUMAN HAZARD POTENTIAL
1,4-Dioxane is absorbed rapidly following oral and inhalation exposure, with much less
absorption occurring from the dermal route. 1,4-Dioxane is primarily metabolized to HEAA,
which is excreted in the urine. Liver and kidney toxicity are the primary noncancer health
effects associated with exposure to 1,4-dioxane in humans and laboratory animals. Several fatal
cases of hemorrhagic nephritis and centrilobular necrosis of the liver were related to
occupational exposure (i.e., inhalation and dermal contact) to 1,4-dioxane (Barber, 1934,
062913; Johnstone, 1959, 062927). Neurological changes were also reported in one case,
including headache, elevation in blood pressure, agitation and restlessness, and coma (Johnstone,
1959, 062927). Perivascular widening was observed in the brain of this worker, with small foci
of demyelination in several regions (e.g., cortex, basal nuclei). Severe liver and kidney
degeneration and necrosis were observed frequently in acute oral and inhalation studies
(> 1,000 mg/kg-day oral, > 1,000 ppm inhalation) (David, 1964, 195954: de Navasquez, 1935,
196174: Drew et al., 1978, 067913: Fairley et al., 1934, 062919: JBRC, 1998, 196242: Kesten et
al., 1939, 194972: Laug et al., 1939, 195055: Schrenk and Yant, 1936, 195076).
Liver and kidney toxicity were the primary noncancer health effects of subchronic and
chronic oral exposure to 1,4-dioxane in animals. Hepatocellular degeneration and necrosis were
observed (Kociba et al., 1974, 062929) and preneoplastic changes were noted in the liver
following chronic administration of 1,4-dioxane in drinking water (Argus et al., 1973, 062912:
JBRC, 1998, 196240: Kano et al., 2009, 594539). Liver and kidney toxicity appear to be related
to saturation of clearance pathways and an increase in the 1,4-dioxane concentration in the blood
(Kociba et al., 1975). Kidney damage was characterized by degeneration of the cortical tubule
cells, necrosis with hemorrhage, and glomerulonephritis (Argus et al., 1965, 017009: Argus et
al., 1973, 062912: Fairley et al., 1934, 062919: Kociba et al., 1974, 062929: NCI, 1978, 062935).
Several carcinogenicity bioassays have been conducted for 1,4-dioxane in mice, rats, and
guinea pigs (Argus et al., 1965, 017009: Argus et al., 1973, 062912: Hoch-Ligeti and Argus,
1970, 029386: Hoch-Ligeti et al., 1970, 062926: JBRC, 1998, 196240: Kano et al., 2009,
594539: Kociba et al., 1974, 062929: NCI, 1978, 062935: Torkelson et al., 1974, 094807). Liver
tumors (hepatocellular adenomas and carcinomas) have been observed following drinking water
exposure in several species and strains of rats, mice, and guinea pigs. Nasal (squamous cell
Note: Hyperlinks to the reference citations throughout this document will take you to the NCEA HERO database
(Health and Environmental Research Online) at http://epa. gov/hero. HERO is a database of scientific literature used
by U.S. EPA in the process of developing science assessments such as the Integrated Science Assessments (ISA)
and the Integrated Risk Information System (IRIS).
120
-------
carcinomas), peritoneal, and mammary tumors were also observed in rats, but were not seen in
mice. With the exception of the NCI (1978, 062935) study, the incidence of nasal cavity tumors
was generally lower than that of liver tumors in the same study population.
Under the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005, 086237),
1,4-dioxane is "likely to be carcinogenic to humans" based on evidence of liver carcinogenicity
in several 2-year bioassays conducted in three strains of rats, two strains of mice, and in guinea
pigs (Argus et al., 1965, 017009: Argus et al., 1973, 062912: Hoch-Ligeti and Argus, 1970,
029386: Hoch-Ligeti et al., 1970, 062926: JBRC, 1998, 196240: Kano et al., 2009, 594539:
Kociba et al., 1974, 062929: NCI, 1978, 062935). Studies in humans found no conclusive
evidence for a causal link between occupational exposure to 1,4-dioxane and increased risk for
cancer; however, only two studies were available and these were limited by small cohort size and
a small number of reported cancer cases (Buffler et al., 1978, 062914: Thiess et al., 1976,
062943).
The available evidence is inadequate to establish a MOA by which 1,4-dioxane induces
liver tumors in rats and mice. The genotoxicity data for 1,4-dioxane is generally characterized as
negative, although several studies may suggest the possibility of genotoxic effects (Galloway et
al., 1987, 007768: Kitchin and Brown, 1990, 062928: Mirkova, 1994, 195062: Morita and
Hayashi, 1998, 195065: Roy et al., 2005, 196094). A MOA hypothesis involving sustained
proliferation of spontaneously transformed liver cells has some support by evidence that suggests
1,4-dioxane is a tumor promoter in mouse skin and rat liver bioassays (King et al., 1973, 029390:
Lundberg et al., 1987, 062933). Some dose-response and temporal evidence support the
occurrence of cell proliferation and hyperplasia prior to the development of liver tumors (JBRC,
1998, 196240: Kociba et al., 1974, 062929). However, the dose-response relationship for the
induction of hepatic cell proliferation has not been characterized, and it is unknown if it would
reflect the dose-response relationship for liver tumors in the 2-year rat and mouse studies.
Conflicting data from rat and mouse bioassays (JBRC, 1998, 196240: Kociba et al., 1974,
062929) suggest that cytotoxicity is not a required precursor event for 1,4-dioxane-induced cell
proliferation. Liver tumors were observed in female rats and female mice in the absence of
lesions indicative of cytotoxicity (JBRC, 1998, 196240: Kano et al., 2009, 594539: NCI, 1978,
062935). Data regarding a plausible dose response and temporal progression from cytotoxicity
to cell proliferation and eventual liver tumor formation are not available.
6.2. DOSE RESPONSE
6.2.1. Noncancer/Oral
The RfD of 3 x 10"2 mg/kg-day was derived based on liver and kidney toxicity in rats
exposed to 1,4-dioxane in the drinking water for 2 years (Kociba et al., 1974, 062929). This
study was chosen as the principal study because it provides the most sensitive measure of
121
-------
adverse effects by 1,4-dioxane. The incidence of liver and kidney lesions was not reported for
each dose group. Therefore, BMD modeling could not be used to derive a POD. Instead, the
RfD is derived by dividing the NOAEL of 9.6 mg/kg-day by a composite UF of 300 (factors of
10 for animal-to-human extrapolation and interindividual variability, and an UF of 3 for database
deficiencies). Information was unavailable to quantitatively assess toxicokinetic or
toxicodynamic differences between animals and humans and the potential variability in human
susceptibility; thus, the interspecies and intraspecies uncertainty factors of 10 were applied. In
addition, a threefold database uncertainty factor was applied due to the lack of information
addressing the potential reproductive toxicity associated with 1,4-dioxane.
The overall confidence in the RfD is medium. Confidence in the principal study (Kociba
et al., 1974, 062929) is medium. Confidence in the database is medium due to the lack of a
multigeneration reproductive toxicity study. Reflecting medium confidence in the principal
study and medium confidence in the database, confidence in the RfD is medium.
6.2.2. Noncancer/Inhalation
No inhalation RfC was derived for 1,4-dioxane. Inhalation data were inadequate and a
route extrapolation from oral toxicity data was not performed, due to direct effects of
1,4-dioxane on the respiratory tract (i.e., respiratory irritation in humans, pulmonary congestion
in animals) (Fairley et al., 1934, 062919: Wirth and Klimmer, 1936, 196105: Yant et al., 1930,
062952) and lack of a suitable kinetic model (Appendix B).
Note that during the development of this assessment, new data regarding the toxicity of
1,4-dioxane through the inhalation route of exposure became available and have not been
included in the current assessment. The IRIS Program will evaluate the more recently published
1,4-dioxane inhalation in a separate assessment.
6.2.3. Cancer/Oral
Under EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005, 086237).
1,4-dioxane is "likely to be carcinogenic to humans" by all routes of exposure. This descriptor is
based on evidence of carcinogenicity from animal studies. An oral CSF for 1,4-dioxane of
0.10 (mg/kg-day)"1 was based on liver tumors in female mice from a chronic study (Kano et al.,
2009, 594539). The available data indicate that the MOA(s) by which 1,4-dioxane induces
peritoneal, mammary, or nasal tumors in rats and liver tumors in rats and mice is unknown (see
Section 4.7.3 for a more detailed discussion of 1,4-dioxane's hypothesized MOAs). Therefore,
based on the U.S. EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005, 086237).
a linear low dose extrapolation was used. The POD was calculated by curve fitting the animal
experimental dose-response data from the range of observation and converting it to a HED
(BMDLsoHED of 4.95 mg/kg-day).
The uncertainties associated with the quantitation of the oral CSF are discussed below.
122
-------
6.2.3.1. Choice of Low-Dose Extrapolation Approach
The range of possibilities for the low-dose extrapolation of tumor risk for exposure to
1,4-dioxane, or any chemical, ranges from linear to nonlinear, but is dependent upon a plausible
MOA(s) for the observed tumors. The MOA is a key consideration in clarifying how risks
should be estimated for low-dose exposure. Exposure to 1,4-dioxane has been observed in
animal models to induce multiple tumor types, including liver adenomas and carcinomas, nasal
carcinomas, mammary adenomas and fibroadenomas, and mesotheliomas of the peritoneal cavity
(Kano et al., 2009, 594539). MOA information that is available for the carcinogenicity of
1,4-dioxane has largely focused on liver adenomas and carcinomas, with little or no MOA
information available for the remaining tumor types. In Section 4.7.3, hypothesized MO As were
explored for 1,4-dioxane. Data are not available to support a carcinogenic MOA for
1,4-dioxane. In the absence of a MOA(s) for the observed tumor types associated with exposure
to 1,4-dioxane, a linear low-dose extrapolation approach was used to estimate human
carcinogenic risk associated with 1,4-dioxane exposure.
In general, the Agency has preferred to use the multistage model for analyses of tumor
incidence and related endpoints because they have a generic biological motivation based on
long-established mathematical models such as the MVK model. The MVK model does not
necessarily characterize all modes of tumor formation, but it is a starting point for most
investigations and, much more often than not, has provided at least an adequate description of
tumor incidence data.
In the studies evaluated (Kano et al., 2009, 594539: Kociba et al., 1974, 062929: NCI,
1978, 062935) the multistage model provided good descriptions of the incidence of a few tumor
types in male (nasal cavity) and female (hepatocellular and nasal cavity) rats and in male mice
(hepatocellular) exposed to 1,4-dioxane (see Appendix D for details). However, the multistage
model did not provide an adequate fit for female mouse liver tumor dataset based upon the
following (U.S. EPA, 2000, 052150):
• Goodness-of-fit/>-value was not greater than 0.10;
• AIC was larger than other acceptable models;
• Data deviated from the fitted model, as measured by their %2 residuals (values were
greater than an absolute value of one).
BMDS software typically implements the guidance in the BMD technical guidance
document (U.S. EPA, 2000, 052150) by imposing constraints on the values of certain parameters
of the models. When these constraints were imposed, the multistage model and most other
models did not fit the incidence data for female mouse liver adenomas or carcinomas.
The log-logistic model was selected because it provides an adequate fit for the female
mouse data (Kano et al., 2009, 594539). A BMR of 50% was used because it is proximate to the
response at the lowest dose tested and the BMDLso was derived by applying appropriate
123
-------
parameter constraints, consistent with recommended use of BMDS in the BMD technical
guidance document (U.S. EPA, 2000, 052150).
The human equivalent oral CSF estimated from liver tumor datasets with statistically
significant increases ranged from 4.2 x 10"4 to 1.0 x 10"1 per mg/kg-day, a range of about three
orders of magnitude, with the extremes coming from the combined male and female data for
hepatocellular carcinomas (Kociba et al., 1974, 062929) and the female mouse liver adenoma
and carcinoma dataset (Kano et al., 2009, 594539).
6.2.3.2. Dose Metric
1,4-Dioxane is known to be metabolized in vivo. However, evidence does not exist to
determine whether the parent compound, metabolite(s), or a combination of the parent compound
and metabolites is responsible for the observed toxicity following exposure to 1,4-dioxane. If the
actual carcinogenic moiety is proportional to administered exposure, then use of administered
exposure as the dose metric is the least biased choice. On the other hand, if this is not the correct
dose metric, then the impact on the CSF is unknown.
6.2.3.3. Cross-Species Scaling
An adjustment for cross-species scaling (BW°75) was applied to address toxicological
equivalence of internal doses between each rodent species and humans, consistent with the
Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005, 086237). It is assumed that equal
risks result from equivalent constant lifetime exposures.
6.2.3.4. Statistical Uncertainty at the POD
Parameter uncertainty can be assessed through confidence intervals. Each description of
parameter uncertainty assumes that the underlying model and associated assumptions are valid.
For the log-logistic model applied to the female mouse data, there is a reasonably small degree of
uncertainty at the 50% excess incidence level (the POD for linear low-dose extrapolation).
6.2.3.5. Bioassay Selection
The study by Kano et al. (2009, 594539) was used for development of an oral CSF. This
was a well-designed study, conducted in both sexes in two species (rats and mice) with a
sufficient number (N=50) of animals per dose group. The number of test animals allocated
among three dose levels and an untreated control group was adequate, with examination of
appropriate toxicological endpoints in both sexes of rats and mice. Alternative bioassays
(Kociba et al., 1974, 062929: NCI, 1978, 062935) were available and were fully considered for
the derivation of the oral CSF.
6.2.3.6. Choice of Species/Gender
The oral CSF for 1,4-dioxane was derived using the tumor incidence data for the female
mouse, which was thought to be more sensitive than male mice or either sex of rats to the
carcinogenicity of 1,4-dioxane. While all data, from both species and sexes reported from the
124
-------
Kano et al. (2009, 594539) study, were suitable for deriving an oral CSF, the female mouse data
represented the most sensitive indicator of carcinogenicity in the rodent model. The lowest
exposure level (66 mg/kg-day [animal dose] or 10 mg/kg-day [HED]) observed a considerable
and significant increase in combined liver adenomas and carcinomas. Additional testing of doses
within the range of control and the lowest dose (66 mg/kg-day [animal dose] or 10 mg/kg-day
[HED]) could refine and reduce uncertainty for the oral CSF.
6.2.3.7. Relevance to Humans
The oral CSF was derived using the tumor incidence in the liver of female mice. A
thorough review of the available toxicological data available for 1,4-dioxane provides no
scientific justification to propose that the liver adenomas and carcinomas observed in animal
models following exposure to 1,4-dioxane are not plausible in humans. Liver adenomas and
carcinomas were considered plausible outcomes in humans due to exposure to 1,4-dioxane.
6.2.3.8. Human Population Variability
The extent of inter-individual variability in 1,4-dioxane metabolism has not been
characterized. A separate issue is that the human variability in response to 1,4-dioxane is also
unknown. Data exploring whether there is differential sensitivity to 1,4-dioxane carcinogenicity
across life stages is unavailable. This lack of understanding about potential differences in
metabolism and susceptibility across exposed human populations thus represents a source of
uncertainty. Also, the lack of information linking a MO A for 1,4-dioxane to the observed
carcinogenicity is a source of uncertainty.
6.2.4. Cancer/Inhalation
Inhalation studies for 1,4-dioxane were not adequate for the determination of an
inhalation unit risk value. No treatment-related tumors were noted in a chronic inhalation study
in rats; however only a single exposure concentration was used (111 ppm 1,4-dioxane vapor for
7 hours/day, 5 days/week for 2 years) (Torkelson et al., 1974, 094807). Route extrapolation
from oral bioassay data was not performed because available kinetic models were not considered
suitable for this purpose.
Note that during the development of this assessment, new data regarding the toxicity of
1,4-dioxane through the inhalation route of exposure became available and have not been
included in the current assessment. The IRIS Program will evaluate the more recently published
1,4-dioxane inhalation data in a separate assessment.
125
-------
7. REFERENCES
ACS (1990). Handbook of Chemical Property Estimation Methods: Environmental Behavior of Organic Compounds.
Washington, DC: American Chemical Society. 004237
Agrawal AK; Shapiro BH (2000). Differential expression of gender-dependent hepatic isoforms of cytochrome P-450 by
pulse signals in the circulating masculine episodic growth hormone profile of the rat. J Pharmacol Exp Ther, 292:
228-237. 196132
Andersen ME; Clewell HJ III; Gargas ML; Smith FA; Reitz RH (1987). Physiologically based pharmacokinetics and the
risk assessment process for methylene chloride. Toxicol Appl Pharmacol, 87: 185-205. doi:10.1016/0041-
008X(87)90281-X 001938
Argus MF; Arcos JC; Hoch-Ligeti C (1965). Studies on the carcinogenic activity of protein-denaturing agents:
Hepatocarcinogenicity of dioxane. JNatl Cancer Inst, 35: 949-958. 017009
Argus MF; Sohal RS; Bryant GM; Hoch-Ligeti C; Arcos JC (1973). Dose-response and ultrastructural alterations in
dioxane carcinogenesis. Influence of methylcholanthrene on acute toxicity. Eur J Cancer, 9: 237-243.
doi:10.1016/0014-2964(73)90088-l 062912
Ashby J (1994). The genotoxicity of 1,4-dioxane. MutatRes, 322: 141-142. doi:10.1016/0165-1218(94)00022-0 195021
Atkinson R (1989). Kinetics and mechanisms of the gas-phase reactions of the hydroxyl radical with organic compounds.
Washington, DC: American Chemical Society. 042876
ATSDR (2007). Toxicological profile for 1,4 dioxane. Draft for public comment. U.S. Department of Health and Human
Services, Public Health Service, Agency for Toxic Substances and Disease. Atlanta,
GA.http://www.atsdr.cdc.gov/toxprofiles/tpl 87.pdf. 196127
Bannasch P (2003). Comments on R. Karbe and R.L. Kerlin (2002) Cystic degeneration/spongiosis hepatis (Toxicol Pathol
30 (2), 216-227). Toxicol Pathol, 31: 566-570. doi: 10.1080/01926230390224700 196140
Barber H (1934). Haemorrhagic nephritis and necrosis of the liver from dioxan poisoning. Guy's Hosp Rep, 84: 267-280.
062913
Braun WH; Young JD (1977). Identification of beta-hydroxyethoxyacetic acid as the major urinary metabolite of 1,4-
dioxane in the rat. Toxicol Appl Pharmacol, 39: 33-38. doi:10.1016/0041-008X(77)90174-0 194370
Bronaugh RL (1982). Percutaneous absorption of cosmetic ingredients. In P Frost; SN Horwitz (Ed.),Principles of
cosmetics for the dermatologist (pp. 277-284). St. Louis, MO: C.V. Mosby. 196146
Brown RP; Delp MD; Lindstedt SL; Rhomberg LR; Beliles RP (1997). Physiological parameter values for physiologically
based pharmacokinetic models. Toxicol Ind Health, 13: 407-484. doi:10.1177/074823379701300401 020304
Buffler PA; Wood SM; Suarez L; Kilian DJ (1978). Mortality follow-up of workers exposed to 1,4-dioxane. J Occup
Environ Med, 20: 255-259. 062914
Bull RJ; Robinson M; Laurie RD (1986). Association of carcinoma yield with early papilloma development in SENCAR
mice. Environ Health Perspect, 68: 11-17. 194336
Burmistrov SO; Arutyunyan AV; StepanovMG; OparinaTI; Prokopenko VM(2001). Effect of chronic inhalation of
toluene and dioxane on activity of free radical processes in rat ovaries and brain. Bull Exp Biol Med, 132: 832-836.
195972
Carpenter SP; Lasker JM; Raucy JL (1996). Expression, induction, and catalytic activity of the ethanol-inducible
cytochrome P450 (CYP2E1) in human fetal liver and hepatocytes. Mol Pharmacol, 49: 260-268. 080660
Clark B; Furlong JW; Ladner A; Slovak AJM (1984). Dermal toxicity of dimethyl acetylene dicarboxylate, N-methyl
pyrrolidone, triethylene glycol dimethyl ether, dioxane and tetralin in the rat. IRCS Med Sci, 12: 296-297. 194970
CRC (2000). Handbook of Chemistry and Physics. Boca Raton, FL: CRC Press LLC. 196090
Note: Hyperlinks to the reference citations throughout this document will take you to the NCEA HERO database
(Health and Environmental Research Online) at http://epa. gov/hero. HERO is a database of scientific literature used
by U.S. EPA in the process of developing science assessments such as the Integrated Science Assessments (ISA)
and the Integrated Risk Information System (IRIS).
126
-------
David H (1964). Electron-microscopic findings in dioxan-dependent nephrosis in rat kidneys. Beitr Pathol Anat, 130: 187-
212. 195954
Derosa CT; Wilbur S; Holler J; Richter P; Stevens YW (1996). Health evaluation of 1,4-dioxane. Toxicol Ind Health, 12: 1-
43. doi:10.1177/074823379601200101 194371
de Navasquez S (1935). Experimental tubular necrosis of the kidneys accompanied by liver changes due to dioxane
poisoning. J Hyg, 35: 540-548. 196174
Drew RT; Patel JM; Lin FN (1978). Changes in serum enzymes in rats after inhalation of organic solvents singly and in
combination. Toxicol Appl Pharmacol, 45: 809-819. doi:10.1016/0041-008X(78)90172-2 067913
Ernstgard L; IregrenA; SjogrenB; Johanson G (2006). Acute effects of exposure to vapours of dioxane in humans. Hum
Exp Toxicol, 25: 723-729. doi:10.1177/0960327106073805 195034
Fairley A; Linton EC; Ford-Moore AH (1934). The toxicity to animals of 1:4 dioxan. J Hyg, 34: 486-501.
doi:10.1017/S0022172400043266 062919
Fisher J; Mahle D; Bankston L; Greene R; Gearhart J (1997). Lactational transfer of volatile chemicals in breast milk. Am
Ind Hyg Assoc J, 58: 425-431. doi:10.1080/15428119791012667 194390
Franke C; Studinger G; Berger G; Bohling S; BruckmannU; Cohors-Fresenborg D; Johncke U (1994). The assessment of
bioaccumulation. Chemosphere, 29: 1501-1514. doi:10.1016/0045-6535(94)90281-X 194356
Frantik E; Hornychova M; Horvath M (1994). Relative acute neurotoxicity of solvents: Isoeffective air concentrations of
48 compounds evaluated in rats and mice. Environ Res, 66: 173-185. doi:10.1006/enrs. 1994.1053 067510
Galloway SM; Armstrong MJ; Reuben C; Colman S; Brown B; Cannon C; Bloom AD; Nakamura F; Ahmed M; Duk S;
Rimpo J; Margolin BH; Resnick MA; Anderson B; Zeiger E (1987). Chromosome aberrations and sister chromatid
exchanges in Chinese hamster ovary cells: Evaluations of 108 chemicals. Environ Mol Mutagen, 10: 1-175.
doi:10.1002/em.28501005Q2 007768
Giavini E; Vismara C; Broccia ML (1985). Teratogenesis study of dioxane in rats. Toxicol Lett, 26: 85-88.
doi:10.1016/0378-4274(85)90189-4 062924
Goldberg ME; Johnson HE; Pozzani UC; Smyth HF Jr (1964). Effect of repeated inhalation of vapors of industrial solvents
on animal behavior: I. Evaluation of nine solvent vapors on pole-climb performance in rats. Am Ind Hyg Assoc J,
25: 369-375. 058035
Goldsworthy TL; Monticello TM; Morgan KT; Bermudez E; Wilson DM; Jackh R; Butterworth BE (1991). Examination of
potential mechanisms of carcinogenicity of 1,4-dioxane in rat nasal epithelial cells and hepatocytes. Arch Toxicol,
65: 1-9. doi:10.1007/BF01973495 062925
Green T; Lee R; Moore RB; Ashby J; Willis GA; Lund VJ; Clapp MJL (2000). Acetochlor-induced rat nasal tumors:
Further studies on the mode of action and relevance to humans. Regul Toxicol Pharmacol, 32: 127-133 .
doi:10.1006/rtph.2000.1413 196210
Grosjean D (1990). Atmospheric chemistry of toxic contaminants. 2. Saturated aliphatics: Acetaldehyde, dioxane, ethylene
glycol ethers, propylene oxide. J Air Waste Manag Assoc, 40: 1522-1531. 196213
Hansch C; Leo A; Hoekman D (1995). Exploring QSAR: Hydrophobic, Electronic, and Steric Constants. Washington, DC:
American Chemical Society. 051424
Haseman JK; Hailey JR (1997). An update of the National Toxicology Program database on nasal carcinogens. Mutat Res,
380: 3-11. doi:10.1016/80027-5107(97)00121-8 195983
Haseman JK; Huff J; Boorman GA (1984). Use of historical control data in carcinogenicity studies in rodents. Toxicol
Pathol, 12: 126-135. doi:10.1177/019262338401200203 020667
Hawley GG; Lewis RJ Sr (2001). Hawley's Condensed Chemical Dictionary . New York, NY: John Wiley & Sons, Inc.
196089
Haworth S; Lawlor T; Mortelmans K; Speck W; Zeiger E (1983). Salmonella mutagenicity test results for 250 chemicals.
Environ Mutagen, 5: 3-142. doi:10.1002/em.2860050703 028947
Hayashi S; Watanabe J; Kawajiri K (1991). Genetic polymorphisms in the 5'-flanking region change transcriptional
regulation of the human cytochrome P450IIE1 gene. J Biochem, 110: 559-565. 196219
HellmerL; Bolcsfoldi G (1992). An evaluation of the E. coliK-12 uvrB/recA DNA repair host-mediated assay. I. In vitro
sensitivity of the bacteria to 61 compounds. Mutat Res, 272: 145-160. doi:10.1016/0165-1161(92)90043-L 194717
127
-------
Hoch-Ligeti C; Argus MF (1970). Effect of carcinogens on the lung of guinea pigs. In P Nettlesheim; MG Hanna Jr; JW
Deatherage Jr (Ed.),Morphology of Experimental Respiratory Carcinogenesis: Proceedings of a Biology Division,
Oak Ridge National Laboratory, Conference held in Gatlinburg, Tennessee, May 13-16, 1970 (pp. 267-279). Oak
Ridge, TN: United States Atomic Energy Comission, Division of Technical Information. 029386
Hoch-Ligeti C; Argus MF; Arcos JC (1970). Induction of carcinomas in the nasal cavity of rats by dioxane. Br J Cancer,
24: 164-167. 062926
HSDB (2007). 1,4-Dioxane. National Library of Medicine, National Toxicology Program, Hazardous Substances Data
Bank. Bethesda, Maryland. 196232
Huang C-Y; Huang K-L; Cheng T-J; Wang J-D; Hsieh L-L (1997). The GST Tl and CYP2E1 genotypes are possible
factors causing vinyl chloride induced abnormal liver function. Arch Toxicol, 71: 482-488.
doi:10.1007/s002040050416 005276
IARC (1999). 1,4-Dioxane. InlARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Volume 71: Re-
evaluation of Some Organic Chemicals, Hydrazine and Hydrogen Peroxide. Part Two - Other Compounds
Reviewed in Plenary Sessions (pp. 589-602). Lyon, France: World Health Organization, International Agency for
Research on Cancer. 196238
ICRP(1975). Report of the task group on reference man: ICRP publication 23. New York, NY: International Commission
of Radiological Protection, Pergamon Press. 196239
JBRC (1998). Two-week studies of 1,4-dioxane in F344 rats and BDF1 mice (drinking water studies). Japan Bioassay
Research Center. Kanagawa, Japan. 196242
JBRC (1998). Two-year studies of 1,4-dioxane in F344 rats and BDF1 mice (drinking water). Japan Bioassay Research
Center. Kanagawa, Japan. 196240
Johnstone RT (1959). Death due to dioxane? AMA Arch Ind Health, 20: 445-447. 062927
Kanada M; Miyagawa M; Sato M; Hasegawa H; Honma T (1994). Neurochemical profile of effects of 28 neurotoxic
chemicals on the central nervous system in rats (1) Effects of oral administration on brain contents of biogenic
amines and metabolites. Ind Health, 32: 145-164. doi:10.2486/indhealth.32.145 078052
Kano H; Umeda Y; Kasai T; Sasaki T; Matsumoto M; Yamazaki K; Nagano K; Arito H; Fukushima S (2009).
Carcinogenicity studies of 1,4-dioxane administered in drinking-water to rats and mice for 2 years. Food Chem
Toxicol, 47: 2776-2784. doi:10.1016/j.fct.2009.08.012 594539
Kano H; Umeda Y; Saito M; Senoh H; Ohbayashi H; Aiso S; Yamazaki K; Nagano K; Fukushima S (2008). Thirteen-week
oraltoxicity of 1,4-dioxane in rats and mice. J Toxicol Sci, 33: 141-153. doi:10.2131/jts.33.141 196245
Karbe E; KerlinRL (2002). Cystic degeneration/spongiosis hepatis in rats. Toxicol Pathol, 30: 216-227.
doi:10.1080/019262302753559551 196246
Kasai T; Kano H; Umeda Y; Sasaki T; Ikawa N; Nishizawa T; Nagano K; Arito H; Nagashima H; Fukushima S (2009).
Two-year inhalation study of carcinogenicity and chronic toxicity of 1,4-dioxane in male rats. Inhal Toxicol, 21:
889-897. doi:l0.1080/08958370802629610 193803
Kasai T; Saito M; Senoh H; Umeda Y; Aiso S; Ohbayashi H; Nishizawa T; Nagano K; Fukushima S (2008). Thirteen-week
inhalation toxicity of 1,4-dioxane in rats. Inhal Toxicol, 20: 961-971. doi: 10.1080/08958370802105397 195044
Kasper P; Uno Y; Mauthe R; Asano N; Douglas G; Matthews E; Moore M; Mueller L; Nakajima M; Singer T; Speit G
(2007). Follow-up testing of rodent carcinogens not positive in the standard genotoxicity testing battery: IWGT
workgroup report. Mutat Res, 627: 106-116. doi:10.1016/j.mrgentox.2006.10.007 195045
Katagiri T; Nagano K; Aiso S; Senoh H; Sakura Y; Takeuchi T; Okudaira M (1998). A pathological study on spontaneous
hepatic neoplasms in BDF1 mice. J Toxicol Pathol, 11: 21-25. doi: 10.1293/tox.l 1.21 193804
KestenHD; Mulinos MG; Pomerantz L (1939). Pathologic effects of certain glycols and related compounds. Arch Pathol,
27: 447-465. 194972
Khudoley VV; Mizgireuv I; Pliss GB (1987). The study of mutagenic activity of carcinogens and other chemical agents
with Salmonella typhimurium assays: Testing of 126 compounds. Arch Geschwulstforsch, 57: 453-462. 194949
King ME; Shefner AM; Bates RR (1973). Carcinogenesis bioassay of chlorinated dibenzodioxins and related chemicals.
Environ Health Perspect, 5: 163-170. 029390
KitchinKT; Brown JL (1990). Is 1,4-dioxane a genotoxic carcinogen? Cancer Lett, 53: 67-71. doi: 10.1016/0304-
3 83 5(90)90012-M 062928
128
-------
Knoefel PK (1935). Narcotic potency of some cyclic acetals. J Pharmacol Exp Ther, 53: 440-444. 195914
Kociba RJ; McCollister SB; Park C; Torkelson TR; Gehring PJ (1974). 1,4-dioxane. I. Results of a 2-year ingestion study
in rats. Toxicol Appl Pharmacol, 30: 275-286. doi:10.1016/0041-008X(74)90099-4 062929
Kociba RJ; Torkelson TR; Young JD; Gehring PJ (1975). 1,4-Dioxane: Correlation of the results of chronic ingestion and
inhalation studies with its dose-dependent fate in rats. In Proceedings of the 6th Annual Conference on
Environmental Toxicology (pp. 345-354). Wright-Patterson Air Force Base, OH: Wright-Patterson Air Force Base,
Air Force Systems Command, Aerospace Medical Division, Aerospace Medical Research Laboratory. 062930
Kurl RN; Poellinger L; Lund J; Gustafsson J-A (1981). Effects of dioxane on RNA synthesis in the rat liver. Arch Toxicol,
49: 29-33. doi:10.1007/BF00352068 195054
Kwan KK; Dutka B J; Rao SS; Liu D (1990). Mutatox test: A new test for monitoring environmental genotoxic agents.
Environ Pollut, 65: 323-332. doi:10.1016/0269-7491(W)90124-U 196078
Laug EP; Calvery HO; Morris HJ; Woodard G (1939). The toxicology of some glycols and derivatives. J Ind Hyg Toxicol,
21: 173-201. 195055
Lesage S; Jackson RE; Priddle MW; Riemann PG (1990). Occurrence and fate of organic solvent residues in anoxic
groundwater at the Gloucester landfill, Canada. Environ Sci Technol, 24: 559-566. doi:10.1021/es00074a016
195913
Leung H-W; Paustenbach DJ (1990). Cancer risk assessment for dioxane based upon a physiologically-based
pharmacokinetic approach. Toxicol Lett, 51: 147-162. 062932
Lewandowski TA; Rhomberg LR (2005). A proposed methodology for selecting a trichloroethylene inhalation unit risk
value for use in risk assessment. Regul Toxicol Pharmacol, 41: 39-54. doi:10.1016/j.yrtph.2004.09.003 626613
Lewis RJ Sr (2000). Sax's Dangerous Properties of Industrial Materials. New York, NY: John Wiley & Sons, Inc. 625540
Lundberg I; Ekdahl M; Kronevi T; Lidums V; Lundberg S (1986). Relative hepatotoxicity of some industrial solvents after
intraperitoneal injection or inhalation exposure in rats. Environ Res, 40: 411-420. doi:10.1016/S0013-
9351(86)80116-5062934
Lundberg I; Hogberg J; Kronevi T; Holmberg B (1987). Three industrial solvents investigated for tumor promoting activity
in the rat liver. Cancer Lett, 36: 29-33. doi:10.1016/0304-3835(87)90099-l 062933
MarzulliFN; Anjo DM; MaibachHI (1981). In vivo skin penetration studies of 2,4-toluenediamine, 2,4-diaminoanisole, 2-
nitro-p-phenylenediamine, p-dioxane and N-nitrosodiethanolamine in cosmetics. Food Cosmet Toxicol, 19: 743-
747. doi:10.1016/0015-6264(81)90530-7 194326
McFee AF; Abbott MG; Gulati DK; Shelby MD (1994). Results of mouse bone marrow micronucleus studies on 1,4-
dioxane. MutatRes, 322: 145-148. 195060
McGregor DB; Brown AG; Howgate S; McBride D; Riach C; Caspary WJ (1991). Responses of the L5178Y mouse
lymphomacell forward mutation assay. V: 27 coded chemicals. Environ Mol Mutagen, 17: 196-219.
doi:10.1002/em.28501703Q9 194381
Merck (2001). The Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals. Whitehouse Station, NJ: Merck
& Co., Inc. 595055
Meylan WM; Howard PH; Boethling RS; Aronson D; Printup H; Gouchie S (1999). Improved method for estimating
bioconcentration/bioaccumulation factor from octanoI/water partition coefficient. Environ Toxicol Chem, 18: 664-
672. doi:10.1002/etc.562018Q412 194377
Mikheev MI; Gorlinskaya Ye P; Solovyova TV (1990). The body distribution and biological action of xenobiotics. J Hyg
Epidemiol Microbiol Immunol, 34: 329-36. 195061
Mirkova ET (1994). Activity of the rodent carcinogen 1,4-dioxane in the mouse bone marrow micronucleus assay. Mutat
Res, 322: 142-144. 195062
Miyagawa M; Shirotori T; Tsuchitani M; Yoshikawa K (1999). Repeat-assessment of 1,4-dioxane in a rat-hepatocyte
replicative DNA synthesis (RDS) test: Evidence for stimulus of hepatocyte proliferation. Exp Toxicol Pathol, 51:
555-558. 195063
Morita T (1994). No clastogenicity of 1,4 dioxane as examined in the mouse peripheral blood micronucleus test.
Mammalian Mutagenicity Study Group Communications, 2: 7-8. 196085
129
-------
Morita T; Hayashi M (1998). 1,4-Dioxane is not mutagenic in five in vitro assays and mouse peripheral blood micronucleus
assay, but is in mouse liver micronucleus assay. Environ Mol Mutagen, 32: 269-280. doi: 10.1002/(SICI)1098-
2280(1998)32:3<269::AID-EM10>3.0.CO;2-8 195065
Mungikar AM; Pawar SS (1978). Induction of the hepatic microsomal mixed function oxidase system in mice by p-dioxane
. Bull Environ Contam Toxicol, 20: 797-804. doi:10.1007/BF01683603 194344
Munoz ER; Barnett BM (2002). The rodent carcinogens 1,4-dioxane and thiourea induce meiotic non-disjunction in
Drosophila melanogaster females. MutatRes, 517: 231-238. doi:10.1016/S1383-5718(02)00083-9 195066
Nannelli A; De Rubertis A; Longo V; Gervasi PG (2005). Effects of dioxane on cytochrome P450 enzymes in liver, kidney,
lung and nasal mucosa of rat. Arch Toxicol, 79: 74-82. doi:10.1007/s00204-004-0590-z 195067
NCI (1978). Bioassay of 1,4-dioxane for possible carcinogenicity. National Cancer Institute. Bethesda, MD. 78-1330
NCICGTR-80. http://ntp.niehs.nih.gov/ntp/htdocs/LT_rpts/tr080.pdf. 062935
NelsonN(1951). Solvent toxicity with particular reference to certain octyl alcohols and dioxanes. Med Bull, 11: 226-238.
196087
Nestmann ER; Otson R; Kowbel DJ; Bothwell PD; Harrington TR (1984). Mutagenicity in a modified Salmonella assay of
fabric-protecting products containing 1,1,1-trichloroethane. EnvironMol Mutagen, 6: 71-80.
doi:10.1002/em.28600601Q9 194339
NRC (1983). Risk assessment in the federal government: Managing the process. National Research Council, National
Academy Press. Washington, DC. PB83-191262; ISBN0309033497.
http://www.nap.edu/openbook.php?record_id=366&page=Rl. 194806
NRC (2009). Science and decisions: Advancing risk assessment. National Research Council. Washington,
DC.http://www.nap.edu/catalog.php?record_id=12209. 628200
Park JH; Hussam A; Couasnon P; Fritz D; Carr PW (1987). Experimental reexamination of selected partition coefficients
from Rohrschneider's data set. Anal Chem, 59: 1970-1976. doi:10.1021/ac00142a016 194328
Platz J; Sehested J; Mogelberg T; Nielsen OJ; Wallington TJ (1997). Atmospheric chemistry of 1,4-dioxane. Faraday Trans
1,93: 2855-2863. doi:10.1039/a700598i 196086
Pozzani UC; Weil CS; Carpenter CP (1959). The toxicological basis of threshold limit values. 5: The experimental
inhalation of vapor mixtures by rats, with notes upon the relationship between single dose inhalation and single
dose oral data. Am Ind Hyg Assoc J, 20: 364-369. doi: 10.1080/00028895909343733 063019
Ramsey JC; Andersen ME (1984). A physiologically based description of the inhalation pharmacokinetics of styrene in rats
and humans. Toxicol Appl Pharmacol, 73: 159-175. doi:10.1016/0041-008X(84)90064-4 063020
Reitz RH; McCroskey PS; Park CN; Andersen ME; Gargas ML (1990). Development of a physiologically based
pharmacokinetic model for risk assessment with 1,4-dioxane. Toxicol Appl Pharmacol, 105: 37-54.
doi:10.1016/0041-008X(90)90357-Z 094806
Rosenkranz HS; Klopman G (1992). 1,4-dioxane: Prediction of in vivo clastogenicity. Mutat Res, 280: 245-251.
doi:10.1016/0165-1218(92)90054-4 004321
Roy SK; Thilagar AK; Eastmond DA (2005). Chromosome breakage is primarily responsible for the micronuclei induced
by 1,4-dioxane in the bone marrow and liver of young CD-I mice. Mutat Res, 586: 28-37.
doi:10.1016/j.mrgentox.2005.05.007 196094
Schrenk HH; Yant WP (1936). Toxicity of dioxan. J Ind Hyg Toxicol, 18: 448-460. 195076
Sheu CW; Moreland FM; Lee JK; Dunkel VC (1988). In vitro BALB/3T3 cell transformation assay of nonoxynol-9 and
1,4-dioxane. Environ Mol Mutagen, 11: 41-48. doi:10.1002/em.2850110106 195078
Silverman L; Schulte HF; First MW (1946). Further studies on sensory response to certain industrial solvent vapors. J Ind
Hyg Toxicol, 28: 262-266. 063013
Sina JF; Bean CL; Dysart GR; Taylor VI; Bradley MO (1983). Evaluation of the alkaline elution/rat hepatocyte assay as a
predictor of carcinogenic/mutagenic potential. MutatRes, 113: 357-391. doi:10.1016/0165-1161(83)90228-5
007323
Smyth HF Jr; Seaton J; Fischer L (1941). The single dose toxicity of some glycols and derivatives. J Ind Hyg Toxicol, 23:
259-268. 060695
130
-------
Stickney JA; Sager SL; Clarkson JR; Smith LA; Locey BJ; Bock MJ; Hartung R; Olp SF (2003). An updated evaluation of
the carcinogenic potential of 1,4-dioxane. Regul Toxicol Pharmacol, 38: 183-195. doi:10.1016/S0273-
2300(03)00090-4 195080
Stoner GD; Conran PB; Greisiger EA; Stober J; Morgan M; Pereira MA (1986). Comparison of two routes of chemical
administration on the lung adenoma response in strain A/J mice. Toxicol Appl Pharmacol, 82: 19-31.
doi:10.1016/0041-008X(86)90433-3 064678
Stott WT; Quast JF; Watanabe PG (1981). Differentiation of the mechanisms of oncogenicity of 1,4-dioxane and 1,3-
hexachlorobutadiene in the rat. Toxicol Appl Pharmacol, 60:287-300. doi:10.1016/0041-008X(91)90232-4 063021
Stroebel P; Mayer F; Zerban H; Bannasch P (1995). Spongiotic pericytoma: A benign neoplasm deriving from the
perisinusoidal (Ito) cells in rat liver. Am J Pathol, 146: 903-913. 196101
Surprenant KS (2002). Dioxane. In Ullmann's Encyclopedia of Industrial Chemistry (p. 543). Weinheim, Germany: Wiley-
VCH Verlag. 196117
Sweeney LM; Thrall KD; Poet TS; Corley RA; Weber TJ; Locey BJ; Clarkson J; Sager S; Gargas ML (2008).
