EPA/635/R-08/006F 

www.epa.gov/iris

TOXICOLOGICAL REVIEW 

OF 

ETHYLENE GLYCOL 

MONOBUTYL ETHER (EGBE) 

(CAS No. 111-76-2) 

In Support of Summary Information on the 

Integrated Risk Information System (IRIS) 

March 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. 

 ii 
CONTENTS—TOXICOLOGICAL REVIEW OF ETHYLENE GLYCOL MONOBUTYL 

ETHER (CAS No. 111-76-2) 

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

LIST OF FIGURES ................................................................................................................... VIII 

LIST OF ABBREVIATIONS AND ACRONYMS ..................................................................... IX 

FOREWORD................................................................................................................................ XI 

AUTHORS, CONTRIBUTORS, AND REVIEWERS ...............................................................XII 

1. INTRODUCTION ...................................................................................................................... 1 

2. CHEMICAL AND PHYSICAL INFORMATION .................................................................... 3 

3. TOXICOKINETICS ................................................................................................................... 4 

3.1. ABSORPTION AND DISTRIBUTION.............................................................................. 4 

3.2. METABOLISM AND ELIMINATION.............................................................................. 5 

4. HAZARD IDENTIFICATION................................................................................................. 15 

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

CONTROLS...................................................................................................................... 15 

4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN 

ANIMALS—ORAL AND INHALATION ...................................................................... 18 

4.2.1. Subchronic Studies...................................................................................................... 18 

4.2.1.1. Oral ...................................................................................................................... 18 

4.2.1.2. Inhalation ............................................................................................................. 24 

4.2.2. Chronic Studies and Cancer Bioassays....................................................................... 28 

4.2.2.1. Inhalation ............................................................................................................. 28 

4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND INHALATION ..... 33 

4.4. OTHER STUDIES............................................................................................................. 37 

4.4.1. Acute and Short-Term Exposure Studies.................................................................... 37 

4.4.2. Dermal Exposure Studies............................................................................................ 41 

4.4.3. Ocular Exposure Studies............................................................................................. 42 

4.4.4. Genotoxicity................................................................................................................ 43 

4.4.5. Immunotoxicity........................................................................................................... 46 

4.4.6. Other In Vitro Studies................................................................................................. 48 

4.5. SYNTHESIS AND EVALUATION OF MAJOR NONCANCER EFFECTS AND 

MODE OF ACTION: ORAL AND INHALATION ....................................................... 50 

4.6. EVALUATION OF CARCINOGENICITY ..................................................................... 55 

4.6.1. Summary of Overall Weight of Evidence................................................................... 55 

4.6.2. Synthesis of Human, Animal, and Other Supporting Evidence.................................. 56 

4.6.3. Mode-of-Action Information ...................................................................................... 58 

4.6.3.1. Hypothesized MOA for Liver Tumor Development in Male Mice..................... 58 

4.6.3.1.1. Temporal association and species specificity. .............................................. 60 

4.6.3.1.2. Dose-response relationships. ........................................................................ 61 

4.6.3.1.3. Biological plausibility and coherence of the database.................................. 62 

4.6.3.1.4. Relevance of the hypothesized MOA to humans.......................................... 64 

 iii 
4.6.3.1.5. Other possible MOAs for liver tumor development in male mice. .............. 65 

4.6.3.2. Hypothesized MOA for Forestomach Tumor Development in Female Mice ..... 67 

4.6.3.2.1. Temporal association. ................................................................................... 69 

4.6.3.2.2. Dose-response relationships. ........................................................................ 69 

4.6.3.2.3. Biological plausibility and coherence of the database.................................. 70 

4.6.3.2.4. Relevance of the hypothesized MOA to humans.......................................... 71 

4.6.3.2.5. Other possible MOAs for forestomach tumor development in female mice.71 

4.6.3.3. Conclusions about the Hypothesized MOAs....................................................... 72 

4.7. SUSCEPTIBLE POPULATIONS ..................................................................................... 73 

4.7.1. Possible Childhood Susceptibility .............................................................................. 75 

4.7.2. Possible Gender Differences....................................................................................... 76 

5. DOSE-RESPONSE ASSESSMENTS...................................................................................... 78 

5.1. INHALATION REFERENCE CONCENTRATION (RFC) ............................................ 78 

5.1.1. Choice of Principal Study and Critical Effect—with Rationale and Justification...... 78 

5.1.2. Methods of Analysis—Including Models (PBPK, BMD, etc.) .................................. 82 

5.1.2.1. BMD Approach Applied to Hemosiderin Staining Data ..................................... 83 

5.1.2.2. Selection of the POD ........................................................................................... 86 

5.1.3. RfC Derivation—Including Application of Uncertainty Factors................................ 87 

5.1.4. RfC Comparison Information ..................................................................................... 89 

5.1.5. Previous Inhalation Assessment ................................................................................. 90 

5.2. ORAL REFERENCE DOSE (RfD)................................................................................... 90 

5.2.1. Choice of Principal Study and Critical Effect—with Rationale and Justification...... 90 

5.2.2. Methods of Analysis—Including Models (PBPK, BMD, etc.) .................................. 93 

5.2.2.1. BMD Approach Applied to Hemosiderin Endpoint ............................................ 93 

5.2.2.2. Route-to-Route Extrapolation from Inhalation Data ........................................... 93 

5.2.2.3. Selection of the POD ........................................................................................... 93 

5.2.3. RfD Derivation—Including Application of UFs ........................................................ 94 

5.2.4. RfD Comparison Information ..................................................................................... 95 

5.2.5. Previous Oral Assessment........................................................................................... 96 

5.3. UNCERTAINTIES IN THE DERIVATION OF THE INHALATION REFERENCE 

CONCENTRATION (RfC) AND ORAL REFERENCE DOSE (RfD)........................... 97 

5.3.1. Choice of Endpoint ..................................................................................................... 98 

5.3.2. Choice of Dose Metric ................................................................................................ 98 

5.3.3. Use of BMC Approach ............................................................................................... 99 

5.3.4. Choice of Model for BMCL Derivations.................................................................... 99 

5.3.5. Choice of Animal to Human Extrapolation Method................................................... 99 

5.3.6. Route-to-Route Extrapolation..................................................................................... 99 

5.3.7. Statistical Uncertainty at the POD ............................................................................ 100 

5.3.8. Choice of Bioassay ................................................................................................... 100 

5.3.9. Choice of Species/Gender......................................................................................... 100 

5.3.10. Human Relevance of Noncancer Responses Observed in Mice............................. 101 

5.3.11. Human Population Variability ................................................................................ 101 

5.4. CANCER ASSESSMENT............................................................................................... 101 

5.4.1. Quantification for Oral and Inhalation Cancer Risk ................................................. 103 

5.4.2. Uncertainties in Cancer Risk Assessment................................................................. 103 

5.4.2.1. Choice of Low-Dose Extrapolation Method...................................................... 104 

5.4.2.2. Human Relevance of Cancer Responses Observed in Mice.............................. 105 

5.5. POTENTIAL IMPACT OF SELECT UNCERTAINTIES ON THE RFC..................... 105 

 iv 
6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HAZARD AND 

DOSE RESPONSE.................................................................................................. 108 

6.1. HUMAN HAZARD POTENTIAL.................................................................................. 108 

6.2. DOSE RESPONSE.......................................................................................................... 110 

6.2.1. Noncancer—Inhalation ............................................................................................. 110 

6.2.2. Noncancer—Oral ...................................................................................................... 111 

6.2.3. Cancer—Oral and Inhalation .................................................................................... 112 

7. REFERENCES ....................................................................................................................... 113 

APPENDIX A. SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC COMMENTS 

AND DISPOSITION............................................................................................... A-1 

APPENDIX B. PBPK MODELS (CORLEY ET AL.[1994;1997;2005]).................................. B-1 

APPENDIX C. RFD AND RFC DERIVATION OPTIONS...................................................... C-1 

 v 
LIST OF TABLES 

Table 2-1. Physical and chemical properties of EGBE...................................................................3 

Table 3-1. Summary of species-specific toxicokinetic parameters ................................................8 

Table 4-1. Hematology and hemosiderin data from the 13-week drinking water exposure to 

EGBE in F344 rats.....................................................................................................21 

Table 4-2. Incidencea and severity of selected histopathological changes from the 13-week 

drinking water exposure to EGBE in F344 rats and mice .........................................23 

Table 4-3. Hematology and hemosiderin data from a 14-week inhalation study of EGBE in F344 

rats..............................................................................................................................26 

Table 4-4. Hematology and hemosiderin data from a 14-week inhalation study of EGBE in 

B6C3F1 mice..............................................................................................................27 

Table 4-5. Selected female and male rat and mouse nonneoplastic effects from the 2-year 

chronic EGBE inhalation study .................................................................................29 

Table 4-6. Comparison of female and male rat and mouse Hct (manual) values from 3- and 12-

month inhalation exposures to EGBE........................................................................31 

Table 4-7. Summary of genotoxicity studies on EGBE, BAL, and BAA ....................................44 

Table 4-8. Incidence of liver hemangiosarcomas and hepatocellular carcinomas in studies of 

NTP chemicals that caused increased hemosiderin in Kupffer cells in male mice ...62 

Table 5-1. Results of candidate studies.........................................................................................79 

Table 5-2. Female and male rat and mouse liver hemosiderin staining incidence and RBC counts 

from subchronic and chronic EGBE inhalation studies.............................................80 

Table 5-3. Summary of PBPK models..........................................................................................83 

Table 5-4. AUC BAA blood concentrations measured at 12 months in both genders of B6C3F1

mice and F344 rats.....................................................................................................84 

Table 5-5. Comparison of BMC/BMCL values for male and female rat liver hemosiderin 

staining data from inhalation chronic study using measured blood AUC (12 months) 

of the EGBE metabolite BAA as a common dose metric..........................................85 

Table 5-6. Comparison of BMC/BMCL values for male and female mouse liver hemosiderin 

staining data from inhalation chronic study using measured blood AUC (12 months) 

of the EGBE metabolite BAA as a common dose metric..........................................86 

Table 5-7. Subchronic 91-day drinking water studies in rats and mice........................................91 

Table 5-8. Summary of uncertainty in the EGBE noncancer and cancer risk assessments..........97 

Table 5-9. Illustrative potency estimates for tumors in mice, using a linear analysis approach 104 

Table B-1. Selected parameters used in the PBPK model for EGBE developed by Corley et al. 

(1997, 041984; 2005, 100100).................................................................................B-6 

Table C-1. Summary of PBPK models.......................................................................................C-2 

Table C-2. Model estimates of BAA blood levels in female rats following inhalation 

exposures .................................................................................................................C-2 

 vi 
Table C-3. Estimated Cmax for BAA in blood for humans continuously exposed to varying 

concentrations of EGBE ..........................................................................................C-3 

Table C-4. Comparison of BMC/BMCL values for female rat RBC count data from a 14-week 

subchronic inhalation studya, using modeled blood Cmax (3 months) of the EGBE 

metabolite BAA as a common dose metric .............................................................C-5 

Table C-5. AUC BAA blood concentrations measured at 12 months in both genders of B6C3F1

mice and F344 rats.................................................................................................C-12 

Table C-6. Comparison of BMC/BMCL values for male and female rat liver hemosiderin 

staining data from inhalation chronic study using measured blood AUC (12 months) 

of the EGBE metabolite BAA as a common dose metric......................................C-13 

Table C-7. Comparison of BMC/BMCL values for male and female mouse liver hemosiderin 

staining data from inhalation chronic study using measured blood AUC (12 months) 

of the EGBE metabolite BAA as a common dose metric......................................C-14 

Table C-8. PBPK model estimates of BAA Cmax blood levels and incidence of forestomach 

epithelial hyperplasia in female mice ....................................................................C-22 

Table C-9. BMDS model estimates of Cmax BMD10 and BMDL10 values for forestomach 

epithelial hyperplasia in female mice ....................................................................C-23 

Table C-10. Female mouse Cmax values for various time points of the NTP (2000, 196293) study 

estimated by the Lee et al. (1998, 041983) model.................................................C-23 

Table C-11. Estimated Cmax for BAA in blood for humans continuously exposed to varying 

drinking water concentrations of EGBE................................................................C-24 

Table C-12. Estimated Cmax for BAA in blood for humans continuously exposed to varying 

concentrations of EGBE ........................................................................................C-24 

Table C-13. Modeled estimates of BAA in human blood exposed to EGBE in water..............C-28 

Table C-14. Model estimates of BAA blood levels in female rats following oral exposures ...C-29 

Table C-15. Comparison of female rat RBC count and MCV BMD/BMDL values from an oral 

subchronic study using modeled blood Cmax (3 months) of the EGBE metabolite 

BAA as a common dose metric .............................................................................C-30 

 vii 
LIST OF FIGURES 

Figure 3-1. Proposed metabolic scheme of EGBE in rats and humans. .........................................6 

Figure 4-1. Simulated concentrations of EGBE, BAL, and BAA in liver tissues of female mice 

exposed via inhalation for 6 hours to 250 ppm EGBE. .............................................66 

Figure 5-1. PODs for selected endpoints with corresponding applied UFs and derived RfC. .....89 

Figure 5-2. PODs for selected endpoints with corresponding applied UFs and derived RfD. .....96 

Figure 5-3. Potential impact of select uncertainties on the RfC for EGBE. ...............................106 

Figure B-1. PBPK model of Corley et al. (1994, 041977). ........................................................B-2 

Figure B-2. PBPK model of Corley et al. (2005, 100100). ........................................................B-5

Figure C-1. Hill Model run of female rat RBC count versus CMAX BAA from a 14-week 

inhalation study........................................................................................................C-6 

Figure C-2. Multistage Model run of hemosiderin deposition in male rats versus AUC BAA at 

12 months from a 2-year inhalation study. ............................................................C-15 

Figure C-3. Log-Logistic Model run of hemosiderin deposition in female rats versus AUC BAA 

at 12 months from a 2-year inhalation study. ........................................................C-18 

Figure C-4. Log-Logistic Model run of forestomach epithelial hyperplasia in female mice versus 

CMAX BAA from a 2-year inhalation study............................................................C-25 

Figure C- 5. Hill Model run of female rat RBC count versus CMAX BAA from a 3-month oral 

study.......................................................................................................................C-31 

 viii 
LIST OF ABBREVIATIONS AND ACRONYMS 

ADH alcohol dehydrogenase 

AIC Akaike information criterion 

ALDH aldehyde dehydrogenase 

AUC area under the curve 

BAA 2-butoxyacetic acid 

BAL butoxyacetaldehyde 

BMC benchmark concentration 

BMCL benchmark concentration, 95% lower bound 

BMD benchmark dose 

BMDL benchmark dose, 95% lower bound 

BMDS benchmark dose software 

BMR benchmark response 

CASRN Chemical Abstracts Service Registry Number 

CHO Chinese hamster ovary 

CHR contact hypersensitivity response 

CI confidence interval 

ClS clearance rate 

Cmax maximum concentration 

con-A concanavalin-A 

COP cardiac output 

DNA deoxyribonucleic acid 

EA ethyl acrylate 

ECETOC European centre for ecotoxicology and toxicology of chemicals

EG ethylene glycol 

EGBE ethylene glycol monobutyl ether 

EGEE ethylene glycol ethyl ether 

EGME ethylene glycol methyl ether 

GD gestational day 

GFR glomerular filtration rate 

G6PD glucose-6-phosphate dehydrogenase 

GSH glutathione 

Hb hemoglobin 

Hct hematocrit 

HEC human equivalent concentration 

HED human equivalent dose 

HH hereditary hemochromatosis 

Hp haptoglobin 

i.p. intraperitoneal 

i.v. intravenous 

IRIS Integrated Risk Information System 

KLH keyhole limpet hemocyanin 

LOAEL lowest-observed-adverse-effect level 

MAA 2-methoxyacetic acid 

MCH mean corpuscular hemoglobin 

MCHC mean corpuscular hemoglobin concentration 

MCV mean cell volume 

 ix 
ME 2-methoxyethanol 

MN micronuclei 

MOA mode of action 

NK natural killer 

NOAEL no-observed-adverse-effect level 

NTP National Toxicology Program 

NZW New Zealand white 

8-OHdG 8-hydroxydeoxyguanosine 

OR osmotic resistance 

OXA oxazolone 

PBPK physiologically based pharmacokinetic 

PFC plaque-forming cell 

POD point of departure 

RBC red blood cell 

RfC reference concentration 

RfD reference dose 

ROS reactive oxygen species 

s.c. subcutaneous 

SCE sister chromatid exchange 

SD standard deviation 

t1⁄2 half-life 

TNFα tumor necrosis factor-alpha 

TNP-LPS trinitrophenyl-lipopolysaccharide 

UF uncertainty factor 

U.S. EPA U.S. Environmental Protection Agency 

Vd volume of distribution 

WBC white blood cell 

 x 
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 ethylene 

glycol monobutyl ether (EGBE). It is not intended to be a comprehensive treatise on the 

chemical or toxicological nature of EGBE. 

The intent of Section 6, Major Conclusions in the Characterization of Hazard and Dose 

Response, is to present the major conclusions reached in the derivation of the reference dose, 

reference concentration and cancer assessment, where applicable, and to characterize the overall 

confidence in the quantitative and qualitative aspects of hazard and dose response by addressing 

the quality of data and related uncertainties. The discussion is intended to convey the limitations 

of the assessment and to aid and guide the risk assessor in the ensuing steps of the risk 

assessment process. 

For other general information about this assessment or other questions relating to IRIS, 

the reader is referred to EPA’s IRIS Hotline at (202) 566-1676 (phone), (202) 566-1749 (fax), or 

hotline.iris@epa.gov (email address). 

 xi 
AUTHORS, CONTRIBUTORS, AND REVIEWERS 

CHEMICAL MANAGERS/AUTHORS 

Paul Reinhart, Ph.D., DABT 

National Center for Environmental Assessment 

U.S. Environmental Protection Agency 

Research Triangle Park, NC 

Jeffrey Gift, Ph.D. 

National Center for Environmental Assessment 

U.S. Environmental Protection Agency 

Research Triangle Park, NC 

Angela Howard, Ph.D. 

National Center for Environmental Assessment 

U.S. Environmental Protection Agency 

Research Triangle Park, NC 

REVIEWERS

This document was 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 

Jane Caldwell, Ph.D. 

National Center for Environmental Assessment 

U.S. Environmental Protection Agency 

Ila Cote, Ph.D., DABT 

National Center for Environmental Assessment 

U.S. Environmental Protection Agency 

Lynn Flowers, Ph.D., DABT 

National Center for Environmental Assessment 

U.S. Environmental Protection Agency 

Gary Foureman, Ph.D. 

National Center for Environmental Assessment 

U.S. Environmental Protection Agency 

 xii 
Jennifer Jinot, Ph.D. 

National Center for Environmental Assessment 

U.S. Environmental Protection Agency 

Connie Meacham, M.S. 

National Center for Environmental Assessment 

U.S. Environmental Protection Agency 

Reeder Sams II, Ph.D. 

National Center for Environmental Assessment 

U.S. Environmental Protection Agency 

John Vandenberg, Ph.D. 

National Center for Environmental Assessment 

U.S. Environmental Protection Agency 

Debra Walsh, M.S. 

National Center for Environmental Assessment 

U.S. Environmental Protection Agency 

John Whalan 

National Center for Environmental Assessment 

U.S. Environmental Protection Agency 

EXTERNAL PEER REVIEWERS 

David Jollow, Ph.D. 

Professor Emeritus 

Medical University of South Carolina 

Michael Pereira, Ph.D. 

College of Medicine and Public Health 

Ohio State University 

Andrew Salmon, Ph.D. 

Senior Toxicologist 

Office of Environmental Health Hazard Assessment 

California Environmental Protection Agency 

Fletcher Hahn, DVM, Ph.D. 

Scientist Emeritus 

Lovelace Respiratory Research Institute 

Rochelle Tyl, Ph.D. 

Senior Fellow 

Life Sciences and Toxicology 

RTI International 

 xiii 
D. Alan Warren, M.PH., Ph.D. 

Academic Program Director 

Environmental Health Sciences 

University of South Carolina Beaufort 

Gregory Travlos, DVM, DACVP 

Veterinary Medical Officer 

National Institute of Environmental Health Sciences 

 xiv 
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 ethylene 

glycol monobutyl ether (EGBE). IRIS Summaries may include oral reference dose (RfD) and 

inhalation reference concentration (RfC) values for chronic and other 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 (MOA). 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, a plausible inhalation unit risk is 

an upper bound on the estimate of risk per μg/m3

 air breathed. 

Development of these hazard identification and dose-response assessments for EGBE has 

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

(NRC, 1983, 194806). U.S. Environmental Protection Agency (U.S. 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 

 

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 
Mixtures (U.S. EPA, 1986, 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 

(U.S. EPA, 2000, 052150), Supplementary Guidance for Conducting Health Risk Assessment of 

Chemical Mixtures (U.S. EPA, 2000, 004421), 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 December 

2008. 

 2 
2. CHEMICAL AND PHYSICAL INFORMATION 

EGBE is also known as 2-butoxyethanol. EGBE is widely used as a solvent in various 

applications, such as in surface coatings, spray lacquer, quick-dry lacquers, enamels, varnishes, 

varnish removers, latex paint, metal cleaners, and in commercially available cleaning products. 

EGBE has been estimated to range in concentration from 1 to 30% (volume/volume) in industrial 

and commercial products. The average concentration of EGBE in household products in 1977 

was 2.6%. EGBE is a high production volume chemical with an estimated 390 million pounds 

produced in the United States in 1992 (NTP, 2000, 196293). Some relevant physical and 

chemical properties of EGBE are shown in Table 2-1. 

Table 2-1. Physical and chemical properties of EGBE 

CASRN 111-76-2 

Empirical formula C4H9-O-CH2CH2-OH 

Molecular weight 118.2 

Vapor pressure 0.88 mm Hg at 25°C (about 1,200 ppm) 

Water solubility Miscible 

Log Kow 0.81 

Henry’s law constant 2.08 × 10-7–2.08 × 10-8 atm-m3

/mole (25°C) 

Flash point 62°C (closed cup); 70°C (open cup) 

Conversion factor 1 ppm = 4.83 mg/m3; 1 mg/m3 = 0.207 ppm 

Source: HSDB (2005, 597537) 

EGBE exists as a colorless liquid at ambient temperature and pressure. Its evaporation 

rate relative to butyl acetate is 0.08; thus, it is considered a “slow evaporator.” It is miscible in 

water and partitions about equally between phases of octanol and water. Considering the 

magnitude of the octanol:water partition coefficient (≈ 7 :1), it is unlikely that EGBE 

bioaccumulates. Based on the magnitude of the Henry’s law constant, partitioning of EGBE 

between water and air greatly favors the water phase. 

 

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). 

 3 
3. TOXICOKINETICS 

3.1. ABSORPTION AND DISTRIBUTION 

EGBE is absorbed and rapidly distributed in humans following inhalation, ingestion, or 

dermal exposure. Kumagai et al. (1999, 100171) examined 10 polar organic solvents, including 

EGBE, during short-term inhalation by humans. Four healthy male research subjects inhaled 

25 ppm EGBE for 10 minutes; the mean uptake was 79.7% in the last 5 minutes of EGBE 

respiration. 

Johanson and Boman (1991, 006759) attempted to define the relative importance of the 

skin to the total absorption of EGBE vapors by humans by comparing mouth-only and body-only 

exposures. Four research subjects were exposed to 50 ppm EGBE mouth-only for 2 hours, 

followed by 1 hour of no exposure, then 2 hours of 50 ppm body-only exposure (i.e., exposed in 

a chamber while breathing fresh air via a respirator). Blood samples were collected periodically 

for analysis of EGBE under the assumption that the finger-prick blood samples represented 

mixed arterial blood. Since the areas under the curve (AUCs) for the concentration of EGBE in 

the subjects’ blood samples following body-only exposures were three- to fourfold greater than 

following mouth-only exposure, the authors concluded that the skin accounted for approximately 

75% of the total uptake of EGBE in a whole-body exposure. 

Corley et al. (1994, 041977) suggested that Johanson and Boman’s (1991, 006759) 

conclusion of greater absorption of EGBE vapor through the skin than from the respiratory tract 

was inconsistent with the physiological differences (relative surface area, blood perfusion, 

barrier thickness) favoring absorption of vapors through the lungs. They reanalyzed the kinetic 

data of Johanson and Boman, assuming that the finger-prick blood samples represented venous 

blood draining the skin prior to mixing systemically. These revised calculations resulted in 

dermal uptake contributing no more than 22% of the total uptake of EGBE in a whole-body 

exposure at average temperature and humidity (skin permeability coefficient of 3 cm/hour), 

assuming no clothing to hinder absorption. 

To provide experimental validation of the skin’s role in the uptake of EGBE vapors, 

Corley et al. (1997, 041984) conducted a study in which human research subjects exposed one 

arm to 50 ppm [13C]-EGBE for 2 hours. Catheters installed in the antecubital vein of the 

unexposed arm served as the primary site for blood collection, which was analyzed for both 

EGBE and 2-butoxyacetic acid (BAA). Finger-prick blood samples were collected from the 

exposed arm at the end of the 2-hour exposure. If Johanson and Boman’s (1991, 006759) 

 

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). 

 4 
assumption that finger-prick blood samples represented systemic arterial blood was correct, then 

the concentrations of EGBE and BAA in the finger-prick blood samples taken from the exposed 

arm at the end of the 2-hour exposure should have been comparable to the corresponding 

catheter sample taken from the unexposed arm. This was not the case, since the concentration of 

EGBE averaged nearly 1,500-fold higher in the finger-prick blood samples than in the samples 

collected from the unexposed arm, confirming the potential for portal of entry to have a major 

effect when using the finger-prick sampling technique. 

3.2. METABOLISM AND ELIMINATION 

The metabolism of EGBE has been studied extensively in rodents, particularly in rats, 

and the large body of literature on this subject has been thoroughly reviewed 

(Commonwealth of Australia, 1996, 594448; ECETOC, 1994, 594447). Proposed pathways for 

the metabolism of EGBE in rats and humans are presented in Figure 3-1. The principal products 

from metabolic processes in rats or humans are BAA (all species) and the glutamine or glycine 

conjugate of BAA (humans). Other potential metabolic products, such as the glucuronide 

conjugate of EGBE, ethylene glycol (EG), butoxyacetaldehyde (BAL), and CO2 are minor 

metabolites or are transitory in nature (e.g., BAL) and do not accumulate in blood, tissues, or 

excreta. 