Physiologically based pharmacokinetic modeling of 1,4-dioxane in rats, mice, and humans. Toxicol Sci, 101: 32-50.
doi:10.1093/toxsci/kfm251 195085
Thiess AM; Tress E; Fleig I (1976). Arbeitsmedizinische Untersuchungsergebnisse von Dioxan-exponierten Mitarbeitern
[Industrial-medical investigation results in the case of workers exposed to dioxane]. Arbeitsmedizin, Sozialmedizin,
Umweltmedizin, 11: 35-46. 062943
ThurmanGB; Simms BG; Goldstein AL; KilianDJ (1978). The effects of organic compounds used in the manufacture of
plastics on the responsivity of murine and human lymphocytes. Toxicol Appl Pharmacol, 44: 617-641.
doi:10.1016/0041-008X(78)90269-7 018767
Tinwell H; Ashby J (1994). Activity of 1,4-dioxane in mouse bone marrow micronucleus assays. MutatRes, 322: 148-150.
195086
Torkelson TR; Leong BKJ; Kociba RJ; Richter WA; Gehring PJ (1974). 1,4-Dioxane. II. Results of a 2-year inhalation
study in rats. Toxicol Appl Pharmacol, 30: 287-298. doi:10.1016/0041-008X(74)90100-8 094807
U. S. EPA (1986). Guidelines for carcinogen risk assessment. National Center for Environmental Assessment; Office of
Research and Development; U.S. Environmental Protection Agency . Washington, DC. EPA/630/R-00/004 .
http://epa.gov/raf/publications/pdfs/CA%20GUIDELINES_l 986.PDF. 199530
U.S. EPA (1986). Guidelines for mutagenicity risk assessment. U.S. Environmental Protection Agency, Risk Assessment
Forum. Washington, DC. EPA/630/R-98/003. http://www.epa.gov/osa/mmoaframework/pdfs/MUTAGEN2.PDF.
001466
U. S. EPA (1986). Guidelines for the health risk assessment of chemical mixtures. U. S. Environmental Protection Agency,
Risk Assessment Forum. Washington, DC. EPA/630/R-98/002.
http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=22567. 001468
U. S. EPA (1988). Recommendations for and documentation of biological values for use in risk assessment. U. S.
Environmental Protection Agency, Environmental Criteria and Assessment Office, Office of Research and
Development, Office of Health and Environmental Assessment. Cincinnati, OH. EPA/600/6-87/008.
http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=34855. 064560
U.S. EPA (1990). Amendments to the Clean Air Act. Sec. 604. Phase-out of production and consumption of class I
substances. Retrieved , from . 196139
U. S. EPA (1991). Guidelines for developmental toxicity risk assessment. U. S. Environmental Protection Agency, Risk
Assessment Forum. Washington, DC. EPA/600/FR-91/001.
http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=23162. 008567
U. S. EPA (1994). Interim policy for particle size and limit concentration issues in inhalation toxicity studies. U. S.
Environmental Protection Agency, Office of Pesticide Products, Health Effects Division. Washington,
DC.http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid= 186068. 076133
U. S. EPA (1994). Methods for derivation of inhalation reference concentrations and application of inhalation dosimetry.
U.S. Environmental Protection Agency, Office of Research and Development, Office of Health and Environmental
Assessment. Washington, DC. EPA/600/8-90/066F. http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=71993.
006488
131
-------
U. S. EPA (1995). The use of the benchmark dose approach in health risk assessment. U. S. Environmental Protection
Agency, Risk Assessment Forum. Washington, DC. EPA/630/R-94/007.
http://cfpub.epa.gov/ncea/raf/recordisplay.cfm?deid=42601. 005992
U. S. EPA (1996). Guidelines for reproductive toxicity risk assessment. U. S. Environmental Protection Agency, Risk
Assessment Forum. Washington, DC. EPA/630/R-96/009.
http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=283 8. 030019
U.S. EPA (1998). Guidelines for neurotoxicity risk assessment. U.S. Environmental Protection Agency, Office of Research
and Development, National Center for Environmental Assessment. Washington, DC. EPA/630/R-95/001F.
http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=l2479. 030021
U.S. EPA (2000). Benchmark dose technical guidance document [external review draft]. U.S. Environmental Protection
Agency, Risk Assessment Forum. Washington, DC. EPA/630/R-OO/OO1.
http://www.epa.gov/raf/publications^enchmark-dose-doc-draft.htm . 052150
U.S. EPA (2000). Science policy council handbook: Risk characterization. U.S. Environmental Protection Agency, Office
of Research and Development, Office of Science Policy. Washington, D.C.. EPA 100-B-00-002.
www.epa.gov/spc/pdfs/rchandbk.pdf. 052149
U. S. EPA (2000). Supplemental guidance for conducting for health risk assessment of chemical mixtures. U. S. EPA.
Washington, DC. 196144
U. S. EPA (2000). Supplementary guidance for conducting health risk assessment of chemical mixtures. Risk Assessment
Forum, U.S. Environmental Protection Agency. Washington, DC. EPA/630/R-00/002.
http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=20533. 004421
U.S. EPA (2002). A review of the reference dose and reference concentration processes. U.S. Environmental Protection
Agency, Risk Assessment Forum. Washington, DC. EPA/630/P-02/0002F.
http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=55365. 088824
U.S. EPA (2002). Toxic Substances Control Act (TSCA) Inventory Update Database. Retrieved February 22, 2010, from
http://www.epa.gov/iur/ . 594597
U.S. EPA (2005). Guidelines for carcinogen risk assessment, final report. Risk Assessment Forum; U.S. Environmental
Protection Agency. Washington, DC. EPA/630/P-03/001F.
http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=l 16283. 086237
U.S. EPA (2005). Supplemental guidance for assessing susceptibility from early-life exposure to carcinogens. U.S.
Environmental Protection Agency, Risk Assessment Forum. Washington, DC. EPA/630/R-03/003F.
http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=l 16283. 088823
U. S. EPA (2006). A framework for assessing health risk of environmental exposures to children (final). U. S. Environmental
Protection Agency, Office of Research and Development, National Center for Environmental Assessment.
Washington, DC. EPA/600/R-05/093F. http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=158363. 194567
U.S. EPA (2006). U.S. Environmental Protection Agency peer review handbook. U.S. Environmental Protection Agency,
Science Policy Council. Washington, DC. EPA/1 OO/B-06/002.
http://www.epa.gov/peerreview/pdfs/peer_review_handbook_2006.pdf. 194566
U.S. EPA (2009). Toxicological review of 1,4-dioxane (CAS No. 123-91-1) in support of summary information on the
Intergrated Risk Information System (IRIS) [External Review Draft]. U.S. Environmental Protection Agency.
Washington, DC. EPA/635/R-09/005. http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=l99330. 628630
U.S. EPA (2010). Toxicological review of 1,4-Dioxane (CAS No. 123-91-1) in support of summary information on the
Integrated Risk Information System (IRIS). U.S. Environmental Protection Agency. Washington, DC. 625580
UNEP (2000). The Montreal Protocol on substances that deplete the ozone layer. United Nations Environment Programme,
Ozone Secretariat. Nairobi,
Kenya.http://www.google.com/url?sa=t&source=web&cd=l&ved=OCBIQFjAA&url=http%3A%2F%2Fwww.unep
.org%2Fozone%2Fpdfs%2Fmontreal-protocol2000.pdf&ei=-c89TPXON9PRngf-i-
jdDg&usg=AFOjCNH40H15inPn5XFcYTvblPPRDZu-fQ&sig2=qqSaM_nuQlXlHc409kBvgw 196125
Uno Y; Takasawa H; Miyagawa M; Inoue Y; Murata T; Yoshikawa K (1994). An in vivo-in vitro replicative DNA synthesis
(RDS) test using rat hepatocytes as an early prediction assay for nongenotoxic hepatocarcinogens screening of 22
known positives and 25 noncarcinogens. MutatRes, 320: 189-205. doi:10.1016/0165-1218(94)90046-9 194385
132
-------
van Delft JH; vanAgenE; van Breda SG; HerwijnenMH; Staal YC; Kleinjans JC (2004). Discrimination of genotoxic
from non-genotoxic carcinogens by gene expression profiling. Carcinogenesis, 25: 1265-1276.
doi:10.1093/carcin/bghl08 195087
Vieira I; Sonnier M; Cresteil T (1996). Developmental expression of CYP2E1 in the human liver: hypermethylation control
of gene expression during the neonatal period. Eur J Biochem, 238: 476-483. doi:10.1111/j.l432-
1033.1996.0476z.x 011956
Watanabe J; Hayashi S; Kawajiri K (1994). Different regulation and expression of the human CYP2E1 gene due to the Rsal
polymorphism in the 5'-flanking region. J Biochem, 116: 321-326. 196099
Waxman DJ; Pampori NA; Ram PA; Agrawal AK; Shapiro BH (1991). Interpulse interval in circulating growth hormone
patterns regulates sexually dimorphic expression of hepatic cytochrome P450. PNAS, 88: 6868-6872. 196102
Wirth W; Klimmer O (1936). [On the toxicology of organic solvents. 1,4 dioxane (diethylene dioxide)] . Archiv fuer
Gewerbepathologie und Gewerbehygiene, 17: 192-206. 196105
Wolfe NL; Jeffers PM (2000). Hydrolysis. In RS Boethling; D Mackay (Ed.),Handbook of Property Estimation Methods
for Chemicals: Environmental and Health Sciences (pp. 311-333). Boca Raton, FL: Lewis Publishers. 196109
Wolford ST; Schroer RA; Gohs FX; Gallo PP; Brodeck M; Falk HB; Ruhren R (1986). Reference range data base for
serum chemistry and hematology values in laboratory animals. J Toxicol Environ Health A Curr Iss, 18: 161-188.
doi: 10.1080/15287398609530859 196112
Woo Y-T; Arcos JC; Argus MF; Griffin GW; NishiyamaK (1977). Structural identification of p-dioxane-2-one as the major
urinary metabolite of p-dioxane. Naunyn Schmiedebergs Arch Pharmacol, 299: 283-287. doi:10.1007/BF00500322
194355
Woo Y-T; Argus MF; Arcos JC (1977). Metabolism in vivo of dioxane: Effect of inducers and inhibitors of hepatic mixed-
function oxidases. Biochem Pharmacol, 26: 1539-1542. doi:10.1016/0006-2952(77)90431-2 062951
Woo Y-T; Argus MF; Arcos JC (1977). Tissue and subcellular distribution of 3H-dioxane in the rat and apparent lack of
microsome-catalyzed covalent binding in the target tissue. Life Sci, 21: 1447-1456. doi: 10.1016/0024-
3205(77)90199-0062950
Woo Y-T; Argus MF; Arcos JC (1978). Effect of mixed-function oxidase modifiers on metabolism and toxicity of the
oncogen dioxane. Can Res, 38: 1621-1625. 194345
Yamamoto S; Ohsawa M; Nishizawa T; Saito A; Kasai T; Noguchi T; Nagano K; Matsushima T (2000). Long-term
toxicology study of 1,4-Dioxane in the F344 rats by multiple-route exposure (drinking water and inhalation). J
Toxicol Sci, 25: 347. 625635
Yamamoto S; Urano K; Koizumi H; Wakana S; Hioki K; Mitsumori K; Kurokawa Y; Hayashi Y; Nomura T (1998).
Validation of transgenic mice carrying the human prototype c-Ha-ras gene as a bioassay model for rapid
carcinogenicity testing. Environ Health Perspect, 106: 57-69. 594544
Yamamoto S; Urano K; Nomura T (1998). Validation of transgenic mice harboring the human prototype c-Ha-ras gene as a
bioassay model for rapid carcinogenicity testing. Toxicol Lett, 102-103: 473-478. doi:10.1016/S0378-
4274(98)00341-5 196114
Yamazaki K (2006). Correspondence between Kazunori Yamazaki and Julie Stickney. 626614
Yamazaki K; Ohno H; Asakura M; Narumi A; Ohbayashi H; Fujita H; Ohnishi M; Katagiri T; Senoh H; Yamanouchi K;
Nakayama E; Yamamoto S; Noguchi T; Nagano K; Enomoto M; Sakabe H (1994). Two-year toxicological and
carcinogenesis studies of 1,4-dioxane in F344 rats and BDF1 mice. In K Sumino; S Sato; NG Shinkokai
(Ed.),Proceedings: Second Asia-Pacific Symposium on Environmental and Occupational Health 22-24 July, 1993:
Kobe (pp. 193-198). Kobe, Japan: Kobe University School of Medicine, International Center for Medical Research.
196120
Yant WP; SchrenkHH; Waite CP; Patty FA (1930). Acute response of guinea pigs to vapors of some new commercial
organic compounds: VI. Dioxan. Public Health Rep, 45: 2023-2032. 062952
Yasuhara A; Shiraishi H; Nishikawa M; Yamamoto T; Uehiro T; Nakasugi O; Okumura T; Kenmotsu K; Fukui H; Nagase
M; Ono Y; Kawagoshi Y; Baba K; Noma Y (1997). Determination of organic components in leachates from
hazardous waste disposal sites in Japan by gas chromatography-mass spectrometry. J Chromatogr A, 774: 321-332.
doi:10.1016/S0021-9673(97)00078-2 195909
Yasuhara A; Tanaka Y; Tanabe A; Kawata K; Katami T (2003). Elution of 1,4-dioxane from waste landfill sites. Bull
Environ Contam Toxicol, 71: 641-647. doi:10.1007/s00128-003-8917-7 195095
133
-------
Yoon JS; Mason JM; Valencia R; Woodruff RC; Zimmering S (1985). Chemical mutagenesis testing in Drosophila. IV.
Results of 45 coded compounds tested for the National Toxicology Program. Environ Mutagen, 7: 349-367.
doi: 10.1002/em.2860070310194373
Young JD; Braun WH; Gehring PJ (1978). The dose-dependent fate of 1,4-dioxane in rats. J Environ Pathol Toxicol, 2:
263-282. doi: 10.1080/15287397809529693 062955
Young JD; Braun WH; Gehring PJ; Horvath BS; Daniel RL (1976). 1,4-Dioxane and beta-hydroxyethoxyacetic acid
excretion in urine of humans exposed to dioxane vapors. Toxicol Appl Pharmacol, 38: 643-646. doi: 10.1016/0041-
008X(76)90195-2 062953
Young JD; Braun WH; Gehring PJ (1978). Dose—dependent fate of 1,4-dioxane in rats(b). J Toxicol Environ Health A
Currlss, 4: 709-726. doi:10.1080/15287397809529693 625640
Young JD; Braun WH; Rampy LW; Chenoweth MB; Blau GE (1977). Pharmacokinetics of 1,4-dioxane in humans. J
Toxicol Environ Health, 3: 507-520. doi:10.1080/15287397709529583 062956
Zimmermann FK; Mayer VW; Scheel I; Resnick MA (1985). Acetone, methyl ethyl ketone, ethyl acetate, acetonitrile and
other polar aprotic solvents are strong inducers of aneuploidy in Saccharomyces cerevisiae. Mutat Res, 149: 339-
351. doi:10.1016/0027-5107(85)90150-2 194343
134
-------
APPENDIX A. SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC
COMMENTS AND DISPOSITION
The Toxicological Review of 1,4-Dioxane has undergone formal external peer review
performed by scientists in accordance with EPA guidance on peer review (U.S. EPA, 2000,
052149; U.S. EPA, 2006, 194566). The external peer reviewers were tasked with providing
written answers to general questions on the overall assessment and on chemical-specific
questions in areas of scientific controversy or uncertainty. A summary of significant comments
made by the external reviewers and EPA's responses to these comments arranged by charge
question follow. In many cases the comments of the individual reviewers have been synthesized
and paraphrased for development of Appendix A. The majority of the specific observations (in
addition to EPA's charge questions) made by the peer reviewers were incorporated into the
document and are not discussed further in this Appendix. Public comments that were received
are summarized and addressed following the peer-reviewers' comments and disposition.
A.l. EXTERNAL PEER REVIEW PANEL COMMENTS
The reviewers made several editorial suggestions to clarify portions of the text. These
changes were incorporated in the document as appropriate and are not discussed further.
In addition, the external peer reviewers commented on decisions and analyses in the
Toxicological Review of 1,4-Dioxane under multiple charge questions, and these comments were
organized and summarized under the most appropriate charge question.
A.1.1. General Charge Questions
1. Is the Toxicological Review logical, clear and concise? Has EPA accurately, clearly and
objectively represented and synthesized the scientific evidence for noncancer and cancer
hazards?
Comment. All reviewers found the Toxicological Review to be logical, clear, and concise.
One reviewer remarked that it was an accurate, open-minded and balanced analysis of the
literature. Most reviewers found that the scientific evidence was presented objectively
and transparently; however, one reviewer suggested two things to improve the objectivity
and transparency (1) provide a clear description of the mode of action and how it feeds
into the choice of the extrapolation for the cancer endpoint and (2) provide a presentation
of the outcome if internal dose was used in the cancer and noncancer assessments.
Note: Hyperlinks to the reference citations throughout this document will take you to the NCEA HERO database
(Health and Environmental Research Online) at http://epa. gov/hero. HERO is a database of scientific literature used
by U.S. EPA in the process of developing science assessments such as the Integrated Science Assessments (ISA)
and the Integrated Risk Information System (IRIS).
A-l
-------
One reviewer commented that conclusions could not be evaluated in a few places
where dose information was not provided (Sections 3.2, 3.3 and 4.5.2.2). The same
reviewer found the MOA schematics, key event temporal sequence/dose-response table,
and the POD plots to be very helpful in following the logic employed in the assessment.
Response. The mode of action analysis and how conclusions from that analysis fed into
the choice of extrapolation method for the cancer assessment are discussed further under
charge questions C2 and C5. Because of the decision not to utilize the PBPK models,
internal doses were not calculated and thus were not included as alternatives to using the
external dose as the POD for the cancer and noncancer assessments.
In the sections noted by the reviewer (3.2, 3.3, and 4.5.2.2) dose information was
added as available. In Section 3.2, Mikheev et al. (1990, 195061) did not report actual
doses, which is noted in this section. All other dose information in this section was found
to be present after further review by the Agency. In Section 3.3, dose information for
Woo et al. (1977, 062951: 1978, 194345) was added to the paragraph. In Section 4.5.2.2,
study details for Nannelli et al. (2005, 195067) were provided earlier in Section 3.3 and a
statement referring the reader to this section was added.
2. Please identify any additional studies that should be considered in the assessment of the
noncancer and cancer health effects of 1,4-dioxane.
Comment Five reviewers stated they were unaware of any additional studies available to
add to the oral toxicity evaluation of 1,4-dioxane. These reviewers also acknowledged
the Kasai et al. (2008, 195044: 2009, 193803) publications that may be of use to derive
toxicity values following inhalation of 1,4-dioxane.
a. Kasai T; Saito H; Senoh Y; et al. (2008, 195044) Thirteen-week inhalation
toxicity of 1,4-dioxane in rats. Inhal Toxicol 20: 961-971.
b. Kasai T; Kano Y; Umeda T; et al. (2009, 193803) Two-year inhalation study of
carcinogenicity and chronic toxicity of 1,4-dioxane in male rats. Inhal Toxicol in
press.
Other references suggested by reviewers include:
c. California Department of Health Services (1989) Risk Specific Intake Levels for
the Proposition 65 Carcinogen 1, 4-dioxane. Reproductive and Cancer Hazard
Assessment Section. Office of Environmental Health Hazard Assessment
d. National Research Council (2009, 628200) Science and Decisions: Advancing
Risk Assessment. Committee on Improving Risk Analysis Approaches Used by
the U.S. EPA. Washington, D.C., National Academy Press.
e. AT SDR (2007,196127) Toxicol ogical Profile for 1,4-dioxane. Agency for
Toxic Substances and Disease Registry. Atlanta, GA.
A-2
-------
f. Stickney JA; Sager SL; Clarkson JR; et al. (2003, 195080) An updated
evaluation of the carcinogenic potential of 1,4-dioxane. Regul Toxicol Pharmacol
38:183-195.
g. Yamamoto S; Ohsawa M; Nishizawa T; et al. (2000, 625635) Long-term
toxicology study of 1,4-dioxane in R344 rats by multiple-route exposure (drinking
water and inhalation). J Toxicol Sci 25: 347.
Response. The references a-b above will be evaluated for derivation of an RfC and IUR,
which will follow as an update to this oral assessment. References c and e noted above
were considered during development of this assessment as to the value they added to the
cancer and noncancer analyses. Reference g listed above is an abstract from conference
proceedings from the 27th Annual Meeting of the Japanese Society of Toxicology;
abstracts are not generally considered in the development of an IRIS assessment.
Reference d reviews EPA's current risk assessment procedures and provides no specific
information regarding 1,4-dioxane. The Stickney et al. (2003, 195080) reference was a
review article and no new data were presented, thus it was not referenced in this
Toxicological Review but the data were considered during the development of this
assessment.
Following external peer review (as noted above) Kano et al. (2009, 594539) was
added to the assessment, which was an update and peer-reviewed published manuscript
of the JBRC (1998, 196240) report.
3. Please discuss research that you think would be likely to increase confidence in the database
for future assessments of 1,4-dioxane.
Comment. All reviewers provided suggestions for additional research that would
strengthen the assessment and reduce uncertainty in several areas. The following is a
brief list of questions that were identified that could benefit from further research. What
are the mechanisms responsible for the acute and chronic nephrotoxicity? Is the acute
kidney injury (AKI) multifactorial? Are there both tubular and glomerular/vascular
toxicities that result in cortical tubule degeneration and evidence for glomerulonephritis?
What are the functional correlates of the histologic changes in terms of assessment of
renal function? What is the exposure in utero and risk to the fetus and newborn? What are
the concentrations in breast milk following maternal exposure to 1,4-dioxane? What is
the risk for use of contaminated drinking water to reconstitute infant formula? What are
the exposures during early human development? What is the pharmacokinetic and
metabolic profile of 1,4-dioxane during development? What are the susceptible
populations (e.g., individuals with decreased renal function or chronic renal disease,
obese individuals, gender, age)?
A-3
-------
Additional suggestions for future research include: evaluation of potential
epigenetic mechanisms of carcinogenicity, additional information on sources of exposure
and biological concentrations as well as human toxicokinetic data for derivation of
parameter to refine PBPK model, studies to determine toxic moiety, focused studies to
inform mode of action, additional inhalation studies and a multigeneration reproductive
toxicity study.
One reviewer suggested additional analyses of the existing data including a
combined analysis of the multiple datasets and outcomes for cancer and non-cancer
endpoints, evaluation of the dose metrics relevant to the MOA to improve confidence in
extrapolation approach and uncertainty factors, and complete a Bayesian analysis of
human pharmacokinetic data to estimate human variability in key determinants of
toxicity (e.g., metabolic rates and partition coefficients).
Response. A number of research suggestions were provided for further research that may
enhance future health assessments of 1,4-dioxane. Regarding the suggested additional
analyses for the existing data, EPA did not identify a MOA in this assessment, thus
combined analysis of the cancer and non-cancer endpoints as well as application of
various dose metrics to a MOA is not applicable. Because the human PBPK model was
not implemented in this assessment for oral exposure to 1,4-dioxane a Bayesian analysis
was not completed. No additional changes to the Toxicological Review of 1,4-Dioxane
were made in response to these research recommendations.
4. Please comment on the identification and characterization of sources of uncertainty in
Sections 5 and 6 of the assessment document. Please comment on whether the key sources of
uncertainty have been adequately discussed. Have the choices and assumptions made in the
discussion of uncertainty been transparently and objectively described? Has the impact of the
uncertainty on the assessment been transparently and objectively described?
Comment. Six reviewers stated Sections 5 and 6 adequately discussed and characterized
uncertainty, in a succinct, and transparent manner. One reviewer suggested adding
additional discussion of uncertainty relating to the critical study used in the cancer
assessment and another reviewer suggested adding more discussion around the
uncertainty of the toxic moiety.
One reviewer made specific comments on uncertainty surrounding the Kociba et
al. (1974, 062929) study as used for derivation of the RfD, choice of the non-cancer dose
metric, and use of a 10%BMR as the basis for the CSF derivation. These comments and
responses are summarized below under their appropriate charge question.
A-4
-------
Response. The majority of the reviewers thought the amount of uncertainty discussion
was appropriate. Since the external review, Kano et al. (2009, 594539) was published
and this assessment was updated accordingly (previously JBRC (1998, 196240). It is
assumed the uncertainly referred to by the reviewer was addressed by the published Kano
et al. (2009, 594539) paper.
Clarification regarding the uncertainty surrounding the identification of the toxic
moiety was added to Section 4.6.3 stating that the mechanism by which 1,4-dioxane
induces tissue damage is not known, nor is it known whether the toxic moiety is
1,4-dioxane or a metabolite of 1,4-dioxane. Additional text was added to Section 4.7.3
clarifying that available data also do not clearly identify whether 1,4-dioxane or one of its
metabolites is responsible for the observed effects. The impact of the lack of evidence to
clearly identify a toxic moiety related to 1,4-dioxane exposure was summarized in
Sections 5.5.1.2 and 6.2.3.2.
A.1.2. Oral reference dose (RfD) for 1,4-dioxane
1. A chronic RfD for 1,4-dioxane has been derived from a 2-year drinking water study (Kociba
et al., 1974, 062929) in rats and mice. Please comment on whether the selection of this study
as the principal study has been scientifically justified. Has the selection of this study been
transparently and objectively described in the document? Are the criteria and rationale for
this selection transparently and objectively described in the document? Please identify and
provide the rationale for any other studies that should be selected as the principal study.
Comment. Seven of the reviewers agreed that the use of the Kociba et al. (1974, 062929)
study was the best choice for the principal study.
One reviewer stated that Kociba et al. (1974, 062929) was not the best choice
because it reported only NOAEL and LOAELs without providing incidence data for the
endpoints. This reviewer also stated that the study should not have been selected based
on sensitivity of the endpoints, but rather study design and adequacy of reporting of the
study results. Additionally, this reviewer suggested a better principal study would be
either the NCI (1978, 062935) or JBRC (1998, 196240) study.
Response. The reviewer is correct that Kociba et al. (1974, 062929) did not provide
incidence data; however, Kociba et al. (1974, 062929) identified a NOAEL (9.6 mg/kg-
day) and LOAEL (94 mg/kg-day) within the text of the manuscript. Kociba et al. (1974,
062929) was a well conducted chronic bioassay (four dose levels, including controls,
with 60 rats/sex/group) and seven of the peer reviewers found this study to be appropriate
as the basis for the RfD. Further support for the selection of the Kociba et al. (1974,
062929) as the principal study comes from comparison of the liver and kidney toxicity
data reported by JBRC (1998, 196240) and NCI (1978, 062935). which was presented in
A-5
-------
Section 5.1. The effects reported by JBRC (1998, 196240) and NCI (1978, 062935) were
consistent with what was observed by Kociba et al. (1974, 062929) and within a similar
dose range. Derivation of an RfD from these datasets resulted in a similar value (Section
5.1.).
2. Degenerative liver and kidney effects were selected as the critical effect. Please comment on
whether the rationale for the selection of this critical effect has been scientifically justified.
Are the criteria and rationale for this selection transparently and objectively described in the
document? Please provide a detailed explanation. Please comment on whether EPA's
rationale regarding adversity of the critical effect for the RfD has been adequately and
transparently described and is scientifically supported by the available data. Please identify
and provide the rationale for any other endpoints that should be considered in the selection of
the critical effect.
Comment. Five of the reviewers agreed with the selection of liver and kidney effects as
the critical effect. One of these reviewers suggested analyzing all datasets following dose
adjustment (e.g., body weight scaling or PBPK model based) to provide a better rationale
for selection of a critical effect.
One reviewer stated that 1,4-dioxane causing liver and kidney organ specific
effects is logical; however, with regards to nephrotoxicity, the models and limited human
data have not addressed the mechanisms of injury or the clinical correlates to the
histologic data. Also, advances in the field of biomarkers have not yet been used for the
study of 1,4-dioxane.
One reviewer found the selection of these endpoints to be 'without merit' because
of the lack of incidence data to justify the NOAEL and LOAEL values identified in the
study. This reviewer suggested selecting the most sensitive endpoint(s) from the NCI
(NCI, 1978, 062935) or JBRC (1998, 196240) studies for the basis of the RfD, but did
not provide a suggestion as to what effect should be selected.
Response. The liver and kidney effects from Kociba et al. (1974, 062929) was supported
as the critical effect by most of the reviewers. PBPK model adjustment was not
performed because the PBPK model was found to be inadequate for use in the
assessment. EPA acknowledges that neither the mechanisms of injury nor the clinical
correlates to histologic data exist for 1,4-dioxane. This type of information could improve
future health assessments of 1,4-dioxane.
As stated above, Kociba et al. (1974, 062929) identified a NOAEL (9.6 mg/kg-
day) and LOAEL (94 mg/kg-day) within the text of the manuscript and was a well
conducted chronic bioassay (four dose levels, including controls, with 60 rats/sex/group).
A-6
-------
3. Kociba et al. (1974, 062929) derived a NOAEL based upon the observation of degenerative
liver and kidney effects and these data were utilized to derive the point of departure (POD)
for the RfD. Please provide comments with regard to whether the NOAEL approach is the
best approach for determining the POD. Has the approach been appropriately conducted and
objectively and transparently described? Please identify and provide rationales for any
alternative approaches for the determination of the POD and discuss whether such
approaches are preferred to EPA's approach.
Comment: Seven reviewers agreed with the NOAEL approach described in the
document. One of these reviewers also questioned whether any attempt was made to
"semi-qualitatively represent the histopathological observations to facilitate a quantitative
analysis".
One reviewer stated that data were not used to derive the POD, but rather a claim
by the authors of Kociba et al. (1974, 062929) of the NOAEL and LOAEL for the
endpoints. This reviewer preferred the use of a BMD approach for which data include
the reported incidence rather than a study reported NOAEL or LOAEL.
Response: The suggestion to "semi-qualitatively represent the histopathological
observations to facilitate a quantitative analysis" was not incorporated into the document
because it is unclear how this would be conducted since Kociba et al. (1974, 062929) did
not provide incidence data and the reviewer did not illustrate their suggested approach.
See responses to Bl and B2 regarding the NOAEL and LOAEL approach. The Agency
agrees that a Benchmark Dose approach is preferred over the use of a NOAEL or
LOAEL for the POD if suitable data (e.g., reflecting the most sensitive sex, species, and
endpoint identified) are available for modeling and, if suitable data are not available, then
NOAEL and LOAEL values are utilized. In this case, the data were not suitable for
BMD modeling and the LOAEL or NOAEL approach was used.
4. EPA evaluated the PBPK and empirical models available to describe kinetics following
inhalation of 1,4-dioxane (Reitz et al., 1990, 094806: Young et al., 1977, 062956: Young et
al., 1978, 625640: Young et al., 1978, 062955). EPA concluded that the use of existing,
revised, and recalibrated PBPK models for 1,4-dioxane were not superior to default
approaches for the dose-extrapolation between species. Please comment on whether EPA's
rationale regarding the decision to not utilize existing or revised PBPK models has been
adequately and transparently described and is supported by the available data. Please identify
and provide the rationale for any alternative approaches that should be considered or
preferred to the approach presented in the toxicological review.
Comment: Six reviewers found the decision not to utilize the available PBPK models to
be appropriate and supported by available data. One of these reviewers suggested
A-7
-------
presenting as part of the uncertainty evaluation an adjustment of the experimental doses
based on metabolic saturation. Another reviewer stated Appendix B was hard to follow
and that the main document should include a more complete description of the model
refinement effort performed by Sweeney et al. (2008, 195085).
Two reviewers noted a complete evaluation of the models was evident; one of the
reviewers questioned the decision not to use the models on the basis that they were
unable to fit the human blood PK data for 1,4-dioxane. This reviewer suggested the rat
model might fit the human blood PK data, thus raising concern in the reliance on the
human blood PK data to evaluate the PBPK model for 1,4-dioxane. Instead, the reviewer
suggested the human urinary metabolite data may be sufficient to give confidence in the
model. One other reviewer also questioned the accuracy of the available human data.
One reviewer commented that the rationale for not using the PBPK model to extrapolate
from high to low dose was questioned. In addition, the reviewer suggested that two
aspects of the model code for Reitz et al. (1990, 094806) need to be verified:
a. In the document, KLC is defined as a first-order rate constant and is scaled by
BW° 7. This is inconsistent when multiplied by concentration does not result
in units of mg/hr. However, if the parameter is actually considered a
clearance constant (zero-order rate constant) then the scaling rule used, as well
as the interpretations provided, would be acceptable.
b. It is unclear as to why AM is calculated on the basis of RAM and not RMEX.
RMEX seems to represent the amount metabolized per unit time.
Response. The USEPA performed a rigorous evaluation of the PBPK models available
for 1,4-dioxane. This effort was extensively described in Section 3.5 and in Appendix B.
In short, several procedures were applied to the human PBPK model to determine if an
adequate fit of the model to the empirical model output or experimental observations
could be attained using biologically plausible values for the model parameters. The re-
calibrated model predictions for blood 1,4-dioxane levels did not come within 10-fold of
the experimental values using measured tissue:air partition coefficients of (Leung and
Paustenbach, 1990, 062932) or (Sweeney et al., 2008, 195085) (Figures B-8 and B-9).
The utilization of a slowly perfused tissue:air partition coefficient 10-fold lower than
measured values produces exposure-phase predictions that are much closer to
observations, but does not replicate the elimination kinetics (Figure B-10). Re-
calibration of the model with upper bounds on the tissue:air partition coefficients results
in predictions that are still six- to sevenfold lower than empirical model prediction or
observations (Figures B-12 and B-13). Exploration of the model space using an
assumption of first-order metabolism (valid for the 50 ppm inhalation exposure) showed
that an adequate fit to the exposure and elimination data can be achieved only when
A-8
-------
unrealistically low values are assumed for the slowly perfused tissue:air partition
coefficient (Figure B-16). Artificially low values for the other tissue:air partition
coefficients are not expected to improve the model fit, as these parameters are shown in
the sensitivity analysis to exert less influence on blood 1,4-dioxane than VmaxC and Km.
In the absence of actual measurements for the human slowly perfused tissue:air partition
coefficient, high uncertainty exists for this model parameter value. Differences in the
ability of rat and human blood to bind 1,4-dioxane may contribute to the difference in Vd.
However, this is expected to be evident in very different values for rat and human
blood:air partition coefficients, which is not the case (Table B-l). Therefore, some other,
as yet unknown, modification to model structure may be necessary.
The results of USEPA's model evaluation were confirmed by other investigators
(Sweeney et al., 2008, 195085). Sweeney et al. (2008, 195085) concluded that the
available PBPK model with refinements resulted in an under-prediction of human blood
levels for 1,4-dioxane by six- to seven fold. It is anticipated that the high uncertainty in
predictions of the PBPK model for 1,4-dioxane would not result in a more accurate
derivation of human health toxicity values.
Because it is unknown whether the parent or the metabolite is the toxic moiety,
analyses were not conducted to adjust the experimental doses on the basis of metabolic
saturation.
The discussion of Sweeney et al. (2008, 195085) was expanded in the main
document in Section 3.5.3. In the absence of evidence to the contrary, the Agency cannot
discount the human blood kinetic data published by Young et al. (1977, 062956). Even
though the PBPK model provided satisfactory fits to the rodent kinetic data, it was not
used to extrapolate from high dose to low dose in the animal because an internal dose
metric was not identified and external doses were utilized in derivation of the toxicity
values.
KLC was implemented by USEPA during the evaluation of the model and should
have been described as a clearance constant (zero-order rate constant) with units of
L/hr/kg0'70. These corrections have been made in the document; however, this does not
impact the model predictions because it was in reference to the terminology used to
describe this constant.
The reviewer is correct that RMEX is the rate of metabolism of 1,4-dioxane per
unit time; however an amount of 1,4-dioxane metabolized was not calculated in the Reitz
et al. (1990, 094806) model code. Thus, AM is the amount of the metabolite (i.e.,
HEAA) in the body rather than the amount metabolized of 1,4-dioxane. RAM was
published by Reitz et al. (1990, 094806) as equation 2 for the change in the amount of
metabolite in the body per unit time. AMEX is the amount of the metabolite excreted in
the urine. While the variables used are confusing, the code describes the metabolism of
A-9
-------
1,4-dioxane as published in the manuscripts. The comments in the model code were
updated to make this description more clear (Appendix B).
5. Please comment on the selection of the uncertainty factors applied to the POD for the
derivation of the RfD. For instance, are they scientifically justified and transparently and
objectively described in the document? If changes to the selected uncertainty factors are
proposed, please identify and provide a rationale(s). Please comment specifically on the
following uncertainty factors:
• An interspecies uncertainty factor of 10 was used to account for uncertainties in
extrapolating from laboratory animals to humans because a PBPK model to support
interspecies extrapolation was not suitable.
• An intraspecies (human variability) uncertainty factor of 10 was applied in deriving the
RfD because the available information on the variability in human response to
1,4-dioxane is considered insufficient to move away from the default uncertainty factor
of 10.
• A database uncertainty factor of 3 was used to account for lack of adequate
reproductive toxicity data for 1,4-dioxane, and in particular absence of a
multigeneration reproductive toxicity study. Has the rationale for the selection of these
uncertainty factors been transparently and objectively described in the document?
Please comment on whether the application of these uncertainty factors has been
scientifically justified.
Comment:
One reviewer noted the uncertainly factors appear to be the standard default choices and
had no alternatives to suggest.
o Five reviewers agreed that the use of an uncertainty factor of 10 for the interspecies
extrapolation is fully supportable. One reviewer suggested using BW3/4 scaling
rather than an uncertainty factor of 10 for animal to human extrapolation. Along
the same lines, one reviewer suggested a steady-state quantitative analysis to
determine the importance of pulmonary clearance and hepatic clearance and stated
that if hepatic clearance scales to body surface and pulmonary clearance is
negligible, then an adjusted uncertainty factor based on body surface scaling would
be more appropriate.
o Seven reviewers stated that the uncertainty factor of 10 for interindividual
variability (intraspecies) is fully supportable.
o Six reviewers commented that the uncertainty factor of 3 for database deficiencies
is fully justifiable. One reviewer suggested adding text to clearly articulate the
science policy for the use of a factor of 3 for database deficiencies.
A-10
-------
Response. The preferred approach to interspecies scaling is the use of a PBPK model;
however, the PBPK models available for 1,4-dioxane are not suitable for use in this
health assessment as outlined elsewhere. Another approach that has been commonly
implemented in the cancer assessments is the use of body weight scaling based on body
surface area (BW3/4 scaling). It is not standard practice to to apply BW3/4 scaling in
noncancer assessments at this time. The current default approach used by the Agency
when PBPK models are not available for extrapolation is the application of an UFA of 10,
which was implemented in this assessment.
The absence of a multigenerational reproductive study is why the uncertainty
factor for database deficiencies (UFD) was retained; however, it was reduced from 10 to
3. In the text in Section 5.1.3 text was included to clearly state that because of the
absence of a multigenerational reproductive study for 1,4-dioxane an uncertainty factor of
3 was used for database deficiencies. No other changes regarding the use of the
uncertainty factors were made to the document.
A.1.3. Carcinogenicity of 1,4-dioxane
1. Under the EPA's 2005 Guidelines for Carcinogen Risk Assessment
(www.epa.gov/iris/backgr-d.htm), the Agency concluded that 1,4-dioxane is likely to be
carcinogenic to humans. Please comment on the cancer weight of evidence characterization.
Has the scientific justification for the weight of evidence descriptor been sufficiently,
transparently and objectively described? Do the available data for both liver tumors in rats
and mice and nasal, mammary, and peritoneal tumors in rats support the conclusion that
1,4-dioxane is a likely human carcinogen?
Comment. All reviewers agreed with the Agency's conclusion that 1,4-dioxane is "likely
to be carcinogenic to humans". However, two reviewers also thought 1,4-dioxane could
be categorized as a potential human carcinogen, since low-dose environmental exposures
would be unlikely to result in cancer. One reviewer also suggested providing a brief
recapitulation of the guidance provided by the 2005 Guidelines for Carcinogen Risk
Assessment regarding classification of a compound as likely to be carcinogenic to
humans and how a chemical falls into this category.
Response. The document includes a weight-of-evidence approach to categorize the
carcinogenic potential of 1,4-dioxane. This was included in Section 4.7.1 based upon
U.S. EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005, 086237).
1,4-Dioxane can be described as likely to be carcinogenic to humans based on evidence
of liver carcinogenicity in several 2-year bioassays conducted in three strains of rats, two
A-ll
-------
strains of mice, and in guinea pigs. Additionally, tumors in other organs and tissues have
been observed in rats due to exposure to 1,4-dioxane.
2. Evidence indicating the mode of action of carcinogenicity of 1,4-dioxane was considered.
Several hypothesized MOAs were evaluated within the Toxicological Review and EPA
reached the conclusion that a MOA(s) could not be supported for any tumor types observed
in animal models. Please comment on whether the weight of the scientific evidence supports
this conclusion. Please comment on whether the rationale for this conclusion has been
transparently and objectively described. Please comment on data available for 1,4-dioxane
that may provide significant biological support for a MOA beyond what has been described
in the Toxicological Review. Considerations should include the scientific support regarding
the plausibility for the hypothesized MOA(s), and the characterization of uncertainty
regarding the MOA(s).
Comment. Three reviewers commented that the weight of evidence clearly supported the
conclusion that a mode of action could not be identified for any of the tumor sites. One
reviewer commented that there is inadequate evidence to support a specific MOA with
any confidence and low-dose linear extrapolation is necessary; this reviewer also pointed
out that EPA should not rule out a metabolite as the toxic moiety.
One reviewer stated this was outside of his/her area of expertise but indicated that
the discussion was too superficial and suggested adding statements as to what the Agency
would consider essential information to make a determination about a MOA.
Two reviewers commented that even though the MOA for 1,4-dioxane is not clear
there is substantial evidence that the MOA is non-genotoxic. One of these reviewers also
suggested that a nonlinear cancer risk assessment model should be utilized.
One reviewer suggested adding more text to the summary statement to fully
reflect the available MOA information which should be tied to the conclusion and choice
of an extrapolation model.
Response. The Agency agrees with the reviewer not to rule out a toxic metabolite as the
toxic moiety. In Section 5.5.1.2 text is included relating that there is not enough
information to determine whether the parent compound, its metabolite(s), or a
combination is responsible for the observed toxicities following exposure to 1,4-dioxane.
It is not feasible to describe the exact data that would be necessary to conclude
that a particular MOA was operating to induce the tumors observed following
1,4-dioxane exposure. In general, the data would fit the general criteria described in the
U.S. EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005, 086237). For
1,4-dioxane, several MOA hypotheses have been proposed and are explored for the
A-12
-------
observed liver tumors in Section 4.7.3. This analysis represents the extent to which data
could provide support for any particular MOA.
One reviewer suggested that the evidence indicating that 1,4-dioxane is not
genotoxic supports a nonlinear approach to low-dose extrapolation. In accordance with
the U.S. EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005, 086237).
the absence of evidence for genotoxicity does not invoke the use of nonlinear low-dose
extrapolation, nor does it define a MOA. A nonlinear low-dose extrapolation can be
utilized when a MOA supporting a nonlinear dose response is identified. For 1,4-dioxane
this is not the case; a cancer MOA for any of the tumor types observed in animal models
has not been elucidated. Therefore, as concluded in the Toxicological Review, the
application of a nonlinear low-dose extrapolation approach was not supported.
Additional text has been added to Section 5.4.3.2 to relay the fact that several
reviewers recommended that the MOA data support the use of a nonlinear extrapolation
approach to estimate human carcinogenic risk associated with exposure to 1,4-dioxane
and that such an approach should be presented in the Toxicological Review. Additional
text has also been added to the summary statement in Section 6.2.3 stating that the weight
of evidence is inadequate to establish a MOA(s) by which 1,4-dioxane induces peritoneal,
mammary, or nasal tumors in rats and liver tumors in rats and mice (see Section 4.7.3 for
a more detailed discussion of 1,4-dioxane's hypothesized MO As).
3. A two-year drinking water cancer bioassay (JBRC, 1998, 196240) was selected as the
principal study for the development of an oral slope factor (OSF). Please comment on the
appropriateness of the selection of the principal study. Has the rationale for this choice been
transparently and objectively described?
Comment.
Seven reviewers agreed with the choice of the JBRC (1998, 196240) study as the
principal study for the development of an OSF. However, two reviewers that agreed with
the choice of JBRC (1998, 196240) also commented on the description and evaluation of
the study. One reviewer commented the evaluation of the study should be separated from
the evaluation/selection of endpoints within the study. The other reviewer suggested that
details on the following aspects should be added to improve transparency of the study:
(1) rationale for selection of doses; (2) temporal information on body weight for
individual treatment groups; (3) temporal information on mortality rates; and (4) dosing
details.
One reviewer thought that the complete rationale for selection of the JBRC (1998,
196240) study was not provided because there was no indication of whether the study
was conducted under GLP conditions, and the study was not peer reviewed or published.
This reviewer noted the NCI (1978, 062935) study was not appropriate for use, but that
A-13
-------
the Kociba et al. (1974, 062929) study may have resulted in a lower POD had they
employed both sexes of mice and combined benign and malignant tumors.
Response. Since the External Peer Review draft of the Toxicological Review of
1,4-Dioxane was released (U.S. EPA, 2009, 628630), the cancer portion of the study
conducted by the JBRC laboratory was published in the peer-reviewed literature as Kano
et al. (2009, 594539). This manuscript was reviewed by EPA. EPA determined that the
data published by Kano et al. (2009, 594539) should be included in the assessment of
1,4-dioxane for several reasons: (1) while the JBRC (1998, 196240) was a detailed
laboratory report, it was not peer-reviewed; (2) the JBRC improved the diagnosis of pre-
and neoplastic lesions in the liver according to the current diagnostic criteria and
submitted the manuscript based on this updated data; (3) the Kano et al. (2009, 594539)
peer-reviewed manuscript included additional information such as body weight growth
curves and means and standard deviations of estimated dose for both rats and mice of
both sexes. Thus, the Toxicological Review was updated to reflect the inclusion of the
data from Kano et al. (2009, 594539), and Appendix E was added for a clear and
transparent display of the data included in the multiple reports.
In response to the peer reviewers, dose information was updated throughout the
assessment and are also provided in detail in Section 4.2.1.2.6, along with temporal
information on body weights and mortality. Text was also added to Section 4.2.1.2.6
regarding the choice of high dose selection as included in the Kano et al. (2009, 594539)
manuscript. Additional discussoin regarding the mortality rates was also added to
Section 5.4.1 in selection of the critical study for the oral cancer assessment.
Documentation that the study was conducted in accordance with Organization for
Economic Co-operation and Development (OECD) Principles of Good Laboratory
Practice (GLP) is provided in the manuscript (Kano et al., 2009, 594539) and this was
also added to the text in Section 4.2.1.2.6.
4. Combined liver tumors (adenomas and carcinomas) in female CjrBDFl mice from the JBRC
(1998, 196240) study were chosen as the most sensitive species and gender for the derivation
of the final OSF. Please comment on the appropriateness of the selections of species and
gender. Please comment on whether the rationale for these selections is scientifically
justified. Has the rationale for these choices been transparently and objectively described?
Comment. Six reviewers agreed the female CjrBDFl mice should be used for the
derivation of the OSF. Five of these reviewers agreed with the rationale for the selection
of the female CjrBDFl mouse as the most sensitive gender and species. However, one
reviewer suggested that the specific rationale (i.e., that the final OSF is determined by
A-14
-------
selecting the gender/species that gives the greatest OSF value) be stated clearly in a
paragraph separate from the other considerations of study selection.
One reviewer was unsure of both the scientific justification for combining benign
and malignant liver tumors, as well as the background incidence of the observed liver
tumors in historical control CjrBDFl male and female mice.
One reviewer commented that the scientific basis for the selection of female
CjrBDFl mice was unclear. This reviewer thought that the rationale for the choice of
this strain/sex compared to all others was not clearly articulated.
Response. Using the approach described in the Guidelines for Carcinogen Risk
Assessment (U.S. EPA, 2005, 086237) studies were first evaluated based on their quality
and suitability for inclusion in the assessment. Once the studies were found to be of
sufficient quality for inclusion in the assessment, the dose-response analysis was
performed with the goal of determining the most appropriate endpoint and species for use
in the derivation of an OSF. These topics are discussed in detail in Section 4.7 and 5.4.
Benign and malignant tumors that arise from the same cell type (e.g.,
hepatocellular) may be combined to more clearly identify the weight of evidence for a
chemical. This is in accordance with the US EPA's 2005 Guidelines for Carcinogen Risk
Assessment as referenced in the Toxicological Review. In the absence of a MO A (MOA
analysis described in detail in Section 4.7.) for 1,4-dioxane carcinogenicity, it is not
possible to determine which species may more closely resemble humans. Text in Section
5.4.4 indicates that the calculation of an OSF for 1,4-dioxane is based upon the dose-
response data for the most sensitive species and gender.
5. Has the scientific justification for deriving a quantitative cancer assessment been
transparently and objectively described? Regarding liver cancer, a linear low-dose
extrapolation approach was utilized to derive the OSF. Please provide detailed comments on
whether this approach to dose-response assessment is scientifically sound, appropriately
conducted, and objectively and transparently described in the document. Please identify and
provide the rationale for any alternative approaches for the determination of the OSF and
discuss whether such approaches are preferred to EPA's approach.
Comment. Four reviewers agreed with the approach for the dose-response assessment.
One reviewer commented that even if a nongenotoxic MOA were identified for
1,4-dioxane it may not be best evaluated by threshold modeling. One reviewer
commented the use of the female mouse data provided an appropriate health protective
and scientifically valid approach.
One reviewer commented that the basic adjustments and extrapolation method for
derivation of the OSF were clearly and adequately described, but disagreed with the
A-15
-------
linear low-dose extrapolation. This reviewer suggested that the lack of certainty regarding
the MOA was not a sufficient cause to default to a linear extrapolation. Another reviewer
commented that the rationale for a linear low-dose extrapolation to derive the OSF was
not clear, but may be in accordance with current Agency policy in the absence of a
known MOA. This reviewer also commented that 1,4-dioxane appears to be non-
genotoxic and nonlinear models should be tested on the available data to determine if
they provide a better fit and are more appropriate.
One reviewer thought that the justification for a linear extrapolation was not
clearly provided and that a disconnect between the MOA summary and the choice of a
linear extrapolation model existed. In addition, this reviewer commented that the
pharmacokinetic information did not support the use of a linear extrapolation approach,
but rather use of animal PBPK models to extrapolate from high to low dose that would
result in a mixture of linear and nonlinear extrapolation models was warranted.
One reviewer suggested consideration of an integrated assessment of the cancer
and noncancer endpoints; however, if linear low-dose extrapolation remains the approach
of choice by the Agency, then the effect of choosing BMRs other than 10% was
recommended to at least be included in the uncertainty discussion. Using BMRs lower
than 10% may allow for the identification of a risk level for which the low-dose slope is
'best' estimated.
Response. The EPA conducted a cancer MOA analysis evaluating all of the
available data for 1,4-dioxane. Application of the framework in the USEPA's Guidelines
for Carcinogen Risk Assessment (2005, 086237) demonstrates that the available evidence
to support any hypothesized MOA for 1,4-dioxane-induced tumors does not exist. In the
absence of a MOA, the USEPA's Guidelines for Carcinogen Risk Assessment (2005,
086237) indicate that a low dose linear extrapolation should be utilized for dose response
analysis (see Section 5.4). Some of the potential uncertainty associated with this
conclusion was characterized in Section 5.5. Note that there is no scientific basis to
indicate that in the absence of evidence for genotoxicity a nonlinear low-dose
extrapolation should be used. As concluded in the Toxicological Review, the application
of a nonlinear low-dose extrapolation approach was not supported.
With regards to the PBPK model available for 1,4-dioxane, it is clear that there
currently exist deficiencies within the model and as such, the model was not utilized for
interspecies extrapolation. Given the deficiencies and uncertainty in the 1,4-dioxane
model it also does not provide support for a MOA.
Lastly, in the absence of a MOA for 1,4-dioxane carcinogenicity it is not possible
to harmonize the cancer and noncancer effects to assess the risk of health effects due to
exposure. However, the choice of the BMDLio, which was more than 15-fold lower than
A-16
-------
the response at the lowest dose (66 mg/kg-day), was reconsidered in response to a public
comment. BMDs and BMDLs were calculated using a BMR of 30 and 50% extra risk
(BMD30, BMDL30, BMD50, and BMDL50). A BMR of 50% was used as it resulted in a
BMDL closest to the response level at the lowest dose tested in the bioassay.
A.2. PUBLIC COMMENTS
Comments on the Toxicological Review of 1,4-Dioxane submitted by the public are summarized
below in the following categories: Oral reference dose for 1,4-dioxane, carcinogenicity of
1,4-dioxane, PBPK modeling, and other comments.
A.2.1. Oral reference dose (RfD) for 1,4-dioxane
Comment: An UF for database deficiencies is not necessary because of considerable
evidence showing no reproductive or developmental effects from 1,4-dioxane exposure.
Response: Due to the lack of a multigenerational reproductive study for 1,4-dioxane an
UF of 3 was retained for database deficiencies. Without clear evidence showing a lack of
reproductive or developmental effects in a multigenerational reproductive study, there is
still uncertainty in this area.
A.2.2. Carcinogenicity of 1,4-dioxane
Comment: Using liver tumors as the basis for the oral CSF is more appropriate than
nasal tumors (1988 IRIS assessment of 1,4-dioxane); however, the use of mouse liver
tumor data is inappropriate because it is inconsistent with other liver models both
quantitatively and in the dose-response pattern. High mortality rates in the study are also
a limitation. Liver tumor data from rats should be used instead, which represents a better
animal model for 1,4-dioxane carcinogenicity assessment.
Response: Even though the dose-response is different for mice and rats, the female mice
were considered to be appropriate for the carcinogenicity assessment for several reasons.
The female mouse liver tumors from the Kano et al. (2009, 594539)report were found to
be the most sensitive species and endpoint. Section 4.2.1.2.6 was updated to include
additional information on mortality rates. The majority of the animals lived past
52 weeks (only 4 females died prior to 52 weeks, 2 in each the mid- and high-dose
groups). The cause of death in the female mice that died between 1 and 2 years was
attributed to liver tumors.
A-17
-------
Comment: The OSF was based on the most sensitive group, Crj :BDF1 mice; however
BDF1 mice have a high background rate of liver tumors. The incidence of liver tumors in
historical controls for this gender/species should be considered in the assessment.
Sensitivity of the test species/gender as well as other criteria should be considered in the
selection of the appropriate study, including internal and external validity as outlined in
Lewandowski and Rhomberg (2005, 626613). The female Crj :BDF1 mice had a low
survival rate that should be considered in the selection of the animal model for
1,4-dioxane carcinogenicity.
Response. Katagiri et al. (1998, 193804) summarized the incidence of hepatocellular
adenomas and carcinomas in control male and female BDF1 mice from ten 2-year
bioassays at the JBRC. For female mice, out of 499 control mice, the incidence rates
were 4.4% for hepatocellular adenomas and 2.0% for hepatocellular carcinomas. Kano
et al. (2009, 594539) reported a 10% incidence rate for hepatocellular adenomas and a
0% incidence rate for hepatocellular carcinomas in control female BDF1. These
incidence rates are near the historical control values and thus are appropriate for
consideration in this assessment. Additional text regarding these historical controls was
added to the study description in Section 4.2.1.2.6.
Comment: Low-dose linear extrapolation for the oral CSF is not appropriate nor justified
by the data. The weight of evidence supports a threshold (nonlinear) MOA when
metabolic pathway is saturated at high doses. Nonlinear extrapolations should be
evaluated and presented for 1,4-dioxane. Oral CSFs should be derived and presented
using both the BW3/4 scaling as well as available PBPK models to extrapolate across
species.
Response: The absence of evidence for genotoxicity/mutagenicity does not indicate the
use of nonlinear low-dose extrapolation. For 1,4-dioxane, a MOA to explain the
induction of tumors does not exist so the nature of the low-dose region of the dose-
response is unknown. The oral CSF for 1,4-dioxane was derived using BW3/4 scaling for
interspecies extrapolation. The PBPK and empirical models available for 1,4-dioxane
were evaluated and found not to be adequate for use in this assessment, described in
detail in Appendix B.
Comment: The POD for the BDF1 female mouse is 15-fold lower than the lowest dose
in the bioassay, thus the POD is far below the lower limit of the data and does not follow
the U.S. EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005, 086237).
A-18
-------
Response. The comment is correct that the animal BMDLio was more than 15-fold
lower than the response at the lowest dose (66 mg/kg-day) in the bioassay. BMDs and
BMDLs were calculated using a BMR of 30 and 50% extra risk (BMD30, BMDL30,
BMD50, and BMDL50). A BMR of 50% was chosen as it resulted in a BMDL closest to
the response level at the lowest dose tested in the bioassay.
Comment. The geometric mean of the oral cancer slope factors (as done with B[a]P &
DDT) should have been used instead of relying on the female BDF1 mouse data, since a
MO A could not be determined for 1,4-dioxane.
Response. In accordance with the BMD technical guidance document (U.S. EPA, 2000,
052150), averaging tumor incidence is not a standard or default approach. Averaging the
tumor incidence response diminishes the effect seen in the sensitive species/gender.
Comment EPA should critically reexamine the choice of JBRC (1998, 196240) as the
principal study since it has not been published or peer-reviewed. A transcript of e-mail
correspondence should be provided.
Response. JBRC (1998, 196240) was published as conference proceedings as Yamazaki
et al. (1994, 196120) and recently in the peer-reviewed literature as Kano et al. (2009,
594539). Additional study information was also gathered from the authors (Yamazaki,
2006, 626614) and is available upon request from the IRIS Hotline. The peer-reviewed
and published data from Kano et al. (2009, 594539) was incorporated into the final
version of the Toxicological Review of 1,4-Dioxane.
Comment. The WOE does not support a cancer descriptor of likely to be carcinogenic to
humans determination, but rather suggestive human carcinogen at the high dose levels
used in rodent studies seems more appropriate for the following reasons: 1) lack of
conclusive human epidemiological data; 2) 1,4-dioxane is not mutagenic; and 3) evidence
at high doses it would act via cell proliferation MOA.
Response: A cancer classification of "likely, " based on evidence of liver carcinogenicity
in several two-year bioassays conducted in three strains of rats, two strains of mice, and
in guinea pigs was chosen. Also, mesotheliomas of the peritoneum, mammary, and nasal
tumors have been observed in rats. The Agency agrees that human epidemiological
studies are inconclusive. The evidence at any dose is insufficient to determine a MOA.
A-19
-------
A.2.3. PBPK Modeling
Comment. EPA should have used and considered PBPK models to derive the oral
toxicity values (rat to human extrapolation) rather than relying on a default method. The
draft did not consider the Sweeney et al. (2008, 195085) model. The PBPK model should
be used for both noncancer and cancer dose extrapolation.
Response: The Agency evaluated the Sweeney et al. (2008, 195085) publication and this
was included in Appendix B of the document. Text was added to the main document in
Section 3.5.2.4 and 3.5.3 regarding the evaluation of Sweeney et al. (2008, 195085). This
model was determined not to be appropriate for interspecies extrapolation. Additionally,
see response to the external peer review panel comment B4.
Comment: EPA should use the modified inhalation inputs used in the Reitz et al. (1990,
094806) model and the updated input parameters provided in Sweeney et al. (2008,
195085) and add a compartment for the kidney
Response: See response to previous comment regarding evaluation of Sweeney et al.
(2008, 195085). Modification of the model to add a kidney compartment is not within
the scope of this assessment.
A.2.4. Other Comments
Comment: EPA should consider the Kasai et al. (2008, 195044: 2009, 193803) studies
for inhalation and MOA relevance.
Response: The 13 week and 2-year inhalation studies by Kasai et al. (2008, 195044;
2009, 193803) were published late in the development stage of this assessment. The IRIS
Program will evaluate these recently published 1,4-dioxane inhalation data for the
potential to derive an RfC in a separate assessment.
Comment: 1,4-Dioxane is not intentionally added to cosmetics and personal care
products - correct sentence on page 4.
Response: This oversight was corrected in the document.
A-20
-------
APPENDIX B. EVALUATION OF EXISTING PBPK MODELS FOR 1,4-DIOXANE
B.I. BACKGROUND
Several pharmacokinetic models have been developed to predict the absorption,
distribution, metabolism, and elimination of 1,4-dioxane in rats and humans. Single
compartment, empirical models for rats (Young et al., 1978, 062955; Young et al., 1978,
625640) and humans (Young et al., 1977, 062956) were developed to predict blood levels of
1,4-dioxane and urine levels of the primary metabolite, p-hydroxyethoxy acetic acid (HEAA).
Physiologically based pharmacokinetic (PBPK) models that describe the kinetics of 1,4-dioxane
using biologically realistic flow rates, tissue volumes and affinities, metabolic processes, and
elimination behaviors, were also developed (Fisher et al., 1997, 194390; Leung and Paustenbach,
1990, 062932; Reitz et al., 1990, 094806).
In developing updated toxicity values for 1,4-dioxane, the available PBPK models were
evaluated for their ability to predict observations made in experimental studies of rat and human
exposures to 1,4-dioxane. The model of Reitz et al. (1990, 094806) was identified for further
consideration to assist in the derivation of toxicity values. Issues related to the biological
plausibility of parameter values in the Reitz et al. (1990, 094806) human model were identified.
The model was able to predict the only available human inhalation data set (50 ppm 1,4 dioxane
for 6 hours; Young et al., 1977, 062956) by increasing (i.e., doubling) parameter values for
human alveolar ventilation, cardiac output, and the blood:air partition coefficient above the
measured values. Furthermore, the measured value for the slowly perfused tissue:air partition
coefficient (i.e., muscle) was replaced with the measured liver value to improve the fit. Analysis
of the Young et al. (1977, 062956) human data suggested that the apparent volume of
distribution (Vd) for 1,4-dioxane was approximately 10-fold higher in rats than humans,
presumably due to species differences in tissue partitioning or other process not represented in
the model. Subsequent exercising of the model demonstrated that selecting a human slowly
perfused tissue:air partition coefficient much lower than the measured rat value resulted in better
agreement between model predictions of 1,4-dioxane in blood and experimental observations.
Based upon these observations, several model parameters (e.g., metabolism/elimination
parameters) were re-calibrated using biologically plausible values for flow rates and tissue:air
partition coefficients.
Note: Hyperlinks to the reference citations throughout this document will take you to the NCEA HERO database
(Health and Environmental Research Online) at http://epa. gov/hero. HERO is a database of scientific literature used
by U.S. EPA in the process of developing science assessments such as the Integrated Science Assessments (ISA)
and the Integrated Risk Information System (IRIS).
B-l
-------
This appendix describes activities conducted in the evaluation of the empirical models
(Young et al., 1977, 062956: Young et al., 1978, 062955: Young et al., 1978, 625640). and re-
calibration and exercising of the Reitz et al. (1990, 094806) PBPK model, and evaluation of the
Sweeney et al. (2008, 195085) model to determine the potential utility of the PBPK models for
1,4-dioxane for interspecies and route-to-route extrapolation.
B.2. SCOPE
The scope of this effort consisted of implementation of the Young et al. (1977, 062956:
1978, 062955: 1978, 625640) empirical rat and human models using the acslXtreme simulation
software, re-calibration of the Reitz et al. (1990, 094806) human PBPK model, and evaluation of
model parameters published by Sweeney et al. (2008, 195085). Using the model descriptions
and equations given in Young et al. (1977, 062956: 1978, 062955: 1978, 625640). model code
was developed for the empirical models and executed, simulating the reported experimental
conditions. The model output was then compared with the model output reported in Young et al.
(1977, 062956: 1978, 062955: 1978, 625640).
The PBPK model of Reitz et al. (1990, 094806) was re-calibrated using measured values
for cardiac and alveolar flow rates and tissue:air partition coefficients. The predictions of blood
and urine levels of 1,4-dioxane and HEAA, respectively, from the re-calibrated model were
compared with the empirical model predictions of the same dosimeters to determine whether the
re-calibrated PBPK model could perform similarly to the empirical model. As part of the PBPK
model evaluation, EPA performed a sensitivity analysis to identify the model parameters having
the greatest influence on the primary dosimeter of interest, the blood level of 1,4-dioxane.
Variability data for the experimental measurements of the tissue:air partition coefficients were
incorporated to determine a range of model outputs bounded by biologically plausible values for
these parameters. Model parameters from Sweeney et al. (2008, 195085) were also tested to
evaluate the ability of the PBPK model to predict human data following exposure to 1,4-dioxane.
B.3. IMPLEMENTATION OF THE EMPIRICAL MODELS IN acslXtreme
The empirical models of Young et al. (1977, 062956: 1978, 062955: 1978, 625640) for
1,4-dioxane in rats and humans were reproduced using acslXtreme, version 2.3 (Aegis
Technologies, Huntsville, AL). Model code files were developed using the equations described
in the published papers. Additional files containing experiment-specific information (i.e., BWs,
exposure levels, and duration) were also generated.
B.3.1. Model Descriptions
The empirical model of Young et al. (1978, 062955: 1978, 625640) for 1,4-dioxane in
rats is shown in Figure B-l. This is a single-compartment model that describes the absorption
and metabolism kinetics of 1,4-dioxane in blood and urine. No information is reported
B-2
-------
describing pulmonary absorption or intravenous (i.v.) injection/infusion of 1,4-dioxane. The
metabolism of 1,4-dioxane and subsequent appearance of HEAA is described by Michaelis-
Menten kinetics governed by a maximum rate (Vmax, ug/mL-hour) and affinity constant (Km,
ug/mL) . Both 1,4-dioxane and HEAA are eliminated via the first-order elimination rate
constants, ke and kme, respectively (hour"1) by which 35% of 1,4-dioxane and 100% of HEAA
appear in the urine, while 65% of 1,4-dioxane is exhaled. Blood concentration of 1,4-dioxane is
determined by dividing the instantaneous amount of 1,4-dioxane in blood by a Vd of 301 mL/kg
BW.
Inhalation (k
i.v. admin
dDioxtody _ Fmax x Diox,^
dt
„ , „ .
K +DioX
dt Km+Diox,
— km
boi]y
Exhaled (65%)
k x Diox
J*
*• Urine (35%)
k xHEAA
•*• Urine
Source: Used with permission from Taylor & Francis, Young et al. (1978, 062955; 1978,
625640).
Figure B-l. Schematic representation of empirical model for 1,4-dioxane in
rats.
Figure B-2 illustrates the empirical model for 1,4-dioxane in humans as described in
Young et al. (1977, 062956). Like the rat model, the human model predicts blood 1,4-dioxane
and urinary 1,4-dioxane and HEAA levels using a single-compartment structure. However, the
metabolism of 1,4-dioxane to HEAA in humans is modeled as a first-order process governed by
a rate constant, KM (hour"1). Urinary deposition of 1,4-dioxane and HEAA is described using the
first order rate constants, ke(diox) and kme(HEAA), respectively. Pulmonary absorption is described
by a fixed rate of 76.1 mg/hour (kiNH). Blood concentrations of 1,4-dioxane and HEAA are
calculated as instantaneous amount (mg) divided by Vd(diox) or Vd(HEAA), respectively (104 and
480 mL/kg BW, respectively).
B-3
-------
Inhalation (k^
Dioxane
K
•M
HEAA
'd(HEAA) X
e (diox)
ne (HEAA)
Urine
Cumulative
Dioxane and
HEAA
Source: Used with permission from Taylor & Francis, Young et al. (1977, 062956).
Figure B-2. Schematic representation of empirical model for 1,4-dioxane in
humans.
B.3.2. Modifications to the Empirical Models
Several modifications were made to the empirical models. The need for the
modifications arose in some cases from incomplete reporting of the Young et al. (1977, 062956;
1978, 062955; 1978, 625640) studies and in other cases from the desire to add capabilities to the
models to assist in the derivation of toxicity values.
For the rat model, no information was given by Young et al. (1978, 062955; 1978,
625640) regarding the parameterization of pulmonary absorption (or exhalation) or i.v.
administration of 1,4-dioxane. Therefore, additional parameters were added to simulate these
processes in the simplest form. To replicate 1,4-dioxane inhalation, a first-order rate constant,
kiNH (hour"1), was introduced. kiNH was multiplied by the inhalation concentration and the
respiratory minute volume of 0.238 L/minute (Young et al., 1978, 062955: 1978, 625640). The
value for kiNn was estimated by optimization against the blood time course data of Young et al.
(1978, 062955; 1978, 625640). Intravenous (i.v.) administration was modeled as instantaneous
appearance of the full dose at the start of the simulation. Rat urinary HEAA data were reported
by Young et al. (1978, 062955: 1978, 625640) in units of concentration. To simulate urinary
HEAA concentration, an estimate of urine volume was required. Since observed urinary
volumes were not reported by Young et al. (1978, 062955: 1978, 625640), a standard rat urine
production rate of 0.00145 L/hour was used.
For humans, Young et al. (1977, 062956) used a fixed 1,4-dioxane inhalation uptake rate
of 76.1 mg/hour, which corresponded to observations during a 50 ppm exposure. In order to
facilitate user-specified inhalation concentrations, pulmonary absorption was modeled. The
modeling was performed identically to the rat model, but using a human minute volume of
7 L/minute. Urinary HEAA data were reported by Young et al. (1977, 062956) as a cumulative
amount (mg) of HEAA. Cumulative amount of HEAA in the urine is readily calculated from the
B-4
-------
rate of transfer of HEAA from plasma to urine, so no modification was necessary to simulate this
dose metric for humans.
Neither empirical model of Young et al. (1977, 062956: 1978, 062955: 1978, 625640)
described oral uptake of 1,4-dioxane. Adequate data to estimate oral absorption parameters are
not available for either rats or humans; therefore, neither empirical model was modified to
include oral uptake.
B.3.3. Results
The acslXtreme implementation of the Young et al. (1978, 062955: 1978, 625640) rat
empirical model simulates the 1,4-dioxane blood levels from the i.v. experiments identically to
the model output reported in the published paper (Figure B-3). However, the acslXtreme version
predicts urinary HEAA concentrations in rats that are approximately threefold lower and reach a
maximum sooner than the predicted levels reported in the paper (Figure B-4). These
discrepancies may be due, at least in part, to the reliance in the acslXtreme implementation on a
constant, standard, urine volume rather than experimental measurements, which may have been
different from the assumed value and may have varied over time. Unreported model parameters
(e.g., lag times for appearance of excreted HEAA in bladder urine) may also contribute to the
discrepancy.
Observations and predictions of 1,4-dioxane in rat blood
following 3 to 1000 mg/kg IV injection
D
acsl version - Youn
(1978a,b) empirical
Young etal. (1978a
observations
et al.
model
b)
20
30 40
Time (hrs)
50
70
Source: Used with permission from Taylor & Francis, Young et al. (1978, 062955: 1978,
625640).
Figure B-3. Output of 1,4-dioxane blood level data from the acslXtreme
implementation (left) and published (right) empirical rat model simulations
of i.v. administration experiments.
B-5
-------
Observations and predictions of HEAA in rat urine
following 10 or 1000 mg/kg IV injection
B)
E
&*z
" i
5<
a
—
D
acsl version- Youn
(1978a, b) empirical
Young etal. (1978a
observations
et al.
model
b)
20 30
Time(hrs)
J— ^= ^T •«^> -4^r lit*
Source: Used with permission from Taylor & Francis, Young et al. (1978, 062955; 1978,
625640).
Figure B-4. Output of HEAA urine level data from acslXtreme
implementation (left) and published (right) empirical rat model simulations
of i.v. administration experiments.
The Young et al. (1978, 062955; 1978, 625640) report did not provide model predictions
for the 50-ppm inhalation experiment. However, the acslXtreme implementation produces blood
1,4-dioxane predictions that are quite similar to the reported observations (Figure B-5). As with
the urine data from the i.v. experiment, the acslXtreme-predicted urinary HEAA concentrations
are approximately threefold lower than the observations, presumably for the same reasons
discussed above for the i.v. predictions.
B-6
-------
Observations and predictions of 1,4-dioxane in rat blood
following a 6 hour 50 ppm inhalation exposure
D
acsl version - Young et al.
(1978a, b) empirical model
Young et al. (1978a, b)
observations
6 8 10
Time (hrs)
Observations and predictions of HEAA in rat urine
following a 6 hour 50 ppm inhalation exposure
25.0
20.0 -
.§15.0 -
10.0 -
5.0 -
0.0
D
acsl version -Young et
al. (1978a, b) empirical
model
Young etal. (1978a, b)
observations
20 30
Time (hrs)
40
50
Source: Used with permission from Taylor & Francis, Young et al. (1978, 062955; 1978,
625640).
Figure B-5. acslXtreme predictions of blood 1,4-dioxane and urine HEAA
levels from the empirical rat model simulations of a 6-hour, 50-ppm
inhalation exposure.
Inhalation data for a single exposure level (50 ppm) are available for humans. The
acslXtreme predictions of the blood 1,4-dioxane observations are identical to the predictions
reported in Young et al. (1977, 062956) (Figure B-6). Limited blood FtEAA data were reported,
and the specimen analysis was highly problematic (e.g., an analytical interference was
sometimes present from which FtEAA could not be separated). For this reason, Young et al.
(1977, 062956) did not compare predictions of the blood HEAA data to observations in their
manuscript.
B-7
-------
Observations and predictions of 1,4-dioxane in human
blood following a 6 hour 50 ppm inhalation exposure
D
acsl version - Young et
al (1977) empirical model
observed
Time(hrs)
9 10 11 12
Source: Used with permission from Taylor & Francis, Young et al. (1978, 062955; 1978,
625640).
Figure B-6. Output of 1,4-dioxane blood level data from the acslXtreme
implementation (left) and published (right) empirical human model
simulations of a 6-hour, 50-ppm inhalation exposure.
Data for cumulative urinary HEAA amounts are provided in Young et al. (1977, 062956),
and no analytical problems for these data were reported. Nevertheless, model predictions for
urinary HEAA were not presented in the manuscript. The acslXtreme prediction of the HEAA
kinetics profile is similar to the observations, although predicted values are approximately 1.5- to
2-fold lower than the observed values (Figure B-7). Unlike urinary HEAA observations in the
rat, human observations were reported as cumulative amount produced, negating the need for
urine volume data. Therefore, discrepancies between model predictions and experimental
observations for humans cannot be attributed to uncertainties in urine volumes in the subjects.
B-8
-------
Observations and predictions of HEAA in human urine
following a 6 hour 50 ppm inhalation exposure
700.0
>. "3
•a
a <
IS
3 I
600.0 -
500.0 -
400.0 -
300.0 -
200.0 -
100.0 -
0.0
D
acsl version - Young et al
(1977) empirical model
observed
0
10 15
Time(hrs)
20
25
Source: Used with permission from Taylor & Francis, Young et al. (1977, 062956).
Figure B-7. Observations and acslXtreme predictions of cumulative HEAA
in human urine following a 6-hour, 50-ppm inhalation exposure.
B.3.4. Conclusions for Empirical Model Implementation
The empirical models described by Young et al. (1977, 062956: 1978, 062955: 1978,
625640) for rats and humans were implemented using acslXtreme. The models were modified to
allow for user-defined inhalation levels by addition of a first-order rate constant for pulmonary
uptake of 1,4-dioxane, fitted to the inhalation data. No modifications were made for oral
absorption as adequate data are not available for parameter estimation. The acslXtreme
predictions of 1,4-dioxane in the blood are identical to the published predictions for simulations
of 6-hour, 50-ppm inhalation exposures in rats and humans and 3 to 1,000 mg/kg i.v. doses in
rats (Figures B-3, B-5, and B-6). However, the acslXtreme version predicts lower urinary
HEAA concentrations in rats appearing earlier than either the Young et al. (1978, 062955: 1978,
625640) model predictions or the experimental observations. The lower predicted urinary
HEAA levels in the acslXtreme implementation for rats is likely due to use of default values for
urine volume in the absence of measured volumes. The reason for differences in time-to-peak
levels is unknown, but may be the result of an unreported adjustment by Young et al. (1978,
062955: 1978, 625640) in model parameter values. For humans, Young et al. (1977, 062956)
did not report model predictions of urinary HEAA levels. The urinary HEAA levels predicted by
acslXtreme were low relative to the observations. However, unlike the situation in rats, these
data are not dependent on unreported urine volumes (observations were reported as cumulative
HEAA amount rather than HEAA concentration), but reflect the model parameter values
B-9
-------
reported by Young et al. (1977, 062956). Presently, there is no explanation for the lack of fit of
the reported urinary HEAA elimination rate constant to the observations.
B.4. INITIAL RE-CALIBRATION OF THE PBPK MODEL
Concern regarding adjustments made to some of the parameter values in Reitz et al.
(1990, 094806) prompted a re-calibration of the Reitz et al. (1990, 094806) human PBPK model
using more biologically plausible values for all measured parameter values. Reitz et al. (1990,
094806) doubled the measured physiological flows and blood:air partition coefficient and
substituted the slowly-perfused tissue:air partition coefficient with the liverair value in order to
attain an adequate fit to the observations. This approach increases uncertainty in these parameter
values, and in the utilization of the model for cross-species dose extrapolation. Therefore, the
model was re-calibrated using parameter values that are more biologically plausible to determine
whether an adequate fit of the model to the available data can be attained.
B.4.1. Sources of Values for Flow Rates
The cardiac output of 30 L/hour/kg0'74 (Table B-l) reported by Reitz et al. (Reitz et al.,
1990, 094806) is approximately double the mean resting value of 14 L/hour/kg0'74 reported in the
widely accepted compendium of Brown et al. (1997, 020304). Resting cardiac output was
reported to be 5.2 L/minute (or 14 L/hour/kg0'74), while strenuous exercise resulted in a flow of
9.9 L/minute (or 26 L/hour/kg0'74) (Brown et al., 1997, 020304). Brown et al. (1997, 020304)
also cite the ICRP (1975, 196239) as having a mean respiratory minute volume of 7.5 L/minute,
which results in an alveolar ventilation rate of 5 L/minute (assuming 33% lung dead space), or
13 L/minute/kg0'74. Again, this is roughly half the value of 30 L/hour/kg0'74 employed for this
parameter by Reitz et al. (1990, 094806). Young et al. (1977, 062956) reported that the human
subjects exposed to 50 ppm for 6 hours were resting inside a walk-in exposure chamber. Thus,
use of cardiac output and alveolar ventilation rates of 30 L/hour/kg074 is not consistent with the
experimental conditions being simulated.
B-10
-------
Table B-l. Human PBPK model parameter values for 1,4-dioxane
Parameter
Reitz et al. (1990,
094806)
Leung and
Paustenbach (1990,
062932)
Sweeney et al.