 5 
Sources: Adapted from Corley et al. (1997, 041984) and Medinsky et al. (1990, 041986). 

Figure 3-1. Proposed metabolic scheme of EGBE in rats and humans. 

The two main oxidative pathways of EGBE metabolism observed in rats are via alcohol 

dehydrogenase (ADH) and O-dealkylation by a cytochrome P450 dealkylase (CYP 2E1). 

Because BAA is excreted in the urine of both rats and humans following EGBE exposure, it has 

been suggested that the production of BAA through the formation of BAL by ADH would be 

applicable to both rats and humans (Corley et al., 1997, 041984; Corley et al., 2005, 100100; 

Medinsky et al., 1990, 041986). However, the other proposed metabolic pathways of EGBE 

may only be applicable to rats, since the metabolites of these pathways (i.e., EG, EGBE 

glucuronide, and EGBE sulfate) have been observed in the urine of rats (Bartnik et al., 1987, 

100086; Ghanayem et al., 1987, 041645), but not in humans (Corley et al., 1997, 041984). In 

addition, Corley et al. (1997, 041984) confirmed an observation of Rettenmeier et al. (1993, 

042198) that approximately two-thirds of the BAA formed by humans is conjugated with 

 6 
glutamine and, to a lesser extent, glycine. The BAA-glutamine and BAA-glycine conjugation 

pathways have not been detected in the rat. 

Carpenter et al. (1956, 066464) first identified BAA as the metabolite responsible for the 

hemolytic toxicity of EGBE by incubating BAA with whole blood from a variety of species. 

Blood from rats, mice, and rabbits was more rapidly hemolyzed than blood from humans, 

monkeys, dogs, or guinea pigs when incubated in vitro at 37.5°C with a saline solution of 0.1% 

of the sodium salt of BAA. These results correlated well with osmotic fragility studies using 

blood from these same species following in vivo inhalation exposures to EGBE. In contrast, a 

much higher concentration (2.5%) of EGBE was required to produce a similar degree of 

hemolysis in vitro. Subsequent investigations have shown that hemolytic blood concentrations 

of BAA can be produced following oral or dermal administration or inhalation of EGBE. 

The uptake and metabolism of EGBE is essentially linear following a 6-hour inhalation 

exposure of up to 438 ppm, a concentration that causes mortality in animals (Sabourin et al., 

1992, 006763). BAA is the primary metabolite in rats following drinking water (Medinsky et al., 

1990, 041986) and inhalation (Dill et al., 1998, 041981) exposures. EGBE is eliminated 

primarily as BAA in urine. Lesser amounts of the glucuronide and sulfate conjugates of EGBE 

have been observed in the urine of rats (Bartnik et al., 1987, 100086; Ghanayem et al., 1987, 

041645) but not humans (Corley et al., 1997, 041984). No significant differences in the urinary 

levels of BAA were found following administration of equivalent doses of EGBE dermally or in 

drinking water (Medinsky et al., 1990, 041986; Sabourin et al., 1993, 597595; Shyr et al., 1993, 

006766). Corley et al. (1997, 041984) reported that the elimination kinetics of EGBE and BAA 

appear to be independent of the route of exposure. Elimination of EGBE and BAA following 

repeated inhalation exposure appears to be dependent on species, gender, age, time of exposure, 

and exposure concentration (Dill et al., 1998, 041981; NTP, 2000, 196293). In rodents, 

dose-dependent clearances of EGBE and BAA have been observed (Corley et al., 1994, 041977; 

Ghanayem et al., 1990, 042016). A summary of species-specific toxicokinetic parameters is 

shown in Table 3-1 followed by a brief summary of key individual studies. 

 7 
Table 3-1. Summary of species-specific toxicokinetic parameters 

EGBE toxicokinetics 

t1/2 in blood (hr) 

Species Gender Route Mean Reference 

Human Male Inhalation 0.65 Johanson (1986, 006758) 

Human Male Dermal vapor 0.53–0.6 Johanson and Boman (1991, 006759) 

Human Male Dermal vapor 0.66 Corley et al. (1997, 041984) 

Human Male/female Inhalation 0.93 Jones and Cocker (2003, 056771) 

F344 rat Male i.v. 0.11–0.17 Ghanayem et al. (1990, 042016) 

F344 rat Male Inhalation 0.13–0.69 Dill et al. (1998, 041981) 

F344 rat Female Inhalation 0.12–0.50 Dill et al. (1998, 041981) 

B6C3F1 mouse Male Inhalation 0.05–0.16 Dill et al. (1998, 041981) 

B6C3F1 mouse Female Inhalation 0.06–0.14 Dill et al. (1998, 041981) 

B6C3F1 mouse Female i.p. 0.16 Poet et al. (2003, 100190) 

B6C3F1 mouse Female Gavage 0.35 Poet et al. (2003, 100190) 

Clearance (mL/min/kg body weight) 

Species Gender Route Mean Reference 

Human Male Inhalation 16.2 Johanson (1986, 006758) 

Guinea pig Female i.v. 128 Johanson (1986, 006758) 

F344 rat Male i.v. 5.9–13.3 Ghanayem et al. (1990, 042016) 

Sprague-Dawley rat Male Inhalation 2.2–2.3 Johanson (1994, 069322) 

BAA toxicokinetics

t1/2 in blood (hr) 

Species Gender Route Mean Reference 

Human Male Inhalation 4.3 Johanson and Johnsson (1991, 100158) 

Human Male Dermal vapor 3.27 Corley et al. (1997, 041984) 

F344 rat Male i.v. 1.5–3.2 Ghanayem et al. (1990, 042016) 

F344 rat Male Inhalation 0.55–4.96 Dill et al. (1998, 041981) 

F344 rat Female Inhalation 0.79–6.6 Dill et al. (1998, 041981) 

B6C3F1 mouse Male Inhalation 0.36–4.0 Dill et al. (1998, 041981) 

B6C3F1 mouse Female Inhalation 0.38–4.5 Dill et al. (1998, 041981) 

B6C3F1 mouse Female i.p. 1.05–1.42 Poet et al. (2003, 100190) 

B6C3F1 mouse Female Gavage 1.55–2.11 Poet et al. (2003, 100190) 

Clearance (mL/min/kg body weight) 

Species Gender Route Mean Reference 

Sprague-Dawley rat Male Inhalation 0.49–0.58 Johanson (1994, 069322) 

i.p. = intraperitoneal; i.v. = intravenous 

Percutaneous absorption of EGBE in rats is rapid and produces measured blood levels of 

BAA sufficient to produce hemolysis (Bartnik et al., 1987, 100086). Metabolism, disposition, 

and pharmacokinetic studies in male F344 rats conducted by Corley et al. (1994, 041977) 

produced hemolytic blood concentrations of BAA (0.5 mM) following a single oral dose of 126 

mg/kg of [14C]-labeled EGBE. Using their physiologically based pharmacokinetic (PBPK) 

 8 
model, they predicted that such hemolytic blood concentrations would also be produced in rats 

following a single 6-hour EGBE inhalation exposure >200 ppm. A report that evaluated the 

National Toxicology Program (NTP, 2000, 196293) inhalation bioassay suggests that BAA 

blood concentrations in female rats, which achieved higher blood concentrations than males, 

exceeded 0.5 mM (approximately 67 μg BAA/g blood), following exposure to 62.5 ppm EGBE 

for both 1-day and 12-month exposure durations (Dill et al., 1998, 041981). 

The metabolic basis for the hematotoxicity of EGBE was studied in male F344 rats by 

using pyrazole and cyanamide as metabolic inhibitors of ADH and aldehyde dehydrogenase 

(ALDH), respectively (Ghanayem et al., 1987, 041608). Male F344 rats, 9–13 weeks old, were 

pretreated with pyrazole or cyanamide followed by administration of 500 mg/kg EGBE by 

gavage. The use of pyrazole protected rats from EGBE-induced hematotoxicity and resulted in a 

10-fold lower ratio of BAA to conjugated EGBE excreted in urine. Cyanamide treatment 

significantly reduced the hematotoxic response in a manner similar to that of pyrazole; it also 

resulted in a high mortality rate in rats given cyanamide and EGBE. This effect was possibly 

due to the increased levels of unmetabolized BAL; the effect was not observed in animals treated 

with cyanamide or EGBE alone. Pyrazole completely blocked the increase in spleen weight to 

body weight ratios seen in EGBE-treated animals. Gavage administration of either BAL or BAA 

at equivalent molar doses to 125 mg/kg EGBE produced identical increased spleen to body 

weight ratios and identical increases in free hemoglobin (Hb) levels in plasma. Pretreatment of 

rats with cyanamide prior to administration of BAL provided significant protection against BALinduced hematotoxicity. These studies confirm the central role of BAA in the hematotoxic 

response elicited in rats. 

The effects of age, dose, and metabolic inhibitors on the toxicokinetics of EGBE were 

studied in male F344 rats (Ghanayem et al., 1990, 042016). Rats aged 3–4 and 12–13 months 

were administered a single gavage dose of 31.2, 62.5, or 125 mg/kg EGBE. Pretreatments 

included pyrazole, cyanamide, or probenecid, an inhibitor of renal anion transport. 

Toxicokinetic parameters for EGBE, including AUC, maximum plasma concentration (Cmax), 

and clearance rate (ClS), were dose dependent; AUC and Cmax increased and ClS decreased as 

dose levels increased. Other measured parameters were unaffected by dose. Age had no effect 

on half-life (t1⁄2), volume of distribution (Vd), or C1S of EGBE, but Cmax and AUC increased with 

age. As expected from previous studies, inhibition of EGBE metabolism by either pyrazole or 

cyanamide resulted in significantly increased t1⁄2 and AUC, as well as decreased ClS. BAA 

toxicokinetics were also altered by dose and age, as well as by administration of metabolic 

inhibitors. Statistically significant, slight increases in Cmax, AUC, and t1⁄2 were seen at higher 

doses; these results were more pronounced in older rats. Probenecid pretreatment at EGBE dose 

levels of 31.2 and 62.5 mg/kg produced no changes in the measured toxicokinetic parameters for 

EGBE. Pretreatment produced two- to threefold increases in AUC, and two- to sixfold increases 

in t1⁄2 for BAA. These results indicate that renal organic acid transport is vital to renal elimination 

 9 
of BAA. The increased Cmax, AUC, and t1⁄2 in older versus younger rats may be due to 

differences in relative contributions of the two primary metabolic pathways previously discussed, 

or to compromised renal clearance. 

Dill et al. (1998, 041981) described the toxicokinetics of EGBE and BAA in male and 

female F344 rats and B6C3F1 mice as part of the 2-year EGBE inhalation toxicity and 

carcinogenicity study conducted by the NTP (2000, 196293). Blood samples were collected 

from a satellite group of animals postexposure (i.e., after the daily 6-hour exposure) after 1 day, 

2 weeks, and 3, 6, 12, and 18 months of exposure to target EGBE concentrations of 0, 31.2 (rats 

only), 62.5, 125, or 250 ppm (mice only) by whole-body inhalation; the samples were assayed 

for EGBE and BAA. Postexposure time points varied from 10 to 720 minutes following 1 day, 

2 weeks, and 3 and 6 months and varied from 10 to 2,880 minutes following 12 months. 

Postexposure 16-hour urine samples were collected after 2 weeks and 3, 6, 12, and 18 months of 

exposure and assayed for BAA. In addition, a separate set of aged mice were kept in the control 

chamber and exposed to EGBE for 3 weeks when they were approximately 19 months old. 

Postexposure blood samples were collected after 1 day and 3 weeks of exposure; 16-hour urine 

samples were collected after 2 weeks of exposure. Overall, mice eliminated both EGBE and 

BAA from blood faster than rats: for example after the 1-day exposure, t1⁄2 for rats (males and 

females, over three concentrations) averaged 8.6 minutes for EGBE, while the t1⁄2 for mice (males 

and females, over three concentrations) averaged 4 minutes for EGBE. In contrast, the rate of 

BAA elimination from blood decreased as the exposure concentration increased. As exposure 

continued, the rates of elimination for both EGBE and BAA decreased in both species, resulting 

in longer residence times in the blood. At 1 day postexposure, t1⁄2 in male rats was 9.4 minutes, 

and at 18 months postexposure, t1⁄2 was 15.8 minutes. Female rats were significantly less 

efficient in clearing BAA from their blood than males, possibly as a result of reduced renal 

clearance in female rats. The aged mice were observed to eliminate BAA from blood >10 times 

slower than young mice after 1 day of exposure, but this difference was less obvious after 3 

weeks of exposure. These findings provide evidence that the elimination kinetics of EGBE and 

BAA, following repeated inhalation exposure to EGBE, appear to be dependent on various 

factors, including species, gender, age, time of exposure, and exposure concentration. 

Green et al. (2002, 041610) explored reasons that female mice develop marked 

hyperkeratosis in the forestomach when given oral doses (1/day for 10 days) of either EGBE or 

BAA. Irritation from the carboxylic acid BAA is hypothesized to cause cell damage followed by 

cell proliferation and eventually the observed hyperkeratosis. Their studies examined the 

activity and localization of ADH and ALDH (the principal enzymes involved in the metabolism 

of EGBE in the stomach tissues of mice and rats) the localization of these enzymes in a human 

stomach sample, and whole body autoradiography of mice exposed to radiolabeled EGBE (see 

Section 4.4.1). Tissue homogenates were prepared from female B6C3F1 mice (n = 30) and rats 

(n = 10; gender and species not specified) and centrifuged at 41,000 × g, with the supernatants 

 10 
used to examine the metabolism of EGBE by ADH and ALDH. The stomachs were separated 

into fore and glandular sections and used to measure the metabolism of EGBE to BAL and BAL 

to BAA by ADH and ALDH, respectively. A marked species difference in ALDH activity was 

observed between rats and mice: 

Rats: Km = 0.29 mM 

Vmax = 1.627 nmol/minute per mg protein, forestomach, Km = 0.73 mM 

Vmax = 2.170 nmol/minute per mg protein, glandular stomach 

Mice: Km = 46.59 mM 

Vmax = 17.094 nmol/minute per mg protein, forestomach, Km = 87.01 mM 

Vmax = 13.986 nmol/minute per mg protein, glandular stomach 

Km values were over two orders of magnitude greater in mice compared to rats. Based 

upon the Km and Vmax values reported, while the mouse ALDH enzyme has a lower affinity than 

the rat enzyme for EGBE, the mouse enzyme has a much greater capacity to metabolize EGBE 

to the intermediate without becoming saturated. The fact that EGBE is held in the forestomach, 

along with the information that rates for the ADH enzyme were of the same order of magnitude 

for rats and mice, suggests that mice are capable of generating more BAA in the forestomach 

than rats for the same dose and exposure duration. 

Green et al. (2002, 041610) also examined the distribution of ALDH and ADH in rat, 

mouse, and human stomach tissue sample from a single individual, using histochemical staining. 

The stratified squamous epithelium of the forestomach of both rats and mice contained the 

highest staining intensity for ALDH and ADH. These enzymes were found throughout the 

mucosa in the human stomach tissue sample; the highest concentration was found in the mucusproducing cells at the surface. Data indicates that the distribution of these enzymes in humans is 

more closely comparable to that found in the rodent glandular stomach than in the rodent 

forestomach. This finding, combined with the difference in ALDH and ADH activity between 

mouse and rat forestomach, suggests that humans are at much lower risk for the tissue irritation 

seen in the mouse forestomach. 

Deisinger and Boatman (2004, 594446) determined the extent of the in vivo formation of 

BAL and BAA from EGBE and their elimination kinetics from blood, liver, and forestomach of 

mice. Male and female B6C3F1 mice (4/gender/time point) were administered oral doses of 

600 mg/kg EGBE dissolved in distilled water. At 5, 15, 45, and 90 minutes following the dose, 

blood, liver, and forestomach tissues, along with forestomach contents, were collected and 

processed to determine EGBE, BAL, and BAA concentrations in the samples. High EGBE 

concentrations were measured at all time points; maximum concentrations occurred 5 minutes 

after dosing, with a mean of 123 mM in females and 129 mM in males. EGBE levels in blood 

 11 
and liver were also at maximum concentrations at 5 minutes postdosing, but at levels that were 

roughly 50-fold lower than in the forestomach. BAA concentrations in all organs were 

substantial in the 5-minute samples, and concentrations continued to increase until leveling off in 

the 45- and 90-minute samples. Concentrations of BAA measured in the forestomach were 

lower than concentrations in blood and liver tissues. Furthermore, BAA was found to be 

associated with forestomach tissues, rather than forestomach contents. BAL levels were highest 

in the initial samples, 5 minutes postdose, and then declined. Levels of BAL measured in the 

forestomach were 10-fold to 100-fold lower than the parent compound or carboxylic acid 

metabolite. No differences between male and female mice were apparent in the parent 

compound or BAA organ concentrations at comparable time points following dosing, but the 

BAL concentrations were up to twofold greater at some time points in the liver and forestomach 

of female mice compared to male mice. 

Using rate constants derived from mouse stomach fractions (Green et al., 2002, 041610) 

and making several assumptions about the use of these enzyme activity data, Corley et al. (2005, 

100100) estimated that 250 ppm EGBE would result in peak Cmax values of 7 μM EGBE, 0.5 μM 

BAL, and 3,250 μM BAA in liver tissue of male mice at the end of a 6-hour inhalation exposure. 

The model includes the metabolism of EGBE to BAL via ADH, and the subsequent metabolism 

of BAL to BAA via ALDH in both the liver and forestomach. The model predicts that the 

concentrations of BAL in gastrointestinal tract tissues of male and female mice at 5 minutes 

postdosing, the time of maximal concentration, would be 18 and 33 μM, respectively, following 

gavage exposure to 600 mg/kg EGBE. This compares well with the levels of BAL actually 

observed in forestomach tissue of male and female mice at 5 minutes postdosing: 19 and 33 μM, 

respectively, following gavage exposure to EGBE at 600 mg/kg (Deisinger and Boatman, 2004, 

594446). 

For humans, the elimination kinetics of EGBE and BAA appear to be independent of the 

route of exposure. For whole-body exposures under exercise conditions, the elimination t1⁄2 for 

EGBE and BAA were 0.66 and 4 hours, respectively (Johanson, 1986, 006758; Johanson and 

Johnsson, 1991, 100158). For dermal exposure to neat liquids, the t1⁄2 for elimination of EGBE 

and BAA were 1.3 and 3.1 hours, respectively (Johanson et al., 1988, 100156). For dermal 

exposure to vapors, the elimination t1⁄2 for EGBE was 0.53–0.6 hours. 

Haufroid et al. (1997, 042040) conducted a study on 31 male workers exposed to low 

levels of EGBE in a beverage package production plant. The average airborne EGBE exposure 

concentration was 2.91 ± 1.30 mg/m3

 (0.59 ± 0.27 ppm). Postshift urine samples showed an 

average BAA concentration of 10.4 mg/g creatinine. One exposed individual who exhibited a 

very low urinary BAA excretion was found to possess a genetic polymorphism for CYP 2E1 that 

produced increased oxidative activity. However, the researchers did not measure BAA 

conjugated to glutamine, an alternative pathway for BAA excretion in humans. Further 

 12 
investigations on the influence of genetic polymorphisms for CYP 2E1 on urinary BAA 

excretion rate are needed before conclusions can be drawn. 

Johanson and Johnsson (1991, 100158) analyzed venous blood samples collected at 0, 2, 

4, and 6 hours postexposure from five healthy, male research subjects exposed to 20 ppm EGBE 

via inhalation for 2 hours during light physical exercise on a bicycle ergometer. Blood samples 

were analyzed for BAA concentrations. An average peak blood concentration of 45 μM BAA 

was reached 2–4 hours after exposure. The range of concentration was from 36 to 60 μM. The 

average t1⁄2 for elimination of BAA from blood was 4.3 hours, with a range from 1.7 to 9.6 hours, 

suggesting that blood levels of BAA would probably not increase following prolonged 

occupational exposures to concentrations of EGBE vapor at or below existing occupational 

exposure limits of 20–25 ppm. Thus, blood levels would not reach those shown to cause adverse 

effects in vitro. The average renal clearance of BAA was 23–39 mL/minute, which was only 

about one-third of the glomerular filtration rate (GFR). The authors suggested that the low 

clearance of BAA relative to the GFR could have been related to the binding of BAA to proteins 

in blood or to a low efficiency in renal tubular secretion. The low pKa of 3.5 estimated by the 

researchers indicates that tubular reabsorption is unlikely, because more than 99% of the BAA in 

normal human urine (pH ~6) is ionized. The Vd averaged 15 L (range 6.5–25 L) based on whole 

blood measurements, and was approximately equal to the volume of extracellular water (13–

16 L), a further indication of binding of BAA to blood proteins. 

Laitinen (1998, 100173) reported BAA levels in a study of eight silkscreen printers 

(gender not specified) exposed to a mixture of EGBE and 2-butoxyethylacetate. Daily mean 8-

hour air concentrations ranged from 0.1 to 0.6 ppm during a 5-day period. Urine samples from 

these workers contained 75 mg BAA/g creatinine immediately after the work shift, and 58 mg/g 

creatinine the following morning, 14–16 hours postshift. Laitinen et al. (1998, 100174) reported 

similar postshift urinary levels of 60 mg BAA/g creatinine in another group of 37 male and 15 

female silkscreen workers exposed to 5 ppm EGBE and its acetate for one 8-hour workday. 

Several PBPK models have been developed for EGBE, each sequentially building upon 

the advances from the previous model. The first model was developed by Johanson (1986, 

006758) to describe the kinetics of EGBE in the blood of human volunteers exposed for 2 hours 

to 20 ppm EGBE in air while exercising. Shyr et al. (1993, 006766) published a model to 

describe the pharmacokinetics of EGBE in male F344 rats based upon the drinking water 

exposure data of Medinsky et al. (1990, 041986) and the inhalation and dermal data of Sabourin 

et al. (1992, 006763; 1993, 597595). Corley et al. (1994, 041977) then extended the Johanson 

(1986, 006758) model to describe the kinetics of EGBE, as well as the major metabolite, BAA, 

in rats and humans and later validated the human dermal exposure model in Corley et al. (1997, 

041984). Lee et al. (1998, 041983) followed with a model that included young and old, male 

and female rats and mice to describe the kinetics of EGBE and BAA in the NTP 2-year 

inhalation bioassay (data in Dill et al., 1998, 041981). Based upon the data of Dill et al. (1998, 

 13 
041981), there were species, gender, age, and exposure concentration-dependent differences in 

the kinetics of BAA. Lee et al. (1998, 041983) made several assumptions about the plasma 

protein binding of BAA, the metabolism of EGBE to BAA and other metabolites, and the renal 

clearance of BAA (all initially described by Corley et al. (1994, 041977) and Shyr et al. (1993, 

006766) in male rats) to describe the kinetic data in female rats and mice as a function of age and 

chronic exposure to EGBE. Corley et al. (2005, 100100) replaced the assumptions used by Lee 

et al. (1998, 041983) with experimental data. This model, along with the Lee et al. (1998, 

041983) rat and mouse model and Corley et al. (1997, 041984) human model, is used in this 

current review to calculate the internal dose of EGBE (Cmax of BAA in blood) used in the 

development of the RfC and RfD. This model is described in more detail in Appendix B. 

 14 
4. HAZARD IDENTIFICATION 

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

CONTROLS 

Carpenter et al. (1956, 066464) conducted three controlled inhalation studies. In the first 

study, a group of two men and six rats were exposed simultaneously for 4 hours to an EGBE 

concentration of 113 ppm in a 1,250 cubic foot room. Effects observed in humans included nasal 

and ocular irritation, a metallic taste, and belching. Erythrocyte osmotic fragility did not change 

for the men, yet rose appreciably for the rats. In a second study, a group of two men, one 

woman, and three rats were exposed to 195 ppm EGBE for two 4-hour periods, separated by a 

30-minute recess, in a 6.5 cubic foot room. There was no change in the subjects’ blood pressure, 

erythrocyte fragility, or pulse rate. They experienced nose and throat irritation, followed by 

ocular irritation and disturbed taste; one subject reported a headache. In the rats, an increase in 

erythrocyte fragility values was noted. In the third study, two men and two women were 

exposed for 8 hours to a 100 ppm EGBE concentration. No changes in blood pressure, 

erythrocyte fragility, or pulse rate were observed. Again, nasal and throat irritation followed by 

ocular irritation and a disturbing metallic taste were experienced. Two subjects reported 

headaches. 

There are a number of case reports of acute ingestion of EGBE, consisting primarily of 

accidental or intentional ingestion. Bauer et al. (1992, 100087) reported the effects of acute 

ingestion of 500 mL of window cleaner containing 9.1% EGBE and 2.5% ethanol by a 53-yearold alcoholic male. He was comatose with metabolic acidosis, shock and noncardiogenic 

pulmonary edema when brought to a hospital, approximately 10 hours after ingestion. He had 

increased heart rate, decreased blood pressure, and transient polyuria and hypoxemia. 

Hypochromic anemia was evident with an Hb concentration of 9.1 g/100 mL, a hematocrit (Hct) 

of 25%, and thrombocytopenia. The patient recovered and was discharged after 15 days. 

Gijsenbergh et al. (1989, 100134) reported that a 23-year-old woman weighing 64 kg 

ingested approximately 25–30 g of EGBE (~400–500 mg/kg) and ethanol (~4:1 ratio) as a 

window cleaner in an apparent suicide attempt. She was comatose when admitted to the 

hospital, exhibiting dilated pupils, obstructive respiration, and metabolic acidosis, including 

depression of blood Hb concentration and hematuria. The presence of EGBE in the blood and 

dialysis fluid was confirmed. Treatment consisted of supportive therapy, forced diuresis, 

bicarbonate administration, and hemodialysis. Her Hb concentration fell from 11.9 g Hb/100 mL 

upon admission to 8.9 g Hb/100 mL. She was discharged after 8 days. 

 

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). 