(2008. 195085)
EPAC
Physiological Flows
Cardiac output (QCC)a
Alveolar ventilation (QPC)a
30
30
-
-
-
-
17.0
17.7
Partition Coefficients (PCs)
Blood:air (PB)
Fat:air (PFA)
Liver:air (PLA)
Rapidly perfused tissue :air (PRA)
Slowly perfused tissue:air (PSA)
3,650
851
1,557
1,557
1,557
1,825 ± 94
851 ±118
1,557 ±114
-
997 ± 254
1,666 ± 287
-
1,862 ± 739b
-
1,348 ± 290b
1,850
851
1,557
1,557
166
Metabolic Constants
Maximum rate for 1,4-dioxane
metabolism (VmaxC)d
Metabolic affinity constant (Km)e
HEAA urinary elimination rate
constant (kme)f
6.35
3.00
0.56
~
-
~
~
-
~
5.49
9.8
0.44
aL/hour/kgBW074
bMeasurement for rat tissue
'Biologically plausible values utilized by EPA in this assessment
dmg/hour/kg BW°75
emg/L
'hour1
Examination of the experimental data of Young et al. (1977, 062956) yields an estimated
alveolar ventilation to be 7 L/minute (or 16 L/hour/kg0'74) for volunteers having a mean BW of
84 kg. This rate is based on the Young et al. (1977, 062956) estimate of 76.1 mg/hour for
1,4-dioxane uptake. Based on these findings, the cardiac output and alveolar ventilation rates of
17.0 and 17.7 L/hour/kga74 were biologically plausible for the experimental subjects. These rate
estimates are based on calculations made using empirical data and are consistent with standard
human values and the experimental conditions (i.e., subject exertion level) reported by Young
et al. (1977, 062956). Therefore, these flow values were chosen for the model re-calibration.
B.4.2. Sources of Values for Partition Coefficients
Two data sources are available for the tissue:air equilibrium partition coefficients for
1,4-dioxane: Leung and Paustenbach (1990, 062932) and Sweeney et al. (2008, 195085). Both
investigators report mean values and standard deviations for human blood:air, rat liverair, and
rat muscle:air (e.g., slowly perfused tissue:air), while Leung and Paustenbach et al. (1990,
062932) also reported values for rat fatair (Table B-l).
B-ll
-------
B.4.3. Calibration Method
The PBPK model was twice re-calibrated using the physiological flow values suggested
values (current EPA assessment, see Table B-l) and the partition coefficients of Leung and
Paustenbach (1990, 062932) and Sweeney et al. (2008, 195085) separately. For each calibration,
the metabolic parameters Vmaxc and Km, were simultaneously fit (using the parameter estimation
tool provided in the acslXtreme software) to the output of 1,4-dioxane blood concentrations
generated by the acslXtreme implementation of the Young et al. (1977, 062956) empirical
human model for a 6 hour, 50 ppm inhalation exposure. Subsequently, the HEAA urinary
elimination rate constant, kme, was fitted to the urine HEAA predictions from the empirical
model. The empirical model predictions, rather than experimental observations, were used to
provide a more robust data set for model fitting, as the empirical model simulation provided 240
data points (one prediction every 0.1 hour) compared with hourly experimental observations, and
to avoid introducing error by calibrating the model to data digitally captured from Young et al.
(1977. 062956).
B.4.4. Results
Results of the model re-calibration are provided in Table B-2. The re-calibrated values
for Vmaxc and kme associated with the Leung and Paustenbach (1990, 062932) or Sweeney et al.
(2008, 195085) tissue:air partition coefficients are very similar. However, the fitted value for Km
using the Sweeney et al. (2008, 195085) partition coefficients is far lower (0.0001 mg/L) than
that resulting from use of the Leung and Paustenbach (1990, 062932) partition coefficients
(2.5 mg/L). This appears to be due to the higher slowly perfused tissue:air partition coefficient
determined by Sweeney et al. (2008, 195085) (1,348 vs. 997), resulting in a higher apparent Vd
than if the Leung and Paustenbach (1990, 062932) value is used. Thus, the optimization
algorithm selects a low Km, artificially saturating metabolism in an effort to drive predicted
blood 1,4-dioxane levels closer to the empirical model output. Saturation of metabolism during a
50 ppm inhalation exposure is inconsistent with the observed kinetics.
B-12
-------
Table B-2. PBPK metabolic and elimination parameter values resulting
from re-calibration of the human model using alternative values for
physiological flow rates3 and tissuerair partition coefficients
Source of Partition Coefficients
Maximum rate for 1,4-dioxane metabolism (VmaxC)b
Metabolic affinity constant (Km)°
HEAA urinary elimination rate constant (kme)d
Leung and Paustenbach (1990,
062932)
16.9
2.5
0.18
Sweeney et al. (2008,
195085)
20.36
0.0001
0.17
,0.74
"Cardiac output = 17.0 L/hour/kg BW074, alveolar ventilation = 17.7 L/hour/kg BW
bmg/hour/kg BW°75
cmg/L
dhouf1
Plots of predicted and experimentally observed blood 1,4-dioxane and urinary HEAA
levels are shown in Figures 4-1 and 4-2. Neither re-calibration resulted in an adequate fit to the
blood 1,4-dioxane data from the empirical model output or the experimental observations. Re-
calibration using either the Leung and Paustenbach (1990, 062932) or Sweeney et al. (2008,
195085) partition coefficients resulted in blood 1,4-dioxane predictions that were at least 10-fold
lower than empirical model predictions or observations.
Observations and predictions of 1,4-dioxane in human blood from
a 6-hour, 50 ppm exposure: VmaxC and Km fit while using PC
values from Leung and Paustenbach (1990)
100.0
10.0-
0.1
6 8 10
Time (hrs)
Observations and predictions of HEAA in human urine from a
6-hour, 50 ppm exposure: kme fit while using PC values from
Leung and Paustenbach (1990)
10 15
Time (hrs)
Source: Used with permission from Elsevier, Ltd., Leung and Paustenbach (1990,
062932).
Figure B-8. Predicted and observed blood 1,4-dioxane concentrations (left)
and urinary HEAA levels (right) following re-calibration of the human PBPK
model with tissue:air partition coefficient values.
The refitted values for kme resulted in HEAA levels in urine that were very similar to the
empirical model output (compare Figures B-7, B-8, and B-9), which was not surprising, given
the fitting of a single parameter to the data.
B-13
-------
Obser\
a 6-
100.0 -
c
o
"ro
X 10.0 -
c
o
0 —
c ^>
5S.
O
9 1.0 -
•^-
•o
o
o
m
0.1 -
Cations and predictions of 1 ,4-dioxane in human blood from
hour, 50 ppm exposure: VmaxC and Km fit while using PC
values from Sweeney et al. (2008)
a observed
J J $ jf • • • • empirical predricted
.r! H
!•
•T , \
r \ H,
\ 1 Jv
) 5 10 15
Time (hrs)
Observations and predictions of HEAA in human urine from a
6-hour, 50 ppm exposure: kme fit while using PC values from
Sweeney et al. (2008)
700.0
Source: Used with permission of Oxford Journals, Sweeney et al. (2008, 195085).
Figure B-9. Predicted and observed blood 1,4-dioxane concentrations (left)
and urinary HEAA levels (right) following re-calibration of the human PBPK
model with tissue:air partition coefficient values.
Outputs of the blood 1,4-dioxane and urinary HEAA levels using the suggested (Table B-
1) parameters are shown in Figure B-10. These outputs rely on a very low value for the slowly
perfused tissue:air partition coefficient (166) that is six- to eightfold lower than the measured
values reported in Leung and Paustenbach (1990, 062932) and Sweeney et al. (2008, 195085).
and 10-fold lower than the value used by Reitz et al. (1990, 094806). While the predicted
maximum blood 1,4-dioxane levels are much closer to the observations, the elimination kinetics
are markedly different, producing higher predicted elimination rates compared to observations
during the post-exposure phase of the experiment.
B-14
-------
Observations and predictions of 1,4-dioxane in
human blood from a 6-hour, 50 ppm exposure:
EPA parameter estimates used
100.0 -
Observations and predictions of HEAA in human
urine from a 6-hour, 50 ppm exposure:
EPA parameter estimates used
700.0 -
Figure B-10. Predicted and observed blood 1,4-dioxane concentrations (left)
and urinary HEAA levels (right) using EPA estimated biologically plausible
parameters (Table B-l).
B.4.5. Conclusions for PBPK Model Implementation
Re-calibration of the human PBPK model was performed using experiment-specific
values for cardiac output and alveolar ventilation (values derived from Young et al., 1977,
062956) and measured mean tissue:air 1,4-dioxane partition coefficients reported by Leung and
Paustenbach (1990, 062932) or Sweeney et al. (2008, 195085). The resulting predictions of
1,4-dioxane in blood following a 6-hour, 50-ppm inhalation exposure were 10-fold (or more)
lower than either the observations or the empirical model predictions, while the predictions of
urinary HEAA by the PBPK and empirical models were similar to each other, but lower than
observed values (Figures B-8 and B-9). Output from the model using biologically plausible
parameter values (Table B-l), Figure B-10 shows that application of a value for the slowly
perfused tissue:air partition coefficient, which is 10-fold lower than the measured value reported
by Leung and Paustenbach (1990, 062932), results in closer agreement of the predictions to
observations during the exposure phase, but not during the elimination phase. Thus, model re-
calibration using experiment-specific flow rates and mean measured partition coefficients does
not result in an adequate fit of the PBPK model to the available data.
B.4.6. SENSITIVITY ANALYSIS
A sensitivity analysis of the Reitz et al. (1990, 094806) model was performed to
determine which PBPK model parameters exert the greatest influence on the outcome of
dosimeters of interest—in this case, the concentration of 1,4-dioxane in blood. Knowledge of
B-15
-------
model sensitivity is useful for guiding the choice of parameter values to minimize model
uncertainty.
B.4.7. Method
A univariate sensitivity analysis was performed on all of the model parameters for two
endpoints: blood 1,4-dioxane concentrations after 1 and 4 hours of exposure. These time points
were chosen to assess sensitivity during periods of rapid uptake (1 hour) and as the model
approached steady state (4 hours) for blood 1,4-dioxane. Model parameters were perturbated 1%
above and below nominal values and sensitivity coefficients were calculated as follows:
,
J \x)
Ax f(x)
where x is the model parameter, f(x) is the output variable, Ax is the perturbation of the
parameter from the nominal value, and f (x) is the sensitivity coefficient. The sensitivity
coefficients were scaled to the nominal value of x and f(x) to eliminate the potential effect of
units of expression. As a result, the sensitivity coefficient is a measure of the proportional
change in the blood 1,4-dioxane concentration produced by a proportional change in the
parameter value, with a maximum value of 1.
B.4.8. Results
The sensitivity coefficients for the seven most influential model parameters at 1 and
4 hours of exposure are shown in Figure B-l 1. The three parameters with the highest sensitivity
coefficients in descending order are alveolar ventilation (QPC) (1.0), the blood:air partition
coefficient (PB) (0.65), and the slowly perfused tissue:air partition coefficient (PSA) (0.51). Not
surprisingly, these were the parameters that were doubled or given surrogate values in the Reitz
et al. (1990, 094806) model in order to achieve an adequate fit to the data. Because of the large
influence of these parameters on the model, it is important to assign values to these parameters in
which high confidence is placed, in order to reduce model uncertainty.
B-16
-------
Sensitivity Coefficients: CV - 1 hr
0.01 0.10 1.00
QPC
PB
PSA
CD
"S
Ł QSC
2
OJ
°- QCC
vmaxc
K™
I
I
I
I
I
I
]
Sensitivity Coefficients: CV - 4 hr
0.01 0.10 1.00
QPC
PB
5 PSA
~(D
E w
OJ vmaxC
OJ
*• K™
PRA
QSC
|
I
|
I
I
I
I
Figure B-ll. The highest seven sensitivity coefficients (and associated
parameters) for blood 1,4-dioxane concentrations (CV) at 1 (left) and 4
(right) hours of a 50-ppm inhalation exposure.
B.5. PBPK MODEL EXERCISES USING BIOLOGICALLY PLAUSIBLE PARAMETER
BOUNDARIES
The PBPK model includes numerous physiological parameters whose values are typically
taken from experimental observations. In particular, values for the flow rates (cardiac output and
alveolar ventilation) and tissue:air partition coefficients (i.e., mean and standard deviations) are
available from multiple sources as means and variances. The PBPK model was exercised by
varying the partition coefficients over the range of biological plausibility (parameter mean ±
2 standard deviations), re-calibrating the metabolism and elimination parameters, and exploring
the resulting range of blood 1,4-dioxane concentration time course predictions. Cardiac output
and alveolar ventilation were not varied because the experiment-specific values used did not
include any measure of inter-individual variation.
B.5.1. Observations Regarding the Volume of Distribution
Young et al. (1978, 062955; 1978, 625640) used experimental observations to estimate a
Vd for 1,4-dioxane in rats of 301 mL, or 1,204 mL/kg BW. For humans, the Vd was estimated to
be 104 mL/kg BW (Young et al., 1977, 062956). It is possible that a very large volume of the
slowly perfused tissues in the body of rats and humans may be a significant contributor to the
estimated 10-fold difference in distribution volumes for the two species. This raises doubt
regarding the appropriateness of using the measured rat slowly perfused tissue:air partition
coefficient as a surrogate values for humans in the PBPK model.
B-17
-------
B.5.2. Defining Boundaries for Parameter Values
Given the possible 10-fold species differences in the apparent Vd for 1,4-dioxane in rats
and humans, boundary values for the partition coefficients were chosen to exercise the PBPK
model across its performance range to either minimize or maximize the simulated Vd. This was
accomplished by defining biologically plausible values for the partition coefficients as the
mean ± 2 standard deviations of the measured values. Thus, to minimize the simulated Vd for
1,4-dioxane, the selected blood:air partition coefficient was chosen to be the mean + 2 standard
deviations, while all of the other tissue:air partition coefficients were chosen to be the mean - 2
standard deviations. This created conditions that would sequester 1,4-dioxane in the blood, away
from other tissues. To maximize the simulated 1,4-dioxane Vd, the opposite selections were
made: blood and other tissue:air partition coefficients were chosen as the mean - 2 standard
deviations and mean + 2 standard deviations, respectively. Subsequently, Vmaxc, Km, and kme
were optimized to the empirical model output data as described in Section B.4.3. This procedure
was performed for both the Leung and Paustenbach (1990, 062932) and Sweeney et al. (2008,
195085) partition coefficients (Table B-l). The two predicted time courses resulting from the re-
calibrated model with partition coefficients chosen to minimize or maximize the 1,4-dioxane Vd
represent the range of model performance as bounded by biologically plausible parameter values.
B.5.3. Results
The predicted time courses for a 6-hour, 50-ppm inhalation exposure for the re-calibrated
human PBPK model with mean (central tendency) and ± 2 standard deviations from the mean
values for partition coefficients are shown in Figure B-12 for the Leung and Paustenbach (1990,
062932) values and Figure B-13 for the Sweeney et al. (2008, 195085) values. The resulting
fitted values for Vmaxc, Km, and kme, are given in Table B-3. By bounding the tissue:air partition
coefficients with upper and lower limits on biologically plausible values from Leung and
Paustenbach (1990, 062932) or Sweeney et al. (2008, 195085). the model predictions are still at
least six- to sevenfold lower than either the empirical model output or the experimental
observations. The range of possible urinary HEAA predictions brackets the prediction of the
empirical model, but this agreement is not surprising, as the cumulative rate of excretion depends
only on the rate of metabolism of 1,4-dioxane, and not on the apparent Vd for 1,4-dioxane.
These data show that the PBPK model cannot adequately reproduce the predictions of blood
1,4-dioxane concentrations of the Young et al. (1977, 062956) human empirical model or the
experimental observations when constrained by biologically plausible values for physiological
flow rates and tissue:air partition coefficients.
B-18
-------
1,4-Dioxane in human blood from a 6-hour, 50 ppm
exposure
Cumulative HEAA in human urine from a 6-hour, 50 ppm
exposure
700
25
Source: Used with permission of Elsevier, Ltd., Leung and Paustenbach (1990, 062932)
Figure B-12. Comparisons of the range of PBPK model predictions from
upper and lower boundaries on partition coefficients with empirical model
predictions and experimental observations for blood 1,4-dioxane
concentrations (left) and urinary HEAA levels (right) from a 6-hour, 50-ppm
inhalation exposure.
1,4-Dioxane in human blood from a 6-hour, 50 ppm
exposure
•inn n
one Concentration
mg/L)
o- I
b c
Blood 1,4-Dio
3 -^
^ b
Young et al. (1977) empirical model
Sweeney etal. (2008) PC -UCL
Sweeney et al. (2008) PC - Central
n ,j U Sweeney etal. (2008) PC -LCL
d % n Young etal. (1977) observation
' ° data
10 S
\
'/ "" \ * t,
^ \\ '*
i \ V nv
0 5 10 15
Time (hrs)
Cumulative HEAA in human urine from a 6-hour, 50 ppm
exposure
700 -,
Cumulative Urinary
HEAA Amount (mg)
a °
a
D
jtjr — — Young et al. (1977) empirical mo
/t Sweeney etal. (2008) PC -UCL
/> Sweeney et al. (2008) PC - LCL
,0* n Young etal. (1977) observation
del
ral
data
1 5 10 15 20 25
Time (hrs)
Source: Used with permission of Oxford Journals, Sweeney et al. (2008, 195085): Used
with permission of Taylor & Francis, Young et al. (1977, 062956).
Figure B-13. Comparisons of the range of PBPK model predictions from
upper and lower boundaries on partition coefficients with empirical model
predictions and experimental observations for blood 1,4-dioxane
concentrations (left) and urinary HEAA levels (right) from a 6-hour, 50-ppm
inhalation exposure.
B-19
-------
Table B-3. PBPK metabolic and elimination parameter values resulting
from recalibration of the human model using biologically plausible values for
physiological flow rates3 and selected upper and lower boundary values for
tissuerair partition coefficients
Source of partition coefficients
Maximum rate for 1,4-dioxane
metabolism (Vmaxc)b
Metabolic dissociation constant
(Km)c
HEAA urinary elimination rate
constant (kme)d
Leung and Pausenbach (1990, 062932)
For maximal Vd
14.95
5.97
0.18
For minimal Vd
18.24
0.0001
0.17
Sweeney et al. (2008, 195085)
For maximal Vd
17.37
4.88
0.26
For minimal Vd
21.75
0.0001
0.19
"Cardiac output = 17.0 L/hour/kg BW074' alveolar ventilation = 17.7 L/hour/kg BW074
bmg/hour/kg BW°75
cmg/L
dhour-1
B.5.4. Alternative Model Parameterization
Since the PBPK model does not predict the experimental observations of Young et al.
(1977, 062956) when parameterized by biologically plausible values, an exercise was performed
to explore alternative parameters and values capable of producing an adequate fit of the data.
Since the metabolism of 1,4-dioxane appears to be linear in humans for a 50-ppm exposure
(Young et al., 1977, 062956), the parameters Vmaxc and Km were replaced by a zero-order, non-
saturable metabolism rate constant, kLc- This rate constant was fitted to the experimental blood
1,4-dioxane data using partition coefficient values of Sweeney et al. (2008, 195085) to minimize
the Vd (i.e., maximize the blood 1,4-dioxane levels). The resulting model predictions are shown
in Figure B-14. As before, the maximum blood 1,4-dioxane levels were approximately
sevenfold lower than the observed values.
B-20
-------
1 ,4-Dioxane in human blood from a 6-hour, 50 ppm
exposure: kLC (3.0) fitted to all observations
Blood 1,4-Dioxane Concentration
(mg/L) _^
ONJ-CiOCOONJ-CiC
D D
D
-i * *
? '
* »
t *
i ^
i *
ID '
f
r^"^ V^
Young et al. (1977) empirical
model
\c - fitted model
D Young etal. (1977)
observation data
%
D
0 24 6 8 10 12 14
Time (hrs)
Figure B-14. Predictions of blood 1,4-dioxane concentration following
calibration of a zero-order metabolism rate constant, RLC? to the
experimental data.
A re-calibration was performed using only the data from the exposure phase of the
experiment, such that the elimination data did not influence the initial metabolism and tissue
distribution. The model predictions from this exercise are shown in Figure B-15. These
predictions are more similar to the observations made during the exposure phase of the
experiment; however, this is achieved at greatly reduced elimination rate (compare Figures B-10
and B-15).
B-21
-------
16.1
14.1
12.1
10.1
J~
"5> 8.1
_Ł_
6.1
4.1
2.1
0.1
1,4-Dioxane in human blood from a 6-hour, 50 ppm
exposure: kLC(0.1) fitted 1 to 6-hour observations
. Young et al. (1977) empirical
model
- fitted model
10
12
14
Time (hrs)
Figure B-15. Predictions of blood 1,4-dioxane concentration following
calibration of a zero-order metabolism rate constant, RLC? to only the
exposure phase of the experimental data.
Finally, the model was re-calibrated by simultaneously fitting kLc and the slowly
perfused tissue:air partition coefficient to the experimental data with no bounds on possible
values (except that they be non-zero). The fitted slowly perfused tissue:air partition coefficient
was an extremely low (and biologically unlikely) value of 0.0001. The resulting model
predictions, however, were closer to the observations than even the empirical model predictions
(Figure B-16). These exercises show that better fits to the observed blood 1,4-dioxane kinetics
are achieved only when parameter values are adjusted in a way that corresponds to a substantial
decrease in apparent Vd of 1,4-dioxane in the human, relative to the rat (e.g., decreasing the
slowly perfused tissue:air partition coefficient to extremely low values, relative to observations).
Downward adjustment of the elimination parameters (e.g., decreasing kLc) increases the
predicted blood concentrations of 1,4-dioxane, achieving better agreement with observations
during the exposure phase of the experiment; however, it results in unacceptably slow
elimination kinetics, relative to observations following cessation of exposure. These
observations suggest that some other process not captured in the present PBPK model structure is
responsible for the species differences in 1,4-dioxane Vd and the inability to reproduce the
human experimental inhalation data with biologically plausible parameter values.
B-22
-------
1,4-Dioxane in human blood from a 6-hour,
50 ppm exposure
rj Young etal. (1977)
observation data
10
12
Time (hrs)
14
Figure B-16. Predictions of blood 1,4-dioxane concentration following
simultaneous calibration of a zero-order metabolism rate constant, kLC, and
slowly perfused tissue:air partition coefficient to the experimental data.
B.6. CONCLUSIONS
The rat and human empirical models of Young et al. (1977, 062956: 1978, 062955: 1978,
625640) were successfully implemented in acslXtreme and perform identically to the models
reported in the published papers (Figures 3-3 through 3-6), with the exception of the lower
predicted HEAA concentrations and early appearance of the peak HEAA levels in rat urine. The
early appearance of peak HEAA levels cannot presently be explained, but may result from
manipulations of kme or other parameters by Young et al. (1978, 062955: 1978, 625640) that
were not reported. The lower predictions of HEAA levels are likely due to reliance on a standard
urine volume production rate in the absence of measured (but unreported) urine volumes. While
the human urinary HEAA predictions were lower than observations, this is due to parameter
fitting of Young et al. (1977, 062956). No model output was published in Young et al. (1977,
062956) for comparison. The empirical models were modified to allow for user-defined
inhalation exposure levels. However, no modifications were made to model oral exposures
because adequate data to parameterize such modifications do not exist for rats or humans.
Several procedures were applied to the human PBPK model to determine if an adequate
fit of the model to the empirical model output or experimental observations could be attained
using biologically plausible values for the model parameters. The re-calibrated model
predictions for blood 1,4-dioxane levels do not come within 10-fold of the experimental values
using measured tissue:air partition coefficients from Leung and Paustenbach (1990, 062932) or
Sweeney et al. (2008, 195085) (Figures B-8 and B-9). Use of a slowly perfused tissue:air
B-23
-------
partition coefficient 10-fold lower than measured values produces exposure-phase predictions
that are much closer to observations, but does not replicate the elimination kinetics
(Figure B-10). Re-calibration of the model with upper bounds on the tissue:air partition
coefficients results in predictions that are still six- to sevenfold lower than empirical model
prediction or observations (Figures B-12 and B-13). Exploration of the model space using an
assumption of first-order metabolism (valid for the 50-ppm inhalation exposure) showed that an
adequate fit to the exposure and elimination data can be achieved only when unrealistically low
values are assumed for the slowly perfused tissue:air partition coefficient (Figure B-16).
Artificially low values for the other tissue:air partition coefficients are not expected to improve
the model fit, because the sensitivity analysis to exert less influence on blood 1,4-dioxane than
Vmaxc and Km. This suggests that the model structure is insufficient to capture the apparent
10-fold species difference in the blood 1,4-dioxane Vd between rats and humans. In the absence
of actual measurements for the human slowly perfused tissue:air partition coefficient, high
uncertainty exists for this model parameter value. Differences in the ability of rat and human
blood to bind 1,4-dioxane may contribute to the difference in Vd. However, this is expected to
be evident in very different values for rat and human blood:air partition coefficients, which is not
the case (Table B-l). Therefore, some other, as yet unknown, modification to model structure
may be necessary.
B.7. RECOMMENDATIONS FOR UTILIZING EXISTING PBPK MODELS
The use of empirical or PBPK models to reduce uncertainty in extrapolation of dose-
responses (in terms of internal dosimetry) requires accurate representation of exposure and
biological reality. In the case of the empirical models of Young et al. (1977, 062956; 1978,
062955; 1978, 625640), the acslXtreme implementations are adequate for predicting blood
1,4-dioxane levels for a variety of inhalation exposure levels in rats and up to 50 ppm in humans.
However, the absence of data with which to evaluate simulated oral absorption in either species
precludes the inclusion of this route of exposure in the models. Therefore, the empirical models
may be useful for assessment of toxicity by inhalation exposure, but not by oral exposure, and
not for route-to-route extrapolation. For the PBPK model, an apparent gap in the model structure
exists such that experimental observations of blood 1,4-dioxane levels in humans during and
following inhalation exposures to 1,4-dioxane cannot be reproduced under the constraints of
biologically plausible parameter values for all parameters. Therefore, the use of the PBPK
model (in its present form) is not recommended for application to the derivation of toxicity
values for 1,4-dioxane.
B-24
-------
B.8. acslXtreme CODE FOR THE YOUNG ET AL. EMPIRCAL MODEL FOR
1,4-DIOXANE IN RATS
PROGRAM: Young (1978, 062955) rat.csl
I
! Created by Michael Lumpkin, Syracuse Research Corporation, 08/06
! This program implements the 1-compartment empirical model for 1,4-dioxane
! in rats, developed by Young et al. 1978a, b. Program was modified to run
! in ACSL Xtreme and to include user-defined i.v. and inhalation concentrations
!(MLumpkin, 08/06)
I
INITIAL
!*****Timing and Integration Commands*****
ALGORITHM IALG=2 ! Gear integration algorithm for stiff systems
IMERROR %%%%=0.01 IRelative error for lead in plasma
NSTEPS NSTP=1000 INumber of integration steps per communication interval
CINTERVAL CINT=0.1 ! Communication interval
CONSTANT TSTART=0. ! Start of simulation (hr)
CONSTANT TSTOP=70. !End of simulation (hr)
! * * * * *MODEL PARAMETERS *****
CONSTANT BW=0.215 !Body weight (kg)
CONSTANT MINVOL=0.238 Irespiratory minute volume (L/min) estimated from Young et al.
(1978)
CONSTANT IVDOSE = 0. !IV dose (mg/kg)!
CONSTANT CONC = 0. !inhalation concentration (ppm)
CONSTANT MOLWT=88.105 !mol weight of 1,4-dioxane
CONSTANT TCHNG=6.0 lExposure pulse 1 width (hr)
CONSTANT TDUR=24.0 lExposure duration (hr)
CONSTANT TCHNG2=120.0 lExposure pulse 2 width (hr)
CONSTANT TDUR2=168.0 lExposure duration 2 (hr)
CONSTANT Vmax=4.008 !(mcg/mL/hr)
CONSTANT Km=6.308 !(mcg/mL)
CONSTANT Kinh=0.43 I pulmonary absorption constant (/hr)
CONSTANT Ke=0.0149 !(/hr)
CONSTANT Kme=0.2593 !(/hr)
CONSTANT Vd=0.3014 !(L)
IV = IVDOSE*BW
AmDIOXi=IV
END ! Of Initial Section
DYNAMIC
B-25
-------
DERIVATIVE
!*** Dioxane inhalation concentration ***
CIZONE=PULSE(0.0, TDUR, TCHNG) * PULSE(0.0, TDUR2, TCHNG2)
!First pulse is hours/day, second pulse is hours/week
CI=CONC*CIZONE*MOLWT/24450. ! Convert to mg/L
!*** Dioxane metabolism/1 st order elimination ***
dAmDIOX=(Kinh*CI*(MINVOL*60))-((Vmax*(AmDIOX))/(Km+(AmDIOX)))-
(Ke*(AmDIOX))
AmDIOX=INTEG(dAmDIOX,AmDIOXi)
ConcDIOX=AmDIOX/Vd ! plasma dioxane concentration (mcg/mL)
AUCDIOX=INTEG(ConcDIOX,0) Iplasma dioxane AUC
!*** HE A A production and 1st order metabolism ***
dAmHEAA=((Vmax*(AmDIOX))/(Km+(AmDIOX)))-(Kme*(AmHEAA))
AmHEAA=INTEG(dAmHEAA,0.)
ConcHEAA=AmHEAA/Vd Iplasma HEAA concentration
! *** 1st order dioxane elimination to urine ***
dAmDIOXu=(Ke*(AmDIOX))*0.35
AmDIOXu=INTEG(dAmDIOXu,0.)
ConcDIOXu=Ke*AmDIOX*0.35/1.45e-3 lurine production approx 1.45e-3 L/hr in SD rats
!*** 1st order dioxane exhaled ***
dAmDIOXex=(Ke*(AmDIOX))*0.65
AmDIOXex=INTEG(dAmDIOXex,0.)
! *** 1st order HEAA elimination to urine ***
dAmHEAAu=(Kme*(AmHEAA))
AmHEAAu=INTEG(dAmHEAAu,0.)
ConcHEAAu=Kme*AmHEAA/1.45e-3 lurine production approx 1.45e-3 L/hr in SD rats
END ! of Derivative Section
DISCRETE
END ! of Discrete Section
TERMT (T .GT. TSTOP)
END ! of Dynamic Section
TERMINAL
END ! of Terminal Section
END ! of Program
B-26
-------
B.9. acslXtreme CODE FOR THE YOUNG ET AL. EMPIRICAL MODEL FOR
1,4-DIOXANE IN HUMANS
PROGRAM: Young (1977, 062956) human.csl
I
! Created by Michael Lumpkin, Syracuse Research Corporation, 01/06
! This program implements the 1-compartment model for 1,4-dioxane in humans,
! developed by Young et al., 1977. Program was modified to run
! in acslXtreme (MLumpkin, 08/06)
I
INITIAL
!*****Timing and Integration Commands*****
ALGORITHM IALG=2 ! Gear integration algorithm for stiff systems
IMERROR %%%%=0.01 IRelative error for lead in plasma
NSTEPS NSTP=1000 INumber of integration steps per communication interval
CINTERVAL CINT=0.1 ! Communication interval
CONSTANT TSTART=0. ! Start of simulation (hr)
CONSTANT TSTOP=120. !End of simulation (hr)
!**** *MODEL PARAMETERS *****
! CONST ANT DATA=1 ! Optimization dataset
CONSTANT MOLWT=88.105 !mol weight for 1,4-dioxane
CONSTANT DOSE=0. IDose (mg/kg
CONSTANT CONC=0. !Inhalation concentration (ppm)
CONSTANT BW=84.1 IBody weight (kg)
CONSTANT MINVOL=7.0 Ipulmonary minute volume (L/min)
CONSTANT F=l .0 IFraction of dose absorbed
CONSTANT kinh=l .06 IRate constant for inhalation (mg/hr); optimized by MHL
CONSTANT ke=0.0033 IRate constant for dioxane elim to urine (hr-1)
CONSTANT km=0.7096 IRate constant for metab of dioxane to HEAA (hr-1)
CONSTANT kme=0.2593 IRate constant for transfer from rapid to blood (hr-1)
CONSTANT VdDkg=0.104 I Volume of distribution for dioxane (L/kg BW)
CONSTANT VdMkg=0.480 I Volume of distribution for HEAA (L/kg BW)
CONSTANT OStart=0. I Time of first oral dose (hr)
CONSTANT OPeriod=120. I Oral Dose pulse period (hr)
CONSTANT OWidth= 1. I Width (gavage/drink time) of oral dose (hr)
CONSTANT IStart=0. I Time of inhalation onset (hr)
CONSTANT IPeriod=120. llnhalation pulse period (hr)
CONSTANT IWidth=6. I Width (duration) of inhalation exposure (hr)
END I Of Initial Section
DYNAMIC
DERIVATIVE
B-27
-------
I****VARIABLES and DEFINED VALUES*****
VdD=BW*VdDkg ! Volume of distribution for dioxane
VdM=BW* VdMkg ! Volume of distribution for HEAA
InhalePulse=PULSE(IStart,IPeriod,IWidth)
Inhale=CONC*InhalePulse*MOLWT/24450. !Convert to mg/L
!*****DIFFERENTIAL EQUATIONS FOR COMPARTMENTS****
!*** Dioxane in the body (plasma) ***
dAMTbD=(Kinh*Inhale*(MINVOL*60))-(AMTbD*km)-(AMTbD*ke)
AMTbD=INTEG(dAMTbD,0.)
CbD=AMTbD/VdD
AUCbD=INTEG(CbD,0)
! * * * HEAA in the body (plasma)* * *
dAMTbM=AMTbD*km-AMTbM*kme
AMTbM=INTEG(dAMTbM,0.)
CbM=AMTbM/VdM
! *** Cumulative Dioxane in the urine ***
dAMTuD=(AMTbD*ke)
AMTuD=INTEG(dAMTuD,0.)
! *** Cumulative HEAA in the urine ***
dAMTuM=(AMTbM*kme)
AMTuM=INTEG(dAMTuM,0.)
END ! Of Derivative Section
DISCRETE
END ! of Discrete Section
TERMT (T .GT. TSTOP)
END ! Of Dynamic Section
TERMINAL
END ! of Terminal Section
END ! of Program
B-28
-------
B.10. acslXtreme CODE FOR THE REITZ ET AL. PBPK MODEL FOR 1,4- DIOXANE
(Reitz et al., 1990, 094806)
PROGRAM: DIOXANE.CSL (Used in Risk Estimation Procedures)
! Added a venous blood compartment and 1st order elim of metab.'
IMass Balance Checked OK for Inhal, IV, Oral, and Water RHR
IDefmed Dose Surrogates for Risk Assessment 01/04/89'
[Modified the Inhal Route to use PULSE for exposure conditions'
!Modifications by GLDiamond, Aug2004, marked as !**
I
IMetabolism of dioxane modified by MLumpkin, Oct2006, to include 1st order
lor saturable kinetics. For 1st order, set VmaxC=0; for M-Menten, set K1C=0.
I
INITIAL
INTEGER I
1=1
! ARRAY TDATA(20) ! CONSTANT TDATA=999, 19*1.OE-6 !**
CONSTANT BW = 0.40 !'Body weight (kg)'
CONSTANT QPC =15. ! 'Alveolar ventilation rate (1/hr)'
CONSTANT QCC=15. !'Cardiac output (1/hr)'
IFlows to Tissue Compartments'
CONSTANT QLC = 0.25 !'Fractional blood flow to liver'
CONSTANT QFC = 0.05 !'Fractional blood flow to fat'
CONSTANT QSC = 0.18 ! 'Fractional blood flow to slow'
QRC = 1.0 - (QFC + QSC + QLC)
CONSTANT SPDC = 1.0 ! diffusion constant for slowly perfused tissues
! Volumes of Tissue/Blood Compartments'
CONSTANT VLC = 0.04 !'Fraction liver tissue'
CONSTANT VFC = 0.07 !'Fraction fat tissue'
CONSTANT VRC = 0.05 !'Fraction Rapidly Perf tissue'
CONSTANT VBC = 0.05 !'Fraction as Blood'
VSC = 0.91 - (VLC + VFC + VRC + VBC)
!Partition Coefficients'
CONSTANT PLA=1557. !Liver/air partition coefficient'
CONSTANT PFA= 851. !'Fat/air partition coefficient'
CONSTANT PSA = 2065. !'Muscle/air (Slow Perf) partition'
CONSTANT PRA=1557. !'Richly perfused tissue/air partition'
CONSTANT PB = 1850. !'Blood/air partition coefficient'
! Other Compound Specific Parameters'
CONSTANT MW = 88.1 I'Molecular weight (g/mol)'
CONSTANT KLC = 12.0 ! temp zero-order metab constant
CONSTANT VMAXC = 13.8 I'Maximum Velocity of Metabol.'
CONSTANT KM = 29.4 !'Michaelis Menten Constant'
CONSTANT ORAL = 0.0 !'Oral Bolus Dose (mg/kg)'
B-29
-------
CONSTANT KA = 5.0 ! 'Oral uptake rate (/hi)'
CONSTANT WATER = 0.0 ! 'Cone in Water (mg/liter, ppm)'
CONSTANT WDOSE=0.0 !Water dose (mg/kg/day) **
CONSTANT IV = 0.0 !'IV dose (mg/kg)'
CONSTANT CONC = 0.0 !'Inhaled concentration (ppm)'
CONSTANT KME = 0.276 ! 'Urinary Elim constant for met (hr-1)'
! Timing commands'
CONSTANT TSTOP = 50 ! 'Length of experiment (hrs)'
CONSTANT TCHNG= 6 !'Length of inhalation exposure (hrs)'
CINTERVAL CINT=0.1
CONSTANT WIDD=24. ! **
CONSTANT PERD=24. ! **
CONSTANT PERW= 168. !**
CONST ANT WIDW= 168. !**
CONSTANT DAT=0.017 !**
! Scaled parameters calculated in this section of Program'
QC=QCC*BW**0.74
QP=QPC*BW**0.74
QL=QLC*QC
QF=QFC*QC
QS=QSC*QC
QR=QRC*QC
VL=VLC*BW
VF=VFC*BW
VS=VSC*BW
VR=VRC*BW
VB=VBC*BW
PL=PLA/PB
PR=PRA/PB
PS=PSA/PB
PF=PFA/PB
KL = KLC*bw**0.7 ! Zero-order metab constant
VMAX = VMAXC*BW**0.7
DOSE = ORAL*BW !'Initial Amount in Stomach'
ABO = IV*BW ! 'Initial Amount in Blood'
!DRINK = 0.102*BW**0.7*WATER/24 ITnput from water (mg/hr)' !**
IDRINKA = 0.102*BW**0.7*WATER/DAT ITnput from water (mg/hr)' ! **
DRINKA=WDOSE*BW/DAT
CV = ABO/VB ! 'Initialize CV
END !'End of INITIAL'
DYNAMIC
ALGORITHM IALG = 2 ! 'Gear method for stiff systems'
TERMT(T .GE. TSTOP )
CR = AR/VR
B-30
-------
CS = AS/VS
CF = AF/VF
BODY = AL + AR + AS + AF + AB + TUMMY
BURDEN = AM + BODY
TMASS = BURDEN + AX + AMEX
!Calculate the Interval Excretion Data here:'
! DAX = AMEX-AMEX2
! IF(DOSE .LE. 0.0 .AND. IV .LE. 0.0 ) GO TO SKIP1
! PCTAX = 100*(AX - AX2)/(DOSE + IV*BW)
! PCTMX = 100*(AMEX - AMEX2)/(DOSE + IV*BW)
! SKIP 1.. CONTINUE
! IF(T .LT. TDATA(I) .OR. I .GE. 20 ) GO TO SKIP
! AX2=AX
! AMEX2=AMEX
1=1+1
SKIP.. CONTINUE
IDISCRETE EXPOSE
! CIZONE = 1.0 ! CALL LOGD(.TRUE.) Turns on inhalation exposure?
!END
IDISCRETE CLEAR
! CIZONE = 0.0 ! CALL LOGD(.TRUE.)
!END
DERIVATIVE
!Use Zero-Crossing Form of DISCRETE Function Here'
! SCHEDULE command must be in DERIVATIVE section'
! DAILY = PULSE (0.0, PERI, TCHNG )
! WEEKLY = PULSE (0.0, PER2, LEN2 )
! SWITCHY = DAILY * WEEKLY
! SCHEDULE EXPOSE .XP. SWITCHY - 0.995
! SCHEDULE CLEAR .XN. SWITCHY - 0.005
DAILY=PULSE(0.0,PERD,WIDD)
WEEKLY=PULSE(0.0,PERW,WIDW)
SWITCHY = DAILY * WEEKLY
CI = CONC*MW724451.0* SWITCHY!**
!CA = Concentration in arterial blood (mg/1)'
CA = (QC*CV+QP*CI)/(QC+(QP/PB))
CX = CA/PB
DRINK=DRINKA* SWITCHY ! * *
! TUMMY = Amount in stomach'
B-31
-------
RTUMMY = -KA*TUMMY
TUMMY = INTEG(RTUMMY,DOSE)
!RAX = Rate of Elimination in Exhaled air'
RAX = QP*CX
AX = INTEG(RAX, 0.0)
! AS = Amount in slowly perfused tissues (mg)'
RAS = SPDC*(CA-CVS) !now governed by diffusion-limited constant, SPDC, instead of QS
AS = INTEG(RAS,0.)
CVS = AS/(VS*PS)
! AR = Amount in rapidly perfused tissues (mg)'
RAR = QR*(CA-CVR)
AR = INTEG(RAR,0.)
CVR = AR/(VR*PR)
! AF = Amount in fat tissue (mg)'
RAF = QF*(CA-CVF)
AF = INTEG(RAF,0.)
CVF = AF/(VF*PF)
! AL = Amount in liver tissue (mg)'
RAL = QL*(CA-CVL) - KL*CVL - VMAX*CVL/(KM+CVL) + KA*TUMMY + DRINK
AL = INTEG(RAL,0.)
CVL = AL/(VL*PL)
IMetabolism comments updated by EDM on 2/1/10
! AM = Amount metabolized (mg)'
RMEX = (KL*CVL)+(VMAX*CVL/(KM+CVL)) IRate of 1,4-dioxane metabolism
RAM = (KL*CVL)+(VMAX*CVL)/(KM+CVL) - KME*AM IRate of change of metabolite
in body
AM = INTEG(RAM, 0.0) I'Amt Metabolite in body
CAM = AM/BW ! 'Cone Metabolite in body'
AMEX = INTEG(KME* AM, 0.0) !'Amt Metabolite Excreted via urine'
! AB = Amount in Venous Blood'
RAB = QF*CVF + QL*CVL + QS*CVS + QR*CVR - QC*CV
AB = INTEG(RAB, ABO)
CV = AB/VB
AUCV = INTEG(CV, 0.0)
[Possible Dose Surrogates for Risk Assessment Defined Here'
CEX = 0.667*CX + 0.333*CI I'Conc in Exhal Air'
AVECON = PLA * (CEX+CI)/2 ! 'Ave Cone in Nose Tissue'
AUCCON = INTEG(AVECON, 0.0) ! 'Area under Curve (Nose)'
AUCMET = INTEG(CAM, 0.0) !'Area under Curve (Metab)'
B-32
-------
CL = AL/VL ! 'Cone Liver Tissue'
AUCL = INTEG(CL, 0.0) !'Area under Curve (Liver)'
AAUCL=AUCL/TIME
! Dose Surrogates are Average Area under Time/Cone Curve per 24 hrs'
IF (T .GT. 0) TIME=T
dayS = TIME/24.0
NOSE = AUCCON/DAYS !'Nasal Turbinates'
LIVER = AUCL/DAYS ! 'Liver Tissues'
METAB = AUCMET/DAYS !'Stable Metabolite'
END !'End of dynamic'
END ! End of TERMINAL
END !'End of PROGRAM
B-33
-------
APPENDIX C. DETAILS OF BMD ANALYSIS FOR ORAL RfD FOR 1,4-DIOXANE
C.I. CORTICAL TUBULE DEGENERATION
All available dichotomous models in the Benchmark Dose Software (version 2.1.1) were
fit to the incidence data shown in Table C-l, for cortical tubule degeneration in male and female
Osborne-Mendel rats exposed to 1,4-dioxane in the drinking water (NCI, 1978, 062935). Doses
associated with a BMR of a 10% extra risk were calculated.
Table C-l. Incidence of cortical tubule degeneration in Osborne-Mendel rats
exposed to 1,4-dioxane in drinking water for 2 years
Males (mg/kg-day)
0
0/3 la
240
20/3 lb
(65%)
530
27/3 3b
(82%)
Females (mg/kg-day)
0
0/3 la
350
0/34
640
10/32b
(31%)
"Statistically significant trend for increased incidence by Cochran-Armitage test (p < 0.05) performed for this
review.
blncidence significantly elevated compared to control by Fisher's exact test (p < 0.05) performed for this review.
Source: NCI (1978. 062935).
As assessed by the %2 goodness-of-fit test, several models in the software provided
adequate fits to the data for the incidence of cortical tubule degeneration in male and female rats
(tf'p > 0.1) (Table C-2). Comparing across models, a better fit is indicated by a lower AIC
value (U.S. EPA, 2000, 052150). As assessed by Akaike's Information Criterion (AIC), the log-
probit model provided the best fit to the cortical tubule degeneration incidence data for male rats
(Table C-2, Figure C-l) and could be used to derive a POD of 38.5 mg/kd-ay for this endpoint.
The Weibull model provided the best fit to the data for female rats (Table C-2, Figure C-5) and
could be used to derive a POD of 452.4 mg/kg-day for this endpoint. For those models that
exhibit adequate fit, models with the lower AIC values are preferred. Differences in AIC values
of less than 1 are generally not considered important. BMDS modeling results for all
dichotomous models are shown in Table C-2.
Note: Hyperlinks to the reference citations throughout this document will take you to the NCEA HERO database
(Health and Environmental Research Online) at http://epa. gov/hero. HERO is a database of scientific literature used
by U.S. EPA in the process of developing science assessments such as the Integrated Science Assessments (ISA)
and the Integrated Risk Information System (IRIS)
C-l
-------
Table C-2. Goodness-of-fit statistics and BMDio and BMDLio values from
models fit to incidence data for cortical tubule degeneration in male and
female Osborne-Mendel rats (NCI, 1978, 062935) exposed to 1,4-dioxane in
drinking water
Model
AIC
p-valuea
Scaled
Residual of
Interest
BMD10
(mg/kg-day)
BMDL10
(mg/kg-day)
Male
Gammab
Logistic
Log-logistic0
Log-probif
Multistage
(2 degree)d
Probit
Weibullb
Quantal-Linear
74.458
89.0147
75.6174
74.168
74.458
88.782
74.458
74.458
0.6514
0.0011
1
0.7532
0.6514
0.0011
0.6514
0.6514
0
-1.902
0
0
0
-1.784
0
0
28.80
88.48
20.85
51.41
28.80
87.10
28.80
28.80
22.27
65.84
8.59
38.53
22.27
66.32
22.27
22.27
Female
Gammab
Logistic
Log-logistic0
Log-probif
Multistage
(2 degree)d
Probit
Weibullb
Quantal-Linear
41.9712
43.7495
41.7501
43.7495
48.1969
43.7495
41.75
52.3035
0.945
0.9996
0.9999
0.9997
0.1443
0.9997
0.9999
0.03
0.064
0
0
0
-1.693
0
0
-2.086
524.73
617.44
591.82
584.22
399.29
596.02
596.45
306.21
437.08
471.92
447.21
436.19
297.86
456.42
452.36
189.49
ap-Value from the %2 goodness-of-fit test for the selected model. Values < 0.1 indicate that the model
exhibited a statistically significant lack of fit, and thus a different model should be chosen.
bPower restricted to > 1.
°Slope restricted to > 1.
dBetas restricted to >0.
Source: NCI (1978, 062935).
C-2
-------
LogProbit Model with 0.95 Confidence Level
T3
CD
"o
I
0.8
0.6
0.4
0.2
100
200
300
400
500
dose
14:4902/01 2010
Source: NCI (1978, 062935).
Figure C-l. BMD Log-probit model of cortical tubule degeneration
incidence data for male rats exposed to 1,4-dioxane in drinking water for 2
years to support the results in Table C-2.
Probit Model. (Version: 3.1; Date: 05/16/2008)
Input Data File: C:\14DBMDS\lnp_nci_mrat_cortdeg_Lnp-BMR10-restrict.(d)
Gnuplot Plotting File: C:\14DBMDS\lnp_nci_mrat_cortdeg_Lnp-BMR10-restrict.plt
Mon Feb 01 14:49:17 2010
BMDS Model Run
The form of the probability function is:
P[response] = Background + (1-Background) * CumNorm(Intercept+Slope*Log(Dose)
where CumNorm(.) is the cumulative normal distribution function
Dependent variable = Effect
Independent variable = Dose
Slope parameter is restricted as slope >= 1
Total number of observations = 3
Total number of records with missing values = 0
Maximum number of iterations =250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
C-3
-------
User has chosen the log transformed model
Default Initial (and Specified) Parameter Values
background = 0
intercept = -5.14038
slope = 1
Asymptotic Correlation Matrix of Parameter Estimates
(*** The model parameter(s) -background -slope have been estimated at a boundary
point, or have been specified by the user, and do not appear in the correlation
matrix)
intercept
intercept 1
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
background 0 NA
intercept -5.22131 0.172682 -5.55976 -4.88286
slope 1 NA
NA - Indicates that this parameter has hit a bound implied by some ineguality
constraint and thus has no standard error.
Analysis of Deviance Table
Model Log(likelihood) # Param's Deviance Test d.f. P-value
Full model -35.8087 3
Fitted model -36.084 1 0.550629 2 0.7593
Reduced model -65.8437 1 60.07 2 <.0001
AIC: 74.168
Dose
0.0000
240.0000
530.0000
Est. Prob.
0.0000
0.6023
0.8535
Goodness of Fit
Expected Observed Size
0.000
18.672
28.166
0.000
20.000
27.000
31
31
33
Scaled
Residual
0.000
0.487
-0.574
ChiA2 = 0.57 d.f. = 2 P-value = 0.7532
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 51.4062
BMDL = 38.5284
C-4
-------
Weibull Model with 0.95 Confidence Level
T3
CD
"o
I
0.5
0.4
0.3
0.2
0.1
Weibull
BMDL
BMD
100
200
300
dose
400
500
600
14:2012/042009
Source: NCI (1978, 062935).
Figure C-2. BMD Weibull model of cortical tubule degeneration incidence
data for female rats exposed to 1,4-dioxane in drinking water for 2 years to
support the results in Table C-2.
Weibull Model using Weibull Model (Version: 2.12; Date: 05/16/2008)
Input Data File: Z:\14Dioxane\BMDS\wei_nci_frat_cortdeg_Wei-BMR10-Restrict.(d)
Gnuplot Plotting File: Z:\14Dioxane\BMDS\wei_nci_frat_cortdeg_Wei-BMR10-Restrict.plt
Fri Dec 04 14:20:41 2009
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background) * [1-EXP (-slope*dose/xpower) ]
Dependent variable = Effect
Independent variable = Dose
Power parameter is restricted as power >=1
Total number of observations = 3
Total number of records with missing values = 0
Maximum number of iterations =250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
C-5
-------
Default Initial (and Specified) Parameter Values
Background = 0.015625
Slope = 1.55776e-010
Power = 3.33993
Asymptotic Correlation Matrix of Parameter Estimates
(*** The model parameter(s) -Background -Power have been estimated at a boundary
point, or have been specified by the user, and do not appear in the correlation
matrix)
Slope
Slope
-1.$
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
Background 0 NA
Slope 1.15454e-051 1.#QNAN 1.#QNAN 1.#QNAN
Power 18 NA
NA - Indicates that this parameter has hit a bound implied by some ineguality
constraint and thus has no standard error.
Model
Full model
Fitted model
Reduced model
Analysis of Deviance Table
Log(likelihood) # Param's Deviance Test d.f. P-value
-19.8748 3
-19.875 1 0.000487728 2 0.9998
-32.1871 1 24.6247 2 <.0001
AIC:
41.75
Dose
0.0000
350.0000
640.0000
Est. Prob.
0.0000
0.0000
0.3125
Goodness of Fit
Expected Observed Size
0.000
0.000
9.999
0.000
0.000
10.000
31
34
32
Scaled
Residual
0.000
-0.016
0.000
=0.00
d.f. = 2
P-value = 0.9999
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 596.445
BMDL = 452.359
C-6
-------
C.2. LIVER HYPERPLASIA
All available dichotomous models in the Benchmark Dose Software (version 2.1.1) were
fit to the incidence data shown in Table C-3, for liver hyperplasia in male and female
F344/DuCrj rats exposed to 1,4-dioxane in the drinking water (JBRC, 1998, 196240: Kano et al.,
2009, 594539). Benchmark doses associated with a BMR of a 10% extra risk were calculated.
Table C-3. Incidence of liver hyperplasia in F344/DuCrj rats exposed to
1,4-dioxane in drinking water3
Males (mg/kg-day)
0
3/40
11
2/45
55
9/3 5a
274
12/22C
Females (mg/kg-day)
0
2/3 8b
18
2/37
83
9/38
429
24/24c
aDose information from Kano et al. (2009, 594539) and incidence data from sacrificed animals from JBRC (1998,
196240).
blncidence significantly elevated compared to control by %2 test (p < 0.05).
Incidence significantly elevated compared to control by %2 test (p < 0.01).
Sources: Kano et al. (2009, 5945391: JBRC (1998, 196240X
For incidence of liver hyperplasia in F344 male rats, the logistic, probit, and
dichotomous-Hill models all exhibited a statistically significant lack of fit (i.e., %2 p-va\ue < 0.1;
see Table C-4), and thus should not be considered further for identification of a POD. All of the
remaining models exhibited adequate fit, but the AIC values for the gamma, multistage, quantal-
linear, and Weibull models were lower than the AIC values for the log-logistic and log-probit
models. Finally, the AIC values for gamma, multistage, quantal-linear, and Weibull models in
Table C-4 are equivalent and, in this case, essentially represent the same model. Therefore,
consistent with the external review draft Benchmark Dose Technical Guidance (U.S. EPA, 2000,
052150), any of them with equal AIC values (gamma, multistage, quantal-linear, or Weibull)
could be used to identify a POD for this endpoint of 23.8 mg/kg-day.
For liver hyperplasias in F344 female rats exposed to 1,4-dioxane, the quantal-linear and
dichotomous-Hill models did not result in a good fit (i.e., %2p-va\ue < 0.1; See Table C-4). The
multistage (3-degree) model had the lowest AIC value and was selected as the best-fitting model.
Therefore, consistent with the BMD technical guidance document (U.S. EPA, 2000, 052150), the
BMDL from the multistage (3-degree) model was selected to yield a POD for this endpoint of
27.1 mg/kg-day.
C-7
-------
Table C-4. Benchmark dose modeling results based on the incidence of liver
hyperplasias in male and female F344 rats exposed to 1,4-dioxane in drinking
water for 2 years
Model
AIC
p-valuea
Scaled
Residual of
Interest
BMD10
(mg/kg-day)
BMDL10
(mg/kg-day)
Male
Gammab
Logistic
Log-logistic0
Log-probif
Multistage"1
(2 degree)
Probit
Weibullb
Quantal-Linear
Dichotomous-Hill
114.172
117.047
115.772
115.57
114.172
116.668
114.172
114.172
117.185
0.3421
0.0706
0.1848
0.1431
0.3421
0.0859
0.3421
0.3421
NCe
0.886
1.869
0.681
1.472
0.886
1.804
0.886
0.886
-0.2398
35.90
83.56
33.39
54.91
35.90
76.69
35.90
35.90
32.01
23.81
63.29
16.96
37.05
23.81
58.57
23.81
23.81
14.84
Female
Gammab
Logistic
Log-logistic0
Log-probif
Multistage11
(2 degree)
Multistage"1
(3 degree)
Probit
Weibullb
Quantal-Linear
Dichotomous-Hill
78.8357
77.0274
78.8357
78.8357
76.9718
76.8351
77.0308
78.8349
87.3833
2972.99
0.9783
0.9174
0.9781
0.9781
0.9563
0.9999
0.9095
0.9995
0.0245
NCe
0
-0.016
0
0
-0.107
0
0.017
0
-1.116
0
70.78
54.66
77.72
74.64
56.06
65.28
52.53
66.47
21.52
NCe
40.51
41.11
51.21
50.97
31.17
27.08
38.44
36.14
15.61
NCe
a/>-Value from the %2 goodness-of-fit test for the selected model. Values < 0.1 indicate that the model
exhibited a statistically significant lack of fit, and thus a different model should be chosen.
bPower restricted to > 1.
°Slope restricted to > 1.
dBetas restricted to >0.
eNC=Not calculated.
Sources: Kano et al. (2009, 594539): JBRC (1998, 196240).
C-8
-------
Gamma Multi-Hit Model with 0.95 Confidence Level
T3
CD
"o
I
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
Gamma Multi-Hit
BMDL
BMD
50
100
150
200
250
dose
14:35 12/042009
Figure C-3. BMD gamma model of liver hyperplasia incidence data for F344
male rats exposed to 1,4-dioxane in drinking water for 2 years to support
results Table C-4.
Gamma Model. (Version: 2.13; Date: 05/16/2008)
Input Data File: Z:\14Dioxane\BMDS\gam_jbrcl998_mrat_liver_hyper_Gam-BMR10-
Restrict.(d)
Gnuplot Plotting File: Z:\14Dioxane\BMDS\gam_jbrcl998_mrat_liver_hyper_Gam-BMR10-
Restrict.pit
Fri Dec 04 14:35:02 2009
BMDS Model Run
The form of the probability function is:
P[response]= background+(1-background)*CumGamma[slope*dose,power],
where CumGamma(.) is the cummulative Gamma distribution function
Dependent variable = Effect
Independent variable = Dose
Power parameter is restricted as power >=1
Total number of observations = 4
Total number of records with missing values = 0
Maximum number of iterations =250
Relative Function Convergence has been set to: le-008
C-9
-------
Parameter Convergence has been set to: le-008
Default Initial (and Specified) Parameter Values
Background = 0.0853659
Slope = 0.00479329
Power = 1.3
Asymptotic Correlation Matrix of Parameter Estimates
(*** The model parameter(s) -Power have been estimated at a boundary point, or have
been specified by the user, and do not appear in the correlation matrix )
Background
Slope
Variable
Background
Slope
Power
Background
1
-0.36
Estimate
0.0569658
0.00293446
1
Slope
-0.36
1
Parameter Estimates
95.0% Wald Confidence Interval
Std. Err. Lower Conf. Limit Upper Conf. Limit
0.0278487 0.00238329 0.111548
0.000814441 0.00133818 0.00453073
NA
NA - Indicates that this parameter has hit a bound implied by some ineguality
constraint and thus has no standard error.
Model
Full model
Fitted model
Reduced model
Analysis of Deviance Table
Log(likelihood) # Param's Deviance Test d.f. P-value
-53.9471 4
-55.0858 2 2.27725 2 0.3203
-67.6005 1 27.3066 3 <.0001
AIC:
114.172
Goodness of Fit
Scaled
0
11
55
274
Dose
.0000
.0000
.0000
.0000
Est
0.
0.
0.
0.
. Prob.
0570
0869
1975
5780
Expected
2.
3.
6.
12.
279
911
913
715
Observed
3.
2.
9.
12.
,000
,000
,000
,000
Size
40
45
35
22
Residual
0
-1
0
-0
.492
.011
.886
.309
=2.15
d.f. = 2
P-value = 0.3421
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 35.9046
BMDL = 23.8065
C-10
-------
Multistage Model with 0.95 Confidence Level
T3
CD
"o
I
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
Multistage
BMDL
BMD
50
100
150
200
250
dose
14:3512/042009
Figure C-4. BMD multistage (2 degree) model of liver hyperplasia incidence
data for F344 male rats exposed to 1,4-dioxane in drinking water for 2 years
to support results Table C-4.
Multistage Model. (Version: 3.0; Date: 05/16/2008)
Input Data File: Z:\14Dioxane\BMDS\mst_jbrcl998_mrat_liver_hyper_Mst-BMR10-
restrict.(d)
Gnuplot Plotting File: Z:\14Dioxane\BMDS\mst_jbrcl998_mrat_liver_hyper_Mst-BMR10-
Restrict.pit
Fri Dec 04 14:35:06 2009
BMDS Model Run
The form of the probability function is:
P [response] = background + (1-background) * [1-EXP (-betal*dose/xl-beta2*dose/x2 ) ]
The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Total number of observations = 4
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
C-ll
-------
Maximum number of iterations =250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.0750872
Beta(l) = 0.00263797
Beta(2) = 0
Asymptotic Correlation Matrix of Parameter Estimates
(*** The model parameter(s) -Beta(2) have been estimated at a boundary point, or have
been specified by the user, and do not appear in the correlation matrix)
Background
Beta(l)
Background
1
-0.49
Beta(l)
-0.49
1
Variable
Background
Beta(l)
Beta(2)
Estimate
0.0569658
0.00293446
0
Parameter Estimates
95.0% Wald Confidence Interval
Std. Err. Lower Conf. Limit Upper Conf. Limit
Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-53.9471
-55.0858
-67.6005
Param's
4
2
1
Deviance Test d.f.
P-value
2.27725
27.3066
0.3203
<.0001
AIC:
114.172
0
11
55
274
Dose
.0000
.0000
.0000
.0000
Est
0.
0.
0.
0.
. Prob.
0570
0869
1975
5780
Expe
2.
3.
6.
12.
Gooc
:cted
279
911
913
715
Inesj
01
3.
2.
9.
12.
3 Of Fit
:> served
,000
,000
,000
,000
Size
40
45
35
22
S
Re
0
-1
0
-0
caled
si dual
.492
.011
.886
.309
=2.15
d.f. = 2
P-value = 0.3421
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0.95
35.9046
23.8065
82.1206
Taken together, (23.8065, 82.1206) is a 90% two-sided confidence interval for the BMD
C-12
-------
Weibull Model with 0.95 Confidence Level
T3
CD
"o
I
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
Weibull
BMDL
BMD
50
100
150
200
250
dose
14:3512/042009
Figure C-5. BMD Weibull model of liver hyperplasia incidence data for F344
male rats exposed to 1,4-dioxane in drinking water for 2 years to support the
results in Table C-4.
Weibull Model using Weibull Model (Version: 2.12; Date: 05/16/2008)
Input Data File: Z:\14Dioxane\BMDS\wei_jbrcl998_mrat_liver_hyper_Wei-BMR10-
Restrict.(d)
Gnuplot Plotting File: Z:\14Dioxane\BMDS\wei_jbrcl998_mrat_liver_hyper_Wei-BMR10-
Restrict.pit
Fri Dec 04 14:35:08 2009
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background) * [1-EXP (-slope*dose/xpower) ]
Dependent variable = Effect
Independent variable = Dose
Power parameter is restricted as power >=1
Total number of observations = 4
Total number of records with missing values = 0
Maximum number of iterations =250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
C-13
-------
Default Initial (and Specified) Parameter Values
Background = 0.0853659
Slope = 0.00253609
Power = 1
Asymptotic Correlation Matrix of Parameter Estimates
(** The model parameter(s) -Power have been estimated at a boundary point, or have
been specified by the user, and do not appear in the correlation matrix )
Background
Slope
Background
1
-0.36
Slope
-0.36
1
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
Background 0.0569661 0.0278498 0.00238155 0.111551
Slope 0.00293445 0.000814445 0.00133816 0.00453073
Power 1 NA
NA - Indicates that this parameter has hit a bound implied by some ineguality
constraint and thus has no standard error.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-53.9471
-55.0858
-67.6005
Param's
4
2
1
Deviance Test d.f.
2.27725
27.3066
P-value
0.3203
<.0001
AIC:
114.172
Goodness of Fit
Scaled
0
11
55
274
Dose
.0000
.0000
.0000
.0000
Est
0.
0.
0.
0.
. Prob.
0570
0869
1975
5780
Expected
2.
3.
6.
12.
279
911
913
715
Observed
3.
2.
9.
12.
,000
,000
,000
,000
Size
40
45
35
22
Residual
0
-1
0
-0
.492
.011
.886
.309
ChiA2 =2.15
d.f. = 2
P-value = 0.3421
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 35.9047
BMDL = 23.8065
C-14
-------
Quantal Linear Model with 0.95 Confidence Level
T3
CD
"o
I
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
Quantal Linear
BMDL
BMD
50
100
150
200
250
dose
14:3512/042009
Figure C-6. BMD quantal-linear model of liver hyperplasia incidence data
for F344 male rats exposed to 1,4-dioxane in drinking water for 2 years to
support the results in Table C-4.
Quantal Linear Model using Weibull Model (Version: 2.12; Date: 05/16/2008)
Input Data File: Z:\14Dioxane\BMDS\qln_jbrcl998_mrat_liver_hyper_Qln-BMR10.(d)
Gnuplot Plotting File: Z:\14Dioxane\BMDS\gln_jbrcl998_mrat_liver_hyper_Qln-BMR10.plt
Fri Dec 04 14:35:09 2009
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(-slope*dose)]
Dependent variable = Effect
Independent variable = Dose
Total number of observations = 4
Total number of records with missing values = 0
Maximum number of iterations =250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial (and Specified) Parameter Values
Background = 0.0853659
Slope = 0.00253609
Power = 1 Specified
C-15
-------
Asymptotic Correlation Matrix of Parameter Estimates
(*** The model parameter(s) -Power have been estimated at a boundary point, or have
been specified by the user, and do not appear in the correlation matrix)
Background
Slope
Background
1
-0.36
Slope
-0.36
1
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
Background 0.0569665 0.02785 0.00238157 0.111551
Slope 0.00293447 0.000814452 0.00133818 0.00453077
Model
Full model
Fitted model
Reduced model
AIC:
Analysis of Deviance Table
Log(likelihood)
-53.9471
-55.0858
-67.6005
114.172
Param's
4
2
1
Deviance Test d.f.
2.27725
27.3066
P-value
0.3203
<.0001
Goodness of Fit
Scaled
0
11
55
274
Dose
.0000
.0000
.0000
.0000
Est
0.
0.
0.
0.
. Prob.
0570
0869
1975
5780
Expected
2.
3.
6.
12.
279
911
913
716
Observed
3.
2.
9.
12.
,000
,000
,000
,000
Size
40
45
35
22
Residual
0
-1
0
-0
.492
.011
.886
.309
ChiA2 =2.15
d.f. = 2
P-value = 0.3421
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 35.9044
BMDL = 23.8065
C-16
-------
Multistage Model with 0.95 Confidence Level
T3
CD
"o
I
0.8
0.6
0.4
0.2 -_,_-,-
10:3005/21 2010
Source: JBRC (1998. 196240).
Figure C-7. BMD log-probit model of liver hyperplasia incidence data for
F344 female rats exposed to 1,4-dioxane in drinking water for 2 years to
support the results in Table C-4.
Multistage Model. (Version: 3.0; Date: 05/16/2008)
Input Data File: H:\14Dioxane\BMDS\mst_jbrcl998_frat_liver_hyper_Mst-BMR10-Restrict-
3deg.(d)
Gnuplot Plotting File: H:\14Dioxane\BMDS\mst_jbrcl998_frat_liver_hyper_Mst-BMR10-
Restrict-Sdeg.plt
Fri May 21 10:30:14 2010
BMDS Model Run
The form of the probability function is:
P [response] = background + (1-background) * [1-EXP (-betal*dose/xl-beta2*dose/x2-
beta3*dose/x3) ]
The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Total number of observations = 4
Total number of records with missing values = 0
Total number of parameters in model = 4
Total number of specified parameters = 0
C-17
-------
Degree of polynomial = 3
Maximum number of iterations =250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0
Beta(l) = 0
Beta(2) = 0
Beta(3) = 1.2696e+012
Asymptotic Correlation Matrix of Parameter Estimates
(*** The model parameter(s) -Beta(l), -Beta(2) have been estimated at a boundary
point, or have been specified by the user, and do not appear in the correlation
matrix)
Background Beta(3)
Background 1 -0.55
Beta(3) -0.55 1
Parameter Estimates
Variable
Background
Beta(l)
Beta(2)
Beta(3)
Estimate
0.0523101
0
0
.78712e-007
Std. Err.
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
Indicates that this value is not calculated.
Model
Full model
Fitted model
Reduced model
Analysis of Deviance Table
Log(likelihood)
-36.4175
-36.4175
-79.9164
# Param' s
4
2
1
Deviance
0.00016582
86.9979
Test d
2
3
P-value
0.9999
<.0001
AIC:
76.8351
Goodness of Fit
Scaled
0
18
83
429
Dose
.0000
.0000
.0000
.0000
Est
0.
0.
0.
1.
. Prob.
0523
0544
2368
0000
Expected
1.
2.
8.
24.
988
013
999
000
Observed
2.
2.
9.
24.
,000
,000
,000
,000
Size
38
37
38
24
Residual
0
-0
0
0
.009
.009
.000
.000
ChiA2 =0.00
d.f. = 2
P-value = 0.9999
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 65.2814
BMDL = 27.0766
BMDU = 91.3457
Taken together, (27.0766, 91.3457) is a 90% two-sided confidence interval for the BMD
C-18
-------
APPENDIX D. DETAILS OF BMD ANALYSIS FOR ORAL CSF FOR 1,4-DIOXANE
Dichotomous models available in the Benchmark Dose Software (BMDS) (version 2.1.1)
were fit to the incidence data for hepatocellular carcinoma and/or adenoma for mice and rats, as
well as nasal cavity tumors, peritoneal mesotheliomas, and mammary gland adenomas in rats
exposed to 1,4-dioxane in the drinking water. Doses associated with a benchmark response
(BMR) of a 10% extra risk were calculated. BMDio and BMDLio values from the best fitting
model, determined by adequate global- fit ($p > 0.1) and AIC values, are reported for each
endpoint (U.S. EPA, 2000, 052150). If the multistage cancer model is not the best fitting model
for a particular endpoint, the best-fitting multistage cancer model for that endpoint is also
presented as a point of comparison.
A summary of the model predictions for the Kano et al. (2009, 594539) study are shown
in Table D-l. The data and BMD modeling results are presented separately for each dataset as
follows:
• Hepatic adenomas and carcinomas in female F344 rats (Tables D-2 and D-3; Figure D-l)
• Hepatic adenomas and carcinomas in male F344 rats (Tables D-4 and D-5; Figures D-2
and D-3)
• Significant tumor incidence data at sites other than the liver (i.e., nasal cavity, mammary
gland, and peritoneal) in male and female F344 rats (Table D-6)
o Nasal cavity tumors in female F344 rats (Table D-7; Figure D-4)
o Nasal cavity tumors in male F344 rats (Table D-8; Figure D-5)
o Mammary gland adenomas in female F344 rats (Table D-9; Figures D-6 and D-7)
o Peritoneal mesotheliomas in male F344 rats (Table D-10; Figures D-8 and D-9)
• Hepatic adenomas and carcinomas in female BDF1 mice (Tables D-l 1, D-l2, and D-13;
Figures D-10, D-l 1, D-12, and D-13)
• Hepatic adenomas and carcinomas in male BDF1 mice (Tables D-l4 and D-l5; Figures
D-14andD-15)
Data and BMD modeling results from the additional chronic bioassays (Kociba et al., 1974,
062929; NCI, 1978, 062935) were evaluated for comparison with the data from Kano et al.
(2009, 594539). These results are presented as follows:
• Summary of BMDS dose-response modeling estimates associated with liver and nasal
tumor incidence data resulting from chronic oral exposure to 1,4-dioxane in rats and mice
(Table D-l 6)
Note: Hyperlinks to the reference citations throughout this document will take you to the NCEA HERO database
(Health and Environmental Research Online) at http://epa. gov/hero. HERO is a database of scientific literature used
by U.S. EPA in the process of developing science assessments such as the Integrated Science Assessments (ISA)
and the Integrated Risk Information System (IRIS)
D-l
-------
• Incidence of hepatocellular carcinoma and nasal squamous cell carcinoma in male and
female Sherman rats (combined) (Kociba et al., 1974, 062929) treated with 1,4-dioxane
in the drinking water for 2 years (Table D-17)
o BMDS dose-response modeling results for incidence of hepatocellular carcinoma in
male and female Sherman rats (combined) (Kociba et al., 1974, 062929) exposed to
1,4-dioxane in drinking water for 2 years (Table D-18; Figures D-16 and D-17)
o BMDS dose-response modeling results for incidence of nasal squamous cell
carcinoma in male and female Sherman rats (combined) (Kociba et al., 1974, 062929)
exposed to 1,4-dioxane in the drinking water for 2 years (Table D-19; Figure D-18)
• Incidence of nasal cavity squamous cell carcinoma and hepatocellular adenoma in
Osborne-Mendel rats (NCI, 1978, 062935) exposed to 1,4-dioxane in the drinking water
(Table D-20)
o BMDS dose-response modeling results for incidence of hepatocellular adenoma in
female Osborne-Mendel rats (NCI, 1978, 062935) exposed to 1,4-dioxane in the
drinking water for 2 years (Table D-21; Figures D-19 and D-20)
o BMDS dose-response modeling results for incidence of nasal cavity squamous cell
carcinoma in female Osborne-Mendel rats (NCI, 1978, 062935) exposed to
1,4-dioxane in the drinking water for 2 years (Table D-22; Figures D-21 and D-22)
o BMDS dose-response modeling results for incidence of nasal cavity squamous cell
carcinoma in male Osborne-Mendel rats (NCI, 1978, 062935) exposed to 1,4-dioxane
in the drinking water for 2 years (Table D-23; Figures D-23 and D-24)
• Incidence of hepatocellular adenoma or carcinoma in male and female B6C3Fi mice
(NCI, 1978, 062935) exposed to 1,4-dioxane in drinking water (Table D-24)
o BMDS dose-response modeling results for the combined incidence of hepatocellular
adenoma or carcinoma in female B6C3Fi mice (NCI, 1978, 062935) exposed to
1,4-dioxane in the drinking water for 2 years (Table D-25; Figure D-25)
o BMDS dose-response modeling results for incidence of combined hepatocellular
adenoma or carcinoma in male B6C3Fi mice (NCI, 1978, 062935) exposed to
1,4-dioxane in the drinking water for 2 years (Table D-26; Figures D-26 and D-27).
D.I. GENERAL ISSUES AND APPROACHES TO BMDS MODELING
D.I.I. Combining Data on Adenomas and Carcinomas
The incidence of adenomas and the incidence of carcinomas within a dose group at a site
or tissue in rodents are sometimes combined. This practice is based upon the hypothesis that
adenomas are a severe endpoint by themselves and most would have developed into carcinomas
if exposure at the same dose was continued (U.S. EPA, 2005, 086237). The incidence at high
doses of both tumors in rat and mouse liver is high in the key study (Kano et al., 2009, 594539).
D-2
-------
The incidence of hepatic adenomas and carcinomas was summed without double-counting them
so as to calculate the combined incidence of either a hepatic carcinoma or a hepatic adenoma in
rodents.
The variable N is used to denote the total number of animals tested in the dose group.
The variable Y is used here to denote the number of rodents within a dose group that have
characteristic X, and the notation Y(X) is used to identify the number with a specific
characteristic X. Modeling was performed on the adenomas and carcinomas separately and the
following combinations of tumor types:
• Y(adenomas) = number of animals with adenomas, whether or not carcinomas are
present;
• Y(carcinomas) = number of animals with carcinomas, whether or not adenomas are also
present;
• Y(either adenomas or carcinomas) = number of animals with adenomas or carcinomas,
not both = Y(adenomas) + Y(carcinomas) - Y(both adenomas and carcinomas);
• Y(neither adenomas nor carcinomas) = number of animals with no adenomas and no
carcinomas = N - Y(either adenomas or carcinomas).
D.1.2. Model Selection Criteria
Multiple models were fit to each dataset. The model selection criteria used in the BMD
technical guidance document (U.S. EPA, 2000, 052150) were applied as follows:
• />-value for goodness-of-fit > 0.10
• AIC smaller than other acceptable models
• %2 residuals as small as possible
• No systematic patterns of deviation of model from data
Additional criteria were applied to eliminate implausible dose-response functions:
• Monotonic dose-response functions, e.g. no negative coefficients of polynomials in MS
models
• No infinitely steep dose-response functions near 0 (control dose), achieved by requiring
the estimated parameters "power" in the Weibull and Gamma models and "slope" in the
log-logistic model to have values > 1.
Because no single set of criteria covers all contingencies, an extended list of preferred models are
presented below in Table D-l.
D.1.3. Summary
The BMDS models recommended to calculate rodent BMD and BMDL values and
corresponding human EMDnED and BMDLnED values are summarized in Table D-l.
D-3
-------
Table D-l. Recommended models for rodents exposed to 1,4-dioxane in
drinking water (Kano et al., 2009, 594539)
Endpoint
Model
selection
criterion
Model Type
AIC
p-value
BMDa
mg/kg-
day
BMDLa
mg/kg-
day
BMDHEDa
mg/kg-
day
BMDLHE
a
D
mg/kg-
day
Female F344 Rat
Hepatic
Tumors
Mammary
Gland
Tumors
Nasal
Cavity
Tumors
Lowest
AIC
Lowest
AIC
Lowest
AIC
Multistage
(2 degree)
LogLogistic
Multistage
(3 degree)
91.5898
194.151
42.6063
0.4516
0.8874
0.9966
79.83
161.01
381.65
58.09
81.91
282.61
19.84
40.01
94.84
14.43
20.35
70.23
Male R344 Rat
Hepatic
Tumors
Peritoneal
Meso-
thelioma
Nasal
Cavity
Tumors
Lowest
AIC
Lowest
AIC
Lowest
AIC
Probit
Probit
Multistage
(3 degree)
147.787
138.869
24.747
0.9867
0.9148
0.9989
62.20
93.06
328.11
51.12
76.32
245.63
17.43
26.09
91.97
14.33
21.39
68.85
Female BDF1 Mouse
Hepatic
Tumors
Lowest
AIC
BMR 50%
LogLogistic
LogLogistic
176.214
176.214
0.1421
0.1421
5.54
49.88b
3.66
32.93b
0.83
7.51b
0.55
4.95b
Male BDF1 Mouse
Hepatic
Tumors
Lowest
AIC
Log-
Logistic
248.839
0.3461
34.78
16.60
5.63
2.68
aValues for BMR 10% unless otherwise noted.
bBMR 50%.
D.2. FEMALE F344 RATS: HEPATIC CARCINOMAS AND ADENOMAS
The incidence data for hepatic carcinomas and adenomas in female F344 rats (Kano et
al., 2009, 594539) are shown in Table D-2.
D-4
-------
Table D-2. Data for hepatic adenomas and carcinomas in female F344 rats
(Kano et al., 2009, 594539)
Tumor type
Hepatocellular adenomas
Hepatocellular carcinomas
Either adenomas or carcinomas
Neither adenomas nor carcinomas
Total number per group
Dose (mg/kg-day)
0
3
0
3
47
50
18
1
0
1
49
50
83
6
0
6
44
50
429
48
10
48
2
50
Source: Used with permission from Elsevier, Ltd., Kano et al. (2009, 594539).
Note that the incidence of rats with adenomas, with carcinomas, and with either
adenomas or carcinomas are monotone non-decreasing functions of dose except for 3 female rats
in the control group. These data therefore appear to be appropriate for dose-response modeling
using HMDS.
The results of the BMDS modeling for the entire suite of models are presented in
Table D-3.
Table D-3. BMDS dose-response modeling results for the combined
incidence of hepatic adenomas and carcinomas in female F344 rats (Kano et
al., 2009, 594539)
Model
Gamma
Logistic
LogLogistic
LogProbitb
Multistage-Cancer
(1 degree)
Multistage-Cancer
(2 degree)'
Multistage-Cancer
(3 degree)
Probit
Weibull
Quantal-Linear
Dichotomous-Hill
AIC
93.1067
91.7017
93.102
93.0762
114.094
91.5898
93.2682
91.8786
93.2255
114.094
4458.37
/7-value
0.3024
0.4459
0.3028
0.3074
0.0001
0.4516
0.2747
0.3839
0.2825
0.0001
NCd
BMD10
mg/kg-day
89.46
93.02
88.34
87.57
25.58
79.83
92.81
85.46
92.67
25.58
NCd
BMDL10
mg/kg-day
62.09
71.60
65.52
66.19
19.92
58.09
59.31
67.84
59.89
19.92
NCd
x23
0.027
0.077
0.016
0.001
-1.827
-0.408
0.077
-0.116
0.088
-1.827
0
BMDiOHED
mg/kg-day
22.23
23.12
21.95
21.76
6.36
19.84
23.06
21.24
23.03
6.36
0
BMDLiOHED
mg/kg-day
15.43
17.79
16.28
16.45
4.95
14.43
14.74
16.86
14.88
4.95
0
aMaximum absolute %2 residual deviation between observed and predicted count. Values much larger than 1 are
undesirable.
bSlope restricted > 1.
'Best-fitting model.
Value unable to be calculated (NC: not calculated) by BMDS.
D-5
-------
Multistage Cancer Model with 0.95 Confidence Level
I
c
o
0.8
0.6
0.4
0.2
Multistage Cancer
Linear extrapolation
350
400
450
07:20 10/262009
Source: Used with permisison of Elservier, Ltd., Kano et al. (2009, 594539).
Figure D-l. Multistage BMD model (2 degree) for the combined incidence of
hepatic adenomas and carcinomas in female F344 rats.
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File: L:\Priv\NCEA_HPAG\14Dioxane\BMDS\msc_kano2009_frat_hepato_adcar_Msc-
BMR10-2poly.(d)
Gnuplot Plotting File:
L:\Priv\NCEA_HPAG\14Dioxane\BMDS\msc_kano2009_frat_hepato_adcar_Msc-BMR10-2poly.plt
Mon Oct 26 08:20:52 2009
BMDS Model Run
The form of the probability function is:
P [response] = background + (1-background) * [1-EXP (-betal*dose/xl-beta2*dose/x2) ]
The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Total number of observations = 4
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.0281572
D-6
-------
Beta(l) = 0
Beta(2) = 1.73306e-005
Asymptotic Correlation Matrix of Parameter Estimates (*** The model parameter(s)
Beta(l)have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
Background
Beta(2)
Variable
Background
Beta(1)
Beta(2)
Background Beta(2)
1 -0.2
-0.2 1
Parameter Estimates
95.0% Wald Confidence Interval
Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
0.0362773 * * *
0 * * *
1.65328e-005 * * *
Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood)
-42.9938
-43.7949
-120.43
91.5898
# Param' s
4
2
1
Deviance
1.60218
154.873
Test d.f.