 15 
Gualtieri et al. (1995, 594443; 2003, 100140) reported a case of a suicide attempt with an 

industrial-strength window cleaner. The 18-year-old male weighed 71 kg; he consumed between 

360 and 480 mL of a concentrated glass cleaner that contained 22% EGBE, a dose equivalent to 

1,131–1,509 mg/kg. He was admitted to the hospital with no abnormalities other than epigastric 

discomfort within 3 hours postingestion. Approximately 10 hours postadmission, the patient was 

noticeably lethargic, weak, and hyperventilating, symptoms consistent with the onset of 

metabolic acidosis. BAA was measured; the highest serum concentration found was 

4.86 mmol/L, collected approximately 16 hours postingestion. The patient was transferred to a 

tertiary care hospital where hemodialysis was initiated at approximately 24 hours postingestion. 

Ethanol therapy was started 30 minutes later. Treatment also consisted of intravenous (i.v.) 

doses of 100 mg thiamine and 50 mg folic acid every 12 hours and 50 mg pyridoxine every 

6 hours. Following 4 hours of dialysis, the patient was alert and remained hemodynamically 

stable. Ten days after discharge, the patient was readmitted following a second ingestion of 

480 mL of the same cleaner, an EGBE dose equivalent to 1,509 mg/kg. Treatment included 

ethanol therapy and hemodialysis, and was initiated within a few hours of ingestion to control the 

metabolic acidosis. Due to this early treatment, ethanol therapy had an impact on the disposition 

of EGBE and BAA. As with the first episode, metabolic acidosis was manifest. This high-dose 

oral ingestion was nearly 1.1–1.5 g EGBE/kg body weight. The highest serum BAA 

concentration was 2.07 mmol/L, collected 22 hours postingestion. No evidence of hemolysis or 

renal abnormalities was detected. 

A 50-year-old woman ingested approximately 250–500 mL of a window cleaner 

containing 12% EGBE, representing ~30–60 mL, in an apparent suicide attempt (RambourgSchepens et al., 1988, 100191). She was diagnosed with metabolic acidosis, hypokalemia, a rise 

in serum creatinine level, and a marked increase in urinary excretion of oxalate crystals. 

Moderate hemoglobinuria appeared on the third day postexposure, and a progressive 

erythropenia was noted. In the absence of more complete hematologic details from this and 

other similar case studies, it is not possible to determine whether these effects were due to 

hemolysis or other factors related to the profound blood chemistry changes observed. The 

clinical status improved gradually and the patient was discharged on the 10th day. 

Burkhart and Donovan (1998, 056375) summarized the case of a 19-year-old male who 

ingested 20–30 ounces (or ~590–885 mL) of a product that contained 25–35% EGBE (an 

exposure equivalent to ~177–265 mL, estimated at >3,000 mg/kg) along with 15–25% propylene 

glycol, 5–10% monoethanolamine, and 1–3% potassium hydroxide. On his arrival at the hospital 

3.5 hours after ingestion, the patient was deeply comatose with severe hypotension. Hematuria 

developed on the second day, with no evidence of renal or hepatic toxicity; however, pulmonary 

toxicity consisting of severe aspiration pneumonia was present. The patient had a significant 

recovery, despite severe neurologic deficits that were slow to resolve. 

 16 
Osterhoudt (2002, 100186) reported on a 16-month-old girl who ingested an unknown 

amount of cleaning solution containing EGBE (10–30%), monoethanolamine (5–10%), 

alkoxylated linear alcohols (1–5%), ethylenediaminetetraacetic acid (1–5%), and potassium 

hydroxide (1–5%). Metabolic acidosis was manifest, and a single dose (15 mg/kg) of the ALDH 

inhibitor fomepizole was administered. Within 2 hours, the metabolic acidosis was completely 

resolved, and there was no evidence of alkaline mucosal injury, hepatic or renal dysfunction, or 

hemolysis. 

Dean and Krenzelok (1991, 597279) reported that 24 children, aged 7 months to 9 years, 

were observed subsequent to oral ingestion of at least 5 mL of glass window cleaner containing 

EGBE in the 0.5–9.9% range. Two children drank more than 15 mL, and were treated by gastric 

lavage. No symptoms of EGBE poisoning, such as metabolic acidosis, and no hemolysis were 

observed in any of the children. 

Raymond et al. (1998, 100193) reported on seven clerical workers who were evaluated 8 

months after they entered a file room where the supervisor believed that EGBE had been applied 

overnight to strip the floor. Exact details of the product used were unknown, but based on 

containers found and exposure symptoms of noted intense eye and respiratory irritation, marked 

dyspnea, nausea, and faintness, the authors suggested that they were exposed to EGBE 

concentrations of 200–300 ppm. Of major concern were skin spots—cherry angiomas—that 

appeared between 4 and 22 weeks after exposure in six of the seven workers. All workers 

continued to experience recurrent eye and tracheobronchial irritation; four had a dry cough. 

Workplace air sampling conducted by a certified industrial hygienist 1 week after the floor 

stripping found no detectable EGBE, although traces (0.1–0.2 ppm) of formaldehyde were 

identified. Five years after the exposure, four of the workers who could be contacted reported 

that they continued to have outbreaks of new cherry angiomas. It should be noted that no other 

studies linking EGBE exposure to outbreaks of cherry angiomas are available in the literature. 

The authors included the observation that, since this report, they had seen three patients who they 

believe were also exposed to EGBE vapor in an unrelated incident, and who did not develop any 

skin spots. Cherry angiomas are the most common cutaneous vascular lesion; they are benign 

and formed by a proliferation of dilated venules. The spots occur more frequently with 

increasing age but can appear in younger individuals. There are reports in the literature of cherry 

angiomas appearing following individual exposure to other chemicals, such as bromides (Cohen 

et al., 2001, 100096), glutaraldehyde (Raymond et al., 1998, 100193), and sulfur mustard gas 

(Firooz et al., 1999, 100115). 

A cross section of 31 male workers, aged 22–45, employed for 1–6 years, who were 

exposed to low levels of EGBE in a beverage packing production plant were monitored by 

Haufroid et al. (1997, 042040). The effect of external EGBE exposure and internal BAA levels 

on erythrocyte lineage were investigated by studying red blood cell (RBC) count, Hb, Hct, mean 

cell volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin 

 17 
concentration (MCHC), haptoglobin (Hp), reticulocyte count, and osmotic resistance (OR), a 

measure of osmotic fragility. Also studied were serum glutamic-oxaloacetic and glutamicpyruvic transaminases and renal creatinine and urinary retinol binding protein parameters. The 

average airborne concentration of EGBE was 2.91 mg/m3

, or 0.6 ppm (standard deviation [SD] 

of ±1.30 mg/m3

 or 0.27 ppm). In addition, there was coexposure to methyl ethyl ketone. Single 

determinations of BAA in postshift urine samples were used to assess exposure to low levels of 

EGBE. No differences were observed for RBC counts, Hb, MCV, MCH, Hp, reticulocyte count, 

or between exposed and control workers. The only statistically significant change observed in 

exposed workers when compared with a matched control group (n = 21) was a 3.3% decrease in 

Hct (p = 0.03) and a 2.1% increase in MCHC (p = 0.02). The implications of these small 

erythroid effects are unclear. Both values are within their corresponding normal clinical ranges 

and, given that no statistically significant changes were observed in other erythroid parameters, 

they do not appear to be related to the more severe effects observed in laboratory animals. 

Furthermore, no correlation was found between any of the nine erythroid parameters measured 

and the parameters of internal exposure. No significant differences were observed in hepatic and 

renal biomarkers. 

Several human studies investigated the dermal absorption of EGBE. Jakasa et al. (2004, 

100151) dermally exposed six male research subjects, ages 22–55, to 50%, 90%, or neat EGBE 

for 4 hours on the forearm over an area of 40 cm2

. The dermal absorption of EGBE from 

aqueous solutions was markedly higher than from neat EGBE. In Jones et al. (2003, 056771), 

four research subjects were exposed via inhalation of 50 ppm EGBE for 2 hours on nine separate 

occasions, with each occasion separated by 3 weeks, at varying temperatures and humidity 

levels. Results show that “baseline” dermal contribution to total body absorption of EGBE vapor 

in appropriately dressed workers was, on average, 11%. Higher temperature (30°C, mean 14%, 

p = 0.03) and greater humidity (65% relative humidity, mean 13%, p = 0.1) both increased 

dermal absorption. The wearing of whole-body overalls did not attenuate absorption (mean 

10%). By combining several factors together in the industrial scenario, dermal absorption of 

vapors was reported to be as high as 39% of the total absorbed dose. 

4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN 

ANIMALS—ORAL AND INHALATION 

4.2.1. Subchronic Studies 

4.2.1.1. Oral 

A number of subchronic studies by the gavage route of exposure have been conducted. 

Krasavage (1986, 066474) conducted a toxicity study using groups of 10 COBS CD (SpragueDawley) BR adult male rats treated by gavage with 222, 443, or 885 mg/kg-day undiluted EGBE 

5 days/week for 6 weeks. Endpoints evaluated throughout the study included body weight, food 

 18 
consumption, clinical signs, and survival. Hematology and serum clinical chemistry parameters 

were determined after the last treatment. Dose-related changes were observed in the RBC counts 

of all treatment groups, including statistically significant decreases in RBC count and Hb 

concentration and a statistically significant increase in MCH. Statistically significant 

hematological changes occurring at 443 and 885 mg/kg-day were increased MCV and decreased 

MCHC. The increased MCV at higher doses is likely due to both an increase in MCV and in the 

number of larger reticulocytes in the circulation following the erythropoietic response (NTP, 

2000, 196293). Based on decreased RBC count and trends in Hb and other hematological 

endpoints, the lowest-observed-adverse-effect level (LOAEL) was determined to be 222 mg/kgday, the lowest dose tested. A no-observed-adverse-effect level (NOAEL) was not identified. 

Nagano et al. (1979, 100179) performed a toxicity study in male JCL/ICR mice 

(five/group) using gavage doses of 0, 357, 714, or 1,430 mg/kg-day EGBE 5 days/week for 

5 weeks. Parameters evaluated at the end of the study were hematology (RBC and white blood 

cell [WBC] counts, MCV, and Hb), absolute and relative weights of testes, and testicular 

histology. Mean RBC counts were significantly lower than the control values in the 357 and 

714 mg/kg-day groups, but WBC counts were not affected. All animals in the 1,430 mg/kg-day 

group died before examinations were performed; mortality was not observed in the lower dose 

groups, and no differences in testes weights or histology were found. The LOAEL for this study, 

based on the reduced RBC count, was 357 mg/kg-day. A NOAEL was not determined. 

Siesky et al. (2002, 042204) investigated whether subchronic exposure to EGBE in F344 

male rats, 8–10 weeks old, and B6C3F1 male mice, 8–10 weeks old, produced an increase in 

oxidative damage and deoxyribonucleic acid (DNA) synthesis in endothelial cells and 

hepatocytes in the mouse liver, the putative cancer target cells. Mice (60/group) were treated via 

gavage with doses of 0, 225, 450, and 900 mg/kg-day and rats (20/group) with 0, 225, and 

450 mg/kg-day for 90 days. A dose-related increase in hemolysis was observed in both rats and 

mice. An increase in the percentage of iron-stained Kupffer cells was observed following 450 

and 900 mg/kg in mice and 225 and 450 mg/kg in rats. An increase in oxidative damage, as 

measured by 8-hydroxydeoxyguanosine (8-OHdG) levels, was seen in mouse livers at 450 and 

900 mg/kg-day after 7 or 90 days, while no increase was seen in rat livers at any dose or time 

point examined. Vitamin E levels were reduced by all doses of EGBE in the mouse and rat liver 

(statistically significant at 7 and 90 days in both mice and rats); however, the basal level of 

vitamin E was ~2.5-fold higher in rat than in mouse liver. The LOAEL for this study was 

450 mg/kg-day in mice and 225 mg/kg-day in rats, based on the percentage of iron-stained 

Kupffer cells. The NOAEL was 225 mg/kg-day in mice, and a NOAEL was not determined in 

rats. 

NTP (1993, 042063) performed a 13-week toxicity study in F344 rats and B6C3F1 mice 

where groups of 10 animals/gender/species received EGBE in drinking water at doses of 0, 750, 

1,500, 3,000, 4,500, and 6,000 ppm in rats and 0, 750, 1,500, 3,000, 4,500, and 6,000 ppm in 

 19 
mice. The corresponding doses in mg/kg-day, based on measured drinking water consumption 

were: 0, 69, 129, 281, 367, or 452 mg/kg-day in male rats; 0, 82, 151, 304, 363, or 470 mg/kgday in female rats; 0, 118, 223, 553, 676, or 694 mg/kg-day in male mice; and 0, 185, 370, 676, 

861, or 1,306 mg/kg-day in female mice. Due to a dose-related decrease in water consumption 

in the 2-week studies, the test chemical was administered at a constant concentration (ppm) in 

the 13-week studies rather than on a mg/kg body weight basis. Hematology was performed on 

rats but not on mice. Complete histological exams were performed on all control animals and all 

animals in the highest dose group. Vaginal cytology and sperm indices were evaluated in rats 

and mice from the control and three highest dose groups. Hematologic changes in both genders 

persisting until or developing by 13 weeks included dose-related indications of mild to moderate 

anemia. Portions of the hematologic results from the NTP 13-week rat drinking-water study are 

presented in Table 4-1. The various results shown in this table are indicative of the various 

degrees of hemolysis caused by exposure to increasing concentrations of EGBE. Overall, the 

dose-related increase in MCV is indicative of erythrocyte swelling that would be expected to 

precede cell lysis and an increase in the number of reticulocytes. Deficits in RBCs as a result of 

lysis manifest through dose-related decreases in the measures of both RBC count and Hb 

concentration. Hct would be expected to decrease but did not. The increases noted both in 

reticulocytes (young RBCs) and, at higher doses, in nucleated erythrocytes (immature and 

prematurely released blood cells) are homeostatic responses that would be anticipated to occur as 

the lysed blood cells are being replaced. More specifically, male rats evaluated at 13 weeks 

showed significantly reduced RBC counts at ≥ 281 mg/kg-day (3,000 ppm) and reduced Hb 

concentration, reduced platelets, and increased bone marrow cellularity at ≥ 367 mg/kg-day 

(4,500 ppm). These data also suggest that female rats are more sensitive to the effects from 

EGBE, since several statistically significant effects occurred at the 750 ppm concentration, the 

lowest level tested in females; males did not show statistically significant effects until two dose 

levels higher (3,000 ppm). In addition, the degree to which these various measures were affected 

was somewhat greater in females than males (indicated as percent control in the tables), 

particularly at the three highest exposure concentrations. 

 20 
Table 4-1. Hematology and hemosiderin data from the 13-week drinking 

water exposure to EGBE in F344 rats 

Endpointa Control 750 ppm 1,500 ppm 3,000 ppm 4,500 ppm 6,000 ppm 

N 

Males 

Females 

8 

10 

10 

10 

10 

10 

10 

10 

10 

10 

9 

9 

Hct (%) 

Males 

Females 

44.8 ± 0.8 

44.8 ± 0.6 

45.0 ± 0.6 (100) 

43.2 ± 0.8 (96) 

44.7 ± 0.4 (99)

42.8 ± 0.7 (95)

44.1 ± 0.7 (98)

43.6 ± 0.7 (97)

42.3 ± 0.6b

 (94) 

44.4 ± 0.7 (99) 

43.4 ± 0.4 (97)

46.1 ± 0.7 (103)

Hb (g/dL) 

Males 

Females 

15.0 ± 0.2 

14.9 ± 0.2 

15.2 ± 0.1 (101) 

14.4 ± 0.2b

 (97) 

14.9 ± 0.1 (99)

13.9 ± 0.2 (93)

14.6 ± 0.1 (97)

14.2 ± 0.2 (95)

14.0 ± 0.1c

 (93) 

14.0 ± 0.2c

 (94) 

13.7 ± 0.2c

 (91)

13.4 ± 0.2c

 (90)

Erythrocytes 

(106

/μL) 

Males 

Females 

8.64 ± 0.15 

8.15 ± 0.09 

8.74 ± 0.10 (101) 

7.59 ± 0.15c

 (93) 

8.54 ± 0.09 

(99) 

7.09 ± 0.14c

(87) 

8.11 ± 0.12b

(94) 

7.00 ± 0.12c

(86) 

7.48 ± 0.12c

(86) 

6.80 ± 0.11c

(83) 

7.18 ± 0.12c

(83) 

6.58 ± 0.14c

(81) 

Reticulocytes 

(106

/μL) 

Males 

Females 

0.14 ± 0.03 

0.12 ± 0.02 

0.24 ± 0.06 

0.17 ± 0.03 

0.15 ± 0.02 

0.19 ± 0.03 

0.18 ± 0.02 

0.28 ± 0.03c

0.22 ± 0.05 

0.28 ± 0.05c

0.46 ± 0.07c

0.27 ± 0.05c

Nucleated 

erythrocytes 

(103

/μL) 

Males 

Females 

0.00 ± 0.00 

0.01 ± 0.01 

0.00 ± 0.00 

0.03 ± 0.02 

0.01 ± 0.01 

0.02 ± 0.01 

0.01 ± 0.01 

0.05 ± 0.02 

0.00 ± 0.00 

0.10 ± 0.03b

0.04 ± 0.02c

0.16 ± 0.04c

MCV (fL) 

Males 

Females 

52.0 ± 0.4 

54.8 ± 0.3 

51.5 ± 0.3 (99) 

57.0 ± 0.4c

 (104) 

52.3 ± 0.4 

(100) 

60.5 ± 0.4c

(110) 

54.4 ± 0.3c

(105) 

62.4 ± 0.6c

(114) 

56.7 ± 0.5c

(109) 

65.3 ± 0.6c 

(119) 

60.6 ± 1.1c

(116) 

70.1 ± 0.9c

(128) 

MCH (pg) 

Males 

Females 

17.4 ± 0.2 

18.3 ± 0.2 

17.4 ± 0.1 

18.9 ± 0.2 

17.5 ± 0.2 

19.7 ± 0.2 

18.0 ± 0.2b

20.2 ± 0.3c

18.7 ± 0.3c

20.6 ± 0.2c

19.1 ± 0.3c

20.4 ± 0.1c

Hemosiderin 

(incidence) 

Males 

Females 

0/10 

0/10 

0/10 

0/10 

0/10 

2/10 

0/10 0/10 

10/10 

7/10 

10/10 10/10 

a

Values listed are mean ± standard error at various EGBE concentrations in ppm for the 13-week time point. 

Percent of control values in parentheses. 

b

Statistically significant difference, p ≤ 0.05. c

Statistically significant difference, p ≤ 0.01. 

Source: NTP (1993, 042063). 

Statistically significant hematologic effects in female rats at week 13 included reduced 

RBC counts and Hb concentrations at ≥ 82 mg/kg-day and increased reticulocytes, decreased 

platelets, and increased bone marrow cellularity at approximately 304 mg/kg-day, all being 

 21 
indicative of hemolysis. There were no histopathological changes in the testes or epididymis at 

≥ 129 mg/kg-day. 

Table 4-2 shows that liver effects, including cytoplasmic alterations, hepatocellular 

degeneration, and pigmentation were observed in the mid- and high-dose groups (129, 281, 367, 

and 452 mg/kg-day for males and 151, 304, 363, and 470 mg/kg-day for females; statistics not 

reported). As with the hematologic effects, these effects appeared to be more severe in females 

than in males. Cytoplasmic alterations of liver hepatocytes, consisting of hepatocytes staining 

more eosinophilic and lacking the basophilic granularity of the cytoplasm present in hepatocytes 

from control animals, were observed in the lowest-dose groups tested (69 mg/kg-day for males 

and 82 mg/kg-day for females). The lack of cytoplasmic granularity or “ground-glass” 

appearance of the hepatocytes suggests that this response was not due to enzyme induction 

(Greaves, 2000, 089176). The cytoplasmic alterations were judged to increase in severity in both 

genders, but especially in females, with the severity in the two highest dose groups being judged 

as “moderate.” Liver pigmentation, colored brown to green and staining strongly positive for 

iron (indicative of hemosiderin accumulation), was noted in the cytoplasm of Kupffer cells in 

both genders of rats. In females, liver pigmentation was noted in 0/10 controls and 0/10 at 

82 mg/kg-day, 2/10 with a severity grade of 1 (minimal) at 151 mg/kg-day, and 10/10 in the 

three highest dose levels; the severities increased from a numerical grade of 1.2 in the 304 

mg/kg-day group to 1.9 the upper two dose groups. In males, the hemosiderin pigmentation was 

noted in animals exposed to the highest dose only (452 mg/kg-day) at an incidence of 7/10 and a 

severity rating of 1 (minimal). No hepatic pigmentation was reported in the mice exposed for 

13 weeks. The hematological (decreased RBC count and Hb) and hepatic changes were doserelated; 69–82 mg/kg-day was considered a LOAEL. A NOAEL was not identified. 

 22 
Table 4-2. Incidencea

 and severity of selected histopathological changes from 

the 13-week drinking water exposure to EGBE in F344 rats and mice 

Control 750 ppm 1,500 ppm 3,000 ppm 4,500 ppm 6,000 ppm 

Rat

N 10 10 10 10 10 10 

Liver cytoplasmic alterations (NR) 

Males 0 4 (1.0) 8 (1.0) 7 (1.1) 10 (2.0) 10 (1.8) 

Females 0 5 (1.4) 9 (2.0) 10 (2.2) 10 (3.0) 10 (3.0) 

Hepatocellular degeneration (NR) 

Males 0 0 0 8 (1.0) 8 (1.0) 8 (1.0) 

Females 0 0 0 10 (1.3) 10 (1.3) 10 (1.1) 

Kupffer cell pigmentation (NR) 

Males 0 0 0 0 0 7 (1.0) 

Females 0 0 2 (1.0) 10 (1.2) 10 (1.9) 10 (1.9) 

Mouse 

N 10 10 10 10 10 10 

Necropsy body weight (g)b

31.1 ± 0.7 31.8 ± 0.8 30.9 ± 1.5 28.0 ± 0.7c 28.4 ± 0.5c 27.8 ± 0.9d Females 

(only) 

Relative kidney weight (right) (mg organ wt/g body wt)b

Females 

(only) 

6.33 ± 0.10 7.69 ± 0.14d 8.06 ± 0.29d 7.47 ± 0.19d 7.55 ± 0.18d 8.21 ± 0.26d

a

Incidences represent the number of animals with lesions. Average severity (in parentheses) is based on the number 

of animals with lesions; 1 = minimal, 2 = mild, 3 = moderate, 4 = marked. 

b

Mean ± standard error. 

c

Statistically significant difference, p ≤ 0.05. 

d

Statistically significant difference, p ≤ 0.01. 

NR = Statistics not reported 

Source: NTP (1993, 042063). 

Female mice showed statistically significant reductions in body weight gain starting at 

3,000 ppm and statistically significant increases in relative kidney weight at all doses. Changes 

at the higher doses followed the reductions in body weight at those dose levels, while the 

increases at lower doses (750 and 1,500 ppm) were due to increased absolute kidney weights at 

those doses. Body weight reduction followed the decreased water consumption data. No 

histopathologic changes were noted at any dose level, even though relative kidney weights 

showed a statistically significant increase at 750 and 1,500 ppm in the absence of reduction in 

body weight gain. 

Keith et al. (1996, 041625) administered EGBE at 120 mg/kg-day for 120 days by gavage 

to transgenic FVB/N mice (25 mice/gender/group) carrying the v-Ha-ras oncogene and observed 

the animals for an additional 120 days. EGBE did not induce an increase in tumors at any site. 

 23 
4.2.1.2. Inhalation 

Wistar rats (23 animals/group, gender not specified) were exposed to 0, 135, or 320 ppm 

EGBE 7 hours/day, 5 days/week for 5 weeks (Werner et al., 1943, 597282). Hematologic 

endpoints—RBC, WBC, differential, and reticulocyte counts and Hb concentration—were 

evaluated. Exposure to 320 ppm EGBE resulted in an increased percentage of circulating 

immature granulocytes, a decreased Hb concentration and RBC count, and an increased 

reticulocyte count. These hematologic changes were not severe; they were reversed 3 weeks 

after discontinuing exposure. No effect on WBC count was observed. In another study, Werner 

et al. (1943, 100219) exposed groups of two dogs of unspecified strain to subchronic inhalation 

doses of 0 or 415 ppm EGBE 7 hours/day, 5 days/week for 12 weeks. Necropsies were 

performed 5 weeks postexposure; hematologic parameters were examined before, during, and 

after the exposure. No statistical analysis was presented. The authors concluded that exposure 

of dogs to EGBE vapors resulted in decreased Hb concentration and RBC count with increased 

hypochromia, polychromatophilia, and microcytosis. These hematologic effects were not severe 

and they were reversed 5 weeks after the end of exposure. 

Carpenter et al. (1956, 066464) studied the hemolytic effects of EGBE vapor inhalation 

in rats, mice, dogs, and monkeys, in addition to humans. An unspecified strain of rats 

(15/gender/group) was exposed via inhalation to 54, 107, 203, 314, or 432 ppm EGBE 7 

hours/day, 5 days/week for 6 weeks. Erythrocyte osmotic fragility was observed in rats 

immediately after a single 7-hour exposure to ≥ 107 ppm. Osmotic fragility in females exceeded 

that for males. In almost all cases, these high fragility values returned to normal after the rats 

rested overnight. In the same study, the authors exposed groups of 10 male C3H mice to 100, 

200, or 400 ppm EGBE 7 hours/day for 30, 60, or 90 days. An increase in erythrocyte osmotic 

fragility occurred at all concentrations and was consistent throughout the exposures. In all 

instances, erythrocyte osmotic fragility was normal after a 17-hour rest period. The LOAELs for 

these rat and mouse studies were 54 and 100 ppm, respectively. NOAELs were not reported. 

The authors reported slight increases in erythrocyte osmotic fragility for a male and a female dog 

(basenji hybrids) exposed to 200 ppm EGBE for 31 days (7 hours/day). RBC counts and Hb 

concentrations were slightly decreased in the female. Erythrocyte permeability, as determined 

by radio-iodine uptake, was increased in both genders, but was not statistically different when 

compared with control values. A female dog succumbed after 8 days of inhalation exposure to 

385 ppm of EGBE (7 hours/day). Symptoms included loss of weight, transitory increases in 

erythrocyte osmotic fragility, nasal and ocular infection, weakness, apathy, anorexia, and 

increased WBC count. Necropsy of this animal revealed severe congestion and hemorrhage of 

the lungs and congestion of the liver and both kidneys. In addition, a severe subcapsular 

hemorrhage was found in one adrenal gland. A male dog survived after 28 days of inhalation 

exposure to 385 ppm of EGBE for 7 hours/day. Effects in the male were similar to the female, 

but developed more slowly. At autopsy, congestion of the kidneys was not observed. In studies 

 24 
on male and female monkeys, occasional rises in erythrocyte osmotic fragility were reported that 

were more frequent in the female than in the male following 90-day inhalation exposure to 100 

ppm of EGBE. 

A 90-day subchronic inhalation study was performed using F344 rats (16/gender/group) 

exposed to EGBE for 6 hours/day, 5 days/week at concentrations of 0, 5, 25, and 77 ppm (Dodd 

et al., 1983, 066465). After 31 completed exposures (6 weeks), the 77 ppm female rats had 

slight but statistically significant decreases in RBC counts (13% below control value) and Hb 

concentrations, accompanied by an 11% increase above the control value in MCH. The 77 ppm 

males exhibited slight (5%) but statistically significant decreases in RBC counts and Hb 

concentration that were accompanied by increases in MCH. At the end of the 90-day study 

(66 exposures), the hematologic effects seen in the 77 ppm exposed animals had either lessened 

or returned to the ranges of control values and were no longer statistically significant. The 

NOAEL was determined to be 25 ppm, and the LOAEL was 77 ppm. 

In the subchronic portion of the inhalation NTP (2000, 196293) study, F344 rats and 

B6C3F1 mice (10/gender) were exposed to EGBE concentrations of 0, 31, 62.5, 125, 250, and 

500 ppm (0, 150, 302, 604, 1,208, and 2,416 mg/m3) 6 hours/day, 5 days/week for 14 weeks. 

Hematologic and hemosiderin staining results are presented in Tables 4-3 and 4-4. These results 

are indicative of the various degrees of hemolysis caused by exposure to increasing 

concentrations of EGBE. Both rat genders exhibited clinical signs at the three highest doses, 

consistent with the hemolytic effects of EGBE, including: (1) deficits in RBCs as a result of 

lysis manifestation through the dose-related decrease in Hct—a finding consistent with decreases 

noted for both RBC count and Hb concentrations; and (2) increases in both reticulocytes and 

nucleated erythrocytes at higher doses—homeostatic responses that would be anticipated to 

occur as the lysed blood cells are being replaced. Female rats may be somewhat more sensitive; 

several statistically significant effects occur at the 31 ppm level in females, as opposed to a 

single parameter for males. In addition, the degree to which these various measures are affected 

is somewhat greater in females than males (indicated as percent control) particularly at the three 

highest concentrations. Hematologic evaluation showed mild-to-moderate regenerative anemia 

at all concentrations in females and at the three highest concentrations in males. Exposurerelated trends were noted for reticulocyte count, RBC count, MCV, Hb concentration, and Hct. 

Liver-to-body-weight ratios increased significantly in males at the two highest concentrations 

and in females at the highest concentration. Histopathologic effects at concentrations >62.5 ppm 

for male rats and >31 ppm for females consisted of excessive splenic congestion in the form of 

extramedullary hematopoiesis, hemosiderin accumulation in Kupffer cells, liver necrosis, 

centrilobular hepatocellular degeneration, renal tubular degeneration, intracytoplasmic Hb and 

hemosiderin deposition, and bone marrow hyperplasia. In addition, five moribund female rats 

were sacrificed from the highest concentrations and one from the 250 ppm group. The LOAEL 

 25 
for hematological alterations was 31 ppm for female rats and 62.5 ppm for male rats. The 31 

ppm exposure level was considered a NOAEL for male rats. 

Table 4-3. Hematology and hemosiderin data from a 14-week inhalation 

study of EGBE in F344 rats 

Endpointa Control 

31 ppm 

(150 mg/m3) 

62.5 ppm 

(302 mg/m3) 

125 ppm 

(604 mg/m3) 

250 ppm 

(1,208 mg/m3) 

500 ppm 

(2,416 mg/m3) 

Hct (%) 

Males 

Females 

46.8 ± 0.5 

48.5 ± 0.5 

45.8 ± 0.6 (98) 

46.0 ± 0.5c

(95) 

47.0 ± 0.4(100)

45.2 ± 0.5c

(93) 

44.5 ± 0.5c

 (95) 

42.9 ± 0.4c

 (88)

41.1 ± 0.3c

 (88) 

40.0 ± 0.3c

 (82) 

37.3 ± 0.4c

 (80) 

36.2 ± 0.6c

 (75) 

Hb (g/dL) 

Males 

Females 

15.5 ± 0.1 

15.6 ± 0.1 

14.8 ± 0.3 (95) 

15.0 ± 0.1c

(96) 

15.4 ± 0.1 (99) 

14.6 ± 0.1c

(94) 

14.5 ± 0.2c

 (94) 

13.6 ± 0.1c

 (87)

13.1 ± 0.1c

 (85) 

12.5 ± 0.1c

 (80) 

11.7 ± 0.1c

 (75) 

10.5 ± 0.3c

 (67) 

Erythrocytes 

(106

/μL) 

Males 

Females 

9.05 ± 0.08 

8.48 ± 0.05 

8.71 ± 0.14b (96)

8.08 ± 0.07c

 (95)

8.91 ± 0.06(98)

7.70 ± 0.08c

(91)

8.01 ± 0.08c

(89)

6.91 ± 0.05c

(81)

7.10 ± 0.07c

 (78) 

6.07 ± 0.04c

 (72) 

5.97 ± 0.05c

(66) 

4.77 ± 0.15c

 (56)

Reticulocytes 

(106

/μL) 

Males 

Females 

0.16 ± 0.02 

0.13 ± 0.02 

0.17 ± 0.03 

0.10 ± 0.01 

0.15 ± 0.02 

0.16 ± 0.02 

0.30 ± 0.04c

0.26 ± 0.04b

0.48 ± 0.06c

0.34 ± 0.04c

0.68 ± 0.07c

0.40 ± 0.11c

Nucleated 

erythrocytes 

(103

/μL) 

Males 

Females 

0.04 ± 0.02 

0.04 ± 0.02 

0.05 ± 0.01 

0.05 ± 0.02 

0.04 ± 0.03 

0.12 ± 0.03b

0.11 ± 0.03 

0.18 ± 0.07 

0.17 ± 0.04c

0.61 ± 0.24c

0.20 ± 0.06c

0.73 ± 0.27c

MCV (fL) 

Males 

Females 

50.4 ± 0.3 

55.1 ± 0.3 

50.2 ± 0.2 (100) 

55.3 ± 0.2 (100)

50.7 ± 0.2 (100)

56.4 ± 0.2 (102)

53.1 ± 0.2c

(105)

58.7 ± 0.2c

(107)

53.8 ± 0.3c

 (107) 

61.6 ± 0.2c

 (112) 

58.5 ± 0.3c

(117) 

66.8 ± 0.9c

 (121)

MCH (pg) 

Males 

Females 

17.1 ± 0.1 

18.4 ± 0.1 

17.0 ± 0.1 

18.6 ± 0.2 

17.3 ± 0.1 

19.0 ± 0.0c

18.1 ± 0.1c

19.6 ± 0.1c

18.4 ± 0.1c

20.6 ± 0.1c

19.5 ± 0.1c

22.0 ± 0.1c

Hemosiderin 

(incidence) 

Males 

Females 

0/10 

0/10 

0/10 

0/10 

0/10 

10/10 

7/10 10/10 

9/9 

10/10 

10/10 5/5 

a

Values listed are mean ± standard error (percent of control). 

b

Statistically significant difference, p ≤ 0.05. c

Statistically significant difference, p ≤ 0.01. 

Source: NTP (2000, 196293). 

 26 
Table 4-4. Hematology and hemosiderin data from a 14-week inhalation 

study of EGBE in B6C3F1 mice 

Endpointa Control 

31 ppm 

(150 mg/m3) 

62.5 ppm 

(302 mg/m3) 

125 ppm 

(604 mg/m3) 

250 ppm 

(1,208 mg/m3) 

500 ppm 

(2,416 mg/m3)

Hct (%) 

Males 

Females 

47.3 ± 1.0 

46.2 ± 0.3 

48.3 ± 0.4(102) 

45.9 ± 0.3(99) 

47.6 ± 0.5(101) 

45.8 ± 0.3(99) 

46.6 ± 0.4(99) 

45.1 ± 0.2c

(98) 

44.2 ± 0.4c

(93) 

42.3 ± 0.4c

(92) 

36.3 ± 1.4c

(77)

37.8 ± 1.0c

(82)

Hb (g/dL) 

Males 

Females 

15.7 ± 0.4 

15.7 ± 0.1 

16.0 ± 0.1(102) 

15.4 ± 0.1b

(98) 

15.9 ± 0.1(101) 

15.4 ± 0.1b

(98) 

15.4 ± 0.1c

(98) 

14.8 ± 0.1c

(94) 

14.4 ± 0.1c

(92) 

13.7 ± 0.1c

(87) 

11.4 ± 0.4c

(73)

11.6 ± 0.1c

(74)

Erythrocytes 

(106

/μL) 

Males 

Females 

9.71 ± 0.22 

9.72 ± 0.05 

10.04 ± 0.08(103)

9.55 ± 0.06b

(98) 

9.77 ± 0.1(101) 

9.51 ± 0.06b

(98)

9.47 ± 0.06b

(98) 

9.18 ± 0.05c

(94) 

8.90 ± 0.07c

(92)

8.57 ± 0.06c(88)

7.21±0.23c

(74)

7.35±0.07c

(76)

Reticulocytes 

(106

/μL) 

Males 

Females 

0.21 ± 0.03 

0.18 ± 0.02 

0.22 ± 0.03 

0.21 ± 0.03 

0.21 ± 0.02 

0.19 ± 0.02 

0.32 ± 0.03b

0.29 ± 0.02c

0.45 ± 0.04c

0.47 ± 0.04c

0.79 ± 0.20c

1.17 ± 0.28c

MCV (fL) 

Males 

Females 

49.1 ± 0.4 

48.3 ± 0.3 

48.5 ± 0.3(99) 

48.8 ± 0.2(101) 

49.0 ± 0.4 (100) 

48.8 ± 0.2 (101) 

49.7 ± 0.4(101) 

49.5 ± 0.5 (102) 

49.8 ± 0.4(101) 

49.0 ± 0.3(101) 

48.3± 0.9(98) 

48.8± 1.0(101)

MCH (pg) 

Males 

Females 

16.2 ± 0.1 

16.1 ± 0.1 

16.0 ± 0.1(99) 

16.0 ± 0.1(99) 

16.2 ± 0.1(100) 

16.2 ± 0.1(101) 

16.2 ± 0.0(100) 

16.1 ± 0.1(100) 

16.2 ± 0.1(100) 

16.0 ± 0.0(99) 

15.8 ± 0.2(98) 

15.8 ± 0.1(98)

Hemosiderin 

(incidence) 

Males 

Females 

0/10 

0/10 

0/10 

0/10 

0/10 

0/10 

0/10 0/10 

10/10 

6/6 

0/10 6/6 

a

Values listed are mean ± standard error (percent of control).

b

Statistically significant difference, p ≤ 0.05. c

Statistically significant difference, p ≤ 0.01. 

Source: NTP (2000, 196293). 

The mice exposed via the inhalation route exhibited clinical signs consistent with the 

hemolytic effects of EGBE at the two highest concentrations for both genders (NTP, 2000, 

196293). Hematologic evaluation indicated a moderate regenerative anemia (marked by 

decreased RBC counts, increased reticulocyte counts, and increased MCV) with an increase in 

platelets at the three highest concentrations in both genders. Histopathological effects consisted 

of excessive extramedullary splenic hematopoiesis, renal tubular degeneration, hemosiderin 

deposition in the spleen and kidney and accumulation in Kupffer cells, and testicular 

degeneration. Forestomach necrosis, ulceration, inflammation, and epithelial hyperplasia were 

observed at concentrations >31 ppm for females and >62.5 ppm for males. In addition, four 

females and four males either died or were sacrificed moribund at the highest concentration. The 

NOAEL for male and female mice was 31 ppm and the LOAEL was 62.5 ppm, based on 

histopathological changes in the forestomach. 

 27 
4.2.2. Chronic Studies and Cancer Bioassays 

4.2.2.1. Inhalation 

NTP (2000, 196293) also completed a two-species, 2-year inhalation study on EGBE in 

both genders of rats and mice. In this chronic study, animals were exposed to EGBE 6 

hours/day, 5 days/week at concentrations of 0, 31, 62.5, and 125 ppm (0, 150, 302, and 604 

mg/m3

) for groups of 50 F344/N rats and 0, 62.5, 125, and 250 ppm (0, 302, 604, and 1,208 

mg/m3

) for groups of 50 B6C3F1 mice. The researchers stated that the highest exposure was 

selected to produce a 10–15% depression in hematologic indices. They reported that no effect on 

survival was observed in rats, but survival was statistically significantly decreased in male mice 

exposed to 125 or 250 ppm, compared with chamber controls (54, 52, and 78% respectively 

(NTP, 2000, 196293)). Although statistics were not reported for mean body weights, the rats 

exposed to 31 and 62.5 ppm had similar mean body weights to the control rats. Mean body 

weights of the exposed mice were generally less than those of controls, with females 

experiencing greater and earlier reductions. From week 17 to the end of the study, the mean 

body weights of 125 ppm female rats were generally less than those of controls. Nonneoplastic 

effects in rats included hyaline degeneration of the olfactory epithelium in males (13/48, 21/49, 

23/49, 40/50) and females (13/50, 18/48, 28/50, 40/49) and Kupffer cell pigmentation in the 

livers of males (23/50, 30/50, 34/50, 42/50) and females (15/50, 19/50, 36/50, 47/50) 

(Table 4-5). The severity of the olfactory lesion was not affected by exposure. The Kupffer cell 

pigmentation is a result of hemosiderin accumulation and is a recognized secondary effect of the 

hemolytic activity of EGBE (NTP, 2000, 196293). 

 28 
Table 4-5. Selected female and male rat and mouse nonneoplastic effects 

from the 2-year chronic EGBE inhalation study 

Control 31 ppm 62.5 ppm 125 ppm 250 ppm 

Rat

Kupffer cell pigmentation, hemosiderin in the liver 

Male 

Female 

23/50 30/50 

15/50 19/50 

34/50a

36/50a

42/50a

47/50a

NT 

NT 

Hyaline degeneration of the olfactory epithelium 

Male 

Female

13/48 21/49a

18/48 

23/49a

28/50a

40/50a

40/49a

NT 

13/50 NT 

Mouse

Kupffer cell pigmentation, hemosiderin in the liver 

Male 

Female 

0/50 NT 

0/50 NT 

0/50 

5/50a

8/49b

25/49b

30/49b

44/50b

Hematopoietic cell proliferation in the spleen 

Male 

Female 

12/50 NT 

24/50 NT 

11/50 

29/50 

26/48b

32/49 

42/50b

35/50a

Hemosiderin in the spleen 

Male 

Female 

0/50 NT 

39/50 NT 

6/50a

44/50 

45/48b

46/49b

44/49b

48/50b

Forestomach ulcers 

Male 

Female 

1/50 NT 

1/50 NT 

2/50 

7/50a

9/49b

13/49b

3/48 

22/50b

Forestomach epithelial hyperplasia 

Male 

Female 

1/50 NT 

6/50 NT 

7/50a

27/50b

16/49b

42/49b

21/48b

44/50b

Hyaline degeneration of the olfactory epithelium 

Females (only) 6/50 NT 14/50 11/49 12/50 

Bone marrow hyperplasia 

Males (only) 0/50 NT 1/50 9/49a 5/50a

a

Statistically significant difference, p ≤ 0.05. 

b

Statistically significant difference, p ≤ 0.01. 

NT = not tested 

Source: NTP (2000, 196293). 

Nonneoplastic, statistically significant effects in mice included forestomach ulcers and 

epithelial hyperplasia, hematopoietic cell proliferation and hemosiderin pigmentation in the 

spleen, Kupffer cell pigmentation in the livers, and bone marrow hyperplasia (males only). 

Hyaline degeneration of the olfactory epithelium (females only) was increased relative to 

chamber controls but was not statistically significant. As in the rats, the Kupffer cell 

pigmentation was considered a secondary effect of the hemolytic activity of EGBE. Bone 

marrow hyperplasia, hematopoietic cell proliferation, and hemosiderin pigmentation in the 

spleen were also attributed to the primary hemolytic effect; it was followed by regenerative 

 29 
hyperplasia of the hematopoietic tissue. The forestomach lesions did not appear to be related to 

the hemolytic effect of EGBE. Incidences of ulcer were significantly increased in all exposed 

female groups, as well as males exposed to 125 ppm. Incidences of epithelial hyperplasia, 

usually focal, were significantly increased in all exposed groups of males and females. The 

hyperplasia was often associated with ulceration, particularly in the females, and consisted of 

thickness of the stratified squamous epithelium and sometimes the keratinized layer of the 

forestomach. Ulceration consisted of a defect in the forestomach wall that penetrated the full 

thickness of the epithelium and frequently contained accumulations of inflammatory cells and 

debris. 

Using the same exposure levels described above, additional groups of rats (27/gender/

exposure group) and mice (30/gender/exposure group) in the 2-year study were examined at 3, 6, 

and 12 months (8–10 animals/time point) for hematologic effects (NTP, 2000, 196293). Nine 

male and nine female rats were exposed to 31 ppm EGBE, specifically to evaluate hematology at 

3 months and to receive a total evaluation at 6 months. Animals were continuously exposed, as 

described above, until their sacrifice at 3, 6, or 12 months. As in the 14-week study, inhalation 

of EGBE by both species resulted in the development of exposure-related hemolytic effects, 

inducing a responsive anemia. In rats, the anemia was persistent and did not progress or 

ameliorate in severity from 3 months to the final blood collection at 12 months. Statistically 

significant (p < 0.05) decreases in automated and manual Hct values, Hb concentrations, and 

RBC counts occurred at 3, 6, and 12 months in the 125 and 250 ppm female mice and the 250 

ppm male mice. Statistically significant decreases in these same endpoints were also observed in 

62.5 ppm females at 6 months and in 125 ppm males at 6 and 12 months (decreases in Hct were 

observed only at 3 and 6 months). MCV was increased in female mice at the highest duration 

(12 months) and exposure (250 ppm) levels. Reticulocyte counts were increased significantly in 

the 125 ppm females at 3 and 6 months and in the 125 ppm males at 6 months of exposure. 

Table 4-6 shows the responses available for a representative measure of the hematologic effects 

from EGBE exposure. Hct levels for male and female rats and mice measured after 3 months or 

12 months are presented. 

 30 
Table 4-6. Comparison of female and male rat and mouse Hct (manual) 

values from 3- and 12-month inhalation exposures to EGBE 

Control 31.2 ppm 62.5 ppm 125 ppm 250 ppm 

Female ratsa

3 mos 

12 mos 

46.5 ± 0.5 

45.4 ± 0.2 

46.1 ± 0.5 (95) 

– 

43.3 ± 0.5c

 (93) 

45.3 ± 0.3 (100) 

42.2 ± 0.5c

 (91) 

42.3 ± 0.4c

 (93) 

– 

– 

Male ratsa

3 mos 

12 mos 

44.9 ± 0.2 

47.8 ± 0.4 

46.9 ± 0.5 (104) 

– 

44.8 ± 0.4 (100) 

45.9 ± 0.8b

 (96) 

42.9 ± 0.5b

 (95) 

42.9 ± 1.2c

 (90) 

– 

– 

Female micea

3 mos 

12 mos 

49.3 ± 0.5 

46.9 ± 0.4 

– 

– 

48.9 ± 0.4 (99) 

46.3 ± 0.4 (99) 

46.2 ± 0.5c

 (94) 

43.8 ± 0.4c

 (93) 

43.7 ± 0.5c

 (89) 

41.8 ± 0.3c

 (89) 

Male micea

3 mos 

12 mos 

47.5 ± 0.3 

47.9 ± 0.4 

– 

– 

47.3 ± 0.5 (100) 

48.7 ± 1.9 (102) 

46.0 ± 0.4b

 (97) 

46.4 ± 1.0 (97) 

43.7 ± 0.2c

 (92) 

42.1 ± 0.4c

 (88) 

a

These results are from a serial sacrifice conducted as a part of the 2-year chronic inhalation study. Values listed 

are mean ± standard error (percent of control). 

b

Statistically significant difference, p ≤ 0.05. c

Statistically significant difference, p ≤ 0.01. 

– = data were not available 

Source: NTP (2000, 196293). 

In vitro studies by Ghanayem (1989, 042014) have shown that the hemolysis caused by 

the EGBE metabolite BAA is preceded by erythrocyte swelling. If the observed increase in 

MCV is in response to cell swelling, it could be a preliminary indicator of the hemolytic effect. 

Other researchers, however, have attributed the increased MCV at all exposures and the 

increased MCH at higher exposures to the erythropoietic response subsequent to hemolysis and 

the corresponding increase in the number of larger reticulocytes in circulation (cited in NTP, 

2000, 196293). Reticulocyte counts were significantly increased in female rats at 62.5 ppm (6 

and 12 months) and in male rats at 125 ppm (3 and 6 months). Since a statistically significant 

increase in reticulocyte count was not observed at any time point in males or females exposed to 

31 ppm or in males exposed to 62.5 ppm, it appears that reticulocyte count alone cannot account 

for the increase in MCV. The observed increases in MCV may be a combined result of both 

erythrocyte swelling prior to, and an increased number of reticulocytes subsequent to, hemolysis; 

the former would be more influential at lower exposure levels, and the latter would have more 

relative impact at higher levels. 

Similar effects indicating anemia were also observed in mice, where females were more 

sensitive. As in rats, the anemia response was observed at slightly higher doses, but was 

persistent and did not progress or ameliorate in severity from 3 months to the final blood 

collection at 12 months. Table 4-6 shows the manual Hct values in male and female rats and 

mice at 3 months and 12 months. Statistically significant (p < 0.05) decreases in automated and 

 31 
manual Hct values and Hb and RBC counts occurred at 3, 6, and 12 months in the 125 ppm 

females and the 250 ppm males and females. Statistically significant decreases in these 

endpoints were also observed in 62.5 ppm females exposed for 6 months and in 125 ppm males 

exposed for 6 and 12 months (decreases in Hct were observed only at 3 and 6 months). No 

changes were observed in the MCV of mice, except for an increase in female mice at the highest 

duration (12 months) and exposure (250 ppm) levels. Reticulocyte counts were increased 

significantly in 125 ppm females at 3 and 6 months and in 125 ppm males at 6 months of 

exposure. 

At the end of the 2-year chronic bioassay (NTP, 2000, 196293), no significant neoplastic 

effects were observed in male or female rats. In female rats, the combined incidence of benign 

and malignant pheochromocytoma of the adrenal medulla was 3/50, 4/50, 1/49, and 8/49. The 

incidence in the high-dose group (16%) does not represent a statistically significant increase over 

the chamber control group (6%), but did exceed the historical control range (6.4 ± 3.5%; range 

2–13%) for this effect. 

Male mice exposed to 125 and 250 ppm EGBE had a low survival rate. A high rate of 

hepatocellular carcinomas was found in these exposure groups (10/50 [control], 11/50, 16/49, 

21/49); the increase at the high-exposure level was statistically significant (p < 0.01). When 

hepatocellular adenomas and carcinomas are combined, no significant increase was observed in 

any exposure group. However, the incidence of hemangiosarcomas in males exposed to 

250 ppm (8%) was significantly increased (p = 0.046) relative to chamber controls (0/50, 1/50, 

2/49, 4/49) and exceeded the range of historical controls (14/968; 1.5 ± 1.5%; range 0–4%). No 

organisms consistent with Helicobacter hepaticus were found in the 14 mice evaluated (NTP, 

2000, 196293). The researchers concluded from this that H. hepaticus was not a factor in the 

development of liver neoplasms. No significant increases in benign or malignant hepatocellular 

tumors or hemangiosarcomas were noted in the female mice, and the incidence of hepatocellular 

adenomas actually decreased significantly (p < 0.05) in relation to the control chamber group 

(16/50, 8/50, 7/49, 8/49). 

Forestomach squamous cell papillomas and carcinomas, combined, were significantly 

increased (trend test = 0.003) in female mice relative to the chamber controls (0/50, 1/50, 2/50, 

6/50). The incidence of these tumor types (12%) at the highest exposure level was statistically 

significant and exceeded the range for the occurrence of these tumors in historical controls 

(0.9 ± 1.1%; range 0–3%). The first incidence of these tumors appeared in the group exposed to 

250 ppm at 582 days, compared with 731 days at 62.5 and 125 ppm. This indicates a decreased 

latency period in the highest exposure group. While the incidence of these types of forestomach 

tumors was not significantly increased over controls in male mice (1/50, 1/50, 2/50, 2/50), the 

incidence of squamous cell papillomas (4%) in the two highest exposure groups exceeded the 

range for historical controls (0.5 ± 0.9%; range 0–2%). The increased incidences of forestomach 

neoplasms in males, as in females, occurred in groups with ulceration and hyperplasia. 

 32 
Section 4.6 has a discussion of the cancer data from this study. With respect to the 

noncancer findings, a NOAEL could not be determined, and a LOAEL of 62.5 ppm was 

determined in mice for hemosiderin deposition. In rats, a NOAEL could not be determined, and 

a LOAEL of 31 ppm was determined for hemosiderin deposition. 

4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND INHALATION 

Due to the known reproductive toxicity, such as effects to male testes and sperm, of two 

other glycol ethers, ethylene glycol methyl ether (EGME; 2-methoxyethanol) and ethylene glycol 

ethyl ether (EGEE; 2-ethoxyethanol), the reproductive toxicity of EGBE was studied in a variety 

of oral studies (Exon et al., 1991, 100112; Foster et al., 1987, 100116; Grant et al., 1985, 

006770; Heindel et al., 1990, 042042; Nagano et al., 1979, 100179; Nagano et al., 1984, 100180; 

NTP, 1993, 042063) and inhalation studies (Dodd et al., 1983, 066465; NTP, 2000, 196293) 

using rats, mice, and rabbits. Several developmental studies have addressed EGBE toxicity from 

conception to sexual maturity, including toxicity to the embryo and fetus, following oral (Sleet et 

al., 1989, 042089; Wier et al., 1987, 042123), inhalation (Nelson et al., 1984, 031878; Tyl et al., 

1984, 100209), and dermal (Hardin et al., 1984, 042030) exposures in rats, mice, and rabbits. In 

many instances, LOAELs and NOAELs were reported for both parental and developmental 

effects; therefore, the developmental studies can also be used to assess systemic toxicity as well 

as developmental toxicity. 