2
3
P-value
= 1.59 d.f. = 2
Benchmark Dose Computation
P-value = 0.4516
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0.95
79.8299
58.085
94.0205
0.4488
<.0001
0
18
83
429
Dose
.0000
.0000
.0000
.0000
Est
0.
0.
0.
0.
. Prob.
0363
0414
1400
9540
Exp
1
2
7
47
Gooc
ected
.814
.071
.001
.701
inesj
01
3.
1.
6.
48.
3 Of Fit
:> served
,000
,000
,000
,000
Size
50
50
50
50
S
Re
0
-0
-0
0
caled
si dual
.897
.760
.408
.202
Taken together, (58.085 , 94.0205) is a 90% two-sided confidence interval for the BMD
Multistage Cancer Slope Factor = 0.00172161
D-7
-------
D.3. MALE F344 RATS: HEPATIC CARCINOMAS AND ADENOMAS
The data for hepatic adenomas and carcinomas in male F344 rats (Kano et al., 2009,
594539) are shown in Table D-4.
Table D-4. Data for hepatic adenomas and carcinomas in male F344 rats
(Kano et al., 2009, 594539)
Tumor type
Hepatocellular adenomas
Hepatocellular carcinomas
Either adenomas or carcinomas
Neither adenomas nor carcinomas
Total number per group
Dose (mg/kg-day)
0
3
0
3
47
50
11
4
0
4
46
50
55
7
0
7
43
50
274
32
14
39
11
50
Source: Used with permission from Elservier, Ltd., Kano et al. (2009, 594539).
Note that the incidence of rats with hepatic adenomas, carcinomas, and with either
adenomas or carcinomas are monotone non-decreasing functions of dose. These data therefore
appear to be appropriate for dose-response modeling using BMDS.
The results of the BMDS modeling for the entire suite of models tested using the data for
hepatic adenomas and carcinomas for male F344 rats are presented in Table D-5.
D-8
-------
Table D-5. BMDS dose-response modeling results for the combined
incidence of adenomas and carcinomas in livers of male F344 rats (Kano et
al., 2009, 594539)
Model
Gamma
Logistic
LogLogistic
LogProbitb
Multistage-Cancer
(1 degree)
Multistage-Cancer
(2 degree)
Multistage-Cancer
(3 degree)
Probif
Weibull
Quantal-Linear
Dichotomous-Hill
AIC
149.884
147.813
149.886
149.913
152.836
149.814
149.772
147.787
149.856
152.836
4441.71
^-value
0.7257
0.9749
0.7235
0.6972
0.0978
0.8161
0.9171
0.9867
0.7576
0.0978
NCd
BMD10
mg/kg-day
62.41
68.74
62.10
61.70
23.82
61.68
63.62
62.20
62.63
23.82
NCd
BMDL10
mg/kg-day
30.79
55.39
34.61
37.49
18.34
28.26
27.49
51.12
30.11
18.34
NCd
x23
-0.03
0.097
-0.021
-0.003
-0.186
-0.063
-0.024
-0.05
-0.039
-0.186
0
BMDjoHED
mg/kg-day
17.49
19.27
17.41
17.29
6.68
17.29
17.83
17.43
17.56
6.68
0
BMDLjoHED
mg/kg-day
8.63
15.53
9.70
10.51
5.14
7.92
7.71
14.33
8.44
5.14
0
"Maximum absolute % residual deviation between observed and predicted count. Values much larger than 1 are
undesirable.
bSlope restricted > 1.
'Best-fitting model.
Value unable to be calculated (NC: not calculated) by BMDS.
D-9
-------
Probit Model with 0.95 Confidence Level
T3
-------
Asymptotic Correlation Matrix of Parameter Estimates
(*** The model parameter(s) -background have been estimated at a boundary point, or
have been specified by the user, and do not appear in the correlation matrix )
intercept
slope
intercept
1
-0.69
slope
-0.69
1
Variable
intercept
slope
Parameter Estimates
95.0% Wald Confidence Interval
Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
1.53138 0.160195 -1.84535 -1.2174
0.00840347 0.000976752 0.00648907 0.0103179
Model
Full model
Fitted model
Reduced model
AIC:
Analysis of Deviance Table
Log(likelihood)
-71.8804
-71.8937
-115.644
147.787
# Param's
4
2
1
Deviance Test d.f.
0.0265818
87.528
P-value
0.9868
<.0001
Goodness of Fit
Dose
0.0000
11.0000
55.0000
274.0000
Est. Prob.
0.0628
0.0751
0.1425
0.7797
Expected
3.142
3.754
7.125
38.985
Observed
3.000
4.000
7.000
39.000
Size
50
50
50
50
Scaled
Residual
-0.083
0.132
-0.050
0.005
= 0.03 d.f. = 2
Benchmark Dose Computation
P-value = 0.9867
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
0.1
Extra risk
0.95
62.1952
51.1158
D-ll
-------
Multistage Cancer Model with 0.95 Confidence Level
T3
-------
Background = 0.0623822
Beta(l) = 0.00142752
Beta(2) = 0
Beta(3) = 5.14597e-008
Asymptotic Correlation Matrix of Parameter Estimates
(*** The model parameter(s) -Beta(2)have been estimated at a boundary point, or have
been specified by the user, and do not appear in the correlation matrix )
Background
Beta(1)
Beta (3)
Background
1
-0. 67
0.58
Beta(l)
-0.67
1
-0.95
Beta(3)
0.58
-0.95
1
Variable
Background
Beta(1)
Beta (2)
Beta(3)
Estimate
0.0619918
0.001449
0
5.11829e-008
Parameter Estimates
95.0% Wald Confidence Interval
Std. Err. Lower Conf. Limit Upper Conf. Limit
Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood)
-71.8804
-71.8858
-115.644
149.772
Param's
4
3
1
Deviance Test d.f.
0.0107754
87.528
P-value
0.9173
<.0001
Goodness of Fit
Scaled
0
11
55
274
Dose
.0000
.0000
.0000
.0000
Est
0.
0.
0.
0.
. Prob.
0620
0769
1412
7799
Expected
3.
3.
7.
38.
100
844
059
997
Observed
3.
4.
7.
39.
,000
,000
,000
,000
Size
50
50
50
50
Residual
-0
0
-0
0
.058
.083
.024
.001
=0.01
d.f. = 1
P-value = 0.9171
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0.95
63.6179
27.4913
123.443
Taken together, (27.4913, 123.443) is a 90% two-sided confidence interval for the BMD
Multistage Cancer Slope Factor = 0.00363752
D-13
-------
D.4. F344 RATS: TUMORS AT OTHER SITES
The data for tumors at sites other than the liver in male and female F344 rats (Kano et al.,
2009, 594539) are shown in Table D-6. Note that the incidence of rats with these endpoints are
monotone non-decreasing functions (except female peritoneal mesotheliomas). These data
therefore appear to be appropriate for dose-response modeling using BMDS.
Table D-6. Data for significant tumors at other sites in male and female F344
rats (Kano et al., 2009, 594539)
Tumor site and type
Nasal cavity squamous cell carcinoma
Peritoneal mesothelioma
Mammary gland adenoma
Total number per group
Dose (mg/kg-day)
Female
0
0
1
6
50
18
0
0
7
50
83
0
0
10
50
429
7
0
16
50
Male
0
0
2
0
50
11
0
2
1
50
55
0
5
2
50
274
3
28
2
50
Source: Used with permission from Elsevier, Ltd., Kano et al., (2009, 594539).
The results of the BMDS modeling for the entire suite of models are presented in Tables
D-7 through Table D-10 for tumors in the nasal cavity, mammary gland, and peritoneal cavity.
D-14
-------
Table D-7. BMDS dose-response modeling results for the incidence of nasal
cavity tumors in female F344 ratsa (Kano et al., 2009, 594539)
Model
Gamma
Logistic
LogLogistic
LogProbif
Multistage-Cancer
(1 degree)
Multistage-Cancer
(2 degree)
Multistage-Cancer
(3 degree)d
Probit
Weibull
Quantal-Linear
Dichotomous-Hill
AIC
44.4964
44.4963
44.4963
44.4963
45.6604
43.0753
42.6063
44.4963
44.4963
45.6604
46.4963
/7-value
1
1
1
1
0.6184
0.9607
0.9966
1
1
0.6184
0.9997
BMD10
mg/kg-day
403.82
421.54
413.69
400.06
375.81
366.07
381.65
414.11
414.86
375.81
413.96
BMDL10
mg/kg-day
269.03
351.74
268.85
260.38
213.84
274.63
282.61
333.31
273.73
213.84
372.57
x2b
0
0
0
0
0.595
0.109
0.021
0
0
0.595
1.64xlO'8
BMD10HED
mg/kg-day
100.35
104.75
102.80
99.42
93.39
90.97
94.84
102.91
103.09
93.39
102.87
BMDL10HED
mg/kg-day
66.85
87.41
66.81
64.71
53.14
68.24
70.23
82.83
68.02
53.14
92.58
aNasal cavity tumors
bMaximum absolute
undesirable.
°Slope restricted > 1.
dBest-fitting model.
in female F344 rats include squamous cell carcinoma and esthesioneuro-epithelioma.
2 residual deviation between observed and predicted count. Values much larger than 1 are
D-15
-------
Multistage Cancer Model with 0.95 Confidence Level
T3
O
c
o
'•g
(0
0.3
0.25
0.2
0.15
0.1
0.05
Multistage Cancer
Linear extrapolation
BMDL
BMD
50
100
150
200 250
dose
300
350
400
450
07:28 10/26 2009
Source: Used with permission from Elsevier, Ltd., Kano et al. (2009, 594539).
Figure D-4. Multistage BMD model (3 degree) for nasal cavity tumors in
female F344 rats.
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File: L:\Priv\NCEA_HPAG\14Dioxane\BMDS\msc_kano2009_frat_nasal_car_Msc-
BMR10-3poly.(d)
Gnuplot Plotting File:
L:\Priv\NCEA_HPAG\14Dioxane\BMDS\msc_kano2009_frat_nasal_car_Msc-BMR10-3poly.plt
Mon Oct 26 08:28:58 2009
BMDS Model Run
The form of the probability function is: P[response] = background + (1-
background) * [1-EXP (-betal*dose/xl-beta2*dose/x2-beta3*dose/x3) ]
The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Total number of observations = 4
Total number of records with missing values = 0
Total number of parameters in model = 4
Total number of specified parameters = 0
Degree of polynomial = 3
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0
Beta(l) = 0
Beta(2) = 0
Beta(3) = 1.91485e-009
D-16
-------
Asymptotic Correlation Matrix of Parameter Estimates
(*** The model parameter(s) -Background -Beta(l) -Beta (2)
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
Beta(3)
Variable
Background
Beta(l)
Beta (2)
Beta (3)
Beta(3)
1
Estimate
0
0
0
1.89531e-009
Parameter Estimates
95.0% Wald Confidence Interval
Std. Err. Lower Conf. Limit Upper Conf. Limit
Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood)
-20.2482
-20.3031
-30.3429
42.6063
# Param's
4
1
1
Deviance Test d.f.
0.109908
20.1894
P-value
0.9906
0.0001551
0
18
83
429
Dose
.0000
.0000
.0000
.0000
Est
0.
0.
0.
0.
. Prob.
0000
0000
0011
1390
Expe
0.
0.
0.
6.
Good
cted
000
001
054
949
ness
01
0.
0.
0.
7.
3 Of Fit
:> served
,000
,000
,000
,000
Size
50
50
50
50
S
Re
0
-0
-0
0
caled
si dual
.000
.024
.233
.021
=0.06
d.f. = 3
P-value = 0.9966
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0.95
381.651
282.609
500.178
Taken together, (282.609, 500.178) is a 90% two-sided confidence interval for the BMD
Multistage Cancer Slope Factor = 0.000353846
D-17
-------
Table D-8. BMDS dose-response modeling results for the incidence of nasal
cavity tumors in male F344 ratsa (Kano et al., 2009, 594539)
Model
Gamma
Logistic
LogLogistic
LogProbif
Multistage-Cancer
(1 degree)
Multistage-Cancer
(2 degree)
Multistage-Cancer
(3 degree)d
Probit
Weibull
Quantal-Linear
Dichotomous-Hill
AIC
26.6968
26.6968
26.6968
26.6968
26.0279
24.9506
24.747
26.6968
26.6968
26.0279
28.6968
/7-value
1
1
1
1
0.8621
0.988
0.9989
1
1
0.8621
0.9994
BMD10
mg/kg-day
299.29
281.06
288.31
303.06
582.49
365.19
328.11
287.96
288.00
582.49
290.52
BMDL10
mg/kg-day
244.10
261.29
245.29
238.86
256.43
242.30
245.63
257.01
246.36
256.43
261.47
x2b
0
0
0
0
0.384
0.073
0.015
0
0
0.384
6.25 xlO'5
BMD10HED
mg/kg-day
83.89
78.78
80.81
84.94
163.28
102.37
91.97
80.72
80.73
163.28
81.44
BMDL10HED
mg/kg-day
68.42
73.24
68.75
66.95
71.88
67.92
68.85
72.04
69.06
71.88
73.29
aNasal cavity tumors in male F344 rats include squamous cell carcinoma, Sarcoma: NOS
esthesioneuro-epithelioma.
bMaximum absolute %2 residual deviation between observed and predicted count. Values
undesirable.
°Slope restricted > 1.
dBest-fitting model.
, rhabdomyosarcoma, and
much larger than 1 are
D-18
-------
Multistage Cancer Model with U.ab Confidence Level
Multistage Cancer
Linear extrapolation
0.15
T3
0
t3
I
C
g
ro
it
0.1
0.05
300
07:34 10/26 2009
Source: Used with permisison from Elsevier, Ltd., Kano et al. (2009, 594539).
Figure D-5. Multistage BMD model (3 degree) for nasal cavity tumors in
male F344 rats.
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File: L:\Priv\NCEA_HPAG\14Dioxane\BMDS\msc_kano2009_mrat_nasal_car_Msc-
BMR10-3poly.(d)
Gnuplot Plotting File:
L:\Priv\NCEA_HPAG\14Dioxane\BMDS\msc_kano2009_mrat_nasal_car_Msc-BMR10-3poly.plt
Mon Oct 26 08:34:20 2009
BMDS Model Run
The form of the probability function is: P[response] = background + (1-background)*[1-
EXP (-betal*dose/xl-beta2*dose/x2-beta3*dose/x3) ]
The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Total number of observations = 4
Total number of records with missing values = 0
Total number of parameters in model = 4
Total number of specified parameters = 0
Degree of polynomial = 3
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0
D-19
-------
Beta(l) = 0
Beta(2) = 0
Beta(3) = 3.01594e-009
Asymptotic Correlation Matrix of Parameter Estimates
(*** The model parameter(s) -Background -Beta(l) -Beta(2)
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
Beta(3)
Beta(3)
1
Parameter Estimates
Variable
Background
Beta(1)
Beta(2)
Beta(3)
Estimate
0
0
0
2.98283e-009
Std. Err.
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood)
-11.3484
-11.3735
-15.5765
24.747
# Param's
4
1
1
Deviance Test d.f.
P-value
0.0502337
8.45625
0.9971
0.03747
0
11
55
274
Dose
.0000
.0000
.0000
.0000
Est
0.
0.
0.
0.
. Prob.
0000
0000
0005
0595
Expe
0.
0.
0.
2.
Good
cted
000
000
025
976
ness
01
0.
0.
0.
3.
3 Of Fit
:> served
,000
,000
,000
,000
Size
50
50
50
50
S
Re
0
-0
-0
0
caled
si dual
.000
.014
.158
.015
=0.03
d.f. = 3
P-value = 0.9989
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0.95
328.108
245.634
1268.48
Taken together, (245.634, 1268.48) is a 90% two-sided confidence interval for the BMD
Multistage Cancer Slope Factor = 0.00040711
D-20
-------
Table D-9. BMDS dose-response modeling results for the incidence of
mammary gland adenomas in female F344 rats (Kano et al., 2009, 594539)
Model
Gamma
Logistic
LogLogisticb
LogProbif
Multistage-Cancer
(1 degree)
Multistage-Cancer
(2 degree)
Multistage-Cancer
(3 degree)
Probit
Weibull
Quantal-Linear
Dichotomous-Hill
AIC
194.222
194.475
194.151
195.028
194.222
194.222
194.222
194.441
194.222
194.222
197.916
/7-value
0.8559
0.7526
0.8874
0.5659
0.8559
0.8559
0.8559
0.7656
0.8559
0.8559
NCd
BMD10
mg/kg-day
176.66
230.35
161.01
270.74
176.66
176.66
176.66
223.04
176.65
176.65
94.06
BMDL10
mg/kg-day
99.13
159.73
81.91
174.66
99.13
99.13
99.13
151.60
99.13
99.13
14.02
x2a
0.465
0.612
0.406
-0.075
0.465
0.465
0.465
0.596
0.465
0.465
3.49xlO"5
BMD10HED
mg/kg-day
43.90
57.24
40.01
67.28
43.90
43.90
43.90
55.43
43.90
43.90
23.37
BMDL10HED
mg/kg-day
24.63
39.69
20.35
43.41
24.63
24.63
24.63
37.67
24.63
24.63
3.48
aMaximum absolute % residual deviation between observed and predicted count. Values much larger than 1 are
undesirable.
bBest-fitting model.
°Slope restricted > 1.
Value unable to be calculated (NC: not calculated) by BMDS.
D-21
-------
Log-Logistic Model with 0.95 Confidence Level
T3
0
O
ro
IL
0.5
0.4
0.3
0.2
0.1
100
150
200
250
300
350
400
45C
dose
11:31,02/01 20
Source: Use with permission from Elsevier, Ltd., Kano et al. (2009, 594539).
Figure D-6. LogLogistic BMD model for mammary gland adenomas in
female F344 rats.
Logistic Model. (Version: 2.12; Date: 05/16/2008)
Input Data File: C:\14DBMDS\lnl_kano2009_frat_mainm_ad_Lnl-BMR10-Restrict.(d)
Gnuplot Plotting File: C:\14DBMDS\lnl_kano2009_frat_mamm_ad_Lnl-BMR10-Restrict.plt
Mon Feb 01 11:31:31 2010
BMDS Model Run
The form of the probability function is:
P[response] = background+(1-background)/[1+EXP(-intercept-slope*Log(dose))]
Dependent variable = Effect
Independent variable = Dose
Slope parameter is restricted as slope >= 1
Total number of observations = 4
Total number of records with missing values = 0
Maximum number of iterations =250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
User has chosen the log transformed model
Default Initial Parameter Values
background = 0.12
intercept = -7.06982
slope = 1
Asymptotic Correlation Matrix of Parameter Estimates
D-22
-------
(*** The model parameter(s) -slope have been estimated at a boundary point, or have
been specified by the user, and do not appear in the correlation matrix )
background
intercept
background
1
-0.53
intercept
-0.53
1
Parameter Estimates
Variable
background
intercept
slope
Estimate
0.130936
-7.2787
1
Std. Err.
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-94.958
-95.0757
-98.6785
# Param's
4
2
1
Deviance Test d.f.
P-value
0.235347
7.4409
0.889
0.0591
AIC:
194.151
0
18
83
429
Dose
.0000
.0000
.0000
.0000
Est
0.
0.
0.
0.
. Prob.
1309
1416
1780
3294
Expe
6.
7.
8.
16.
Gooc
cted
547
080
901
472
Inesj
01
6.
7.
10.
16.
3 Of Fit
:> served
,000
,000
,000
,000
Size
50
50
50
50
S
Re
-0
-0
0
-0
caled
si dual
.229
.032
.406
.142
= 0.24
d.f. = 2
P-value = 0.8874
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 161.012
BMDL = 81.9107
D-23
-------
Multistage Cancer Model with 0.95 Confidence Level
T3
-------
Asymptotic Correlation Matrix of Parameter Estimates
Background Beta(l)
Background 1 -0.58
Beta(l) -0.58 1
Parameter Estimates
Variable
Background
Beta(l)
Estimate
.133161
0.000596394
Std. Err.
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood)
-94.958
-95.111
-98.6785
194.222
# Param' s
4
2
1
Deviance
0.305898
7.4409
Test d.f.
2
3
P-value
0.8582
0.0591
Goodness of Fit
Dose
0.0000
18.0000
83.0000
429.0000
Est. Prob.
0.1332
0.1424
0.1750
0.3288
Expected
6. 658
7.121
8.751
16.442
Observed
6.000
7.000
10.000
16.000
Size
50
50
50
50
Scaled
Residual
-0.274
-0.049
0.465
-0.133
ChiA2 = 0.31
d.f. = 2
P-value = 0.8559
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0.95
176.663
99.1337
501.523
Taken together, (99.1337, 501.523) is a 90% two-sided confidence interval for the BMD
Multistage Cancer Slope Factor = 0.00100874
D-25
-------
Table D-10. BMDS dose-response modeling results for the incidence of
peritoneal mesotheliomas in male F344 rats (Kano et al., 2009, 594539)
Model
Gamma
Logistic
LogLogistic
LogProbitb
Multistage-Cancer
(1 degree)
Multistage-Cancer
(2 degree)
Multistage-Cancer
(3 degree)
Probif
Weibull
Quantal-Linear
Dichotomous-Hill
AIC
140.701
139.016
140.699
140.69
140.826
140.747
140.747
138.869
140.709
140.826
2992
/7-value
0.9189
0.8484
0.9242
0.9852
0.3617
0.8135
0.8135
0.9148
0.8915
0.3617
NCd
BMD10
mg/kg-day
73.52
103.52
72.56
70.29
41.04
77.73
77.73
93.06
74.77
41.04
NCd
BMDL10
mg/kg-day
35.62
84.35
36.37
52.59
30.51
35.43
35.43
76.32
35.59
30.51
NCd
x2a
0.018
0.446
0.014
0.001
-1.066
0.067
0.067
0.315
0.027
-1.066
0
BMD10HED
mg/kg-day
20.61
29.02
20.34
19.70
11.50
21.79
21.79
26.09
20.96
11.50
0
BMDL10HED
mg/kg-day
9.98
23.65
10.19
14.74
8.55
9.93
9.93
21.39
9.97
8.55
0
""Maximum absolute jf residual deviation between observed and predicted count. Values much larger than
undesirable.
bSlope restricted > 1.
'Best-fitting model.
"Value unable to be calculated (NC: not calculated) by BMDS.
1 are
D-26
-------
Probit Model with 0.95 Confidence Level
T3
0
t3
I
o
0.7
0.6
0.5
0.4
0.3
0.2
0.1
Probit
BMDL BMD
50
100
150
200
250
dose
07:41 10/262009
Source: Used with permission from Elsevier, Ltd., Kano et al. (2009, 594539).
Figure D-8. Probit BMD model for peritoneal mesotheliomas in male F344
rats.
Probit Model. (Version: 3.1; Date: 05/16/2008)
Input Data File: L:\Priv\NCEA_HPAG\14Dioxane\BMDS\pro_kano2009_mrat_peri_meso_Prb-
BMR10.(d)
Gnuplot Plotting File:
L:\Priv\NCEA_HPAG\14Dioxane\BMDS\pro_kano2009_mrat_peri_meso_Prb-BMR10.plt
Mon Oct 26 08:41:29 2009
BMDS Model Run
The form of the probability function is: P[response] = CumNorm(Intercept+Slope*Dose),
where CumNorm(.) is the cumulative normal distribution function
Dependent variable = Effect
Independent variable = Dose
Slope parameter is not restricted
Total number of observations = 4
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial (and Specified) Parameter Values
background = 0 Specified
intercept = -1.73485
slope = 0.00692801
Asymptotic Correlation Matrix of Parameter Estimates
D-27
-------
(*** The model parameter(s) -background have been estimated at a boundary point,
have been specified by the user, and do not appear in the correlation matrix )
intercept
slope
Variable
intercept
slope
intercept
1
-0.75
Estimate
-1.73734
0.00691646
slope
-0.75
1
Model
Full model
Fitted model
Reduced model
AIC:
Parameter Estimates
95.0% Wald Confidence Interval
Std. Err. Lower Conf. Limit Upper Conf. Limit
0.18348 -2.09695 -1.37772
0.000974372 0.00500672 0.00882619
Analysis of Deviance Table
Log(likelihood) # Param's Deviance Test d.f. P-value
-67.3451 4
-67.4344 2 0.178619 2 0.9146
-95.7782 1 56.8663 3 <.0001
138.869
Goodness of Fit
Dose
0.0000
11.0000
55.0000
274.0000
Est. Prob.
0.0412
0.0483
0.0874
0.5627
Expected
2.058
2.417
4.370
28.134
Observed
2.000
2.000
5.000
28.000
Size
50
50
50
50
Scaled
Residual
-0.041
-0.275
0.315
-0.038
= 0.18 d.f. = 2
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 93.0615
BMDL = 76.3242
P-value = 0.9148
D-28
-------
Multistage Cancer Model with 0.95 Confidence Level
c
o
'•8
ro
0.7
0.6
0.5
0.4
0.3
0.2
0.1
Multistage Cancer
Linear extrapolation
BMDL
BMD
50
100
150
200
250
dose
07:41 10/262009
Source: Used with permission from Elsevier, Ltd., Kano et al. (2009, 594539).
Figure D-9. Multistage BMD (2 degree) model for peritoneal
mesotheliomas in male F344 rats.
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File: L:\Priv\NCEA_HPAG\14Dioxane\BMDS\msc_kano2009_mrat_peri_meso_Msc-
BMR10-2poly.(d)
Gnuplot Plotting File:
L:\Priv\NCEA_HPAG\14Dioxane\BMDS\msc_kano2009_mrat_peri_meso_Msc-BMR10-2poly.plt
Mon Oct 26 08:41:28 2009
BMDS Model Run
The form of the probability function is:
P [response] = background + (1-background) * [1-EXP (-betal*dose/xl-beta2*dose/x2) ]
The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Total number of observations = 4
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
D-29
-------
Default Initial Parameter Values
Background = 0.0358706
Beta(l) = 0.000816174
Beta(2) = 7.47062e-006
Asymptotic Correlation Matrix of Parameter Estimates
Background
Beta (1)
Beta (2)
Background
1
-0. 67
0.59
Beta(l)
-0.67
1
-0.98
Beta(2)
0.59
-0.98
1
Parameter Estimates
95.0% Wald Confidence Interval
Variable
Background
Beta(l)
Beta (2)
Estimate
0.0366063
0.000757836
7.6893e-006
Std. Err.
Lower Conf. Limit
*
*
Upper Conf. Limit
* - Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood) # Param's Deviance Test d.f.
-67.3451 4
-67.3733 3 0.056567 1
-95.7782 1 56.8663 3
140.747
P-value
0.812
<.0001
Goodness of Fit
Scaled
0
11
55
274
Dose
.0000
.0000
.0000
.0000
Est
0.
0.
0.
0.
. Prob.
0366
0455
0972
5605
Expected
1
2
4
28
.830
.275
.859
.027
Observed
2,
2,
5,
28,
.000
.000
.000
.000
Size
50
50
50
50
Residual
0
-0
0
-0
.128
.186
.067
.008
ChiA2 =0.06
d.f. = 1
P-value = 0.8135
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0.95
77.7277
35.4296
118.349
Taken together, (35.4296, 118.349) is a 90% two-sided confidence interval for the BMD
Multistage Cancer Slope Factor = 0.0028225
D-30
-------
D.5. FEMALE BDF1 MICE: HEPATIC CARCINOMAS AND ADENOMAS
Data for female BDF1 mouse hepatic carcinomas and adenomas are shown in Table D-
11. Note that the incidence of carcinomas and the incidence of either adenomas or carcinomas
are monotone non-decreasing functions of dose. These data therefore appear to be appropriate
for dose-response modeling using BMDS. However, the incidence of adenomas clearly reaches
a peak value at 66 mg/kg-day and then decreases sharply with increasing dose. This cannot be
modeled by a multistage model using only non-negative coefficients. To some extent the
incidence of "either adenomas or carcinomas" retains some of the inverted-U shaped dose-
response of the adenomas, which dominate based on their high incidence at the lowest dose
groups (66 and 278 mg/kg-day), thus is not well characterized by any multistage model.
Table D-ll. Data for hepatic adenomas and carcinomas in female BDF1 mice
(Kano et al., 2009, 594539)
Tumor type
Hepatocellular adenomas
Hepatocellular carcinomas
Either adenomas or carcinomas
Neither adenomas nor carcinomas
Total number per group
Dose (mg/kg-day)
0
5
0
5
45
50
66
31
6
35
15
50
278
20
30
41
9
50
964
3
45
46
4
50
Source: Used with permission from Elsevier, Ltd., Kano et al. (2009, 594539).
The results of the BMDS modeling for the entire suite of models for hepatic adenomas
and carcinomas in female BDF1 mice are presented in Table D-12. The multistage models did
not provide reasonable fits to the incidence data for hepatocellular adenoma or carcinoma in
female BDF1 mice. The log-logistic model provided the best-fit to the data as indicated by the
AIC and/>-value as was chosen as the best-fitting model to carry forward in the analysis;
however, this model resulted in a BMDLio much lower than the response level at the lowest dose
in the study (Kano et al., 2009, 594539). Thus, the log-logistic model was run for BMRs of 30
and 50%. The output from these models are shown in Figures D-l 1 and D-12. A summary of
the BMD results for BMRs of 10, 30, and 50% are shown in Table D-13. Using a higher BMR
resulted in BMDLs closer to the lowest observed response data, and a BMR of 50% was chosen
to carry forward in the analysis.
The graphical output from fitting these models suggested that a simpler model obtained
by dropping the data point for the highest dose (964 mg/kg-day) might also be adequate. This
was tested and the results did not affect the choice of the model, nor significantly affect the
resulting BMDs and BMDLs.
D-31
-------
Table D-12. BMDS dose-response modeling results for the combined
incidence of hepatic adenomas and carcinomas in female BDF1 mice (Kano
et al., 2009, 594539)
Model
Gamma
Logistic
LogLogisticb
LogProbif
Multistage-Cancer
(1 degree)
Multistage-Cancer
(2 degree)
Multistage-Cancer
(3 degree)
Probit
Weibull
Quantal-Linear
Dichotomous-Hill
AIC
203.331
214.951
176.214
198.354
203.331
203.331
203.331
217.671
203.331
203.331
7300.48
/7-value
0
0
0.1421
0
0
0
0
0
0
0
NCd
BMD10
mg/kg-day
26.43
58.05
5.54
26.37
26.43
26.43
26.43
69.89
26.43
26.43
NCd
BMDL10
mg/kg-day
19.50
44.44
3.66
19.57
19.50
19.50
19.50
56.22
19.50
19.50
NCd
x2a
-2.654
3.201
-0.121
-1.166
-2.654
-2.654
-2.654
3.114
-2.654
-2.654
0
BMDiOHED
mg/kg-day
3.98
8.74
0.83
3.97
3.98
3.98
3.98
10.5
3.98
3.98
0
BMDLiOHED
mg/kg-day
2.94
6.69
0.55
2.95
2.94
2.94
2.94
8.46
2.94
2.94
0
aMaximum absolute %2 residual deviation between observed and predicted count. Values much larger than 1 are
undesirable.
bBest-fitting model, lowest AIC value.
°Slope restricted > 1.
Value unable to be calculated (NC: not calculated) by BMDS.
Table D-13. BMDS LogLogistic dose-response modeling results using BMRs
of 10, 30, and 50% for the combined incidence of hepatic adenomas and
carcinomas in female BDF1 mice (Kano et al., 2009, 594539).
BMR
10%
30%
50%
AIC
176.214
176.214
176.214
^-value
0.1421
0.1421
0.1421
BMD
mg/kg-day
5.54
21.38
49.88
BMDL
mg/kg-day
3.66
14.11
32.93
f
-0.121
-0.121
0
BMDaED
mg/kg-day
0.83
3.22
7.51
BMDLaED
mg/kg-day
0.55
2.12
4.95
aMaximum absolute ^ residual deviation between observed and predicted count. Values much larger than 1 are
undesirable.
D-32
-------
Log-Logistic Model with 0.95 Confidence Level
T3
CD
"o
I
0.8
0.6
0.4
0.2
Log-Logistic
° BiMDLBMD
0 200 400 600 800 1000
dose
11:2605/122010
Source: Used with permission from Elsevier, Ltd., Kano et al. (2009, 594539).
Figure D-10. LogLogistic BMD model for the combined incidence of hepatic
adenomas and carcinomas in female BDF1 mice with a BMR of 10%.
Logistic Model. (Version: 2.12; Date: 05/16/2008)
Input Data File:
L:\Priv\NCEA_HPAG\14Dioxane\BMDS\lnl_kano2009_fmouse_hepato_adcar_Lnl-BMR10-
Restrict.(d)
Gnuplot Plotting File:
L:\Priv\NCEA_HPAG\14Dioxane\BMDS\lnl_kano2009_fmouse_hepato_adcar_Lnl-BMR10-
Restrict.pit
Wed May 12 11:26:35 2010
BMDS Model Run
The form of the probability function is:
P[response] = background+(1-background)/[1+EXP(-intercept-slope*Log(dose))]
Dependent variable = Effect
Independent variable = Dose
Slope parameter is restricted as slope >= 1
Total number of observations = 4
Total number of records with missing values = 0
Maximum number of iterations =250
D-33
-------
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
User has chosen the log transformed model
Default Initial Parameter Values
background = 0.1
intercept = -4.33618
slope = 1
Asymptotic Correlation Matrix of Parameter Estimates
(*** The model parameter(s) -slope have been estimated at a boundary point, or have
been specified by the user, and do not appear in the correlation matrix )
background
intercept
Variable
background
intercept
slope
background
1
-0.32
intercept
-0.32
1
Parameter Estimates
95.0% Wald Confidence Interval
Estimate
0.105265
-3.90961
1
Std. Err.
Lower Conf. Limit
*
Upper Conf. Limit
* - Indicates that this value is not calculated.
Analysis of Deviance Table
Deviance Test d.f.
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood)
-84.3055
-86.107
-131.248
176.214
# Param's
4
2
1
P-value
3.6029
93.8853
0.1651
<.0001
0
66
278
964
Dose
.0000
.0000
.0000
.0000
Est
0.
0.
0.
0.
. Prob.
1053
6149
8639
9560
Exp
5
30
43
47
Gooc
ected
.263
.743
.194
.799
inesj
01
5.
35.
41.
46.
3 Of Fit
:> served
,000
,000
,000
,000
Size
50
50
50
50
S
Re
-0
1
-0
-1
caled
si dual
.121
.237
.905
.240
=3.90
d.f. = 2
P-value = 0.1421
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 5.54218
BMDL = 3.65848
D-34
-------
Log-Logistic Model with 0.95 Confidence Level
T3
CD
"o
I
0.8
0.6
0.4
0.2
Log-Logistic
liMDLBMD
200
400
600
800
1000
dose
11:2605/122010
Source: Used with permission from Elsevier, Ltd., Kano et al. (2009, 594539).
Figure D-ll. LogLogistic BMD model for the combined incidence of hepatic
adenomas and carcinomas in female BDF1 mice with a BMR of 30%.
Logistic Model. (Version: 2.12; Date: 05/16/2008)
Input Data File:
L:\Priv\NCEA_HPAG\14Dioxane\BMDS\lnl_kano2009_fmouse_hepato_adcar_Lnl-BMR30-
Restrict.(d)
Gnuplot Plotting File:
L:\Priv\NCEA_HPAG\14Dioxane\BMDS\lnl_kano2009_fmouse_hepato_adcar_Lnl-BMR30-
Restrict.pit
Wed May 12 11:26:36 2010
BMDS Model Run
The form of the probability function is:
P[response] = background+(1-background)/[1+EXP(-intercept-slope*Log(dose))]
Dependent variable = Effect
Independent variable = Dose
Slope parameter is restricted as slope >= 1
Total number of observations = 4
Total number of records with missing values = 0
Maximum number of iterations =250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
User has chosen the log transformed model
D-35
-------
Default Initial Parameter Values
background = 0.1
intercept = -4.33618
slope = 1
Asymptotic Correlation Matrix of Parameter Estimates
(*** The model parameter(s) -slope have been estimated at a boundary point, or have
been specified by the user, and do not appear in the correlation matrix)
background
intercept
background
1
-0.32
intercept
-0.32
1
Variable
background
intercept
slope
Estimate
0.105265
-3.90961
1
Parameter Estimates
Std. Err.
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-84.3055
-86.107
-131.248
Param's
4
2
1
Deviance Test d.f.
P-value
3.6029
93.8853
0.1651
<.0001
AIC:
176.214
0
66
278
964
Dose
.0000
.0000
.0000
.0000
Est
0.
0.
0.
0.
. Prob.
1053
6149
8639
9560
Exp
5
30
43
47
Gooc
ected
.263
.743
.194
.799
inesj
01
5.
35.
41.
46.
3 Of Fit
:> served
,000
,000
,000
,000
Size
50
50
50
50
S
Re
-0
1
-0
-1
caled
si dual
.121
.237
.905
.240
ChiA2 =3.90
d.f. = 2
P-value = 0.1421
Benchmark Dose Computation
Specified effect = 0.3
Risk Type = Extra risk
Confidence level = 0.95
BMD = 21.377
BMDL = 14.1113
D-36
-------
Log-Logistic Model with 0.95 Confidence Level
T3
CD
"o
I
Log-Logistic
0.8
0.6
0.4
0.2
0 : BMpL|JBMD
0 200 400 600 800 1000
dose
11:2605/122010
Source: Used with permission from Elsevier, Ltd., Kano et al. (2009, 594539).
Figure D-12. LogLogistic BMD model for the combined incidence of hepatic
adenomas and carcinomas in female BDF1 mice with a BMR of 50%.
Logistic Model. (Version: 2.12; Date: 05/16/2008)
Input Data File:
L:\Priv\NCEA_HPAG\14Dioxane\BMDS\lnl_kano2009_fmouse_hepato_adcar_Lnl-BMR50-
Restrict.(d)
Gnuplot Plotting File:
L:\Priv\NCEA_HPAG\14Dioxane\BMDS\lnl_kano2009_fmouse_hepato_adcar_Lnl-BMR50-
Restrict.pit
Wed May 12 11:26:36 2010
BMDS Model Run
The form of the probability function is:
P[response] = background+(1-background)/[1+EXP(-intercept-slope*Log(dose))]
Dependent variable = Effect
Independent variable = Dose
Slope parameter is restricted as slope >= 1
Total number of observations = 4
Total number of records with missing values = 0
Maximum number of iterations =250
D-37
-------
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
User has chosen the log transformed model
Default Initial Parameter Values
background = 0.1
intercept = -4.33618
slope = 1
Asymptotic Correlation Matrix of Parameter Estimates
(*** The model parameter(s) -slope have been estimated at a boundary point, or have
been specified by the user, and do not appear in the correlation matrix)
background
intercept
background
1
-0.32
intercept
-0.32
1
Variable
background
intercept
slope
Estimate
0.105265
-3.90961
1
Parameter Estimates
Std. Err.
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
Indicates that this value is not calculated.
Model
Full model
Fitted model
Reduced model
Analysis of Deviance Table
Log(likelihood)
-84.3055
-86.107
-131.248
# Param's
4
2
1
Deviance Test d.f.
P-value
3.6029
93.8853
0.1651
<.0001
AIC:
176.214
Goodness of Fit
Scaled
0
66
278
964
Dose
.0000
.0000
.0000
.0000
Est
0.
0.
0.
0.
. Prob.
1053
6149
8639
9560
Expected
5
30
43
47
.263
.743
.194
.799
Observed
5.
35.
41.
46.
,000
,000
,000
,000
Size
50
50
50
50
Residual
-0
1
-0
-1
.121
.237
.905
.240
=3.90
d.f. = 2
P-value = 0.1421
Benchmark Dose Computation
Specified effect = 0.5
Risk Type = Extra risk
Confidence level = 0.95
BMD = 49.8797
BMDL = 32.9263
D-38
-------
Multistage Cancer Model with 0.95 Confidence Level
T3
CD
"o
I
0.8
0.6
0.4
0.2
Multistage Cancer
Linear extrapolation
BMDLBMD
200
400
600
800
1000
dose
11:2605/122010
Source: Used with permission from Elsevier, Ltd., Kano et al. (2009, 594539).