EGBE did not cause significant effects in the reproductive organs, including testes, in any 

study. In a two-generation reproductive toxicity study, fertility was reduced in mice only at very 

high, maternally toxic doses (>1,000 mg/kg). Maternal toxicity, related to the hematologic 

effects of EGBE, and relatively minor developmental effects have been reported in 

developmental studies and are discussed below. No teratogenic effects were noted in any of the 

studies. It can be concluded from these studies that EGBE is not significantly toxic to the 

reproductive organs of adult males or females, or to the developing fetuses of laboratory animals. 

As discussed in Section 4.2, Nagano et al. (1979, 100179) performed a toxicity study in 

male JCL/ICR mice (five/group), using gavage doses of 0, 357, 714, or 1,430 mg/kg-day EGBE 

5 days/week for 5 weeks. A LOAEL of 357 mg/kg-day based on reduced RBC count was 

identified, but no changes in testes weight or histology were observed. In another study, Nagano 

et al. (1984, 100180) used the same dosing regimen up to 2,000 mg/kg-day to test EGBE and 

other glycol ethers. Testicular atrophy was observed for EGEE and EGME, but not for EGBE. 

Grant et al. (1985, 006770) exposed male F344 rats (six/group) to gavage doses of 0, 500, 

or 1,000 mg/kg-day EGBE and EGME for 4 days. Severe testicular atrophy was observed in rats 

fed 500 mg/kg-day EGME, but no significant effect was noted in rats fed up to 1,000 mg/kg-day 

EGBE. 

Krasavage (1986, 066474) conducted a toxicity study using groups of 10 COBS 

CD(Sprague-Dawley)BR adult male rats treated by gavage with 222, 443, or 885 mg/kg-day 

 33 
undiluted EGBE 5 days/week for 6 weeks. They found no effects on testicular weight and no 

histopathological lesions in the testes, seminal vesicles, epididymides, or prostate gland at any 

exposure level. 

Foster et al. (1987, 100116) fed Alpk/AP (Wistar-derived) male rats single gavage doses 

of 0, 174, 434, or 868 mg/kg BAA. Occasional significant decreases in the weight of the 

prostate gland and seminal vesicles were observed, but the decreases were not time- nor doserelated. No treatment-related lesions were noted following histologic examination of the testes, 

epididymides, or prostate. BAA did not produce any changes in testicular cell populations when 

introduced in vitro at 5 mM. Simultaneous testing of the acids of EGME and EGEE resulted in 

significant spermatocyte cell loss and damage in vivo and in vitro. 

Subchronic reproductive studies were conducted using male and female Swiss CD-1 

mice. Heindel et al. (1990, 042042) exposed them to EGBE in drinking water at doses of 0, 700, 

1,300, and 2,000 mg/kg-day for 7 days premating; subsequently, they exposed the mice for 98 

days while cohoused as breeding pairs. The higher two dose levels resulted in mortality: 13/20 

died during the study in the 2,000 mg/kg-day group and 6/20 died in the 1,300 mg/kg-day dose 

group, as compared with 1 each in the 700 mg/kg-day group and the control group. Statistically 

significant toxic effects seen in the 1,300 and 2,000 mg/kg-day dose groups with adult mice 

included decreased body weight gain, increased kidney and liver weights, and dose-related 

decreases in water consumption. Statistically significant developmental effects observed in the 

1,300 and 2,000 mg/kg-day dose groups included decreased pup weight and fewer and smaller 

litters produced per pair. A significant reduction (5%) in live pup weight was also observed in 

the 700 mg/kg-day dose group. No statistically significant effect on fertility was observed in the 

700 mg/kg-day dose group. 

At the completion of the 98-day continuous breeding phase, F0 breeding pairs were 

separated and housed individually, while exposure to EGBE continued. When the last litter was 

weaned, a 1-week crossover mating trial was performed to determine effects by gender. 

F0 males and females from the 1,300 mg/kg-day dose group were mated with male and female 

control animals. The exposed mice had significantly lower body weights and increased relative 

kidney weights, but reproductive organ weights, sperm motility and morphology, and estrous 

cycle length and frequency did not differ from control mice. In the only histopathological 

examination carried out on treated females, no kidney lesions were observed. The proportion of 

successful copulation was equivalent in all groups, and no developmental effects were observed 

in any offspring. However, the number of fertile females was significantly reduced in the group 

where treated females were mated with control males, suggesting that fertility effects were 

primarily due to effects on the female mice. 

A final phase of this study assessed the fertility and reproductive effects of EGBE in firstgeneration (F1) pups. There were insufficient numbers of offspring to assess the two highest 

dose groups, and no statistically significant effect on fertility was noted when offspring of the 

 34 
low, 700 mg/kg-day dose group were mated. Thus, the researchers concluded that the 700 and 

1,300 mg/kg-day dose levels are considered to be NOAEL and LOAEL values, respectively, for 

both maternal and reproductive effects. A minimal LOAEL for developmental effects was 

700 mg/kg-day, where a very slight decrease in pup weight was observed. 

A study by Exon et al. (1991, 100112), also discussed in Section 4.4.5, looked at 

reproductive parameters in male rats. Groups of six Sprague-Dawley rats were exposed to 

EGBE in drinking water at doses of 0, 180, or 506 mg/kg-day (males) for 21 days. While 

testicular atrophy and necrosis and a reduced number of spermatogenic cells were observed in 

males exposed to EGME, no statistically significant effects on fertility parameters were seen in 

males at any of the tested doses of EGBE. 

NTP (1993, 042063) evaluated the effects of EGBE on the reproductive systems of male 

and female B6C3F1 mice (five/gender/group) following 2-week drinking water exposure to doses 

of 93, 148, 210, 370, or 627 mg/kg-day for males and 150, 237, 406, 673, or 1,364 mg/kg-day 

for females. No deaths were reported, and there were no effects on body weight. Thymus 

weights were decreased in the highest male dose group. There were no treatment-related gross 

lesions in any of the reproductive organs and histopathological examinations were not 

performed. NTP (1993, 042063) also exposed male and female F344 rats (five/gender/group) to 

EGBE for 2 weeks in drinking water. Male rats received doses of 73, 108, 174, 242, or 

346 mg/kg-day, and females received 77, 102, 152, 203, or 265 mg/kg-day. No treatment-related 

deaths occurred during the study, and no changes in body weight were observed in male rats that 

could be related to treatment. However, female rats had lower weight gain in the highest dose 

group. Water consumption was lowest in the highest dose group in both genders, and no 

treatment-related gross lesions of reproductive organs were reported. 

Dodd et al. (1983, 066465) (also discussed in Section 4.2) performed a 90-day subchronic 

inhalation study on F344 rats. Male and female rats (16/gender/group) were exposed to EGBE 

for 6 hours/day, 5 days/week at concentrations of 0, 5, 25, and 77 ppm. They reported no 

changes in testicular weight or in the pathology of the epididymides or testes of male rats at any 

exposure level; reproductive organs of the female rats were not examined histologically. 

NTP (2000, 196293) performed chronic and subchronic inhalation studies of EGBE in 

F344 rats and B6C3F1 mice in which reproductive organs were examined. In the subchronic 

portion of the NTP (2000, 196293) studies, rats and mice (10/gender/group) were exposed to 

concentrations of 0, 31, 62.5, 125, 250, and 500 ppm of EGBE 6 hours/day, 5 days/week for 

14 weeks. Testicular degeneration was reported in 2/4 mice from the 500 ppm group that died or 

were killed moribund. No other effects were noted in the reproductive organs of rats or mice. 

Exposure concentrations were 0, 31, 62.5, and 125 ppm for groups of 50 F344/N rats and 0, 62.5, 

125, and 250 ppm for groups of 50 B6C3F1 mice. No effects were noted in the reproductive 

organs of either species, but the researchers reported that survival was significantly decreased in 

male mice at 125 and 250 ppm (54.0 and 53.1%, respectively). 

 35 
Prenatal and postnatal developmental toxicity tests were conducted in CD-1 mice by 

Wier et al. (1987, 042123). Animals received 0, 350, 650, 1,000, 1,500, or 2,000 mg/kg-day via 

gavage on gestational days (GDs) 8–14. Maternal toxicity included mortality of 3/6 animals in 

the 1,500 mg/kg-day group and 6/6 in the 2,000 mg/kg-day group. Treatment-related clinical 

observations were lethargy, abnormal breathing, and green or red vaginal discharge (the latter at 

≥ 1,500 mg/kg-day). Based on clinical signs in the prenatal study, the LOAEL for maternal 

effects was 650 mg/kg-day. The LOAEL for developmental toxicity was determined to be 

1,000 mg/kg-day based on a statistically significant increase in the number of resorptions and a 

reduced number of viable fetuses. The corresponding NOAEL for prenatal effects was 

350 mg/kg-day. In the postnatal study, reproductive effects were evaluated in offspring of 

CD-1 mice administered EGBE via gavage at 0, 650, or 1,000 mg/kg-day on GDs 8–14. 

Maternal body weight was lowered at 1,000 mg/kg-day. Survival and body weight gain of 

offspring were unaffected by treatment. No statistically significant developmental effects were 

observed. In a simultaneous study with EGEE, developmental toxicity was noted at doses below 

maternal toxicity levels. 

Developmental toxicity was investigated following the administration of EGBE in 

distilled water by gavage to groups of 28–35 pregnant F344 rats at doses of 0, 30, 100, or 

200 mg/kg-day on GDs 9–11, or doses of 0, 30, 100, or 300 mg/kg-day on GDs 11–13 (Sleet et 

al., 1989, 042089). GDs 9–13 were the most critical periods of fetal cardiovascular 

development. Food and water measurements, body and organ weights, clinical signs, 

hematologic analyses of dams, amount of corpora lutea, uterine contents, and number of dead 

and live fetuses were monitored. Maternal effects of EGBE given in either dosing sequence 

included marked, dose-related reductions in body weight and/or weight gain, increases in kidney 

and spleen weights, severe hematotoxicity as evidenced by a decrease in HCT, Hb, and RBC 

counts, and an increase in reticulocytes at doses ≥ 100 mg/kg-day. No indications of 

developmental toxicity were observed at the two lower doses. Viability of embryos was reduced 

by EGBE treatment at the 200 mg/kg-day dose (GDs 9–11) but not at 300 mg/kg-day (GDs 9–

13). A decreased platelet count was noted in the fetuses at 300 mg/kg-day (GDs 9–13). 

Cardiovascular or other types of malformations were not found at any dose. The LOAEL for 

maternal toxicity was 100 mg/kg-day based on signs of hematotoxicity, with a NOAEL 

established at 30 mg/kg-day. The LOAEL for developmental toxicity was 200 mg/kg-day based 

on decreased viability of embryos, with a NOAEL for this endpoint at 100 mg/kg-day. 

Sprague-Dawley rats (15/group) were exposed to 0, 150, or 200 ppm EGBE via 

inhalation for 7 hours/day on GDs 7–15 (Nelson et al., 1984, 031878). Rats exposed to 200 ppm 

showed some evidence of hematuria on the first day of exposure; no significant effects were 

noted thereafter, or at any time in offspring. The LOAEL was 200 ppm for slight maternal 

toxicity; a NOAEL was identified at 150 ppm. The NOAEL for developmental toxicity was 

 36 
200 ppm. Simultaneous testing revealed that 50 ppm exposures to EGME were toxic at all levels 

of embryonic and fetal development. 

Pregnant F344 rats (36/group) and New Zealand white (NZW) rabbits (24/group) were 

exposed to 0, 25, 50, 100, or 200 ppm EGBE via inhalation for 6 hours/day on GDs 6–15 for rats 

or GDs 6–18 for rabbits (Tyl et al., 1984, 100209). Fetuses were weighed and evaluated for 

viability, body weight, and morphological development, including external, visceral, and skeletal 

malformations. In rats, fetotoxicity was observed at 100 and 200 ppm in the form of retarded 

skeletal ossification of vertebral arches or centra, sternebrae, or phalanges. Maternal toxicity 

was also evident at 100 and 200 ppm by increased incidence of hematuria, reduced RBC count, 

decreased weight gain, and reduced food consumption. For both maternal and developmental 

toxicity in the rat, the NOAEL and LOAEL values were 50 and 100 ppm, respectively. In 

rabbits, fetal skeletal ossification of sternebrae and rudimentary ribs was delayed at 200 ppm. 

Maternal toxicity was also evident at 200 ppm as an increased incidence of clinical signs, 

reduced gravid uterine weight, and decreased weight gain. For both maternal and developmental 

effects in the rabbit, the NOAEL and LOAEL values were 100 and 200 ppm, respectively. 

Reproductive toxicity tests were performed on female Sprague-Dawley rats (number not 

specified) via dermal administration during GDs 6–15, 4 times/day at 1,800 and 5,400 mg/kgday (Hardin et al., 1984, 042030). In the highest dose group, 10/11 rats died between days 3 and 

7 of treatment. Signs associated with treatment included red-stained urine, ataxia, inactivity, 

rough coats, and necrosis of the tail tip. At the lower dose, body weight was slightly reduced, yet 

there was no evidence of embryo- or fetotoxicity or gross malformations or variations. 

4.4. OTHER STUDIES 

4.4.1. Acute and Short-Term Exposure Studies 

Ghanayem et al. (1987, 066470) conducted acute toxicity studies designed to assess the 

effect of age on toxicity by comparing effects in treated young (4–5 weeks old) and adult rats (9–

13 weeks, 5–6 months, and/or 16 months old). The researchers exposed male F344 rats 

(six/group) using single gavage doses of EGBE in water (99% purity) to concentrations of 0, 32, 

63, 125, 250, 250, or 500 mg/kg-day. Evaluations included total RBC and WBC counts, urine 

Hb concentration, organ weights, and histology of the liver, spleen, bladder, kidney, and testes. 

Focal necrosis of the liver was observed in adult rats exposed at a dose of 250 or 500 mg/kg. 

Hematologic effects were found to be dose- and age-dependent, with older rats being more 

sensitive than younger rats. Significant decreases in RBC counts, Hct, and Hb and increases in 

free plasma Hb occurred at 125 mg/kg-day in both young and adult rats, with the younger rats 

exhibiting significantly less pronounced responses. The incidence of hemoglobinuria was also 

dose- and age-dependent. Concentrations of free Hb in urine were also significantly higher in 

older rats than in younger rats at all doses. These researchers suggested that the metabolic basis 

 37 
of the age-dependent toxicity of EGBE may be due to a reduced ability in older rats to 

metabolize the toxic metabolite BAA to CO2 and a diminished ability to excrete BAA in the 

urine. Based on increased Hb concentrations in the urine and associated hemolytic effects at 

higher doses, the LOAEL for this study was determined to be 32 mg/kg-day for both young and 

adult rats. A NOAEL was not identified. 

Ghanayem and Sullivan (1993, 041609) performed acute oral toxicity studies in male 

F344 rats (N not specified), using single gavage doses of 250 mg/kg-day EGBE in tap water. 

MCV and Hct values increased immediately after treatment and decreased with time following 

exposure. Hemolysis and decreases in Hb concentrations and RBC counts were reported. 

Grant et al. (1985, 006770) gavaged groups of 24 male F344 rats with EGBE (purity 

99.9%) in water at doses of 0, 500, or 1,000 mg/kg-day for 4 days. Six rats per dose were 

examined at 1, 4, 8, and 22 days after the last dose. The animals were evaluated for changes in 

body weight, hematology, organ weight, and histology. Hematology evaluations showed marked 

dose-related effects on circulating RBCs and WBCs. Changes at 500 and 1,000 mg/kg-day on 

postdosing day 1 included significant dose-related decreases in Hb concentration and total WBC 

and lymphocyte counts and increases in MCV, reticulocyte counts, and MCH. Hct was also 

reduced at 1,000 mg/kg-day. Most of the RBC changes subsequently returned to normal, 

although MCV and MCH remained increased at day 22. Body weight gain was sufficiently 

reduced throughout the posttreatment period at 1,000 mg/kg-day. Changes in relative organ 

weights were evident on posttreatment day 1, including increased liver and spleen weights at 500 

and 1,000 mg/kg-day and increased kidney and reduced thymus weights at 1,000 mg/kg-day. 

These changes returned to normal by posttreatment day 22, except for liver and spleen weights at 

1,000 mg/kg-day, which increased somewhat (~5 and ~20%, respectively). The authors 

determined that EGBE appears to be relatively inactive as a bone marrow toxicant due to the 

observed proliferative response and the lack of hemorrhage at any time in the bone marrow of 

EGBE treated animals. Based on hemolytic anemia with associated reticulocytosis and increased 

hematopoiesis, a LOAEL was established at 500 mg/kg-day, the lowest dose tested. A NOAEL 

was not identified. 

Ghanayem et al. (1992, 100128) administered EGBE to male F344 rats (six/group) via 

gavage for 12 days at dose levels of 0 and 125 mg/kg-day. These investigators identified effects 

of EGBE exposure similar to those identified above. Significant hemolysis occurred, becoming 

more pronounced up to the third day of dosing. Gradual recovery was observed up to day 12. 

MCV, ATP concentration, reticulocyte counts, and relative spleen-to-body weight ratios 

increased up to the sixth day of dosing and declined thereafter. Liver-to-body-weight ratios were 

slightly lowered on days 3 and 6 and slightly increased on day 12. 

Several studies investigated EGBE-induced effects on specific organs and cells. Four 

male and four female F344 rats were exposed to two, three, or four daily doses of EGBE at 

250 mg/kg-day. Ezov et al. (2002, 100113) investigated hemolytic anemia and disseminated 

 38 
thrombosis in rats by investigating the organs for hemolysis and histopathologic evidence of 

disseminated thrombosis. Significant morphological changes in erythrocytes were noted in both 

genders of rats, while disseminated thrombosis and infarction were seen mainly in females and 

consisted of tissue necrosis in the brain, liver, bones, eyes, lungs, and heart. Renal tubular 

necrosis associated with Hb casts was seen in both genders. Koshkaryev et al. (2003, 100168) 

measured changes in adherence, aggregability, and deformability of RBCs. EGBE exposure did 

not affect RBC aggregability, and its effect on deformability was inconclusive; however, the 

exposure enhanced RBC adherence to endothelial cells, with adherence highest at day 2 (the first 

day examined), after which it decreased sharply with time. Shabat et al. (2004, 100200) studied 

bone marrow injury and reported extensive vascular thrombosis resulting in necrosis of bone 

marrow cells, bone-lining cells, and cortical and trabecular osteocytes. The authors concluded 

that, in EGBE-treated rats, interactions of several factors may generate a thrombotic crisis, such 

as the release of procoagulant factors from destroyed erythrocytes; they further concluded that 

disturbed blood flow may result from alterations in the rheology of erythrocytes, including selfaggregation, deformation, and adherence to the endothelium of the blood vessel wall. Redlich et 

al. (2004, 100194) investigated the dental effects from EGBE-induced hemolysis and 

thrombosis. Odontoblastic necrosis in the dental pulp of incisors and molars and muscle-cell 

damage in the tongue were observed; the most severe changes occurred in females. These 

effects were probably the result of ischemic events in the blood vessels supplying these tissues, 

rather than a direct cytotoxic effect of EGBE. 

Corley et al. (1999, 042003) conducted a series of studies in B6C3F1 mice investigating 

aspects of EGBE toxicity, including the occurrence of forestomach lesions in both oral and 

inhalation exposure routes, the dose-response of forestomach irritation, and the occurrence of 

forestomach lesions as a consequence of systemic-only exposure. To determine the extent that 

activities during inhalation exposures (e.g., grooming) could contribute to observed forestomach 

lesions, groups of female mice were exposed for 6 hours to target concentrations of 250 ppm 

EGBE via whole-body (n = 20) and nose-only (n = 20) exposures and concentrations on the fur 

that were available for oral consumption via grooming measured. For whole-body exposures, 

9.2 ± 2.9 mg/kg was available compared with 7.5 ± 2.3 mg/kg for the nose-only exposures. 

Little difference was detected in the internal dose of EGBE from whole-body versus nose-only 

exposures, as measured by the analysis of EGBE and in BAA detected in blood and urine 

postexposure. To inform the dose response of toxicity in the forestomach tissues in mice, neat 

EGBE was administered to male and female mice (five/gender/dose) via gavage (no vehicle) for 

1 week at doses of 100, 400, or 800 mg/kg-day. The dose in the 100 mg/kg-day group was 

increased to 1,200 mg/kg-day after 2 days. Severe hemolysis and mortality were seen, and the 

2-week study was terminated after only four doses. Forestomach lesions consisting of focal 

areas of irritation and epithelial hyperplasia were seen at all exposure levels in this study. Next, 

the researchers administered saline solutions of EGBE to groups of three mice by either 

 39 
intraperitoneal (i.p.) or subcutaneous (s.c.) injection at 400 or 600 mg/kg-day for 3 consecutive 

days or 0 and 400 mg/kg-day for 5 consecutive days. Focal irritation in the forestomach, similar 

to that seen in gavage and inhalation studies, was seen in the three mice administered EGBE by 

i.p. injection at 600 mg/kg for 3 days, while 1/3 mice at 400 mg/kg i.p. and at 400 and 600 mg/kg 

s.c. for 3 days also had forestomach lesions, minimal effects. At 400 mg/kg (5-day study), 

1/6 mice (i.p.) and 2/6 mice (s.c.) also had minimal lesions. From these results, it can be 

concluded that: (1) the contribution to forestomach exposure from grooming activities, etc., 

during whole-body inhalation exposures is incidental; (2) the exposure of forestomach lesions to 

EGBE is similar from gavage and inhalation exposures; and (3) forestomach tissues show a 

similar irritative response whether EGBE exposure is systemic or portal of entry. 

In another series of studies, male and female B6C3F1 mice (16/gender/group) were 

exposed by gavage to 0, 400, 800, or 1,200 mg/kg-day of neat EGBE for 2 days (Poet et al., 

2003). A high level of mortality was seen; the dose was reduced by half and the dosing 

discontinued when survival did not improve after two additional doses. Lesions, including 

epithelial hyperplasia and inflammation of the forestomach, were seen at the higher dose levels 

in both males and females, and minimal-to-mild forestomach epithelial hyperplasias were seen in 

both genders of the lower-dose groups. In a study similar to that performed by Corley et al. 

(1999, 042003), female mice were exposed, either by whole-body or nose-only inhalation, to a 

single 6-hour exposure of 250 ppm EGBE; the concentrations on the fur available for oral 

consumption via grooming were measured. An average of 205 ± 69 μg of EGBE was detected 

on the fur of the mice exposed whole-body, while an average of 170 ± 52 μg was detected on the 

fur of the mice exposed nose-only (Poet et al., 2003, 100190). 

Green et al. (2002, 041610) conducted a series of experiments to examine the effects and 

distribution of EGBE in vivo. First, female B6C3F1 mice (five/group) were given a single, daily 

gavage dose of either EGBE or BAA (0, 50, 150, or 500 mg/kg) for 10 days. Eighteen hours 

after the last dose, animals were sacrificed to look at cell proliferation in the forestomach and 

glandular stomach. The only dose-dependent finding was a thickening of the keratinized layers 

or hyperkeratosis of the forestomach (statistics not reported). A NOAEL of 150 mg/kg EGBE 

and 50 mg/kg BAA was observed. No effects were seen in the glandular stomachs at any dose 

levels. In the next set of studies, female B6C3F1 mice (n = 12) were exposed whole-body to 

250 ppm 2- butoxy[1-14C]ethanol (specific activity 0.365 mCi/mmol) for 6 hours. Following the 

exposure, animals were given free access to food and water; they were terminated (four per time 

point) at 5 minutes and 24 and 48 hours after exposure, and whole body autoradiography was 

performed. Female B6C3F1 mice (n = 12) were given a single i.v. injection of 10 mg/kg 

2-butoxy[1-14C]ethanol (850 μCi/kg). The animals were then given free access to food and water 

and terminated (four per time point) at 4, 24, and 48 hours after dosing, and whole body 

autoradiograms were prepared. These studies showed that whether EGBE was delivered by 

inhalation or by i.v., radiolabeled EGBE was found in the buccal cavity, esophagus, and 

 40 
stomach. This suggests that EGBE somehow enters the stomach via the buccal cavity and 

esophagus following inhalation exposure. 

Administration of a 2,000 mg/kg oral dose of EGBE to guinea pigs caused complete 

mortality of females and 60% mortality of males (Shepard, 1994, 594430; Shepard, 1994, 

594428), but a dose of 1,000 mg/kg caused only a 20% mortality of either gender. Clinical signs 

and gross necropsy indicated toxicity was due to irritation of the stomach. There was no 

evidence of hemolytic toxicity. 

Gingell et al. (1998, 100135) performed acute oral and inhalation toxicity studies in the 

guinea pig. A dose of 2,000 mg/kg EGBE was administered by gavage to five males and five 

females. After excessive mortality (3/5 males, 5/5 females) was observed at this dose, reduced 

doses of 500 and 1,000 mg/kg were administered. No animals died at 500 mg/kg, and 1/5 males 

and 1/5 females died at 1,000 mg/kg. The acute oral median lethal dose for both genders was 

1,414 mg/kg (95% confidence interval [CI] = 1,020–1,961 mg/kg). Clinical signs in the guinea 

pigs included slight-to-severe weakness, salivation and staining of face or abdomen hair, and 

respiratory difficulties in a few males. No evidence of RBC toxicity or hemolysis was observed. 

In the inhalation study, male and female guinea pigs were exposed for 1 hour to 633 ppm and 

691 ppm, respectively. No mortality, clinical signs of toxicity, or exposure-related pathological 

signs were noted. Thus, the median lethal concentration for a 1-hour exposure for guinea pigs 

was >633 ppm in males and >691 ppm in females. 

4.4.2. Dermal Exposure Studies 

EGBE appears to be readily absorbed after contact with animal skin. Rats and rabbits 

exhibit varying degrees of hematotoxicity following dermal application of EGBE (Allen, 1993, 

594452; Allen, 1993, 594455; Allen, 1993, 594459; Allen, 1993, 594460; Bartnik et al., 1987, 

100086; Tyler, 1984, 006769). Bartnik et al. (1987, 100086) performed acute dermal toxicity 

tests using Wistar rats (six/gender/group). A single application of 200, 260, 320, 375, or 

500 mg/kg EGBE was placed on the dorsal shaved skin of rats and covered with a glass capsule. 

Hemolytic and/or hemoglobinuria effects were observed at 500 mg/kg EGBE within 6 hours of 

application. No effects were observed at 200 mg/kg. 

Repeated occluded application of EGBE either neat or as a dilute aqueous solution to 

NZW rabbits (five/gender/group) at 18, 90, 180, or 360 mg/kg (6 hours/day, nine consecutive 

applications) produced hemoglobinuria in males at 360 mg/kg and in females at 180 or 

360 mg/kg (Tyler, 1984, 006769). Only female rabbits showed decreased RBC counts, 

decreased Hb and MCHC, and increased MCH concentrations at the highest treatment level. 

Recovery was noted after 14 days. In a separate 13-week study, occluded dermal administration 

of EGBE to NZW rabbits (10/gender/group) at exposure levels of 10, 50, or 150 mg/kg for 

6 hours/day, 5 days/week produced no observable hematological effects (Tyler, 1984, 006769). 

 41 
Occlusion or semi-occlusion of the site of EGBE administration was also a determining 

factor. For example, some studies have shown no clinical signs of hematotoxicity in SpragueDawley rats (five/gender/group) administered EGBE dermally at 2,000 mg/kg (24-hour 

exposure) either semi-occluded or occluded (Allen, 1993, 594452; Allen, 1993, 594455). 

However, clinical signs of systemic toxicity were noted following the occluded exposure. In 

similar studies in NZW rabbits (five/gender/group), red-stained urine was reported at semioccluded doses of 2,000 mg/kg EGBE, along with other clinical signs of systemic toxicity 

(Allen, 1993, 594459; Allen, 1993, 594460). Similar effects occurred at occluded doses of 500, 

707, and 1,000 mg/kg in this species; deaths occurred at the 500 and 1,000 mg/kg exposures. 

Thus, hematotoxicity varied from nonexistent to severe. In guinea pigs, dermal administration of 

EGBE at 2,000 mg/kg produced no deaths, clinical signs of toxicity, or treatment-related signs of 

organ toxicity (Gingell et al., 1998, 100135; Shepard, 1994, 594428). 

In an assessment of immune parameters, female BALB/c mice (five/group) were 

topically exposed to EGBE at 100, 500, 1,000, and 1,500 mg/kg-day for 4 consecutive days 

(Singh et al., 2001, 100204). A statistically significant increase in spleen-to-body-weight ratio, 

and a 29% increase in splenic cellularity was observed at 1,500 mg/kg. Splenic proliferative 

responses to the T-cell mitogen, concanavalin-A (con-A), were significantly decreased by 32% at 

500 mg/kg-day and 35% at 1,000 mg/kg-day. Allogeneic antigen-driven lymphoproliferative 

responses in the mixed lymphocyte response were significantly reduced by 55% at 500 mg/kgday and 56% at 1,000 mg/kg-day. However, natural killer (NK) cell activity, cytotoxic Tlymphocyte activity, and the T-dependent plaque-forming cell (PFC) response were not 

significantly affected by EGBE exposure. A dose of 100 mg/kg-day was a NOAEL. 

Singh et al. (2002, 100203) exposed female BALB/c mice (five/group) via gavage to 50, 

150, or 400 mg/kg EGBE, or topically on the ear to 0.25, 1.0, 4.0, or 16.0 mg EGBE. The 

researchers measured the oxazolone (OXA)-induced contact hypersensitivity response (CHR). 

Mice that received the gavage doses of EGBE for 10 consecutive days did not exhibit a 

significantly altered OXA-induced CHR as measured by ear swelling 24 hours postchallenge. In 

contrast, topical exposure to EGBE significantly suppressed the OXA-induced CHR at a dose of 

4.0 mg EGBE/ear, but not at any other dose. 

The studies indicate that while the dermal route of exposure can be expected to contribute 

to overall exposure, the concentrations at which effects occur in animals are higher than those 

found following oral and inhalation exposure. In humans, toxicokinetic studies have shown that 

dermal absorption of EGBE vapors do contribute to the total body burden, showing the 

importance of the dermal route of exposure. 

4.4.3. Ocular Exposure Studies 

EGBE has been found to be an irritant when instilled in rabbit (Jacobs and Martens, 

1989, 100150; Kennah et al., 1989, 054156). Kennah et al. (1989, 054156) performed the Draize 

 42 
eye irritation test in rabbits. The percent EGBE concentration and corresponding scores by the 

Texaco single-digit toxicity classification system were 100%—66, 70%—49, 30%—39, 20%—

2, and 10%—1. In an assessment that measured corneal thickness, the highest concentration was 

classified as severely irritating, the 70% concentration was moderately irritating, and the others 

were mildly irritating. Jacobs and Marten (1989, 100150) conducted ocular tests on NZW 

rabbits (n = 6) to determine the effects of EGBE (100 μL, 99% pure) on eye irritation. The 

undiluted chemical was dropped onto the lower lid of one eye; the other eye served as a control. 

The eyes were examined and graded for ocular reactions at 4, 24, 48, 72, 96, and 168 hours 

postinstillation. The authors determined that EGBE should be classified as an irritant based on 

the mean erythema scores and percent corneal thickening. 

4.4.4. Genotoxicity 

Although weakly genotoxic responses have been obtained in two laboratories (Elias et al., 

1996, 042011; Hoflack et al., 1995, 100147), EGBE is not expected to be mutagenic or 

clastogenic based on the available data (summarized in Table 4-7). The NTP reported negative 

responses for mutagenicity when EGBE was tested in Salmonella typhimurium strains TA97, 

TA98, TA100, TA1535, and TA1537 at up to 10 mg/plate with and without metabolic activation 

(Zeiger et al., 1992, 095748). However, Hoflack et al. (1995, 100147) reported that at 38 

μmol/plate (4.5 mg/plate), EGBE induced a weak mutagenic response in salmonella tester strain 

TA97a in the absence of S9 mix (Hoflack et al., 1995, 100147). The work of Hoflack and 

colleagues was repeated by Gollapudi et al. (1996, 100137), and EGBE was found to be negative 

in these tester strains when evaluated at 0.5, 1.0, 2.5, 5.0, 8.5, and 10 mg/plate in the presence 

and absence of Aroclor-induced rat liver S9 mix. Thus, the weak positive result reported in 

salmonella TA97a by Hoflack et al. (1995, 100147) is unconfirmed. A plausible explanation put 

forth by Gollapudi et al. (1996, 100137) is that, given the sensitivity of the Ames test, perhaps 

the weak positive result reported by Hoflack et al. (1995, 100147) is attributed to an impurity in 

their test material. 

 43 
Table 4-7. Summary of genotoxicity studies on EGBE, BAL, and BAA 

Type of test, test species Dosea Result Reference 

In vitro tests: EGBE 

Reverse mutation, S. typhimurium, 

TA97, TA98, TA100, TA1535, 

TA1537 

10 mg/plate Negative (w/ and w/o 

metabolic activation) 

Zeiger et al. (1992, 

095748) 

(work done for NTP) 

Reverse mutation, S. typhimurium his–

TA 98, TA 100, TA 102

115 μmol/plate 

(14.0 mg/plate) 

Negative (w/ and w/o 

metabolic activation) 

Hoflack et al. (1995, 

100147) 

Reverse mutation, S. typhimurium hisTA97a 38 μmol/plate 

(4.5 mg/plate) 

Weakly positive (w/o 

metabolic activation) 

Hoflack et al. (1995, 

100147) 

Reverse mutation, S. typhimurium hisTA97a, TA 100; Escherichia coli

WP2uvrA 

10 mg/plate Negative (w/ and w/o 

metabolic activation) 

Gollapudi et al. (1996, 

100137) 

Sister chromatid exchanges (SCEs), 

micronuclei (MN) and aneuploidy (AP) 

in V79 cells 

10–100 mM (SCE) 

8.46 mM (MN) 

16.8 mM (AP) 

Weakly positive (w/o 

metabolic activation) 

Elias et al. (1996, 

042011)

b

Potentiation of clastogenicity induced 

by methyl methanesulfonate 

8.5 mM Positive (w/o metabolic 

activation) 

Elias et al. (1996, 

042011)

b

Chromosomal aberrations, V79 cells 

and human lymphocytes 

Not available Negative (w/o metabolic 

activation) 

Elias et al. (1996, 

042011)

b

38.1 mMc

(4.5 mg/mL) 

Gene mutation, Chinese hamster ovary 

cells 

Negative (w/o metabolic 

activation) 

Chiewchanwit and Au 

(1995, 041999) 

DNA damage, SVEC4-10 mouse 

endothelial cells 

10 mM Negative Klaunig and 

Kamendulis (2005, 

100165) 

In vitro tests: BAL 

Reverse mutation, S. typhimurium hisTA 97a, TA 98, TA 100 and TA 102 43 μmol/plate 

(5.0 mg/plate) 

Negative (w/ and w/o 

metabolic activation) 

Hoflack et al. (1995, 

100147) 

Chromosomal aberrations, V79 cells 

and human lymphocytes 

0.1–1 mM; 

cytotoxicity not 

reported 

Positive (w/o metabolic 

activation) 

Elias et al. (1996, 

042011)

b

DNA damage, SVEC4-10 mouse 

endothelial cells 

1 mM Negative Klaunig and 

Kamendulis (2005, 

100165) 

 44 
Type of test, test species Dosea Result Reference 

In vitro tests: BAA

Reverse mutation, S. typhimurium hisTA 97a, TA 98, TA 100 and TA 102 8 μmol/plate 

(0.9 mg/plate) 

Negative (w/ and w/o 

metabolic activation); dose 

limited by toxicity 

Hoflack et al. (1995, 

100147) 

SCEs and CAs, V79 cells 0.8 mM Negative (w/o metabolic 

activation) 

Elias et al. (1996, 

042011)

b

Aneuploidy, V79 cells 0.38 mM Weakly positive (w/o 

metabolic activation) 

Elias et al. (1995, 

100147)

b

MN assay, V79 cells 10 mM Positive (w/o metabolic 

activation 

Elias et al. (1995, 

100147)

b

DNA damage, SVEC4-10 mouse 

endothelial cells 

10 mM Negative Klaunig and 

Kamendulis (2005, 

100165) 

In vivo tests: EGBE 

MNs, bone marrow erythrocytes of 

male mice or rats 

550 mg/kg-day, mice

450 mg/kg-day, rats 

Negative 

Negative 

NTP (1996, 042064) 

DNA adducts FVB/N mice 

Sprague-Dawley rats 

120 mg/kg-day, mice 

and rats 

No changes in DNA 

methylation 

Keith et al. (1996, 

041625) 

a

Doses are either the lowest effective dose or the highest ineffective dose. 

b

All in vitro assays were performed without the addition of an exogenous metabolic activation system. c

The authors found that this dose was cytotoxic. 

Elias et al. (1996, 042011) reported that EGBE did not induce chromosomal aberrations 

in Chinese hamster V79 fibroblast cells but that EGBE, at treatment concentrations of ≥ 8.5 mM, 

weakly induced sister chromatid exchanges (SCEs) and micronuclei and potentiated the 

clastogenicity induced by methyl methanesulfonate. Elias et al. (1996, 042011) also reported 

that EGBE weakly induced aneuploidy (numerical chromosomal anomalies) in V79 cells; 

however, this response was found at very high concentrations (16.8 mM EGBE). 

When tested at doses nearing toxicity, EGBE and its metabolite BAL were not mutagenic 

in an in vitro gene mutation assay using Chinese hamster ovary (CHO) cells (CHO-AS52) 

(Chiewchanwit and Au, 1995, 041999). In contrast, Elias et al. (1996, 042011) reported that 

both EGBE and BAL weakly induced gene mutations in Chinese hamster V79 cells only at high 

treatment concentrations (≥ 7.5 mg/mL). It should be noted that Chiewchanwit and Au (1995, 

041999) reported high cytotoxicity at 38.1 mM EGBE (4.5 mg/mL). The gene mutation data 

presented by Elias et al. (1996, 042011) is in graphic form only with mean values and no SDs 

presented. The presence or absence of cytotoxicity was not reported. BAL was also tested for 

induction of DNA damage in the mouse endothelial cell line, SVEC4-10, using the comet assay. 

BAL failed to produce a statistically significant increase in DNA strand breaks at any of the 

concentrations or time points examined (Klaunig and Kamendulis, 2004, 594442; Klaunig and 

Kamendulis, 2005, 100165; Reed et al., 2003, 594436). Other lines of evidence indicate that 

direct interaction of BAL with the DNA molecules does not play a significant role in the 

carcinogenic activity of EGBE. First, BAL causes cytotoxicity at levels associated with 

 45 
chromosome effects, and cytotoxicity itself can have effects that result in chromosome damage, 

such as reduction in the repair of SCEs. Second, acetaldehyde is recognized as “weakly 

mutagenic” and structural comparisons of the aldehyde metabolites of glycol ethers shows that 

longer-chain aldehydes such as BAL are less mutagenic (Chiewchanwit and Au, 1995, 041999). 

Third, if BAL were a stable mutagenic metabolite in any of the in vitro assays exposed to EGBE, 

one would expect them to give positive results; however, the results were generally negative. 

Elias et al. (1996, 042011) suggested that the V79 cells possess neither ALDH nor ADH. The 

relevance of these studies, or of any systems that lack these enzymes, is of limited value in 

elucidating the MOA of toxicity in biological systems that possess these enzymes. BAA has 

been found negative for reverse mutations in S. typhimurium his- with and without metabolic 

activation (Hoflack et al., 1995, 100147). Concentrations of up to 8 μmol/plate were tested, and 

dose was limited by toxicity. BAA (up to 10 mM) was also found negative for induction of 

DNA damage in SVEC4-10 mouse endothelial cells (Klaunig and Kamendulis, 2005, 100165) 

and in an SCE assay in V79 cells (Elias et al., 1996, 042011). BAA was weakly positive for 

aneuploidy in V79 cells at 0.38 mM and positive for micronuclei induction in the same cell line 

at 10 mM, as reported by Elias et al. (1996, 042011). As noted above, the data means are 

presented in graphic form without SDs and cannot be critically evaluated; no cytotoxicity data 

are reported. 

EGBE did not increase the incidence of micronuclei in the bone marrow cells of male 

mice or rats (NTP, 1996, 042064). Animals were given three i.p. injections of EGBE 24 hours 

apart and sacrificed 24 hours after the last injection; rats were dosed at 0, 7, 14, 28, 56, 112.5, 

225, or 450 mg/kg and mice were dosed at 0, 17, 34, 69, 137.5, 275, or 550 mg/kg (NTP, 1996, 

042064). There was high mortality (2/5 mice survived) in mice injected with 1,000 mg/kg doses 

of EGBE. Keith et al. (1996, 041625) treated Sprague-Dawley rats and transgenic FVB/N mice 

carrying the v-Ha-ras oncogene with a single oral dose of 120 mg/kg EGBE; there was no 

increase in DNA adducts in the brain, liver, kidney, testes, or spleen of the rats, and no changes 

in DNA methylation patterns in either species. 

In conclusion, EGBE has been tested in conventional genotoxicity tests for its potential to 

induce gene mutations in systems and cytogenetic damage both in vitro and in vivo. Available 

data do not support a mutagenic or clastogenic mechanism for EGBE. Two laboratories (Elias et 

al., 1996, 042011; Hoflack et al., 1995, 100147) reported weak genotoxicity responses in vitro at 

toxic doses. These results, however, are questionable given limited published information. 

Elliott and Ashby (1997, 100111) reviewed the results of the available genotoxicity studies on 

EGBE and concluded that the data indicate that EGBE has no significant genotoxic activity. 

4.4.5. Immunotoxicity 

Based on the results of the Exon et al. (1991, 100112) study, it appears that the immune 

system is not a sensitive target of EGBE. Groups of six Sprague-Dawley rats were exposed to 

 46 
EGBE in drinking water at doses of 0, 180, or 506 mg/kg-day (males) or 0, 204, or 444 mg/kgday (females) for 21 days. All rats were injected s.c. with heat-aggregated aqueous keyhole 

limpet hemocyanin (KLH), a T-cell dependent antigen, on days 7 and 13 following the start of 

dosing. Endpoints evaluated on day 21 included body weight, absolute and relative organ 

weights (spleen, thymus, liver, kidney, testis), and histology of thymus, liver, kidney, and testis. 

Splenic histology was not assessed because this tissue was used as a source of cells for immune 

function assays. Immune function assays included NK cell cytotoxicity, antibody response to a 

T-cell dependent antigen as determined by measurement of KLH-specific serum 

immunoglobulin G levels, delayed-type hypersensitivity reaction, interleukin-2 and interferon 

production, and spleen cell counts. Terminal body weights were somewhat lower than controls 

in all exposed groups, and the decreases were statistically significant in all groups except the 

180 mg/kg-day males. No dose-related changes in organ weights or histology were observed. 

NK cell cytotoxic responses were significantly enhanced in males at 180 mg/kg-day and females 

at 204 mg/kg-day. At the highest dose tested in males and females, the NK cell cytotoxic 

response was increased above control, but this finding was not statistically significant. 

A decreased NK cell cytotoxic response is an indication of compromised nonspecific immune 

system integrity. Given that this study showed an increased response and no dose-response 

relationship, these findings are not considered to be an indication of an adverse response. No 

significant alterations in other immune parameters were noted. 

Smialowicz et al. (1992, 004389) reported on results of experiments that examined the 

primary antibody response to a T-cell independent antigen (trinitrophenyl-lipopolysaccharide 

[TNP-LPS]) measured with a PFC assay to determine the immunotoxic potential of a variety of 

glycol ethers. This author had previously reported that the shorter-chain glycol ether 2-

methoxyethanol (ME) and its principal metabolite 2-methoxyacetic acid (MAA) suppressed the 

antibody response to TNP-LPS as measured by the PFC assay in F344 rats but not CD-1 mice 

(Smialowicz et al., 1992, 100205). Having established the sensitivity of F344 rats to suppression 

of the antibody response to the T-cell independent antigen TNP-LPS by ME and MAA, the 

authors examined other glycol ethers, including 2-butoxyethanol, in the same dose range, for 

their ability to suppress the antibody response to TNP-LPS by using the same PFC assay. Male 

F344 rats were immunized with a single i.v. injection of 0.5 mL of 40 μg/mL TNP-LPS, then 

dosed (six/dose group) by gavage with 50–400 mg/kg-day of various glycol ethers, including 

EGBE (0, 50, 100, 200, 400 mg/kg-day) for 2 days. All rats exposed to 400 mg/kg-day EGBE 

died, and the 200 mg/kg-day EGBE dose resulted in one dead and one moribund rat. This 

finding was not unexpected, as the hematotoxicity of EGBE in older rats has been reported in the 

literature (Carpenter et al., 1956, 066464; Ghanayem et al., 1987, 066470; Tyler, 1984, 006769). 

EGBE did not suppress the primary antibody response to TNP-LPS in the PFC assay. 

In an assessment of immune parameters, female BALB/c mice (five/group) were 

topically exposed to EGBE at 100, 500, 1,000, and 1,500 mg/kg-day for 4 consecutive days 

 47 
(Singh et al., 2001, 100204). A statistically significant increase in spleen-to-body-weight ratio 

was observed at 1,500 mg/kg, and splenic cellularity was increased by 29% at this dose. Splenic 

proliferative responses to the T-cell mitogen, con-A, were significantly decreased by 32% at 

500 mg/kg-day and 35% at 1,000 mg/kg-day. Allogeneic antigen-driven lymphoproliferative 

responses in the mixed lymphocyte response were significantly reduced by 55% at 500 mg/kgday and 56% at 1,000 mg/kg-day. However, NK cell activity, cytotoxic T-lymphocyte activity, 

and the T-dependent PFC response were not significantly affected by EGBE exposure. For those 

immune parameters measured, 100 mg/kg-day was a NOAEL. 

Singh et al. (2002, 100203) exposed female BALB/c mice (five/group) via gavage to 50, 

150, or 400 mg/kg EGBE or topically to 0.25, 1.0, 4.0, or 16.0 mg EGBE on the ear and 

measured the OXA-induced CHR. Mice that received the gavage doses of EGBE for 10 

consecutive days did not exhibit a significantly altered OXA-induced CHR as measured by ear 

swelling 24 hours postchallenge. In contrast, topical exposure to EGBE significantly suppressed 

the OXA-induced CHR at a dose of 4.0 mg EGBE/ear, but not at any other dose. 

4.4.6. Other In Vitro Studies 

Ghanayem (1989, 042014) compared the metabolic and cellular basis of EGBE-induced 

hemolysis of rat and human erythrocytes in vitro. EGBE is not metabolized when incubated with 

blood from male F344 rats and causes no hemolysis or metabolic alterations at concentrations of 

up to 10 mM. A concentration of 20 mM EGBE was required to produce significant hemolysis 

of rat blood. This may be due to a nonspecific effect occurring at a concentration that is not 

physiologically relevant. In contrast, incubation of rat blood with BAL or BAA at 

concentrations of 0.5, 1.0, or 2.0 mM caused a time- and concentration-dependent increase in 

cell swelling (i.e., increased Hct) followed by hemolysis. This response was more pronounced 

for BAA, with nearly complete hemolysis observed after a 4-hour incubation at 2.0 mM. BAL 

produced only slight hemolysis under the same conditions. The addition of ADH (with nicotine 

adenine dinucleotide cofactor) to rat blood followed by BAL produced a potentiation of the 

hemolytic effects. Addition of cyanamide, an ADH inhibitor, significantly decreased the effects 

with or without added ADH. Both BAL and BAA caused a time- and concentration-dependent 

decrease in blood ATP concentrations, although this effect may be secondary to the swelling and 

lysis observed. Addition of exogenous ATP failed to reverse the hemolytic effects. Neither 

EGBE nor its metabolites, BAL and BAA, caused any detectable changes in the concentrations 

of glutathione (GSH) or glucose-6-phosphate dehydrogenase (G6PD) in rat erythrocytes. Blood 

from male and female human volunteers was unaffected by 4-hour incubations with BAA at 

concentrations of up to 4.0 mM. At 8 mM, slight but significant hemolysis of human blood was 

observed: blood from female volunteers showed a slightly greater sensitivity. These studies 

show that the erythrocyte membrane is the likely target for BAA, and that humans of both 

genders are relatively insensitive to the hemolytic effects of BAA, as compared with rats. 

 48 
Udden (2000, 042110) compared effects on RBC morphology in male F344 rats 

(five/group) dosed with 125 or 250 mg/kg EGBE via gavage with the effects of incubation of rat 

erythrocytes in vitro with BAA (1 or 2 mM). In vivo exposure resulted in stomatoacytosis and 

spherocytosis in blood smears and cup-shaped cells and spherocytosis in fixed samples. In vitro 

incubation resulted in erythrocytes with cup shapes and spherocytosis in the fixed samples. 

Since in vivo and in vitro exposure caused similar changes in RBC morphology in rats, this study 

provides additional evidence for the role of BAA in causing the hemolytic effects from EGBE 

exposure in rats. 

Udden (2002, 042111) performed in vitro comparisons of sub-hemolytic and hemolytic 

effects in rat and human RBCs in response to exposure of high BAA, using samples from 

hospitalized adults, hospitalized children, and well adults. Erythrocyte parameters measured 

included deformability, size distribution, density, MCV, count, osmotic fragility, and hemolysis. 

Exposure for 4 hours resulted in loss of deformability, although at concentrations 150-fold 

different; the first significant alterations noted in cells exposed was at 0.05 mM BAA in rats and 

at 7.5 mM BAA in humans. Significant increases in MCV after a 4-hour exposure to BAA were 

noted at 0.05 mM in rats and at 10.0 mM in humans, a 200-fold difference. A similar differential 

in concentration range was noted in osmotic fragility. Testing of erythrocytes for changes in 

MCV and percent hemolysis from in vitro exposure to either 0 or 10 mM BAA showed no 

differences among the well adults or the hospitalized children. The percent hemolysis seen in the 

average response of the hospitalized adults to 10 mM BAA was significantly increased, from 

0.54 to 0.69%. Significant increases in MCV were noted for all three of these groups, including 

the well adults. The mechanisms underlying erythrocyte damage and the resistance of human 

cells to these effects remain unknown. It should be noted, however, that although the subhemolytic responses were similar for human and rat erythrocytes, other aspects of the 

erythrocytes, including morphology and cell density, were altered in the rats but not in human 

cells. These differences between species suggest that the mechanisms underlying the observed 

prehemolytic effects may be different between rats and humans. 

The possibility exists that certain human subpopulations, including the aged and those 

predisposed to hemolytic disorders, might be at increased risk from EGBE exposure. Udden 

(1994, 042109; 1996, 042209) investigated this possibility using blood from the elderly (mean 

age 71.9; range 64–79 years; five men and four women), from seven patients with sickle cell 

disease, and from four subjects with hereditary spherocytosis, three of whom were studied 

postsplenectomy and one studied presplenectomy. Using a sensitive assay for erythrocyte 

deformability (Udden, 1994, 042109; Udden and Patton, 1994, 056374), it was shown that blood 

from all of these potentially sensitive groups was unaffected by incubations of up to 4 hours with 

2 mM BAA. 

Udden and Patton (2005, 100213) examined the role of osmolarity and cation 

composition of the cell suspension buffers in the mechanism of BAA-induced hemolysis of rat 

 49 
RBCs. Adding sucrose to the cell suspension media or replacing external sodium with potassium 

protected rat RBCs from BAA-induced hemolysis. The authors also observed that adding as 

little as 0.05 mM CaCl2 to the buffer delayed the time course of the hemolytic response, while 

adding MgCl2 had no effect. Use of the calcium-activated potassium channel inhibitor 

charybdotoxin blocked the protective effect of calcium. From this, the authors suggest that BAA 

causes sodium and calcium to enter the cell. While calcium initially has a protective effect via a 

loss of potassium through the calcium-activated potassium channel, compensating for the 

osmotic effect of increased cell sodium, calcium may subsequently have other deleterious effects 

through activation of proteases and other calcium-activated processes. 