Figure D-13. Multistage BMD model (1 degree) for the combined incidence of
hepatic adenomas and carcinomas in female BDF1 mice.
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
L:\Priv\NCEA_HPAG\14Dioxane\BMDS\msc_kano2009_fmouse_hepato_adcar_Msc-BMR10-lpoly.(d)
Gnuplot Plotting File:
L:\Priv\NCEA_HPAG\14Dioxane\BMDS\msc_kano2009_fmouse_hepato_adcar_Msc-BMR10-lpoly.plt
Wed May 12 11:26:31 2010
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(-betal*dose/xl)]
The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Total number of observations = 4
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations =250
D-39
-------
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.51713
Beta(l) = 0.00201669
Asymptotic Correlation Matrix of Parameter Estimates
Background
Beta(l)
Variable
Background
Beta(l)
Background
1
-0.65
Beta(l)
-0. 65
1
Parameter Estimates
95.0% Wald Confidence Interval
Estimate
0.265826
0.00398627
Std. Err.
Lower Conf. Limit
*
Upper Conf. Limit
Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood)
-84.3055
-99.6653
-131.248
203.331
# Param' s
4
2
1
Deviance
30.7195
93.8853
Test d
2
3
Goodness of Fit
P-value
2.1346928e-007
<.0001
Dose
0.0000
66.0000
278.0000
964.0000
Est. Prob.
0.2658
0.4357
0.7576
0.9843
Expected
13.291
21.783
37.880
49.213
Observed
5.000
35.000
41.000
46.000
Size
50
50
50
50
Scaled
Residual
-2.654
3.770
1.030
-3.651
ChiA2 = 35.65
d.f. = 2
P-value = 0.0000
Benchmark Dose Computation
Specified effect = 0.1
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
Extra risk
0.95
26.4309
19.5045
37.5583
Taken together, (19.5045, 37.5583) is a 90% two-sided confidence interval for the BMD
Multistage Cancer Slope Factor = 0.00512702
D-40
-------
D.6. MALE BDF1 MICE: HEPATIC CARCINOMAS AND ADENOMAS
Data for hepatic carcinomas and adenomas in male BDF1 mice (Kano et al., 2009,
594539) are shown in Table D-14. Note that the incidence of carcinomas and the incidence of
either adenomas or carcinomas are monotone non-decreasing functions of dose. These data
therefore appear to be appropriate for dose-response modeling using BMDS. However, the
incidence of adenomas clearly reaches a peak value at 191 mg/kg-day and then decreases sharply
with increasing dose. This cannot be modeled by a multistage model using only non-negative
coefficients. To some extent the incidence of "either adenomas or carcinomas or both" retains
some of the inverted-U shaped dose-response of the adenomas, which dominate based on their
high incidence at the lowest dose groups (49 and 191 mg/kg-day), thus is not well characterized
by any multistage model.
Table D-14. Data for hepatic adenomas and carcinomas in male BDF1 mice
(Kano et al., 2009, 594539)
Tumor type
Hepatocellular adenomas
Hepatocellular carcinomas
Either adenomas or carcinomas
Neither adenomas nor carcinomas
Total number per group
Dose (mg/kg-day)
0
9
15
23
27
50
49
17
20
31
19
50
191
23
23
37
13
50
677
11
36
40
10
50
Source: Used with permission from Elsevier, Ltd., Kano et al. (2009, 594539).
The results of the BMDS modeling for the entire suite of models for hepatic adenomas
and carcinomas in male BDF1 mice are presented in Table D-15.
D-41
-------
Table D-15. BMDS dose-response modeling results for the combined
incidence of hepatic adenomas and carcinomas in male BDF1 mice (Kano et
al., 2009, 594539)
Model
Gamma
Logistic
LogLogisticb
LogProbif
Multistage-Cancer
(1 degree)
Multistage-Cancer
(2 degree)
Multistage-Cancer
(3 degree)
Probit
Weibull
Quantal-Linear
Dichotomous-Hill
AIC
250.551
251.187
248.839
252.244
250.551
250.551
250.551
251.326
250.551
250.551
250.747
/7-value
0.1527
0.112
0.3461
0.0655
0.1527
0.1527
0.1527
0.1048
0.1527
0.1527
NCd
BMD10
mg/kg-day
70.99
91.89
34.78
133.53
70.99
70.99
70.99
97.01
70.99
70.99
11.60
BMDL10
mg/kg-day
44.00
61.98
16.60
78.18
44.00
44.00
44.00
67.36
44.00
44.00
1.63
x2a
0.605
0.529
0.656
0.016
0.605
0.605
0.605
0.518
0.605
0.605
-1.25xlO'5
BMDjoHED
mg/kg-day
11.48
14.86
5.63
21.60
11.48
11.48
11.48
15.69
11.48
11.48
1.88
BMDLjoHED
mg/kg-day
7.12
10.02
2.68
12.64
7.12
7.12
7.12
10.90
7.12
7.12
0.26
"Maximum absolute % residual deviation between observed and predicted count. Values much larger than 1 are
undesirable.
bBest-fitting model.
°Slope restricted > 1.
Value unable to be calculated (NC: not calculated) by BMDS.
D-42
-------
Log-Logistic Model with 0.95 Confidence Level
0.9
I
0.7
0.6
0.5
0.4
0.3
Log-Logistic
BMDL BMD
100
200
300
400
500
600
700
dose
07:30 10/26 2009
Source: Used with permission from Elsevier, Ltd., Kano et al. (2009, 594539).
Figure D-14. LogLogistic BMD model for the combined incidence of hepatic
adenomas and carcinomas in male BDF1 mice.
Logistic Model. (Version: 2.12; Date: 05/16/2008)
Input Data File:
L:\Priv\NCEA_HPAG\14Dioxane\BMDS\lnl_kano2009_inmouse_hepato_adcar_Lnl-BMR10-
Restrict.(d)
Gnuplot Plotting File:
L:\Priv\NCEA_HPAG\14Dioxane\BMDS\lnl_kano2009_mmouse_hepato_adcar_Lnl-BMR10-
Restrict.pit
Thu Nov 12 09:09:36 2009
BMDS Model Run
The form of the probability function is:
P[response] = background+(1-background)/[1+EXP(-intercept-siope*Log(dose))]
Dependent variable = Effect
Independent variable = Dose
Slope parameter is restricted as slope >= 1
Total number of observations = 4
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
User has chosen the log transformed model
D-43
-------
Default Initial Parameter Values
background = 0.46
intercept = -5.58909
slope = 1
Asymptotic Correlation Matrix of Parameter Estimates
(*** The model parameter(s) -slope have been estimated at a boundary point, or have
been specified by the user, and do not appear in the correlation matrix )
background
intercept
background
1
-0.69
intercept
-0. 69
1
Variable
background
intercept
slope
Estimate
0.507468
-5.74623
1
Parameter Estimates
Std. Err.
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-121.373
-122.419
-128.859
# Param's
4
2
1
Deviance Test d.f.
2.09225
14.9718
P-value
0.3513
0.001841
AIC:
248.839
0
49
191
677
Dose
.0000
.0000
.0000
.0000
Est
0.
0.
0.
0.
. Prob.
5075
5741
6941
8443
Exp
25
28
34
42
Gooc
ected
.373
.707
.706
.214
inesj
01
23.
31.
37.
40.
3 Of Fit
:> served
,000
,000
,000
,000
Size
50
50
50
50
S
Re
-0
0
0
-0
caled
si dual
.671
.656
.704
.863
ChiA2 =2.12
d.f. = 2
P-value = 0.3461
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 34.7787
BMDL = 16.5976
D-44
-------
Multistage Cancer Model with 0.95 Confidence Level
0.9
0.8
0.7
0.6
0.5
0.4
0.3
Multistage Cancer
Linear extrapolation
BMDL
BMD
100
200
300
400
500
600
700
dose
07:30 10/26 2009
Source: Used with permission from Elsevier, Ltd., Kano et al. (2009, 594539).
Figure D-15. Multistage BMD model (1 degree) for the combined incidence of
hepatic adenomas and carcinomas in male BDF1 mice.
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
L:\Priv\NCEA_HPAG\14Dioxane\BMDS\msc_kano2009_iranouse_hepato_adcar_Msc-BMR10-lpoly.(d)
Gnuplot Plotting File:
L:\Priv\NCEA_HPAG\14Dioxane\BMDS\msc_kano2009_iranouse_hepato_adcar_Msc-BMR10-lpoly.plt
Mon Oct 26 08:30:50 2009
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(-betal*dose/xl)]
The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Total number of observations = 4
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
D-45
-------
Background = 0.573756
Beta(l) = 0.00123152
Asymptotic Correlation Matrix of Parameter Estimates
Background Beta(l)
Background 1 -0.58
Beta(l) -0.58 1
Parameter Estimates
Variable
Background
Beta(1)
Estimate
0.545889
0.00148414
Std. Err.
*
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood)
-121.373
-123.275
-128.859
250.551
# Param' s
4
2
1
Deviance
3.80413
14.9718
Test d
2
3
P-value
0.1493
0.001841
0
49
191
677
Dose
.0000
.0000
.0000
.0000
Est
0.
0.
0.
0.
. Prob.
5459
5777
6580
8337
Exp
27
28
32
41
Gooc
ected
.294
.887
.899
. 687
inesj
01
23.
31.
37.
40.
3 Of Fit
:> served
,000
,000
,000
,000
Size
50
50
50
50
S
Re
-1
0
1
-0
caled
si dual
.220
.605
.223
.641
= 3.76
d.f. = 2
P-value = 0.1527
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0.95
70.9911
44.0047
150.117
Taken together, (44.0047, 150.117) is a 90% two-sided confidence interval for the BMD
Multistage Cancer Slope Factor = 0.00227248
D-46
-------
D.7. BMD MODELING RESULTS FROM ADDITIONAL CHRONIC BIOASSAYS
Data and BMDS modeling results for the additional chronic bioassays (Kociba et al.,
1974, 062929: NCI, 1978, 062935) were evaluated for comparison with the Kano et al. (2009,
594539) study. These results are presented in the following sections.
The BMDS dose-response modeling estimates and HEDs that resulted are presented in
detail in the following sections and a summary is provided in Table D-16.
Table D-16. Summary of BMDS dose-response modeling estimates associated
with liver and nasal tumor incidence data resulting from chronic oral
exposure to 1,4-dioxane in rats and mice
Endpoint
Model
selection
criterion
Model
Type
AIC
P-
value
BMD10
mg/kg-
day
BMDL10
mg/kg-
day
BMD10HED
mg/kg-
day
BMDL10HED
mg/kg-day
Kociba et al., (1974, 0629291
Male and Female (combined) Sherman Rats
Hepatic
Tumors3
Nasal
Cavity
Tumorsb
Lowest
AIC
Lowest
AIC
Probit
Multistage
(3 degree)
84.3126
26.4156
0.606
0.9999
1113.94
1717.16
920.62
1306.29
290.78
448.24
240.31
340.99
NCI, (1978, 062935)
Female Osborne-Mendel Rats
Hepatic
Tumors0
Nasal
Cavity
Tumorsb
Lowest
AIC
Lowest
AIC
LogLogistic
LogLogistic
84.2821
84.2235
0.7333
0.2486
111.46
155.32
72.41
100.08
28.75
40.07
18.68
25.82
NCI, (1978, 062935)
Male Osborne-Mendel Rats
Nasal
Cavity
Tumorsb
Lowest
AIC
LogLogistic
92.7669
0.7809
56.26
37.26
16.10
10.66
NCI, (1978, 062935)
Female B6C3FJ Mice
Hepatic
Tumorsd
Lowest
AIC,
Multistage
model
Multistage
(2 degree)
85.3511
1
160.68
67.76
23.12
9.75
NCI, (1978, 062935)
Male B6C3FJ Mice
Hepatic
Tumors'1
Lowest
AIC
Gamma
177.539
0.7571
601.69
243.92
87.98
35.67
Incidence of hepatocellular carcinoma.
blncidence of nasal squamous cell carcinoma.
Incidence of hepatocellular adenoma.
Incidence of hepatocellular adenoma or carcinoma.
D-47
-------
D.7.1. Hepatocellular Carcinoma and Nasal Squamous Cell Carcinoma (Kociba et al., 1974,
062929)
The incidence data for hepatocellular carcinoma and nasal squamous cell carcinoma are
presented in Table D-17. The predicted BMDio HED and BMDLio HED values are also presented in
Tables D-18 and D-19 for hepatocellular carcinomas and nasal squamous cell carcinomas,
respectively.
Table D-17. Incidence of hepatocellular carcinoma and nasal squamous cell
carcinoma in male and female Sherman rats (combined) (Kociba et al., 1974,
062929) treated with 1,4-dioxane in the drinking water for 2 years
Animal Dose (mg/kg-day)
(average of male and female dose)
0
14
121
1307
Incidence of hepatocellular
carcinoma3
l/106b
0/110
1/106
10/66d
Incidence of nasal
squamous cell carcinoma"
0/106C
0/110
0/106
3/66d
"Rats surviving until 12 months on study.
bp < 0.001; positive dose-related trend (Cochran-Armitage test).
°p < 0.01; positive dose-related trend (Cochran-Armitage test).
dp < 0.001; Fisher's Exact test.
Source: Used with permission from Elsevier, Ltd., Kociba et al. (1974, 062929).
D-48
-------
Table D-18. BMDS dose-response modeling results for the incidence of
hepatocellular carcinoma in male and female Sherman rats (combined)
(Kociba et al., 1974, 062929) exposed to 1,4-dioxane in the drinking water for
2 years
Model
Gamma
Logistic
LogLogistic
LogProbitb
Multistage-Cancer
(1 degree)
Multistage-Cancer
(2 degree)
Multistage-Cancer
(3 degree)
Probif
Weibull
Quantal-Linear
Dichotomous-Hill
AIC
86.2403
84.3292
86.2422
84.4246
85.1187
86.2868
86.2868
84.3126
86.2443
85.1187
1503.63
/7-value
0.3105
0.6086
0.3103
0.5977
0.3838
0.3109
0.3109
0.606
0.3104
0.3838
NCd
BMD10
mg/kg-day
985.13
1148.65
985.62
1036.97
940.12
1041.72
1041.72
1113.94
998.33
940.12
NCd
BMDL10
mg/kg-day
628.48
980.95
611.14
760.29
583.58
628.56
628.56
920.62
629.93
583.58
NCd
x2a
-0.005
-0.004
-0.005
-0.011
0.279
-0.006
-0.006
-0.005
-0.005
0.279
0
BMD10HED
mg/kg-day
257.15
299.84
257.28
270.68
245.40
271.92
271.92
290.78
260.60
245.40
0
BMDLiOHED
mg/kg-day
164.05
256.06
159.53
198.46
152.33
164.07
164.08
240.31
164.43
152.33
0
aMaximum absolute ^ residual deviation between observed and predicted count. Values much larger than 1 are
undesirable.
bSlope restricted > 1.
'Best-fitting model.
Value unable to be calculated (NC: not calculated) by BMDS.
D-49
-------
Probit Model with 0.95 Confidence Level
T3
-------
slope = 0.0012323
Asymptotic Correlation Matrix of Parameter Estimates
(*** The model parameter(s) -background have been estimated at a boundary point, or
have been specified by the user, and do not appear in the correlation matrix )
intercept
slope
intercept
1
-0.82
slope
-0.82
1
Parameter Estimates
Variable
intercept
slope
95.0% Wald Confidence Interval
Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
-2.55961 0.261184 -3.07152 -2.0477
0.00117105 0.000249508 0.000682022 0.00166008
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-39.3891
-40.1563
-53.5257
Param's
4
2
1
Deviance Test d.f.
1.53445
28.2732
P-value
0.4643
<.0001
AIC:
1.3126
Goodness of Fit
Scaled
0
14
121
1307
Dose
.0000
.0000
.0000
.0000
Est
0.
0.
0.
0.
. Prob.
0052
0055
0078
1517
Expected
0.
0.
0.
10.
555
604
827
014
Observed
1.
0.
1.
10.
,000
,000
,000
,000
Size
106
110
106
66
Residual
0
-0
0
-0
.598
.779
.191
.005
ChiA2 =1.00
d.f. = 2
P-value = 0.6060
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
0.1
Extra risk
0.95
1113.94
920.616
D-51
-------
Multistage Cancer Model with 0.95 Confidence Level
T3
0
t3
c
o
t5
ro
0.25
0.2
0.15
0.1
0.05
Multistage Cancer
Linear extrapolation
BMDL
BMD
200
400
600
dose
800
1000
1200
11:54 10/27 2009
Source: Used with permission from Elsevier, Ltd., Kociba et al. (1974, 062929).
Figure D-17. Multistage BMD model (1 degree) for the incidence of
hepatocellular carcinoma in male and female Sherman rats exposed to
1,4-dioxane in drinking water.
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File: L:\Priv\NCEA_HPAG\14Dioxane\BMDS\msc_kociba_mf_rat_hepato_car_Msc-
BMRlO-lpoly.(d)
Gnuplot Plotting File:
L:\Priv\NCEA_HPAG\14Dioxane\BMDS\msc_kociba_mf_rat_hepato_car_Msc-BMR10-lpoly.plt
Tue Oct 27 12:54:10 2009
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(-betal*dose/xl)]
The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Total number of observations = 4
total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
D-52
-------
Default Initial Parameter Values
Background = 0.000925988
Beta(l) = 0.000124518
Asymptotic Correlation Matrix of Parameter Estimates
Background Beta(l)
Background 1 -0.44
Beta(l) -0.44 1
Parameter Estimates
Variable
Background
Beta(l)
Estimate
0.0038683
0.000112071
Std. Err.
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood)
-39.3891
-40.5594
-53.5257
85.1187
# Param' s
4
2
1
Deviance
2.34056
28.2732
Test d.f.
2
3
P-value
0.3103
<.0001
Goodness of Fit
Dose
0.0000
14.0000
121.0000
1307.0000
Est. Prob.
0.0039
0.0054
0.0173
0.1396
Expected
0.410
0.597
1.832
9.213
Observed
1.000
0.000
1.000
10.000
Size
106
110
106
66
Scaled
Residual
0.923
-0.775
-0.620
0.279
ChiA2 =1.92
d.f. = 2
P-value = 0.3838
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0.95
940.124
583.576
1685.88
Taken together, (583.576, 1685.88) is a 90% two-sided confidence interval for the BMD
Multistage Cancer Slope Factor = 0.000171357
D-53
-------
Table D-19. BMDS dose-response modeling results for the incidence of nasal
squamous cell carcinoma in male and female Sherman rats (combined)
(Kociba et al., 1974, 062929) exposed to 1,4-dioxane in the drinking water for
2 years
Model
Gamma
Logistic
LogLogistic
LogProbitb
Multistage-Cancer
(1 degree)
Multistage-Cancer
(2 degree)
Multistage-Cancer
(3 degree)0
Probit
Weibull
Quantal-Linear
Dichotomous-Hill
AIC
28.4078
28.4078
28.4078
28.4078
27.3521
26.4929
26.4156
28.4078
28.4078
27.3521
30.4078
/7-value
1
1
1
1
0.9163
0.9977
0.9999
1
1
0.9163
0.9997
BMD10
mg/kg-day
1572.09
1363.46
1464.77
1644.38
3464.76
1980.96
1717.16
1419.14
1461.48
3464.76
1465.77
BMDL10
mg/kg-day
1305.86
1306.67
1306.06
1305.49
1525.36
1314.37
1306.29
1306.44
1306.11
1525.35
1319.19
x23
0
0
0
0
0.272
0.025
0.002
0
0
0.272
5.53 xlO'7
BMDiOHED
mg/kg-day
410.37
355.91
382.35
429.24
904.42
517.10
448.24
370.44
381.50
904.42
382.62
BMDL10HED
mg/kg-day
340.87
341.09
340.93
340.78
398.17
343.10
340.99
341.03
340.94
398.17
344.35
aMaximum absolute ^ residual deviation between observed and predicted count. Values much larger than 1 are
undesirable.
bSlope restricted > 1.
'Best-fitting model.
D-54
-------
Multistage Cancer Model with 0.95 Confidence Level
c
o
'•8
ro
0.14
0.12
0.1
0.08
0.06
0.04
0.02
Multistage Cancer
Linear extrapolation
BMDL
BMC)
200
400
600
800 1000
dose
1200
1400
1600
1800
06:25 10/27 2009
Figure D-18. Multistage BMD model (3 degree) for the incidence of nasal
squamous cell carcinoma in male and female Sherman rats exposed to
1,4-dioxane in drinking water.
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File: L:\Priv\NCEA_HPAG\14Dioxane\BMDS\msc_kociba_mf_rat_nasal_car_Msc-
BMR10-3poly.(d)
Gnuplot Plotting File:
L:\Priv\NCEA_HPAG\14Dioxane\BMDS\msc_kociba_mf_rat_nasal_car_Msc-BMR10-3poly.plt
Tue Oct 27 07:25:02 2009
BMDS Model Run
The form of the probability function is:
P [response] = background + (1-background) * [1-EXP (-betal*dose/xl-beta2*dose/x2-
beta3*dose/"3) ]
The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Total number of observations = 4
Total number of records with missing values = 0
Total number of parameters in model = 4
Total number of specified parameters = 0
Degree of polynomial = 3
D-55
-------
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0
Beta(l) = 0
Beta(2) = 0
Beta(3) = 2.08414e-011
Asymptotic Correlation Matrix of Parameter Estimates
(*** The model parameter(s) -Background -Beta(l) -Beta(2)
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
Beta(3)
Beta(3)
Parameter Estimates
Variable
Background
Beta(l)
Beta(2)
Beta (3)
Estimate
0
0
0
2.08088e-011
Std. Err.
*
*
*
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood)
-12.2039
-12.2078
-17.5756
26.4156
# Param's
4
1
1
Deviance Test d.f.
P-value
0.00783284
10.7433
0.9998
0.0132
0
14
121
1307
Dose
.0000
.0000
.0000
.0000
Est
0.
0.
0.
0.
. Prob.
0000
0000
0000
0454
Expe
0.
0.
0.
2.
Good
cted
000
000
004
996
ness
01
0.
0.
0.
3.
3 Of Fit
:> served
,000
,000
,000
,000
Size
106
110
106
66
S
Re
0
-0
-0
0
caled
si dual
.000
.003
.063
.002
=0.00
d.f. = 3
P-value = 0.9999
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0.95
1717.16
1306.29
8354.46
Taken together, (1306.29, 8354.46) is a 90% two-sided confidence interval for the BMD
Multistage Cancer Slope Factor = 7.65529e-005
D-56
-------
D.7.2. Nasal Cavity Squamous Cell Carcinoma and Liver Hepatocellular Adenoma in
Osborne-Mendel Rats (NCI, 1978, 062935)
The incidence data for hepatocellular adenoma (female rats) and nasal squamous cell
carcinoma (male and female rats) are presented in Table D-20. The log-logistic model
adequately fit both the male and female rat nasal squamous cell carcinoma data, as well as
female hepatocellular adenoma incidence data. For all endpoints and genders evaluated in this
section, compared to the multistage models, the log-logistic model had a higher/»-value, as well
as both a lower AIC and lower BMDL. The results of the BMDS modeling for the entire suite of
models are presented in Tables D-21 through D-23.
Table D-20. Incidence of nasal cavity squamous cell carcinoma and
hepatocellular adenoma in Osborne-Mendel rats (NCI, 1978, 062935)
exposed to 1,4-dioxane in the drinking water
Male rat Animal Dose (mg/kg-day)a
Nasal cavity squamous cell carcinoma
0
0/3 3C
240b
12/26d
530
16/33d
Female rat Animal Dose (mg/kg-day)a
Nasal cavity squamous cell carcinoma
Hepatocellular adenoma
0
0/34C
0/3 lc
350
10/30d
10/30d
640
8/29d
ll/29d
"Tumor incidence values were adjusted for mortality (animals surviving to 52 weeks, presented in text of
NCI, 1978, 062935).
bGroup not included in statistical analysis by NCI (1978, 062935) because the dose group was started a
year earlier without appropriate controls.
°p < 0.001; positive dose-related trend (Cochran-Armitage test).
dp < 0.001; Fisher's Exact test.
Source: NCI (1978, 062935).
D-57
-------
Table D-21. BMDS dose-response modeling results for the incidence of
hepatocellular adenoma in female Osborne-Mendel rats (NCI, 1978, 062935)
exposed to 1,4-dioxane in the drinking water for 2 years
Model
Gamma
Logistic
LogLogisticb
LogProbit
Multistage-Cancer
(1 degree)
Multistage-Cancer
(2 degree)
Probit
Weibull
Quantal-Linear
AIC
84.6972
92.477
84.2821
85.957
84.6972
84.6972
91.7318
84.6972
84.6972
/7-value
0.5908
0.02
0.7333
0.3076
0.5908
0.5908
0.0251
0.5908
0.5908
BMD10
mg/kg-day
132.36
284.09
111.46
209.47
132.36
132.36
267.02
132.36
132.36
BMDL10
mg/kg-day
94.06
220.46
72.41
160.66
94.06
94.06
207.18
94.06
94.06
^
0
1.727
0
1.133
0
0
1.7
0
0
BMD10HED
mg/kg-day
34.144
73.29
28.75
54.04
34.14
34.14
68.88
34.14
34.14
BMDL10HED
mg/kg-day
24.26
56.87
18.68
41.45
24.26
24.26
53.44
24.26
24.26
"Maximum absolute
undesirable.
'Best-fitting model.
residual deviation between observed and predicted count. Values much larger than 1 are
D-58
-------
Log-Logistic Model with 0.95 Confidence Level
c
o
'•8
ro
0.5
0.4
0.3
0.2
0.1
Log-Logistic
BMDL
BMD
100
200
300
dose
400
500
600
06:32 10/27 2009
Source: NCI (1978, 062935).
Figure D-19. LogLogistic BMD model for the incidence of hepatocellular
adenoma in female Osborne-Mendel rats exposed to 1,4-dioxane in drinking
water.
Logistic Model. (Version: 2.12; Date: 05/16/2008)
Input Data File: L:\Priv\NCEA_HPAG\14Dioxane\BMDS\lnl_nci_frat_hepato_ad_Lnl-BMR10-
Restrict.(d)
Gnuplot Plotting File: L:\Priv\NCEA_HPAG\14Dioxane\BMDS\lnl_nci_frat_hepato_ad_Lnl-
BMRlO-Restrict.plt
Tue Oct 27 07:32:13 2009
BMDS Model Run
The form of the probability function is:
P[response] = background+(1-background)/[1+EXP(-intercept-siope*Log(dose))]
Dependent variable = Effect
Independent variable = Dose
Slope parameter is restricted as slope >= 1
Total number of observations = 3
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
User has chosen the log transformed model
D-59
-------
Default Initial Parameter Values
background = 0
intercept = -6.62889
slope = 1
Asymptotic Correlation Matrix of Parameter Estimates
(*** The model parameter(s) -background -slope have been estimated at a boundary
point, or have been specified by the user, and do not appear in the correlation
matrix)
intercept
intercept
1
Variable
background
intercept
slope
Estimate
0
-6.91086
1
Parameter Estimates
95.1
Wald Confidence Interval
Std. Err.
Lower Conf. Limit Upper Conf. Limit
Indicates that this value is not calculated.
Model
Full model
Fitted model
Reduced model
Analysis of Deviance Table
Log(likelihood)
-40.8343
-41.141
-50.4308
Param' s
3
1
1
Deviance
0.613564
19.1932
Test d
2
2
P-value
0.7358
<.0001
AIC:
Dose
0.0000
350.0000
640.0000
Est. Prob.
0.0000
0.2587
0.3895
Goodness of Fit
Expected Observed Size
0.000
8.536
12.464
0.000
10.000
11.000
31
33
32
Scaled
Residual
0.000
0.582
-0.531
=0.62
d.f. = 2
P-value = 0.7333
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
0.1
Extra risk
0.95
111.457
72.4092
D-60
-------
Multistage Cancer Model with 0.95 Confidence Level
T3
O
0.5
0.4
0.3
0.2
0.1
Multistage Cancer
Linear extrapolation
BMDL
BMD
100
200
300
dose
400
500
600
06:32 10/27 2009
Source: NCI (1978, 062935).
Figure D-20. Multistage BMD model (1 degree) for the incidence of
hepatocellular adenoma in female Osborne-Mendel rats exposed to
1,4-dioxane in drinking water.
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File: L:\Priv\NCEA_HPAG\14Dioxane\BMDS\msc_nci_frat_hepato_ad_Msc-BMR10-
Ipoly.(d)
Gnuplot Plotting File: L:\Priv\NCEA__HPAG\14Dioxane\BMDS\msc_nci_frat_hepato_ad_Msc-
BMRlO-lpoly.plt
Tue Oct 27 07:32:16 2009
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(-betal*dose/xl)]
The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
D-61
-------
Default Initial Parameter Values
Background = 0.0385912
Beta(l) = 0.000670869
Asymptotic Correlation Matrix of Parameter Estimates
(*** The model parameter(s) -Background have been estimated at a boundary point, or
have been specified by the user, and do not appear in the correlation matrix)
Beta(l)
Beta(l)
1
Variable
Background
Beta(1)
Estimate
0
0.00079602
Parameter Estimates
95.0% Wald Confidence Interval
Std. Err. Lower Conf. Limit Upper Conf. Limit
Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood)
-40.8343
-41.3486
-50.4308
84.6972
# Param' s
3
1
1
Deviance
1.02868
19.1932
Test d.f.
2
2
P-value
0.5979
<.0001
Dose
0.0000
350.0000
640.0000
Est. Prob.
0.0000
0.2432
0.3992
Goodness of Fit
Expected Observed Size
0.000
8.024
12.774
0.000
10.000
11.000
31
33
32
Scaled
Residual
0.000
0.802
-0.640
=1.05
d.f. = 2
P-value = 0.5908
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0.95
132.359
94.0591
194.33
Taken together, (94.0591, 194.33 ) is a 90% two-sided confidence interval for the BMD
Multistage Cancer Slope Factor = 0.00106316
D-62
-------
Table D-22. BMDS dose-response modeling results for the incidence of nasal
cavity squamous cell carcinoma in female Osborne-Mendel rats (NCI, 1978,
062935) exposed to 1,4-dioxane in the drinking water for 2 years
Model
Gamma
Logistic
LogLogisticb
LogProbif
Multistage-Cancer
(1 degree)
Multistage-Cancer
(2 degree)
Probit
Weibull
Quantal-Linear
AIC
84.7996
92.569
84.2235
87.3162
84.7996
84.7996
91.9909
84.7996
84.7996
/7-value
0.1795
0.0056
0.2486
0.0473
0.1795
0.1795
0.0064
0.1795
0.1795
BMD10
mg/kg-day
176.28
351.51
155.32
254.73
176.28
176.28
328.46
176.28
176.28
BMDL10
mg/kg-day
122.27
268.75
100.08
195.76
122.27
122.27
251.31
122.27
122.27
x23
1.466
2.148
0
1.871
1.466
1.466
2.136
1.466
1.466
BMDjoHED
mg/kg-day
45.47
90.68
40.07
65.71
45.47
45.47
84.73
45.47
45.47
BMDLjoHED
mg/kg-day
31.54
69.33
25.82
50.50
31.54
31.54
64.83
31.54
31.54
"Maximum absolute
undesirable.
'Best-fitting model.
°Slope restricted > 1.
residual deviation between observed and predicted count. Values much larger than 1 are
D-63
-------
Log-Logistic Model with 0.95 Confidence Level
T3
0
c
o
t5
ro
0.5
0.4
0.3
0.2
0.1
Log-Logistic
BMDL
BMD
100
200
300
dose
400
500
600
06:30 10/272009
Source: NCI (1978, 062935).
Figure D-21. LogLogistic BMD model for the incidence of nasal cavity
squamous cell carcinoma in female Osborne-Mendel rats exposed to
1,4-dioxane in drinking water.
Logistic Model. (Version: 2.12; Date: 05/16/2008)
Input Data File: L:\Priv\NCEA_HPAG\14Dioxane\BMDS\lnl_nci_frat_nasal_car_Lnl-BMR10-
Restrict.(d)
Gnuplot Plotting File: L:\Priv\NCEA__HPAG\14Dioxane\BMDS\lnl_nci_frat_nasal_car_Lnl-
BMRlO-Restrict.plt
Tue Oct 27 07:30:09 2009
BMDS Model Run
The form of the probability function is:
P[response] = background+(1-background)/[1+EXP(-intercept-siope*Log(dose))]
Dependent variable = Effect
Independent variable = Dose
Slope parameter is restricted as slope >= 1
Total number of observations = 3
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
D-64
-------
User has chosen the log transformed model
Default Initial Parameter Values
background = 0
intercept = -6.64005
slope = 1
Asymptotic Correlation Matrix of Parameter Estimates
(*** The model parameter(s) -background -slope have been estimated at a boundary
point, or have been specified by the user, and do not appear in the correlation
matrix)
intercept
intercept
1
Variable
background
intercept
slope
Estimate
0
-7.24274
1
Parameter Estimates
Std. Err.
*
95.0% Wald Confidence Interval
Lower Conf. Limit
Indicates that this value is not calculated.
Upper Conf. Limit
*
Model
Full model
Fitted model
Reduced model
Analysis of Deviance Table
Log(likelihood)
-39.7535
-41.1117
-47.9161
# Param' s
3
1
1
Deviance
2.71651
16.3252
Test d.f.
2
2
P-value
0.2571
0.0002851
AIC:
1.2235
Dose
0.0000
350.0000
640.0000
Est. Prob.
0.0000
0.2002
0.3140
Goodness of Fit
Expected Observed Size
0.000
7.008
10.992
0.000
10.000
8.000
34
35
35
Scaled
Residual
0.000
1.264
-1.090
ChiA2 = 2.78
d.f. = 2
P-value = 0.2486
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
0.1
Extra risk
0.95
155.324
100.081
D-65
-------
Multistage Cancer Model with 0.95 Confidence Level
0.5
0.4
0.3
0.2
0.1
Multistage Cancer
Linear extrapolation
BMDL
BMD
100
200
300
dose
400
500
600
06:30 10/27 2009
Source: NCI (1978, 062935).
Figure D-22. Multistage BMD model (1 degree) for the incidence of nasal
cavity squamous cell carcinoma in female Osborne-Mendel rats exposed to
1,4-dioxane in drinking water.
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File: L:\Priv\NCEA_HPAG\14Dioxane\BMDS\msc_nci_frat_nasal_car_Msc-BMR10-
Ipoly.(d)
Gnuplot Plotting File: L:\Priv\NCEA__HPAG\14Dioxane\BMDS\msc_nci_frat_nasal_car_Msc-
BMRlO-lpoly.plt
Tue Oct 27 07:30:12 2009
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(-betal*dose/xl)]
The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
D-66
-------
Default Initial Parameter Values
Background = 0.0569154
Beta(l) = 0.00042443
Asymptotic Correlation Matrix of Parameter Estimates
(*** The model parameter(s) -Background have been estimated at a boundary point, or
have been specified by the user, and do not appear in the correlation matrix)
Beta(l)
Variable
Background
Beta(l)
Beta(l)
1
Estimate
0
0.000597685
Parameter Estimates
Std. Err.
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
* - Indicates that this value is not calculated.
Analysis of Deviance Table
Model Log (likelihood) # Param's
Full model
Fitted model
Reduced model
-39.7535
-41.3998
-47.9161
3
1
1
Deviance Test d.f.
3.29259
16.3252
2
2
P-value
0.1928
0.0002851
AIC:
1.7996
Dose
0.0000
350.0000
640.0000
Est. Prob.
0.0000
0.1888
0.3179
Goodness of Fit
Expected Observed Size
0.000
6.607
11.125
0.000
10.000
8.000
34
35
35
Scaled
Residual
0.000
1.466
-1.134
ChiA2 =3.44
d.f. = 2
P-value = 0.1795
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 176.281
BMDL = 122.274
BMDU = 271. 474
Taken together, (122.274, 271.474) is a 90% two-sided confidence interval for the BMD
Multistage Cancer Slope Factor = 0.000817837
D-67
-------
Table D-23. BMDS dose-response modeling results for the incidence of nasal
cavity squamous cell carcinoma in male Osborne-Mendel rats (NCI, 1978,
062935) exposed to 1,4-dioxane in the drinking water for 2 years
Model
Gamma
Logistic
LogLogisticb
LogProbif
Multistage-Cancer
(1 degree)
Multistage-Cancer
(2 degree)
Probit
Weibull
Quantal-Linear
AIC
93.6005
103.928
92.7669
95.0436
93.6005
93.6005
103.061
93.6005
93.6005
/7-value
0.5063
0.0061
0.7809
0.2373
0.5063
0.5063
0.0078
0.5063
0.5063
BMD10
mg/kg-day
73.94
179.05
56.26
123.87
73.94
73.94
168.03
73.94
73.94
BMDL10
mg/kg-day
54.724
139.26
37.26
95.82
54.72
54.72
131.61
54.72
54.72
x2a
0
2.024
0
1.246
0
0
2.024
0
0
BMD10HED
mg/kg-day
21.17
51.25
16.10
35.46
21.16
21.16
48.10
21.17
21.17
BMDLjoHED
mg/kg-day
15.66
39.86
10.66
27.43
15.66
15.66
37.67
15.66
15.66
"Maximum absolute
undesirable.
bBest-fitting model.
°Slope restricted > 1.
residual deviation between observed and predicted count. Values much larger than 1 are
D-68
-------
Log-Logistic Model with 0.95 Confidence Level
T3
i
0.7
0.6
0.5
0.4
0.3
0.2
0.1
500
06:27 10/27 2009
Source: NCI (1978, 062935).
Figure D-23. LogLogistic BMD model for the incidence of nasal cavity
squamous cell carcinoma in male Osborne-Mendel rats exposed to
1,4-dioxane in drinking water.
Logistic Model. (Version: 2.12; Date: 05/16/2008)
Input Data File: L:\Priv\NCEA_HPAG\14Dioxane\BMDS\lnl_nci_mrat_nasal_car_Lnl-BMR10-
Restrict.(d)
Gnuplot Plotting File: L:\Priv\NCEA__HPAG\14Dioxane\BMDS\lnl_nci_mrat_nasal_car_Lnl-
BMRlO-Restrict.plt
Tue Oct 27 07:27:57 2009
BMDS Model Run
The form of the probability function is:
P[response] = background+(1-background)/[1+EXP(-intercept-siope*Log(dose))]
Dependent variable = Effect
Independent variable = Dose
Slope parameter is restricted as slope >= 1
Total number of observations = 3
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
User has chosen the log transformed model
D-69
-------
Default Initial Parameter Values
background = 0
intercept = -6.08408
slope = 1
Asymptotic Correlation Matrix of Parameter Estimates
(*** The model parameter(s) -background -slope have been estimated at a boundary
point, or have been specified by the user, and do not appear in the correlation
matrix)
intercept
Variable
background
intercept
slope
intercept
1
Estimate
0
-6.2272
1
Parameter Estimates
95.0% Wald Confidence Interval
Std.
*
Err.
Lower Conf.
*
Limit
Upper Conf. Limit
*
Indicates that this value is not calculated.
Model
Full model
Fitted model
Reduced model
Analysis of Deviance Table
Log(likelihood)
-45.139
-45.3835
-59.2953
Param's
3
1
1
Deviance Test d.f.
0.488858
28.3126
P-value
0.7832
<.0001
AIC:
92
.7669
Goodness of Fit
Scaled
0
240
530
Dose
.0000
.0000
.0000
Est
0.
0.
0.
. Prob.
0000
3216
5114
Expected
0.
10.
17.
000
612
388
Observed
0.
12.
16.
,000
,000
,000
Size
33
33
34
Residual
0
0
-0
.000
.517
.476
ChiA2 = 0.49 d.f. = 2
Benchmark Dose Computation
P-value = 0.7809
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
0.1
Extra risk
0.95
56.2596
37.256
D-70
-------
Multistage Cancer Model with 0.95 Confidence Level
T3
o>
0.7
0.6
0.5
0.4
0.3
0.2
0.1
Multistage Cancer
Linear extrapolation
100
200
300
400
500
dose
06:28 10/27 2009
Source: NCI (1978, 062935).
Figure D-24. Multistage BMD model (1 degree) for the incidence of nasal
cavity squamous cell carcinoma in male Osborne-Mendel rats exposed to
1,4-dioxane in drinking water.
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File: L:\Priv\NCEA_HPAG\14Dioxane\BMDS\msc_nci_mrat_nasal_car_Msc-BMR10-
Ipoly.(d)
Gnuplot Plotting File: L:\Priv\NCEA__HPAG\14Dioxane\BMDS\msc_nci_mrat_nasal_car_Msc-
BMRlO-lpoly.plt
Tue Oct 27 07:28:00 2009
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(-betal*dose/xl)]
The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
D-71
-------
Background = 0.0578996
Beta(l) = 0.00118058
Asymptotic Correlation Matrix of Parameter Estimates
(*** The model parameter(s) -Background have been estimated at a boundary point, or
have been specified by the user, and do not appear in the correlation matrix)
Beta(l)
Variable
Background
Beta(1)
Beta (1)
1
Estimate
0
0.00142499
Parameter Estimates
95.0% Wald Confidence Interval
Std. Err. Lower Conf. Limit Upper Conf. Limit
* - Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood) # Param's Deviance Test d.f.