Corthals et al. (2006, 100102) tested EGBE, BAL, and BAA in the comet assay to 

determine their ability to induce DNA damage in SVEC4-10 mouse endothelial cells. EGBE (up 

to 10 mM), BAL (up to 1 mM), and BAA (up to 10 mM) did not produce significant increases in 

DNA damage relative to controls at any of the time points examined (2, 4, and 24 hours). The 

researchers next tested the effect of hemolyzed mouse RBC lysate and ferrous sulfate in the same 

system and found that the hemolyzed RBCs produced a statistically significant increase in DNA 

damage at the highest concentration tested (10 × 106 hemolyzed RBCs) for 4 hours. No other 

time points were significant. Ferrous sulfate produced statistically significant increases in DNA 

damage at the highest time point and the lowest concentration tested (24 hours, 0.1 μM) and at 

all time points (2, 4, and 24 hours) in the mid- and high doses tested (0.5 μM and 1.0 μM). The 

next experiment examined the ability of EGBE, BAA, ferrous sulfate, and hemolyzed RBCs to 

stimulate tumor necrosis factor-alpha (TNFα) release from cultured mouse macrophages 

(RAW 264.7 cells). Hemolyzed RBCs (10 × 106 cells) resulted in a statistically significant 

increase (p < 0.05) in TNFα release following a 4-hour treatment. Treatment with EGBE, BAA, 

or ferrous sulfate did not result in increased TNFα release. Finally, the authors report that 

macrophages activated with hemolyzed RBCs (10 × 106

) for 4 hours were able to increase DNA 

synthesis in mouse endothelial cells through co-culturing for 24 hours. These macrophages were 

not, however, able to increase endothelial cell DNA damage (after 4- or 24-hour treatment) as 

measured by the comet assay. The authors did find that LPS activated macrophages, after a 

4-hour treatment, produced statistically significant increases (p < 0.05) in endothelial cell DNA 

damage, as measured by the comet assay. 

4.5. SYNTHESIS AND EVALUATION OF MAJOR NONCANCER EFFECTS AND 

MODE OF ACTION: ORAL AND INHALATION 

Hemolytic anemia is the primary response elicited in sensitive species following 

inhalation, oral, or dermal administration of EGBE. The carboxylic acid metabolite of EGBE, 

BAA, has been shown to be the causative agent in this hemolysis. The mechanisms underlying 

the hemolytic events are unknown. In vitro tests have shown that BAA produces a 

concentration- and time-dependent swelling of rat erythrocytes, and changes the normal 

 50 
erythrocyte morphology from the typical discocyte form to a spherocyte form prior to lysis. One 

potential mechanism that could explain the RBC effects of BAA is the activation of membrane 

phospholipid scrambleases. The outer leaflet of the RBC membrane normally has a net positive 

charge due to the abundance of phosphatidylcholine while the cytoplasmic side contains 

phosphatidylserine. Activation of phospholipid scrambleases results in the translocation of 

phosphatidylserine to the outer leaf while shuffling phosphatidylcholine to the cytoplasmic side 

of the membrane. The externalization of the phosphatidylserine changes the outer membrane net 

charge to negative. The perturbations in membrane homeostasis may result in a loss of 

membrane integrity leading to a loss of deformability of the erythrocyte. The fate of the 

damaged red cells, whether by direct lysis in the circulation or splenic sequestration, is unknown. 

It is likely that under low EGBE exposure conditions, splenic sequestration will predominate but 

at higher exposure conditions, splenic spillage and/or frank intravascular lysis will occur. Heme 

transport mechanisms would then be overloaded and iron containing fragments will accumulate 

in the phagocytic cells of the liver (Kupffer cells) as hemosiderin. It is interesting to note that 

phosphatidylserine externalization has been associated with impending apoptosis in nucleated 

cells. Older erythrocytes are more sensitive to the hemolytic effects of BAA than are younger 

cells or newly formed reticulocytes. Hemolysis can be induced in vivo following administration 

of EGBE or in vitro following addition of BAA to either whole blood or washed erythrocytes. 

Additionally, it has been reported that EGBE exposure to rats caused disseminated 

thrombosis and infarction (Ezov et al., 2002, 100113; Nyska et al., 1999, 042747; Nyska et al., 

1999, 042746; Ramot et al., 2007, 100192; Yoshizawa et al., 2005, 100224). The mechanism by 

which this occurs is presently unknown but several possibilities exist. The changes in 

erythrocyte morphology and decrease in deformability could result in intravascular occlusion. 

Secondly, the lysis of the erythrocyte would result in release of procoagulants. Finally, the 

thrombotic response could be the result of the appearance of anionic phospholipids, particularly 

phosphatidylserine, on the cell surface activating the prothrombinase complex resulting in the 

formation of thrombin (Bevers et al., 1982, 100088; Connor et al., 1989, 100097). Nevertheless, 

thrombosis only occurs at higher EGBE exposure levels and is not the most sensitive endpoint. 

The primary response of hemolysis is indicated via dose-related clinical observations of 

decreases in Hct, Hb concentration, and RBC count in the blood of laboratory animals exposed 

to EGBE. The hemolysis-related events of macrocytosis and increased MCV were also observed 

in the rat, considered to be a sensitive species, and are attributed at least partly to the increased 

number of larger reticulocytes in the circulation following the erythropoietic compensatory 

response (NTP, 2000, 196293). These alterations were persistent throughout the chronic animal 

exposures but do not appear to progress with extended exposure (from 3 months to 1 year for 

rats). These changes persist despite functioning, compensatory, homeostatic mechanisms. 

Liver effects were noted in the NTP (2000, 196293) reports of subchronic and chronic 

inhalation studies in rats and mice and in the subchronic drinking water study in rats. These 

 51 
included statistically significant increases in the iron-staining hepatic pigmentation attributed to 

hemosiderin, the storage protein for insoluble iron, believed to be derived from the Hb released 

during hemolysis. Nyska et al. (2004, 056379) examined the possible association between 

chemically induced hemosiderin deposition and hemangiosarcomas in the liver of mice from 130 

NTP bioassays, and found a highly significant association between liver hemangiosarcoma and 

apparent Kupffer cell pigmentation (see Roberts et al. (2007, 100196) for an overview of the role 

of Kupffer cells in hepatotoxicity and carcinogenicity). The cause for the hemosiderin 

deposition in all cases was the erythrocyte hemolytic effect of the compounds. However, in an 

NTP (1993, 042063) subchronic drinking water study, hepatocellular cytoplasmic changes were 

observed in male rats at an exposure level (750 ppm) below the level at which hematological 

changes were recorded (1,500 ppm). This finding raises the possibility of a direct, primary 

hepatic toxicity due to either EGBE or an EGBE metabolite. Similar liver effects observed in 

female rats at the 750 ppm exposure level were accompanied by hematological effects. 

In the same NTP (1993, 042063) report, no liver lesions were reported in mice exposed to 

drinking water containing up to 6,000 ppm EGBE. The lesions reported in rats consisted of 

cytoplasmic alterations, hepatocellular degeneration, and pigmentation. The cytoplasmic 

alterations, the only lesion observed at the 750 ppm exposure level (corresponding to a 

consumption of roughly 55 mg/kg-day EGBE for adult male rats), were described as hepatocytes 

staining more eosinophilic and lacking the amphophilic-to-basophilic granularity of the 

cytoplasm present in hepatocytes from control animals. Greaves (2000, 089176) suggested that 

the lack of cytoplasmic granularity or ground-glass appearance of the hepatocytes is an 

indication that the response does not involve enzyme induction. The hepatocellular degeneration 

and pigmentation observed at the higher exposure levels in both genders was centrilobular, 

which is consistent with the Kupffer cell pigmentation and hemosiderin deposition reported in 

the NTP (2000, 196293) inhalation studies. This information, along with the observation that all 

other rat and mouse oral and inhalation studies of EGBE report hemolysis at or below exposure 

levels that result in liver effects, suggests that at least these cytoplasmic hepatocellular changes 

in male rats reported in the NTP (1993, 042063) drinking water study may reflect adaptation to a 

subclinical level of hemolysis. However, focal necrosis of the liver observed in male rats 

following gavage administration of 250 and 500 mg/kg EGBE (Ghanayem et al., 1987, 041608) 

was judged to be inconsistent with typical anoxic centrilobular necrosis associated with anemia 

(Edmonson and Peters, 1985, 042168). The effects observed in the Ghanayem et al. (1987, 

041608) study may be associated with the high bolus exposures employed. 

The liver alterations documented throughout these studies suggest a defined progression 

of pathological events with increasing doses of EGBE with increasing levels of hemolysis. In 

particular, hepatic hemosiderin deposition in the liver is a dose-related sequela of the hemolytic 

activity caused by EGBE exposure. This deposition was noted to follow a dose-response 

relationship as well as to increase in severity in the chronic rat and mouse NTP studies; it shows 

 52 
a statistically significant increase relative to the chamber controls. Although some hemosiderin 

deposition in the spleen and liver may be expected with increasing age, the extent of hemosiderin 

deposits noted by NTP in the livers of EGBE exposed animals is not normal. For these reasons, 

hemosiderin deposition in the liver has been considered a pathological finding (Muller et al., 

2006, 597291). The following issues relate to the relevance of these hemolytic and hepatic 

effects to humans and to the MOA of EGBE. 

The weight of evidence from a variety of studies in animals and humans suggests that 

certain species are more susceptible to the hemolytic effects of EGBE. It appears that humans 

are less sensitive to the hemolytic effects of EGBE than rats and mice. On one end of the 

sensitivity range is the guinea pig, which displays no hemolytic effects from EGBE at exposure 

levels as high as an oral dose of 1,000 mg/kg or a dermal dose of 2,000 mg/kg. The rat, 

conversely, displays increased osmotic fragility of erythrocytes at single inhalation exposures 

below 100 ppm and single oral exposures below 100 mg/kg EGBE. No hemolysis was observed 

in controlled laboratory acute inhalation exposures of human volunteers at up to 195 ppm EGBE. 

Some reversible hemolytic effects have been described in addition to more debilitating effects in 

humans who consumed single oral doses of 400–1,500 mg/kg EGBE in cleaning formulations 

(see Section 4.1). Effects in humans from chronic exposure to EGBE have not been studied. 

With respect to gender sensitivity, it has been consistently noted (Carpenter et al., 1956, 

066464; Dodd et al., 1983, 066465; Ezov et al., 2002, 100113; NTP, 2000, 196293) that female 

rats are more sensitive to EGBE-induced hemolysis than males. This gender difference is 

consistent with toxicokinetic data for male and female rats reported by the NTP (2000, 196293) 

2-year study. Female rats eliminated BAA, the toxic metabolite of EGBE, more slowly from the 

blood, resulting in a larger AUC for the blood concentration of BAA versus time. This may be a 

result of the reduced renal excretion observed in female versus male rats. NTP (2000, 196293) 

also reported that, like female rats, female mice tended to have greater blood concentrations of 

BAA at any given time than males. This may explain the slight increase in incidence and 

severity of the anemic response found in female mice, as compared to males. However, unlike 

female rats, female mice excrete slightly more BAA than male mice; no significant difference 

between female and male mice has been noted in the overall rate of elimination or the t1⁄2 of 

BAA. 

Some studies (Ghanayem et al., 1987, 066470; Ghanayem et al., 1990, 042016) were 

designed to assess the effect of age on the toxicokinetics and hemolytic effects in young and 

adult rats treated with single EGBE gavage exposures. Both blood retention (Ghanayem et al., 

1990, 042016) and hematologic effects (Ghanayem et al., 1987, 066470) were found to be doseand age-dependent; older rats retained more of the EGBE metabolite BAA in their blood and 

were more sensitive than younger rats. The increased blood retention of BAA (as measured by 

increased Cmax, AUC, and t1⁄2) in older rats versus younger rats may be due to metabolic 

differences or compromised renal clearance. The researchers suggested that the pharmacokinetic 

 53 
basis of the age-dependent toxicity of EGBE may be due to a reduced ability of older rats to 

metabolize the toxic metabolite BAA to CO2, and a diminished ability to excrete BAA in the 

urine. No comparable studies exist for an analysis of liver effects. 

While older rats appear to be more severely affected by acute doses of EGBE, continuous 

exposures appear to impart a certain level of tolerance to rats and mice over time. Apparent 

tolerance to EGBE-induced hemolysis in rats and mice has been seen in subchronic (Grant et al., 

1985, 006770; Krasavage, 1986, 066474) and chronic (NTP, 2000, 196293) studies. Ghanayem 

et al. (1990, 042016; 1992, 100128) investigated this effect in the male F344 rat. Daily gavage 

administration of EGBE at 125 mg/kg for 12 days resulted initially in hemolytic anemia, which 

was more pronounced following the third day, but the animals recovered gradually to near 

pretreatment levels by day 12. Additionally, rats treated for 3 days at 125 mg/kg followed by a 

7-day recovery period were significantly less sensitive to subsequent treatment with EGBE at 

either 125 or 250 mg/kg, as were rats that were bled and subsequently treated. Ghanayem et al. 

(1990, 042016; 1992, 100128) proposed that the tolerance to hemolysis following repetitive 

dosing is not due to changes in EGBE metabolism, but to the replacement of older and more 

susceptible erythrocytes with younger, less susceptible cells. However, chronic studies in rats 

and mice (NTP, 2000, 196293) have shown that any increased resistance imparted by these 

immature erythrocytes diminishes with age. Rats and mice chronically exposed to EGBE 

experienced anemia that persisted with no apparent progression or amelioration of severity for 9 

months, up to the final blood collection at 12 months of age. There may be a balance in these 

rodents between the release of reticulocytes to the circulation and the aging process, so that the 

level of susceptible cells and severity of anemia remains relatively constant. 

A number of secondary effects resulting from the hemolytic toxicity of EGBE have been 

reported in studies with rats, mice, and rabbits. In the rat, the organs generally affected include 

most prominently the liver (see discussions above) but also the kidneys, spleen, bone marrow, 

and, to a lesser extent, the thymus (Exon et al., 1991, 100112; Grant et al., 1985, 006770; NTP, 

1993, 042063; Shabat et al., 2004, 100200). Typically, increased liver and kidney weights are 

observed with corresponding decreases in body weights at doses that produce a hematotoxic 

response. Accompanying this is hepatocellular degeneration, hemosiderin deposition in the liver, 

and congested spleens. Renal damage is often reported, accompanied by hemosiderin 

accumulation, renal tubular degeneration, and intracytoplasmic Hb. Often these effects are more 

pronounced in females. Hematopoiesis in bone marrow and spleen, increased cellularity of bone 

marrow, and splenic congestion are all secondary to the hematotoxicity of EGBE and develop as 

a compensatory response to hemolysis. In addition, intact erythrocytes have been observed 

histopathologically in spleens from EGBE-treated rats, but not in spleens from control animals. 

This suggests an increased rate of removal of damaged erythrocytes in EGBE-treated rats 

(Ghanayem et al., 1987, 066470). Mild lymphopenia and neutrophilia were observed at 

hemolytic doses of EGBE (Ghanayem et al., 1987, 066470) and were reported to be consistent 

 54 
with a “stress” leukogram produced by the release of endogenous corticosteroids (Wintrobe et 

al., 1981, 100222). Neutrophilia, commonly associated with acute hemolysis or hemorrhage 

(Wintrobe, 1981, 594424), was also observed. 

In the NTP (2000, 196293) study, the incidence of hyaline degeneration of the olfactory 

epithelium was significantly increased in all exposed groups of male rats and in the 62.5 and 

125 ppm groups of females. Hyaline degeneration is the accumulation of intracytoplasmic 

globules of highly eosinophilic material in the epithelial cells. While not unique to EGBE, 

hyaline degeneration has been shown to occur in rodents exposed to other gases and vapors like 

dimethylamine (Buckley et al., 1985, 062668) and pyridine (Nikula et al., 1995, 100182). It also 

has been shown to develop in unexposed aged animals (St Clair and Morgan, 1992, 594426). 

In conclusion, humans are significantly less sensitive to the hemolytic toxicity of EGBE 

than are typical laboratory species such as mice, rats, or rabbits, although human erythrocytes do 

appear capable of responding similarly to the causative EGBE metabolites, albeit at much higher 

exposures. This marked species difference in sensitivity has been demonstrated in several 

laboratory studies and through the use of in vitro studies using either whole blood or washed 

erythrocytes. Based on the results of in vitro testing, blood concentrations of the hemolytically 

active metabolite BAA must reach levels in human blood in excess of 7.5 mM for prehemolytic 

changes to occur. Comparable effects in rat blood occur at in vitro concentrations approximately 

150-fold lower. In addition, blood from potentially sensitive individuals, including the elderly or 

those with congenital hemolytic disorders, does not show an increased hemolytic response when 

incubated with up to 2 mM BAA for 4 hours. Based on simulations from PBPK modeling, 

6-hour whole-body exposure of humans to saturated atmospheres of EGBE will result in 

maximum blood concentrations of BAA below those needed to produce hemolysis (Corley et al., 

2005, 100100). 

Most of the liver alterations documented throughout the EGBE exposure database are 

related directly to hemolysis. Prominent among these alterations is hemosiderin deposition, a 

pathological finding whose occurrence is related to hemolysis (Muller et al., 2006, 597291). 

Humans also experience hemosiderin deposition in the liver, principally in hepatocytes, as a 

consequence of excessive hemolysis, such as with thalassemia, a hereditary form of hemolytic 

anemia (Iancu et al., 1976, 100149). Hemosiderin deposition in the liver is a pathological 

response that follows the precursor hematologic effects, which, as a group, do not appear to 

progress with changes in duration of exposure from subchronic to chronic. 

4.6. EVALUATION OF CARCINOGENICITY 

4.6.1. Summary of Overall Weight of Evidence 

Under the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005, 086237), EGBE 

is deemed “not likely to be carcinogenic to humans” at environmental concentrations below or 

 55 
equivalent to the RfD and RfC, based on laboratory animal evidence, mode-of-action 

information, and limited human study information. The available data indicate that carcinogenic 

effects from EGBE are not likely to occur in humans in the absence of the critical noncancer 

effects, including hepatic hemosiderin staining and irritant effects at the portal of entry, and are 

not likely to be carcinogenic to humans exposed to levels at or below the RfC and RfD values 

derived in this assessment. Carpenter et al. (1956, 066464) (see Section 4.1) reported that no 

changes in erythrocyte osmotic fragility were found in human subjects exposed to up to 195 ppm 

(942 mg/m3; ~600 times the RfC) for two 4-hour periods separated by a 30-minute break. At 

oral doses of 400–500 mg/kg with a one-time bolus dose (see Section 4.1), hematuria has been 

noted in two human case reports. This dose is 3,000–3,500 times the RfD and would need to be 

sustained for a significant period of time to produce hemosiderin deposition. This is unlikely to 

occur because the primary response of humans to high oral doses of EGBE, as shown in the case 

studies in Section 4.1, is metabolic acidosis, which, if not treated, can lead to shock and 

eventually death. No information is available on the carcinogenic effects of EGBE via the oral 

or inhalation route in humans. A 2-year inhalation bioassay with mice and rats (NTP, 2000, 

196293) reported tumors of the liver in male mice, forestomach tumors in female mice, and 

tumors of the adrenal medulla in female rats. Nonneoplastic effects in rats included hyaline 

degeneration of the olfactory epithelium and Kupffer cell pigmentation. Nonneoplastic effects in 

mice included forestomach ulcers and epithelial hyperplasia, hematopoietic cell proliferation, 

Kupffer cell pigmentation, hyaline degeneration of the olfactory epithelium (females only), and 

bone marrow hyperplasia (males only). 

EGBE has been tested in conventional genotoxicity tests for its potential to induce gene 

mutations in vitro and cytogeneticity in both in vitro and in vivo, and the available data do not 

support a mutagenic or clastogenic mechanism for EGBE. Two laboratories (Elias et al., 1996, 

042011; Hoflack et al., 1995, 100147) did report weak genotoxicity responses in vitro at high 

treatment concentrations, but results were not replicated in five other labs reporting negative 

results. 

The hypothesized MOA for the tumors observed following EGBE treatment involves 

exposure to high doses for prolonged periods of time. This MOA is described in the sections that 

follow. The weight of evidence indicates that EGBE is not likely to be carcinogenic to humans 

at expected environmental concentrations. 

4.6.2. Synthesis of Human, Animal, and Other Supporting Evidence 

NTP (2000, 196293) conducted a 2-year inhalation study on EGBE in both genders of 

F344/N rats and B6C3F1 mice. Rats (50/gender/group) were exposed to concentrations of 0, 31, 

62.5, and 125 ppm (0, 150, 302, and 604 mg/m3) and mice (50/gender/group) were exposed to 

concentrations of 0, 62.5, 125, and 250 ppm (0, 302, 604, and 1,208 mg/m3

). The NTP report 

 56 
stated that the highest exposure was selected to produce a 10–15% depression in hematologic 

indices, and survival was significantly decreased in male mice at 125 and 250 ppm (54.0 and 

53.1%, respectively). While the NTP researchers report that no effect on survival was observed 

in rats, the female rats appeared to show a trend toward decreased survival that may have been 

attributable to the hematological effects. Mean body weights of rats exposed to 31 and 62.5 ppm 

were similar to those of control animals. Mean body weights of the exposed mice were generally 

less than those of controls, with females experiencing greater and earlier reductions. From 

week 17 to the end of the study, the mean body weights of 125 ppm female rats were generally 

less than those of controls. 

At the end of the 2-year chronic bioassay (NTP, 2000, 196293), neoplastic effects were 

observed in female rats and in male and female mice. In female rats, the combined incidence of 

benign and/or malignant pheochromocytoma of the adrenal medulla was 3/50, 4/50, 1/49, and 

8/49. The incidence in the high-dose group (16%) did not represent a statistically significant 

increase over the chamber control group (6%), but it exceeded the historical control (6.4 ± 3.5%; 

range 2–13%) for this effect. 

The low survival rate in male mice exposed to 125 and 250 ppm EGBE may have been 

due to carcinogenic effects in the liver. A high rate of hepatocellular carcinomas was found in 

these exposure groups (10/50 [control], 11/50, 16/50, 21/50); the increase at the high-exposure 

level was statistically significant (p < 0.01). However, when hepatocellular adenomas and 

carcinomas were combined, no significant increase was observed in any exposure group. The 

incidence of hemangiosarcomas in males exposed to 250 ppm (8%) was also significantly 

increased (p = 0.046) relative to chamber controls (0/50, 1/50, 2/49, 4/49) and exceeded the 

range of historical controls (14/968; 1.5 ± 1.5%; range 0–4%). No significant increases in 

benign or malignant hepatocellular tumors or hemangiosarcomas were noted in the female mice, 

and the incidence of hepatocellular adenomas actually decreased significantly (p < 0.05) in 

relation to the control chamber group (16/50, 8/50, 7/49, 8/49). It should be noted that in light of 

the high survival rate of the exposed female mice relative to controls (29/50, 31/50, 33/50, 

36/50), the high exposure of 250 ppm may not have provided the maximum tolerated dose. 

Forestomach squamous cell papillomas and carcinomas, combined, were significantly 

increased (trend test = 0.003) in female mice relative to the chamber control group (0/50, 1/50, 

2/50, 6/50). The incidence of these tumor types (12%) at the highest exposure level was also 

statistically significant and exceeded the range for the occurrence of these tumors in historical 

controls (0.9 ± 1.1%; range 0–3%). The first incidence of these tumors appeared in the group 

exposed to 250 ppm at 582 days, as compared to 731 days at 62.5 and 125 ppm, indicating a 

decreased latency period in the highest exposure group. While the incidence of these types of 

forestomach tumors was not significantly increased over controls in male mice (1/50, 1/50, 2/50, 

2/50), the incidence of squamous cell papillomas (4%) in the two highest exposure groups 

exceeded the range for historical controls (0.5 ± 0.9%; range 0–2%). The increased incidence of 

 57 
forestomach neoplasms in males, as in females, occurred in groups with ulceration and 

hyperplasia. 

The NTP (2000, 196293) study concluded that there was no evidence showing 

carcinogenic activity in male F344/N rats, and equivocal evidence of carcinogenic activity in 

female F344/N rats based on increased combined incidences of benign (mainly) and malignant 

pheochromocytoma of the adrenal medulla. The researchers reported some evidence of 

carcinogenic activity in male B6C3F1 mice based on increased incidences of hemangiosarcoma 

of the liver and an increase in the incidence of hepatocellular carcinoma, as well as some 

evidence of carcinogenic activity in female B6C3F1 mice based on increased incidence of 

forestomach squamous cell papilloma (mainly) or carcinoma. 

With respect to the pheochromocytomas reported in female rats, while the data showed a 

positive trend (p = 0.044) and the high-dose tumor frequencies (16%) were above the upper 

range of historical controls (13%), the tumor incidence data were not statistically significant. 

Further, the NTP (2000, 196293) report noted that pheochromocytomas can be difficult to 

distinguish from nonneoplastic adrenal medullary hyperplasia. The presence of mild-tomoderate compression of the adjacent tissue is a primary criterion used to distinguish 

pheochromocytomas from medullary hyperplasia; most tumors observed were small and not 

substantially larger than the more severe grades of adrenal medullary hyperplasia. Interpretation 

of these tumors should be done cautiously. Given the marginal dose response, lack of tumor 

evidence in any other organ system of the rats, and reported difficulties in distinguishing 

pheochromocytomas from nonneoplastic adrenal medullary hyperplasia, this tumor type was not 

given significant weight in the qualitative or quantitative assessment of EGBE cancer potential. 