-45.139 3
-45.8002 1 1.32238 2
-59.2953 1 28.3126 2
93.6005
Goodness of Fit
P-value
0.5162
<.0001
Dose
0.0000
240.0000
530.0000
Est. Prob.
0.0000
0.2896
0.5301
Expected
0.000
9.558
18.024
Observed
0.000
12.000
16.000
Size
33
33
34
Scaled
Residual
-0.000
0.937
-0.695
ChiA2 = 1.36
d.f. = 2
P-value = 0.5063
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 73.9379
BMDL = 54.7238
BMDU = 103.07
Taken together, (54.7238, 103.07 ) is a 90% two-sided confidence interval for the BMD
Multistage Cancer Slope Factor = 0.00182736
D-72
-------
D.7.3. Hepatocellular Adenoma or Carcinoma in B6C3F! Mice (NCI, 1978, 062935)
The incidence data for hepatocellular adenoma or carcinoma in male and female
mice are presented in Table D-24. The 2-degree polynomial model (betas restricted > 0)
was the lowest degree polynomial that provided an adequate fit to the female mouse data
(Figure D-25), while the gamma model provided the best fit to the male mouse data
(Figure D-26). The results of the BMDS modeling for the entire suite of models are
presented in Tables D-25 and D-26 for the female and male data, respectively.
Table D-24. Incidence of hepatocellular adenoma or carcinoma in male and
female B6C3Fi mice (NCI, 1978, 062935) exposed to 1,4-dioxane in drinking
water
Male mouse Animal Dose (mg/kg-day)a
0
8/49b
720
19/50d
830
28/47c
Female mouse Animal Dose (mg/kg-day)a
0
0/50b
380
21/48C
860
35/37c
aTumor incidence values were not adjusted for mortality.
bp < 0.001, positive dose-related trend (Cochran-Armitage test).
°p < 0.001 by Fisher's Exact test pair-wise comparison with controls.
dp = 0.014.
Source: NCI (1978, 062935).
D-73
-------
Table D-25. BMDS dose-response modeling results for the combined
incidence of hepatocellular adenoma or carcinoma in female B6C3Fi mice
(NCI, 1978, 062935) exposed to 1,4-dioxane in the drinking water for 2 years
Model
Gamma
Logistic
LogLogistic
LogProbitb
Multistage-Cancer
(1 degree)
Multistage-Cancer
(2 degree)'
Probit
Weibull
Quantal-Linear
AIC
85.3511
89.1965
85.3511
85.3511
89.986
85.3511
88.718
85.3511
89.986
/7-value
1
0.0935
1
1
0.0548
1
0.1165
1
0.0548
BMD10
mg/kg-day
195.69
199.63
228.08
225.8
49.10
160.68
188.24
161.77
49.10
BMDL10
mg/kg-day
105.54
151.35
151.16
150.91
38.80
67.76
141.49
89.27
38.80
x23
0
0.675
0
0
0
0
-1.031
0
0
BMDjoHED
mg/kg-day
28.16
28.72
32.82
32.49
7.06
23.12
27.08
23.28
7.065
BMDLjoHED
mg/kg-day
15.19
21.78
21.75
21.71
5.58
9.75
20.36
12.84
5.58
aMaximum absolute ^ residual deviation between observed and predicted count. Values much larger than 1 are
undesirable.
bSlope restricted > 1.
"Best-fitting model.
D-74
-------
Multistage Cancer Model with 0.95 Confidence Level
T3
-------
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0
Beta(l) = 2.68591e-005
Beta(2) = 3.91383e-006
Asymptotic Correlation Matrix of Parameter Estimates
(*** The model parameter(s) -Background have been estimated at a boundary point, or
have been specified by the user, and do not appear in the correlation matrix)
Beta(l)
Beta(2)
Beta(l)
1
-0.92
Beta(2)
-0.92
1
Variable
Background
Beta(1)
Beta (2)
Estimate
0
2.686e-005
3.91382e-006
Parameter Estimates
Std. Err.
*
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood)
-40.6756
-40.6756
-91.606
85.3511
# Param's
3
2 3.20014e-010
1 101.861
Deviance Test d.f.
P-value
<.0001
Dose
0.0000
380.0000
860.0000
Est. Prob.
0.0000
0.4375
0.9459
Goodness of Fit
Expected Observed Size
0.000
21.000
35.000
0.000
21.000
35.000
50
48
37
Scaled
Residual
0.000
0.000
0.000
=0.00
d.f. = 1
P-value = 1.0000
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 160.678
BMDL = 67.7635
BMDU = 186.587
Taken together, (67.7635, 186.587) is a 90% two-sided confidence interval for the BMD
Multistage Cancer Slope Factor = 0.00147572
D-76
-------
Table D-26. BMDS dose-response modeling results for the combined
incidence of hepatocellular adenoma or carcinoma in male B6C3Fi mice
(NCI, 1978, 062935) exposed to 1,4-dioxane in drinking water
Model
Gammab
Logistic
LogLogistic
LogProbitd
Multistage-Cancer
(1 degree)
Multistage-Cancer
(2 degree)
Probit
Weibull
Quantal-Linear
AIC
177.539
179.9
179.443
179.443
180.618
179.483
179.984
179.443
180.618
/7-value
0.7571
0.1189
NCC
NCC
0.0762
0.1554
0.1128
NCC
0.0762
BMD10
mg/kg-day
601.69
252.66
622.39
631.51
164.29
354.41
239.93
608.81
164.29
BMDL10
mg/kg-day
243.92
207.15
283.04
305.44
117.37
126.24
196.90
249.71
117.37
x2a
-0.233
0.214
0
0
0.079
0.124
0.191
0
0.079
BMDjoHED
mg/kg-day
87.98
36.94
91.01
92.34
24.02
51.82
35.08
89.02
24.02
BMDLjoHED
mg/kg-day
35.67
30.29
41.39
44.66
17.16
18.46
28.79
36.51
17.16
""Maximum absolute ^ residual deviation between observed and predicted count. Values much larger than 1 are
undesirable.
bBest-fitting model.
0Value unable to be calculated (NC: not calculated) by BMDS.
dSlope restricted > 1.
D-77
-------
Gamma Multi-Hit Model with 0.95 Confidence Level
T3
0
c
o
'•8
ro
ul
0.7
0.6
0.5
0.4
0.3
0.2
0.1
Gamma Multi-Hit
i.
BMDL
BMD
100 200 300 400 500 600 700
dose
800
06:34 10/272009
Source: NCI (1978, 062935).
Figure D-26. Gamma BMD model for the incidence of hepatocellular
adenoma or carcinoma in male B6C3Fi mice exposed to 1,4-dioxane in
drinking water.
Gamma Model. (Version: 2.13; Date: 05/16/2008)
Input Data File: L:\Priv\NCEA_HPAG\14Dioxane\BMDS\gam_nci_mmouse_hepato_adcar_Gam-
BMRlO-Restrict.(d)
Gnuplot Plotting File:
L:\Priv\NCEA_HPAG\14Dioxane\BMDS\gam_nci_mmouse_hepato_adcar_Gam-BMR10-Restrict.plt
Tue Oct 27 07:34:35 2009
BMDS Model Run
The form of the probability function is:
P[response]= background+(1-background)*CumGamma[siope*dose,power],
where CumGamma(.) is the cummulative Gamma distribution function
Dependent variable = Effect
Independent variable = Dose
Power parameter is restricted as power >=1
Total number of observations = 3
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial (and Specified) Parameter Values
Background = 0.17
Slope = 0.000671886
Power = 1.3
D-78
-------
Asymptotic Correlation Matrix of Parameter Estimates
(*** The model parameter(s) -Power have been estimated at a boundary point, or have
been specified by the user, and do not appear in the correlation matrix)
Background
Slope
Variable
Background
Slope
Power
Background
1
-0.52
Estimate
0.160326
0.0213093
18
Slope
-0.52
1
Parameter Estimates
Std. Err.
0.0510618
0.000971596
NA
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
0.060247 0.260405
0.019405 0.0232136
NA - Indicates that this parameter has hit a bound implied by some ineguality
constraint and thus has no standard error.
Model
Full model
Fitted model
Reduced model
Analysis of Deviance Table
Log(likelihood)
-86.7213
-86.7693
-96.715
# Param's
3
2
1
Deviance Test d.f.
0.096042
19.9875
P-value
0.7566
<.0001
AIC:
177.539
Goodness of Fit
Dose
0.0000
720.0000
830.0000
Est. Prob.
0.1603
0.3961
0.5817
Expected
7.856
19.806
27.339
Observed
8.000
19.000
28.000
Size
49
50
47
Scaled
Residual
0.056
-0.233
0.196
= 0.10 d.f. = 1
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 601.692
BMDL = 243.917
P-value = 0.7571
D-79
-------
Multistage Cancer Model with 0.95 Confidence Level
T3
-------
Background =
Beta(l) =
Beta(2) =
0.131156
0
9.44437e-007
Asymptotic Correlation Matrix of Parameter Estimates
(*** The model parameter(s) -Beta(l) have been estimated at a boundary point, or have
been specified by the user, and do not appear in the correlation matrix)
Background
Beta(2)
Background
1
-0.72
Beta(2)
-0.72
1
Variable
Background
Beta(1)
Beta (2)
Estimate
0.1568
0
8.38821e-007
Parameter Estimates
Std. Err.
*
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
* - Indicates that this value is not calculated.
Model
Full model
Fitted model
Reduced model
AIC:
Analysis of Deviance Table
Log(likelihood)
-86.7213
-87.7413
-96.715
179.483
# Param's
3
2
1
Deviance Test d.f.
2.04001
19.9875
P-value
0.1532
<.0001
Goodness of Fit
Dose
0.0000
720.0000
830.0000
Est. Prob.
0.1568
0.4541
0.5269
Expected
7.683
22.707
24.764
Observed
8.000
19.000
28.000
Size
49
50
47
Scaled
Residual
0.124
-1.053
0.946
=2.02
d.f. = 1
P-value = 0.1554
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0.95
354.409
126.241
447.476
Taken together, (126.241, 447.476) is a 90% two-sided confidence interval for the BMD
Multistage Cancer Slope Factor = 0.000792138
D-81
-------
APPENDIX E. COMPARISON OF SEVERAL DATA REPORTS FOR THE JBRC
2-YEAR 1,4-DIOXANE DRINKING WATER STUDY
As described in detail in Section 4.2.1.2.6 of this Toxicological Review of1,4-Dioxane,
the JBRC conducted a 2-year drinking water study on the effects of 1,4-dioxane in both sexes of
rats and mice. The results from this study have been reported three times, once as conference
proceedings (Yamazaki et al., 1994, 196120), once as a detailed laboratory report (JBRC, 1998,
196240), and once as a published manuscript (Kano et al., 2009, 594539). After the External
Peer Review draft of the Toxicological Review of 1,4-Dioxane (U.S. EPA, 2009, 628630) had
been released, the Kano et al. (2009, 594539) manuscript was published; thus, minor changes to
the Toxicological Review of 1,4-Dioxane occurred.
The purpose of this appendix is to provide a clear and transparent comparison of the
reporting of this 2-year 1,4-dioxane drinking water study. The variations included: (1) the level
of detail on dose information reported; (2) categories for incidence data reported (e.g., all
animals or sacrificed animals); and (3) analysis of non- and neoplastic lesions. Even though the
data contained in the reports varied, the differences were minor and did not did not significantly
affect the qualitative or quantitative cancer assessment.
Tables contained within this appendix provide a comparison of the variations in the
reported data (JBRC, 1998, 196240: Kano et al., 2009, 594539: Yamazaki et al., 1994, 196120).
Tables E-l and E-2 show the histological nonneoplastic findings provided for male and female
F344 rats, respectively. Tables E-3 and E-4 show the histological neoplastic findings provided
for male and female F344 rats, respectively. Tables E-5 and E-6 show the histological
nonneoplastic findings provided for male and female F344 rats, respectively. Tables E-7 and E-8
show the histological neoplastic findings provided for male and female Crj:BDFl mice,
respectively.
Note: Hyperlinks to the reference citations throughout this document will take you to the NCEA HERO database
(Health and Environmental Research Online) at http://epa. gov/hero. HERO is a database of scientific literature used
by U.S. EPA in the process of developing science assessments such as the Integrated Science Assessments (ISA)
and the Integrated Risk Information System (IRIS)
E-l
-------
Table E-l. Nonneoplastic lesions: Comparison of histological findings
reported for the 2-year JBRC drinking water study in male F344 rats
Nasal All animals
respiratory
epithelium; Sacrificed
nuclear animals
enlargement
Nasal All animals
respiratory
epithelium; Sacrificed
squamous cell animals
metaplasia
Nasal All animals
respiratory
epithelium; Sacrificed
squamous cell animals
hyperplasia
Nasal gland; All animals
proliferation
Sacrificed
animals
Nasal olfactory All animals
epithelium'
nuclear ' Sacrificed
enlargement animals
Nasal olfactory All animals
epithelium'
respiratory' Sacrificed
metaplasia animals
Nasal olfactory All animals
atrophy Sacrificed
animals
Lamina propria; All animals
change Sacrificed
animals
Lamina propria; All animals
Sacrificed
animals
Nasal.cavity; All animals
adhesion
Sacrificed
animals
Nasal cavity; All animals
inflammation
Sacrificed
animals
Hyperplasia; All animals
liver
Sacrificed
animals
Yamazaki et al. (1994)a
JBRC (1998)
Kanoetal.(2009)
Drinking water concentration (ppm)
0 200 1,000 5,000
0 200 1,000 5,000
0 200 1,000 5,000
Calculated Dose (Intake [mg/kg-day])b,c
Not reported
Not reported
Not reported
0/50 0/50 0/50 31/50
Not reported
0/50 0/50 0/50 2/50
Not reported
0/50 0/50 0/50 5/50
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
3/50 2/10 10/50 24/50
Not reported
Control 8-24 41-121 209-586
(0) (16) (81) (398)
0/50 0/50 0/50 26/50
0/40 0/45 0/35 12/22e
0/50 0/50 0/50 31/50
0/40 0/45 0/35 15/22e
0/50 0/50 0/50 2/50
0/40 0/45 0/35 1/22
Not reported
Not reported
0/50 0/50 5/50 38/50
0/40 0/45 4/35 20/22e
12/50 11/50 20/50 43/50
10/40 11/45 17/35 22/22e
0/50 0/50 0/50 36/50
0/40 0/45 0/35 17/22e
0/50 0/50 0/50 46/50
0/40 0/45 0/35 20/22e
0/50 0/50 1/50 44/50
0/40 0/45 1/35 20/22e
0/50 0/50 0/50 48/50
0/40 0/45 0/35 21/22e
0/50 0/50 0/50 13/50
0/40 0/45 0/35 7/22e
3/50 2/50 10/50 24/50
3/40 2/45 9/35f 12/22e
0 11±1 55±3 274±18
0/50 0/50 0/50 26/50e
Not reported
0/50 0/50 0/50 31/50e
Not reported
0/50 0/50 0/50 2/50
Not reported
Not reported
Not reported
0/50 0/50 5/50 38/50e
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
E-2
-------
Spongiosis All animals
hepatis' liver
Sacrificed
animals
Clear cell foci; All animals
liver
Sacrificed
animals
Acidophilic cell All animals
foci' liver
Sacrificed
animals
Basophilic cell All animals
Sacrificed
animals
Mixed-cell foci; All animals
Sacrificed
animals
Nuclear All animals
enlaroement'
kidney proximal Sacrificed
tubule animals
Yamazaki et al. (1994)"
12/50 20/50 25/50 40/50
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
JBRC (1998)
12/50 20/50 25/50 40/50
12/40 20/45 21/35f 21/22e
3/50 3/50 9/50 8/50
3/40 3/45 9/35f 7/22e
Not reported
Not reported
7/50 11/50 6/50 16/50
7/40 11/45 6/35 8/22f
2/50 8/50 14/50 13/50
2/40 8/45 14/35e 22/22e
0/50 0/50 0/50 50/50
0/40 0/45 0/35 22/22e
Kanoetal.(2009)
Not reported
Not reported
3/50 3/50 9/50 8/50
Not reported
12/50 8/50 7/50 5/50
Not reported
7/50 11/50 8/50 16/50f
Not reported
2/50 8/50 14/50e 13/50e
Not reported
Not reported
Not reported
aDose rates (mg/kg-day) were not provided in Yamazaki et al. (1994). Drinking water concentrations of 1,4-dioxane were used to
identify the dose groups. Statistical test results were not reported.
bJBRC (1998,196240) reported an estimated chemical intake range (of doses) for the animals, and the midpoint of the range (shown
in parentheses) was used in the external peer review draft of this document (U.S. EPA, 2009, 628630).
°Kano et al. (2009,594539) reported a mean intake dose for each group ± standard deviation. The mean shown in this table was
used in this final document (U.S. EPA, 2010, 625580).
dJBRC did not report statistical significance for the "All animals" comparison.
ep < 0.01 by i2 test.
fp< 0.05 by i2 test.
Table E-2. Nonneoplastic lesions: Comparison of histological findings
reported for the 2-year JBRC drinking water study in female F344 rats
Nasal respiratory M wimiik
epithelium; nuclear cQ,,,.jfj,,pH
enlargement sacrmcea
emargemeni animals
Nasal respiratory All animals
squamous cell Sacrificed
metaplasia animals
Nasal respiratory All animals
squamous cell Sacrificed
hyperplasia animals
Nasal gland; All animals
Yamazaki et al. (1994)a
JBRC (1998)
Kanoetal.(2009)
Drinking water concentration (ppm)
0 200 1,000 5,000
0 200 1,000 5,000
0 200 1,000 5,000
Calculated Dose (Intake [mg/kg-day])b,c
Not reported
Not reported
Not reported
0/50 0/50 0/50 35/50
Not reported
0/50 0/50 0/50 5/50
Not reported
0/50 0/50 0/50 11/50
Control 12-29 56-149 307-720
(0) (21) (103) (514)
0/50 0/50 0/50 13/50
0/38 0/37 0/38 7/24e
0/50 0/50 0/50 35/50
0/38 0/37 0/38 18/24e
0/50 0/50 0/50 5/50
0/38 0/37 0/38 4/24f
0/50 0/50 0/50 11/50
0 18±3 83±14 429±69
0/50 0/50 0/50 13/50e
Not reported
0/50 0/50 0/50 35/50e
Not reported
0/50 0/50 0/50 5/50
Not reported
Not reported
E-3
-------
proliferation Sacrificed
animals
Nasal olfactory M wimals
epitnelium; nuclear carrifjrPH
enlargement JHf1
Nasal olfactory All animals
respiratory Sacrificed
metaplasia animals
, , „ All animals
epithelium; atrophy Sacrificed
animals
All animals
hydropic change Sacrificed
animals
All animals
slerosis Sacrificed
animals
„ , . All animals
adhesion Sacrificed
animals
„ , . All animals
inflammation Sacrificed
animals
All animals
Liver; nyperpiasia Sacriflced
animals
All animals
hepatis Sacrificed
animals
All animals
formation Sacrificed
animals
All animals
Liver; dear ceil toci Sacriflced
animals
., ,.,. All animals
cell foci Sacrificed
animals
All animals
cell foci Sacrificed
animals
. , „ All animals
foci ' Sacrificed
animals
Kidney proximal M wimals
tubule; nuclear CarrifWH
enlargement Sjf
Yamazaki et al. (1994)a
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
3/50 2/50 11/50 47/50
Not reported
0/50 0/50 1/50 20/50
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
JBRC (1998)
0/38 0/37 0/38 8/24e
0/50 0/50 28/50 39/50
0/38 0/37 24/38e 22/24e
2/50 0/50 2/50 42/50
1/38 0/37 1/38 24/24e
0/50 0/50 1/50 40/50
0/38 0/37 1/38 22/24e
0/50 0/50 0/50 46/50
0/38 0/37 0/38 23/24e
0/50 0/50 0/50 48/50
0/38 0/37 0/38 23/24e
0/50 0/50 0/50 46/50
0/38 0/37 0/38 24/24e
0/50 0/50 1/50 15/50
0/38 0/37 1/38 7/24e
3/50 2/50 11/50 47/50
2/38 2/37 9/38 24/24e
0/50 0/50 1/50 20/50
0/38 0/37 1/38 14/24e
0/50 1/50 1/50 8/50
0/38 1/37 0/38 5/24f
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
1/50 1/50 3/50 11/50
1/38 1/37 3/38 7/24f
0/50 0/50 6/50 39/50
0/38 0/37 6/38 22/24e
Kanoetal.(2009)
Not reported
0/50 0/50 28/50e 39/50e
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
1/50 1/50 5/50 4/50
Not reported
1/50 1/50 1/50 1/50
Not reported
23/50 27/50 31/50 8/50e
Not reported
1/50 1/50 3/50 ll/50f
Not reported
Not reported
Not reported
E-4
-------
Yamazaki et al. (1994)a
JBRC (1998)
Kanoetal.(2009)
aDose rates (mg/kg-day) were not provided in Yamazaki et al. (1994). Drinking water concentrations of 1,4-dioxane were used to identify
the dose groups. Statistical test results were not reported.
bJBRC (1998,196240) reported an estimated chemical intake range (of doses) for the animals; and the midpoint of the range (shown in
parentheses) was used in the external peer review draft of this document (U.S. EPA, 2009, 628630).
°Kano et al. (2009, 594539) reported a mean intake dose for each group ± standard deviation. The mean shown in this table was used in
this final document (U.S. EPA, 2010, 625580).
dJBRC did not report statistical significance for the "All animals" comparison.
ep< 0.01 by i2 test.
fp< 0.05 by i2 test.
Table E-3. Neoplastic lesions: Comparison of histological findings reported
for the 2-year JBRC drinking water study in male F344 rats
Yamazaki et al. (1994)a
JBRC (1998)
Kano et al. (2009)
Drinking water concentration (ppm)
0 200 1,000 5,000
0 200 1,000 5,000
0 200 1,000 5,000
Calculated Dose (Intake [mg/kg-day])b'c
Not reported
Control 8-24 41-121 209-586
(0) (16) (81) (398)
0 11±1 55±3 274±18
Nasal cavity
Sauamous cell carcinoma M animals
Sacrificed
animals
Sarcoma NOS Allanimals
Sacrificed
animals
Rabdomvosarcoma Allanimals
Sacrificed
animals
Esthesioneuroepithelioma M ammals
Sacrificed
animals
0/50 0/50 0/50 3/50
Not reported
0/50 0/50 0/50 2/50
Not reported
0/50 0/50 0/50 1/50
Not reported
0/50 0/50 0/50 1/50
Not reported
0/50 0/50 0/50 3/50e
Not reported
0/50 0/50 0/50 2/50
Not reported
0/50 0/50 0/50 1/50
Not reported
0/50 0/50 0/50 1/50
Not reported
0/50 0/50 0/50 3/50e
Not reported
0/50 0/50 0/50 2/50
Not reported
0/50 0/50 0/50 1/50
Not reported
0/50 0/50 0/50 1/50
Not reported
Liver
Hepatocellular adenoma M animals
Sacrificed
animals
Heoatocellular carcinoma Allammals
Sacrificed
animals
0/50 2/50 4/50 24/50
Not reported
0/50 0/50 0/50 14/50
Not reported
0/50 2/50 4/49 24/50d'e
Not reported
0/50 0/50 0/49 14/50d'e
Not reported
3/50 4/50 7/50 32/50d'e
Not reported
0/50 0/50 0/50 14/50d'e
Not reported
E-5
-------
All animals
Hepatocellular adenoma
or carcinoma Sacrificed
animals
Yamazaki et al. (1994)a
Not reported
Not reported
JBRC (1998)
0/50 2/50 4/49 33/50d'e
Not reported
Kano et al. (2009)
3/50 4/50 7/50 39/50d'e
Not reported
Tumors at other sites
Peritoneum All animals
Sacrificed
animals
Subcutis fibroma Allanimals
Sacrificed
animals
Mammary gland All animals
Sacrificed
animals
Mammary gland All animals
Sacrificed
animals
Mammary gland Allanimals
fibroadenoma _ ... ,
or adenoma Sacrificed
animals
2/50 2/50 5/50 28/50
Not reported
5/50 3/50 5/50 12/50
Not reported
1/50 1/50 0/50 4/50
Not reported
0/50 0/50 0/50 0/50
Not reported
Not reported
Not reported
2/50 2/50 5/50 28/50d'e
Not reported
5/50 3/50 5/50 12/50e
Not reported
1/50 1/50 0/50 4/50e
Not reported
Not reported
Not reported
Not reported
Not reported
2/50 2/50 5/50 28/50d'e
Not reported
5/50 3/50 5/50 12/50e
Not reported
1/50 1/50 0/50 4/50e
Not reported
0/50 1/50 2/50 2/50
Not reported
1/50 2/50 2/50 6/50e
Not reported
"Dose rates (mg/kg-day) were not provided in Yamazaki et al. (1994). Drinking water concentrations of 1,4-dioxane were used to
identify the dose groups. Statistical test results were not reported.
bJBRC (1998,196240) reported an estimated chemical intake range (of doses) for the animals; and the midpoint of the range (shown in
parentheses) was used in the external peer review draft of this document (U.S. EPA, 2009, 628630).
°Kano et al. (2009, 594539) reported a mean intake dose for each group ± standard deviation. The mean shown in this table was used in
this final document (U.S. EPA, 2010, 625580).
dp < 0.01 by Fisher's Exact test.
'Significantly increased by Peto test for trend p < 0.01.
Table E-4. Neoplastic lesions: Comparison of histological findings
reported for the 2-year JBRC drinking water study in female F344 rats
Yamazaki et al. (1994)a
JBRC (1998)
Kano etal. (2009)
Drinking water concentration (ppm)
0 200 1,000 5,000
0 200 1,000 5,000
0 200 1,000 5,000
Calculated Dose (Intake [mg/kg-day])b,c
Not Reported
Control 12-29 56-149 307-720
(0) (21) (103) (514)
0 18±3 83±14 429±69
Nasal cavity
Squamous cell All animals
carcinoma
Sacrificed
animals
Sarcoma NOS Allanimals
Sacrificed
animals
0/50 0/50 0/50 7/50
Not reported
0/50 0/50 0/50 0/50
Not reported
0/50 0/50 0/50 7/50d'f
Not reported
Not reported
Not reported
0/50 0/50 0/50 7/50e'f
Not reported
0/50 0/50 0/50 0/50
Not reported
E-6
-------
Rabdomyosarcoma All animals
Sacrificed
animals
Esthesioneuroepithelio All animals
ma
Sacrificed
animals
Yamazaki et al. (1994)a
0/50 0/50 0/50 0/50
Not reported
0/50 0/50 0/50 1/50
Not reported
JBRC (1998)
Not reported
Not reported
0/50 0/50 0/50 1/50
Not reported
Kanoetal.(2009)
0/50 0/50 0/50 0/50
Not reported
0/50 0/50 0/50 1/50
Not reported
Liver
Hepatocellular All animals
Sacrificed
animals
Hepatocellular All animals
Sacrificed
animals
Hepatocellular . All animals
adenoma or carcinoma
Sacrificed
animals
1/50 0/50 5/50 38/50
Not reported
0/50 0/50 0/50 10/50
Not reported
Not reported
Not reported
1/50 0/50 5/50 38/50e'f
Not reported
1/50 0/50 0/50 10/50e'f
Not reported
1/50 0/50 5/50 40/50e'f
Not reported
3/50 1/50 6/50 48/50e'f
Not reported
0/50 0/50 0/50 10/50e'f
Not reported
3/50 1/50 6/50 48/50e'f
Not reported
Tumors at other sites
Peritoneum All animals
mesothelioma
Sacrificed
animals
Subcutis fibroma All animals
Sacrificed
animals
Mammary gland All animals
Sacrificed
animals
Mammary gland All animals
Sacrificed
animals
Mammary gland All animals
fibro adenoma
or adenoma Sacrificed
animals
1/50 0/50 0/50 0/50
Not reported
0/50 2/50 1/50 0/50
Not reported
3/50 2/50 1/50 3/50
Not reported
6/50 7/50 10/50 16/50
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
6/50 7/50 10/50 16/50d'f
Not reported
Not reported
Not reported
1/50 0/50 0/50 0/50
Not reported
0/50 2/50 1/50 0/50
Not reported
3/50 2/50 1/50 3/50
Not reported
6/50 7/50 10/50 16/50d'f
Not reported
8/50 8/50 11/50 18/50d'f
Not reported
"Dose rates (mg/kg-day) were not provided in Yamazaki et al. (1994). Drinking water concentrations of 1,4-dioxane were used to identify
the dose groups. Statistical test results were not reported.
bJBRC (1998,196240) reported an estimated chemical intake range (of doses) for the animals; and the midpoint of the range (shown in
parentheses) was used in the external peer review draft of this document (U.S. EPA, 2009, 628630).
°Kano et al. (2009, 594539) reported a mean intake dose for each group ± standard deviation. The mean shown in this table was used in
this final document (U.S. EPA, 2010, 625580).
dp < 0.05 by Fisher's Exact test.
ep < 0.01 by Fisher's Exact test.
'Significantly increased by Peto test for trend p < 0.01.
E-7
-------
Table E-5. Nonneoplastic lesions: Comparison of histological findings
reported for the 2-year JBRC drinking water study in male CrjrBDFl mice
All
Nasal respiratory epithelium; animals
nuclear enlargement _ .- ,
Sacrificed
animals
All
Nasal olfactory epithelium; animals
nuclear enlargement _ .- ,
Sacrificed
animals
All
Nasal olfactory epithelium; animals
atrophy _ .- ,
Sacrificed
animals
All
animals
Nasal cavity inflammation
Sacrificed
animals
All
animals
Tracheal epithelium' atrophy
Sacrificed
animals
All
Tracheal epithelium; nuclear animals
enlargement _ .- ,
Sacrificed
animals
All
Bronhcial epithelium; nuclear animals
enlargement „ .- ,
Sacrificed
animals
All
animals
Bronchial epithelium' atrophy
Sacrificed
animals
All
Lung/bronchial; accumlation of animals
foamy cells „ .- ,
Sacrificed
animals
All
animals
Liver an°iectasis
Sacrificed
animals
Yamazaki et al. (1994)
JBRC (1998)d
Kanoetal.(2009)
Drinking water concentration (ppm)
0 500 2,000 8,000
0 500 2,000 8,000
0 500 2,000 8,000
Calculated Dose (Intake [mg/kg-day])b,c
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Control 37-94 144-358 451-1086
0 (66) (251) (768)
0/50 0/50 0/50 31/50
0/31 0/33 0/25 19/26e
0/50 0/50 9/50 49/50
0/31 0/33 7/25e 26/26e
0/50 0/50 1/50 48/50
0/31 0/33 0/25 26/26e
1/50 2/50 1/50 25/50
1/31 1/33 1/25 15/26e
0/50 0/50 0/50 42/50
0/31 0/33 0/25 24/26e
0/50 0/50 0/50 17/50
0/31 0/33 0/25 12/26e
0/50 0/50 0/50 41/50
0/31 0/33 0/25 24/26e
0/50 0/50 0/50 43/50
0/31 0/33 0/25 26/26e
1/50 0/50 0/50 27/50
1/31 0/33 0/25 22/26e
2/50 3/50 4/50 16/50
2/31 2/33 3/25 8/26f
0 49±5 191±21 677±74
0/500/50 0/50 31/50e
Not reported
0/500/50 9/50e 49/50e
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
E-8
-------
All
Kidney proximal tubule; animals
nuclear enlargement _ .- ,
Sacrificed
animals
All
animals
Testis' mineralization
Sacrificed
animals
Yamazaki et al. (1994)
Not reported
Not reported
Not reported
Not reported
JBRC (1998)d
0/50 0/50 0/50 39/50
0/31 0/33 0/25 22/26e
40/50 42/50 38/50 34/50
28/31 30/33 24/25f 21/26f
Kanoetal.(2009)
Not reported
Not reported
Not reported
Not reported
aDose rates (mg/kg-day) were not provided in Yamazaki et al. (1994). Drinking water concentrations of 1,4-dioxane were used to identify
the dose groups. Statistical test results were not reported.
bJBRC (1998,196240) reported an estimated chemical intake range (of doses) for the animals; and the midpoint of the range (shown in
parentheses) was used in the external peer review draft of this document (U.S. EPA, 2009, 628630).
°Kano et al. (2009, 594539) reported a mean intake dose for each group ± standard deviation. The mean shown in this table was used in
this final document (U.S. EPA, 2010, 625580).
dJBRC did not report statistical significance for the "All animals" comparison.
ep< 0.01 by i2 test.
fp< 0.05 by i2 test.
E-9
-------
Table E-6. Nonneoplastic lesions: Comparison of histological findings
reported for the 2-year JBRC drinking water study in female CrjrBDFl mice
Nasal respiratory M animals
epithelium; Nuclear CnrrifjrPH
e&argement Hf1
Nasal olfactory M animals
epitneimm; Nuclear Sacrificed
enlargement animals
All animals
epithelium; Atrophy Sacrificed
animals
, , „ All animals
epithelium; Atrophy Sacrificed
animals
All animals
Inflammation Sacrificed
animals
, , . , ,. All animals
Atrophy Sacrificed
animals
, . , . , ,. All animals
Nuclear enlargement ' Sacrificed
animals
All animals
Atrophy Sacrificed
animals
Luna/bronchial; M animals
ACCUinlatlOn Ot QarrifirpH
foamy cells JHf1
Kidnev proximal M animals
tubule; Nuclear QarrifirrH
enlargement aacrmced
eniargemem animals
Yamazaki et al. (1994)a
JBRC (1998)b
Kano et al. (2009)
Drinking water concentration (ppm)
0 500 2,000 8,000
0 500 2,000 8,000
0 500 2,000 8,000
Calculated Dose (Intake [mg/kg-day])b>c
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
rnntrn, 45- 192- 759-
Control 1Q9 454 13?4
u (77) (323) (1066)
0/50 0/50 0/50 41/50
0/29 0/29 0/17 5/5e
0/50 0/50 41/50 33/50
0/29 0/29 17/17e 1/5
0/50 0/50 0/50 26/50
0/29 0/29 0/17 1/5
0/50 0/50 1/50 42/50
0/29 0/29 0/17 5/5e
2/50 0/50 7/50 42/50
0/29 0/29 5/17e 5/5e
0/50 0/50 2/50 49/50
0/29 0/29 1/17 5/5e
0/50 1/50 22/50 48/50
0/29 1/29 13/17e 5/5e
0/50 0/50 7/50 50/50
0/29 0/29 3/17 5/5e
0/50 1/50 4/50 45/50
0/29 1/29 3/17 5/5e
0/50 0/50 0/50 8/50
0/29 0/29 0/17 0/5
n 66 ± 278 ± 964 ±
U 10 40 88
0/50 0/50 0/50 41/50e
Not reported
0/50 0/50 41/50e 33/50e
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
aDose rates mg/kg-day]) were not provided in Yamazaki et al. (1994,196120). Drinking water concentrations (ppm) of 1,4-dioxane
were used to identify the dose groups. Statistical test results were not reported.
bStatistical analysis was not performed for data on 'All animals' in the JBRC (1998,196240) report.
°JBRC (1998,196240) reported an estimated chemical intake range (of doses) for the animals; and the midpoint of the range (shown in
parentheses) was used in the external peer review draft of this document (U.S. EPA, 2009, 628630).
dKano et al. (2009, 594539) reported a mean intake dose for each group ± standard deviation. The mean shown in this table was used in
this final document (U.S. EPA, 2010, 625580).
ep < 0.01 by chi-square test.
E-10
-------
Table E-7. Neoplastic lesions: Comparison of histological findings reported
for the 2-year JBRC drinking water study in male CrjrBDFl mice
Yamazaki et al. (1994)a
JBRC (1998)
Kano et al. (2009)
Drinking water concentration (ppm)
0 500 2,000 8,000
0 500 2,000 8,000
0 500 2,000 8,000
Calculated Dose (Intake [mg/kg-day])b>c
Not reported
Control 37-94 If J51-
0 (66) (251) (768)
0 49±5 191±21 677±74
Nasal cavity
All Animals
Esthesioneuroepithelioma _ .- ,
Sacrificed
animals
All Animals
Adenocarcinoma „ .- ,
Sacrificed
animals
0/50 0/50 0/50 1/50
Not reported
0/50 0/50 0/50 0/50
Not reported
0/50 0/50 0/50 1/50
Not reported
Not reported
Not reported
0/50 0/50 0/50 1/50
Not reported
0/50 0/50 0/50 0/50
Not reported
Liver
All Animals
Hepatocellular adenomas _ .- ,
Sacrificed
animals
All Animals
Hepatocellular carcinomas „ .- ,
Sacrificed
animals
All Animals
Either adenoma
or carcinoma Sacrificed
animals
7/50 16/50 22/50 8/50
Not reported
15/50 20/50 23/50 36/50
Not reported
Not reported
Not reported
7/50 16/50 22/50e 8/50
Not reported
15/50 20/50 23/50 36/50d'e
Not reported
21/50 31/50 37/50 39/50d'e
Not reported
9/50 17/50 23/50e 11/50
Not reported
15/50 20/50 23/50 36/50e'f
Not reported
23/50 31/50 37/50d 40/50e'f
Not reported
aDose rates (mg/kg-day) were not provided in Yamazaki et al. (1994). Drinking water concentrations of 1,4-dioxane were used to
identify the dose groups. Statistical test results were not reported.
bJBRC (1998,196240) reported an estimated chemical intake range (of doses) for the animals; and the midpoint of the range (shown in
parentheses) was used in the external peer review draft of this document (U.S. EPA, 2009, 628630).
°Kano et al. (2009, 594539) reported a mean intake dose for each group ± standard deviation. The mean shown in this table was used in
this final document (U.S. EPA, 2010, 625580).
dp < 0.05 by Fisher's Exact test.
'Significantly increased by Peto test for trend p < 0.01.
fp < 0.01 by Fisher's Exact test.
E-ll
-------
Table E-8. Neoplastic lesions: Comparison of histological findings reported
for the 2-year JBRC drinking water study in female CrjrBDFl mice
Yamazaki et al. (1994)a
JBRC (1998)
Kano et al. (2009)
Drinking water concentration (ppm)
0 500 2,000 8,000
0 500 2,000 8,000
0 500 2,000 8,000
Calculated Dose (Intake [mg/kg-day])b'c
Not reported
rnntrn, 45- 192- 759-
Control 1Q9 454 13?4
u (77) (323) (1066)
n 66 ± 278 ± 964 ±
U 10 40 88
Nasal Cavity
All animals
Esthesioneruoepithelioma Sacrificed
animals
All animals
Aaenocarcmoma Sacrificed
animals
0/50 0/50 0/50 0/50
Not reported
0/50 0/50 0/50 1/50
Not reported
Not reported
Not reported
0/50 0/50 0/50 1/50
Not reported
0/50 0/50 0/50 0/50
Not reported
0/50 0/50 0/50 1/50
Not reported
Liver
All animals
Hepatocellular adenomas Sacrificed
animals
All animals
Hepatocellular carcinomas Sacrificed
animals
All animals
or carcinoma Sacrificed
animals
4/50 30/50 20/50 2/50
Not reported
0/50 6/50 30/50 45/50
Not reported
Not reported
Not reported
4/50 30/50d 20/50d 2/50e
Not reported
0/50 6/50f 30/50d 45/50d'8
Not reported
4/50 34/50d 41/50d 46/50d'8
Not reported
5/50 31/50d 20/50d 3/50
Not reported
0/50 6/50f 30/50d 45/50d'8
Not reported
5/50 35/50d 41/50d 46/50d'8
Not reported
aDose rates (mg/kg-day) were not provided in Yamazaki et al. (1994,196120). Drinking water concentrations (ppm) of 1,4-dioxane
were used to identify the dose groups. Statistical test results were not reported.
bJBRC (1998,196240) reported an estimated chemical intake range (of doses) for the animals; and the midpoint of the range (shown in
parentheses) was used in the external peer review draft of this document (U.S. EPA, 2009, 628630).
GKano et al. (2009, 594539) reported a mean intake dose for each group ± standard deviation. The mean shown in this table was used
in this final document (U.S. EPA, 2010, 625580).
dp < 0.01 by Fisher's Exact test.
'Significantly decreased by Cochran-Armitage test for trend p < 0.05
f p < 0.05 by Fisher's Exact test.
Significantly increased by Peto test for trend p < 0.01
E-12
------- |