4.6.3. Mode-of-Action Information 

4.6.3.1. Hypothesized MOA for Liver Tumor Development in Male Mice 

The hypothesized MOA for EGBE-induced liver tumors in male mice is believed to 

involve iron accumulation and subsequent oxidative stress due to the hemolytic effects of a 

metabolite of EGBE. Male mice developed hepatocellular carcinomas and hemangiosarcomas in 

the liver that appeared to be exposure-related. The incidence of hemangiosarcomas was 

statistically significant and increased over both concurrent and historical control groups. The 

hepatocellular carcinomas were within the range of historical controls for male mice, but are also 

considered in this discussion because the dose-response trend is significant and because a similar 

MOA has been suggested for this tumor (Klaunig and Kamendulis, 2005, 100165). The 

incidences in the high dose group of these two types of tumors were only slightly higher than the 

upper end of the range for historical controls. Furthermore, these two tumor types were not 

increased in other organs (e.g., bone, bone marrow) and were not noted in either rats or female 

mice. The hypothesized MOA involves the hemolysis of RBCs, the accumulation of 

hemosiderin, and subsequent oxidative stress that leads to neoplasia in the two cell types 

 58 
believed to be the origin for these two tumors: hepatocytes for hepatocellular carcinoma and 

endothelial cells for hemangiosarcomas. Only the male mice developed these tumor types, and 

this is hypothesized to be due to their susceptibility to oxidative stress. Mice are known to be 

more susceptible to oxidative stress than rats because of their lower antioxidant capacity 

(Bachowski et al., 1997, 042161; Siesky et al., 2002, 042204). The available studies suggest that 

iron accumulation from the hemolytic effects of EGBE produces liver oxidative damage that is 

more severe in mice and increased DNA synthesis in both endothelial cells and hepatocytes that 

may be unique to mice (Corthals et al., 2006, 100102; Siesky et al., 2002, 042204). From this 

and reported differences in antioxidant capacity2 and background rates of these tumors3 between 

male and female mice, it can be hypothesized that events leading to oxidative stress could 

contribute to the development of hemangiosarcomas and hepatocellular carcinomas in male 

mice. A series of events that may lead to the formation of liver tumors follows: 

(1) EGBE is metabolized to BAL, which is subsequently oxidized to BAA; 

(2) BAA causes RBC swelling, triggering sequestration in the spleen by resident 

macrophages. When the capacity of these macrophages becomes overwhelmed, the 

damaged RBCs make their way into the liver; 

(3) Excess Hb from damaged RBCs is taken up by phagocytic (Kupffer) cells of the liver and 

stored as hemosiderin; 

(4) Oxidative damage and increased synthesis of endothelial and hepatocyte DNA are 

initiated by one or more of the following events: 

(a) Generation of reactive oxygen species (ROS) from Hb-derived iron within 

Kupffer cells and perhaps from within hepatocytes and sinusoidal endothelial cells; 

and 

(b) Activation of Kupffer cells to produce cytokines/growth factors that suppress 

apoptosis and promote cell proliferation. 

(5) ROS results in oxidative DNA damage to hepatocytes and endothelial cells; 

(6) ROS modulates hepatocyte and endothelial cell gene expression; 

(7) ROS stimulates hepatocyte and endothelial cell proliferation; 

(8) ROS promotes initiation of hepatocyte and endothelial cells; and 

(9) ROS promotes neoplasm formation. 

 

2

While the reason for the sex difference in liver tumor susceptibility between male and female mice is not clear, it has been shown that 

estrogens can be protective through their antioxidant capacities and through their modulation of the activities of other antioxidants (Nyska 

et al., 2004, 056379). 

3

NTP has observed liver hemangiosarcomas in 105/4183 (2.51%) male versus just 35/4177 (0.84%) female historical controls (Klaunig 

and Kamendulis, 2005, 100165; NTP, 2000, 196293). In addition, other chemicals reported by NTP to cause both early onset hemosiderin 

buildup and liver tumors have also exhibited this male specificity (U.S. EPA, 2005, 594432). 

 59 
The first two steps, the metabolism of EGBE to BAA and the association of BAA with 

hemolytic effects, have been established in many studies, both in vitro and in vivo, with 

sensitivities ranging from extreme sensitivity as in rats, mice, rabbits and dogs, to moderate to 

extreme insensitivity seen in monkeys, guinea pigs, and humans (see Section 3). The third step 

has been seen in both genders of rats and mice exposed to EGBE in multiple studies that 

observed hemosiderin within Kupffer cells and hepatocytes after RBC breakdown (Ghanayem 

and Sullivan, 1993, 041609; Ghanayem et al., 1987, 041645; Ghanayem et al., 1987, 041608; 

Kamendulis et al., 1999, 042182; Krasavage, 1986, 066474; NTP, 2000, 196293; Siesky et al., 

2002, 042204). A number of studies (Kamendulis et al., 1999, 042182; Park et al., 2002, 

030770; Siesky et al., 2002, 042204) provide support for step 4a by showing that in vivo 

exposure to EGBE increases 8-OHdG levels (an indicator of oxidative damage) in mice, but not 

rats, and decreases vitamin E levels in rats and mice. Using rat and mouse hepatocytes, Park et 

al. (2002, 030770) showed that FeSO4 produced dose-related changes in these same indicators in 

mouse hepatocytes, but not in rat hepatocytes, and that treatment with EGBE or BAA did not 

produce changes in these oxidative stress parameters. Additionally, Nyska et al. (2004, 056379) 

analyzed 130 2-year carcinogenicity studies of B6C3F1 mice from NTP bioassays and concluded 

that a significantly increased risk of inducing hepatic hemangiosarcomas in male B6C3F1 mice 

exists in studies with compounds that caused increased tissue burdens of ROS. Klaunig and 

Kamendulis (2005, 100165) and Corthals et al. (2006, 100102) provided support for step 4b, by 

showing that the activation of Kupffer cells, either through RBC hemolytic components and/or 

iron accumulation in the Kupffer cells, results in the production of cytokines such as TNFα. The 

comet assay has been used to assess DNA damage to endothelial cells from ROS (step 5) 

(Klaunig and Kamendulis, 2004, 594442; Klaunig and Kamendulis, 2005, 100165; Reed et al., 

2003, 594436). While step 6 has not been shown directly for endothelial cells or hepatocytes 

exposed to EGBE, induction of oxidative damage has been shown to modify gene expression in 

mammalian cells. In addition, ROS production can stimulate cell proliferation and the inhibition 

of apoptosis (Klaunig and Kamendulis, 2005, 100165; Nyska et al., 2004, 056379). Siesky et al. 

(2002, 042204) observed increased DNA synthesis in endothelial cells and hepatocytes in vivo in 

mice, but not in rats, at doses that produced hemangiosarcomas in the mouse liver (NTP, 2000, 

196293) (step 7). Steps 8 and 9 are consistent with the lack of direct genotoxicity of EGBE (see 

Section 4.4.4) and the high rate of spontaneous endothelial neoplasms in the male mouse liver 

relative to the rat (Klaunig and Kamendulis, 2005, 100165). The observation of decreased 

antioxidant capacity and increased 8-OHdG levels in male mice also lends support to the 

proposed steps of initiation and promotion of neoplasms by ROS (Klaunig et al., 1998, 024780). 

4.6.3.1.1. Temporal association and species specificity. Key steps in the proposed MOA (i.e., 

hemolysis, hepatic hemosiderin buildup, and oxidative damage) have all been observed in 

 60 
subchronic or shorter-duration rat and mouse studies of EGBE (Kamendulis et al., 1999, 042182; 

NTP, 2000, 196293; Siesky et al., 2002, 042204) well in advance of tumor formation. 

Differences between rats and mice with respect to these responses may help to explain the 

relative sensitivity of male mice to the formation of liver tumors following EGBE exposure. In 

mice, Siesky et al. (2002, 042204) observed a dose-dependent increase in levels of liver 

oxidative stress indicators at exposure days 7 and 90, increased endothelial cell DNA synthesis at 

exposure days 7 and 14, and increased hepatocyte DNA synthesis at 90 days. No increase in 

ROS or the DNA synthesis of either cell type was observed in rats at any time point. 

4.6.3.1.2. Dose-response relationships. Five chemicals have been determined by the NTP to 

cause hemosiderin buildup in the livers of mice. As shown in Table 4-8, all three studies that 

reported hemosiderin buildup in Kupffer cells of male mice within 13 weeks of exposure also 

showed an increased incidence of hemangiosarcomas and hepatocellular carcinomas following 

chronic exposure. The dose responses for endpoints describing possible precursor effects, 

splenic hematopoietic cell proliferation, and liver hemosiderin accumulation appear to be doserelated and coincident to the formation of tumors. Dose-responses for several hemolytic effects 

were also observed in rats exposed to EGBE, but liver tumors were not increased in rats at any 

dose. However, the high dose used in the EGBE rat study was only half that of the high dose 

used in the mouse study, and the 2-year duration of these studies represents a smaller fraction of 

the rat lifespan, leaving the possibility that similar responses could have been observed in rats if 

higher and longer EGBE exposures had been administered. 

 61 
Table 4-8. Incidence of liver hemangiosarcomas and hepatocellular 

carcinomas in studies of NTP chemicals that caused increased hemosiderin 

in Kupffer cells in male mice 

Chemical (NTP TR #) Hemosiderin SCa Hemangiosarcoma Hepatocarcinoma Type 

2-Butoxyethanol 

(EGBE) (TR-484) 

0/50, 0/50, 8/49c, 

30/49c

Yes 0/50, 1/50, 2/49, 4/49b 10/50, 11/50, 16/49, 

21/49c

I 

p-Chloroaniline 

hydrochloride (TR-351) 

0/50, 0/49, 0/50, 50/50d Yes 2/50, 2/49, 1/50, 6/50 3/50, 7/49, 11/50b, 17/50c G 

p-Nitroaniline 

(TR-418) 

1/50, 1/50, 8/50b, 

50/50c

Yes 0/50, 1/50, 2/50, 4/50 10/50, 12/50, 13/50, 6/50 G 

C.I. Pigment Red 3 

(TR-407)d 

0/50, 5/50, 30/50, 41/50 No 0/50, 1/50, 1/50, 0/50 5/50, 10/50, 8/50, 4/50 F 

o-Nitroanisole 

(TR-416) 

0/50, 0/50, 3/50, 16/50c No 2/50, 2/50, 1/50, 0/50 7/50, 12/50, 11/50, 7/50 F 

a

Chemicals that caused hemosiderin accumulation in Kupffer cells following subchronic (SC) exposure are 

identified with a “yes” in this column. 

b

Statistically significant difference, p ≤ 0.05. c

Statistically significant difference, p ≤ 0.01. 

d

Statistics not reported. 

F = feed; G = gavage; I = inhalation 

Of the five chemicals listed in Table 4-8, liver hemangiosarcomas were observed with 

only three chemicals that induced hemosiderin buildup by week 13, but not with the other two 

chemicals for which hemosiderin buildup was not observed until the end of the 2-year study 

(Gift, 2005, 100133; U.S. EPA, 2005, 594432). Two of the three chemicals that induced early 

liver hemosiderin accumulation and increased hemangiosarcoma incidence, EGBE and pchloroaniline hydrochloride, also induced an increase in hepatocellular carcinomas. Early 

buildup of hemosiderin combined with early increases in endothelial cell and hepatocyte DNA 

synthesis would result in a longer exposure of cells to oxidative damage via iron-generated 

radicals (step 4). This would be consistent with a mechanism involving a continuing cycle of 

damage and repair and accumulation of DNA mutations (steps 5 and 6). In addition to an earlier 

onset of hemosiderin buildup, mice also show evidence of a more sustained hemolytic response 

to EGBE than rats.4

4.6.3.1.3. Biological plausibility and coherence of the database. Oxidative damage plays an 

important role in the pathogenesis of several diseases, including cancer and cardiovascular 

disease (Djordjevic, 2004, 100106; Klaunig et al., 1998, 024780; Lesgards et al., 2002, 042183). 

In support of the proposed hypothesis, increased ROS are known to accompany the release of 

 

4

Mice experienced an increase in liver and splenic hematopoietic cell proliferation throughout the 2-year NTP (2000, 196293) study, while 

rats tended to compensate for the effects of EGBE after a few months. This increased tolerance in rats is evidenced by a lack of induction 

of splenic hematopoiesis at the end of the 2-year NTP (2000, 196293) study. 

 62 
large amounts of iron from hemolysis (Ziouzenkova et al., 1999, 024904). If EGBE causes 

oxidative stress via hemolysis, then the production of protein and DNA damage would be 

expected, including the production of 8-OHdG, accompanied by a decrease in antioxidant levels, 

such as Vitamin E (Houglum et al., 1997, 024777; Wang et al., 1995, 024797; Yamaguchi et al., 

1996, 024876). These effects were verified by both Siesky et al. (2002, 042204) and 

Kamendulis et al. (1999, 042182), who measured a dose-dependent increase in levels of ROS 

indicators, including 8-OHdG and malondialdehyde, and a decrease in vitamin E levels in the 

livers of mice, but not in rats, after acute and subchronic exposure to EGBE. The fact that mice 

appear more susceptible than rats to ROS from EGBE exposure is consistent with the proposed 

MOA, and is a reasonable explanation for why hemangiosarcomas are not observed in rats 

following chronic EGBE exposure, despite the fact that rats are at least as sensitive as mice to the 

hemolytic effects of EGBE. 

Iron, which is known to accumulate in cells of rodent livers following EGBE exposure, 

can produce hydroxyl radicals in combination with oxidative by-products via the Fenton reaction 

(Kamendulis et al., 1999, 042182). The damaging effects of iron overload to liver sinusoidal 

cells have been shown in rats following a single i.p. injection of 200 mg iron/kg (Junge et al., 

2001, 042180). In addition, endothelial cells appear to be relatively sensitive to oxidative stress 

(DeLeve, 1998, 100104; Spolarics, 1999, 024792). Liver hemangiosarcomas develop from the 

endothelial cell component of the vascular sinusoidal cells of the liver (Frith and Ward, 1980, 

024775). 

In vivo studies have indicated that pretreatment of rats with an ALDH inhibitor, pyrazole, 

prior to a single 125 mg EGBE/kg gavage exposure protected against hemolysis Ghanayem et al. 

(1987, 041608), presumably by blocking the production of both BAL and BAA. Pretreatment of 

rats with an ADH inhibitor, cyanamide, prior to a single 125 mg EGBE/kg gavage exposure, 

reduced hemolytic responses, but increased RBC swelling, increased mortality, decreased BAA 

formation and excretion in the urine, and increased the urinary excretion of EGBE conjugates 

with glucuronide and sulfate (Ghanayem et al., 1987, 041608). This hematotoxicity in the 

presence of cyanamide may be due to BAL, but residual BAA may also be a factor. EGBE + 

cyanamide decreased BAA concentrations in rats; however, some BAA was formed and the 

BAA t1⁄2 was increased (Ghanayem et al., 1990, 042016), and, when Ghanayem et al. (1987, 

041608) administered a gavage dose of 125 mg BAL/kg + cyanamide to rats, the researchers 

observed almost no hemolytic activity. Furthermore, gavage administration to rats of 125 mg 

EGBE/kg and the molar equivalent of BAL and BAA resulted in no significant difference 

between the hemolytic effects of the three chemicals between 2 and 24 hours postexposure 

(Ghanayem et al., 1987, 041608). These data suggest that EGBE’s hemolytic activity (without 

coexposures) is due to BAA and that the metabolism of EGBE and BAL to BAA takes place 

rapidly and completely. 

 63 
4.6.3.1.4. Relevance of the hypothesized MOA to humans. The occurrence of liver tumors in 

mice exposed to EGBE is hypothesized to occur through an MOA that requires first a dosage of 

EGBE that is high enough to cause sustained hemolysis of RBCs and, second, leads to sufficient 

buildup of hemosiderin in the Kupffer cells of the liver to produce ROS and subsequent 

neoplasm formation. 

Several studies have examined the susceptibility of RBCs to BAA-induced hemolysis and 

have found a range in sensitivity from the sensitive (rats, mice, rabbits, and dogs) to the less 

sensitive (monkeys, guinea pigs, and humans). Human volunteers experienced no hemolysis 

from controlled laboratory acute inhalation exposures (up to 195 ppm), a dosage which caused 

significant erythrocyte fragility in rats (Carpenter et al., 1956, 066464). Only mild hemolytic 

effects have been observed in humans acutely exposed to oral doses of EGBE (400–1,500 

mg/kg), doses that have been shown to cause marked hemolytic effects in rats (Ghanayem et al., 

1987, 066470; Grant et al., 1985, 006770). RBCs from populations that were potentially more 

sensitive to hemolysis in general—the elderly, and individuals with sickle cell anemia and 

hereditary spherocytosis—were tested in vitro and not found to exhibit hemolysis after exposure 

to concentrations 40-fold higher (the highest tested in the study) than those shown to induce 

hemolysis in rat RBCs (Udden, 1994, 042109; Udden and Patton, 1994, 056374). In an in vitro 

study of RBCs from hospitalized children and adults, concentrations of up to 150-fold higher 

than those used in rat studies did not produce hemolysis (Udden, 2002, 042111). The resistance 

of human RBCs to the initial event of hemolysis makes it unlikely that they would experience the 

subsequent effects of increased hemosiderin deposition through this pathway, and consequently, 

humans would not be at increased risk of tumor development through this MOA. 

 64 
4.6.3.1.5. Other possible MOAs for liver tumor development in male mice. Although certain 

key events in EGBE’s MOA for the development of liver tumors in male mice are fairly welldescribed and plausible, some alternative considerations (also supported by scientific literature) 

may be involved. ROS can potentially be derived from two sources: iron overloading in the 

liver (through Fenton and Haber-Weiss reactions) and Kupffer cell activation. Via either source, 

oxygen radicals can induce oxidative damage to DNA and lipids as documented in the liver 

following EGBE treatment (Siesky et al., 2002, 042204). The activation of Kupffer cells, 

through phagocytosis of RBC hemolytic components or iron in the Kupffer cell, results in the 

production of cytokines, possibly including vascular endothelial growth factor that may elicit a 

growth response on endothelial cells (Corthals et al., 2006, 100102). In addition to the 

production of oxidative DNA damage, ROS, whether derived from Kupffer cell activation or 

other biological processes, can alter gene expression (e.g., MAP kinase/AP-1 and NFκB), 

resulting in stimulation of cell proliferation and/or inhibition of apoptosis (Klaunig and 

Kamendulis, 2004, 594442). 

Another recognized mechanism for the development of chemically-induced liver 

hemangiosarcomas involves direct interaction with DNA. This MOA is recognized for vinyl 

chloride and thorotrast, two agents that are known to induce hemangiosarcomas in humans. The 

EGBE metabolite BAL is considered to have the greatest potential to interact with DNA, since it 

has been shown to cause in vitro SCE at concentrations ranging from 0.2 to 1 mM (Elliott and 

Ashby, 1997, 100111). However, high ADH activity in the liver, as in the forestomach, is 

expected to result in very short residence time and low Cmax liver tissue concentrations of BAL. 

Corley et al. (2005, 100098) extended their 1994 model (Appendix B) to include the metabolism 

of EGBE to BAL via ALDH and the subsequent metabolism of BAL to BAA via ADH in both 

the liver and forestomach. As shown in Figure 4-1, using rate constants derived from mouse 

stomach fractions (Green et al., 2002, 041610) and making several assumptions about the use of 

these enzyme activity data, Corley et al. (2005, 100100) estimated that 250 ppm EGBE (the 

highest concentration used in the NTP (2000, 196293) study) would result in peak Cmax values of 

7 μM EGBE, 0.5 μM BAL, and 3,250 μM BAA in liver tissue of male mice at the end of a 6-

hour exposure period. 

 65 
BE = EGBE 

Source: Adapted from Corley et al. (2005, 100098). 

Figure 4-1. Simulated concentrations of EGBE, BAL, and BAA in liver 

tissues of female mice exposed via inhalation for 6 hours to 250 ppm EGBE. 

Thus, the Corley et al. (2005, 100098) PBPK model suggests that the high cytotoxic 

concentrations of BAL that showed some evidence of clastogenicity may not be relevant to the 

target organ where lower concentrations of BAL would exist in the presence of metabolizing 

enzymes. A recent gavage study performed by Deisinger and Boatman (2004, 594446) provided 

support for the Corley et al. (2005, 100098) model and the predicted low levels of the BAL 

metabolite in liver tissue.5

 In addition, as discussed in Section 4.4.4, evidence from in vivo and 

in vitro genotoxicity assays does not suggest that BAL would have any significant genotoxicity 

in vivo.6

 Furthermore, the MOA for hemangiosarcoma induction by genotoxins such as vinyl 

chloride and thorotrast involves the initiation of hepatocellular and sinusoidal cell hyperplasia 

and sinusoidal compression, leading to the development of fibrous septa, generally in the 

periportal area, out of which eventually develop multiple areas of angiosarcomas (Foster, 2000, 

042172). EGBE exposure does not generate this same pattern of effects prior to the development 

of cancer in mice. 

 

5

The Corley et al. (2005, 100098) model predicts that the concentrations of BAL in liver tissues of male and female mice would be 17 and 

29 μM, respectively, following oral gavage exposure to 600 mg/kg EGBE. The levels of BAL actually observed in the liver tissue of male 

and female mice, following oral gavage exposure to EGBE at 600 mg/kg, 3 and 4 μM, respectively, were even lower than the predicted 

values (Deisinger and Boatman, 2004, 594446). 

6

The exposure concentrations that would be necessary to cause these effects in humans, if attainable at all, are likely to be much higher 

than the RfC and RfD. 

 66 
4.6.3.2. Hypothesized MOA for Forestomach Tumor Development in Female Mice 

A significant increase over controls (experimental and historical) of papillomas and one 

carcinoma of the forestomach (6/50; 12%) was reported by NTP (2000, 196293) in female mice 

exposed for 2 years to 250 ppm EGBE by inhalation. An increase was also seen in squamous 

cell papillomas in male mice, although this did not reach the level of statistical significance. 

Significant increases in forestomach papillomas and carcinomas were not observed in rats of 

either gender. The study also showed statistically significant, dose-dependent increases in 

hyperplasia for both male and female mice, and for ulceration in female mice. Male mice 

showed significant increases in ulceration at the 125 ppm exposure. The process of irritation, 

hyperplasia, and ulceration is thought to be a part of the cell injury and regeneration process 

elicited by irritant chemicals such as EGBE, and the study authors hypothesized that the 

neoplasia may occur due to an increase in the cell replication associated with regeneration. 

A hypothesized series of events that may lead to the formation of forestomach tumors is 

as follows: 

 (1) Deposition of EGBE/BAA in the stomach and forestomach via consumption or 

reingestion of EGBE laden mucus, salivary excretions, and fur material; 

(2) Retention of EGBE/BAA in food particles of the forestomach long after being cleared 

from other organs; 

(3) Metabolism of EGBE to BAL, which is rapidly metabolized to BAA systemically and in 

the forestomach; 

(4) Irritation of target cells by BAA leading to hyperplasia and ulceration; 

(5) Continued injury by BAA and degeneration leading to high cell proliferation and 

turnover; and 

(6) High levels of cell proliferation and turnover leading to clonal growth of spontaneously 

initiated forestomach cells. 

There are a number of studies that have demonstrated steps 1 and 2 (i.e., the deposition 

and retention of EGBE and BAA in the forestomach). Studies have shown this occurs following 

whole-body exposure (Green et al., 2002, 041610; Poet et al., 2002, 594439), nose-only 

inhalation (Poet et al., 2002, 594439), i.v. exposure (Bennette, 2001, 041810; Green et al., 2002, 

041610; Poet et al., 2002, 594439), i.p. exposure (Corley et al., 1999, 042003; Poet et al., 2002, 

594439), s.c. exposure (Corley et al., 1999, 042003), and gavage exposures (Ghanayem et al., 

1987, 041645; Ghanayem et al., 1987, 041608; Green et al., 2002, 041610; Poet et al., 2002, 

594439). It is of note that following i.v. and inhalation exposures in mice, EGBE metabolites 

rapidly accumulate in salivary secretions and are swallowed (Bennette, 2001, 041810; Green et 

al., 2002, 041610), leading to the collection and retention of the chemical(s) in the forestomach. 

 67 
The same process is likely to occur through other systemic exposures. The metabolism of EGBE 

to BAA (step 3) has been shown in both in vitro and in vivo tests with rats, mice, rabbits, guinea 

pigs, dogs, monkeys, and humans, (see Section 3) and is further supported by the EGBE PBPK 

model developed by Corley et al. (2005, 100098). Step 4, the irritation of target cells, has been 

seen in both genders of B6C3F1 mice, (Green et al., 2002, 041610; NTP, 2000, 196293; Poet et 

al., 2002, 594439), with irritation and a compensatory proliferative response reported following 

exposure to EGBE. Female mice were shown to have more extensive and severe forestomach 

lesions than male mice and were observed in the NTP (2000, 196293) study to have statistically 

significant increases in forestomach tumors. This suggests the importance of the continued 

damage and high cell proliferation (step 5) that is associated with tumor formation. Green et al. 

(2002, 041610) found that the number of cells in S-phase (an indication of cell turnover) 

increased in a dose-dependent fashion after exposure to EGBE and BAA, even though none of 

the changes were statistically significant due to the high turnover for the control group. Step 6, 

high levels of cell proliferation and turnover, leads to clonal growth of spontaneously initiated 

cells and is supported by the continuum of effects observed in the mice (Green et al., 2002, 

041610; NTP, 2000, 196293) and the effects seen with other irritant compounds (Kroes and 

Wester, 1986, 041626). 

Green et al. (2002, 041610) also provided relevant information regarding step 3 through 

examination of the activity and localization of ALDH and ADH in the stomach tissues of mice, 

rats, and a human sample. Whole body autoradiography of mice that had been exposed to 

radiolabeled EGBE was also performed. Histochemical staining of stomach tissues from the 

rodent species showed the dehydrogenase enzymes to be heavily concentrated in the stratified 

squamous epithelium of the forestomach of both rats and mice, whereas their distribution in the 

glandular stomach was more diffuse. By comparison, histochemical analysis of a human 

stomach tissue sample showed both enzymes to be present and evenly distributed throughout the 

epithelial cells of the entire stomach mucosa. A marked species difference in ALDH activity in 

the forestomach was observed between rats (Km = 0.29 mM; Vmax = 1.627 nmol/minute per mg 

protein) and mice (Km = 46.59 mM; Vmax = 17.094 nmol/minute per mg protein) with Km values 

up to one order of magnitude greater in mice compared to rats. These differences indicate that 

mice forestomach tissues would have the capacity to metabolize appreciably larger amounts of 

EGBE to BAL, and subsequently to BAA, than would the rat forestomach. Whole body 

autoradiography of mice exposed to EGBE demonstrated selective accumulation in the 

forestomach, which would provide substrate for these enzymes. Collectively, these data 

demonstrate several points regarding the observed forestomach toxicity in mice following 

exposure to EGBE, including: (1) the accumulation of EGBE in the target tissue, the 

forestomach, of mice; (2) a high degree of localization in the forestomach (as compared to the 

glandular stomach) tissues of both rats and mice of the enzymes that metabolize EGBE to the 

corresponding carboxylic acid; and (3) kinetic differences in these enzymes consistent with mice 

 68 
being capable of metabolizing more EGBE to BAA than rats. The data also suggest that human 

stomach tissues do not have a high localization of the EGBE metabolizing enzymes; the diffuse 

distribution of these enzymes in the human stomach sample examined is more similar to the 

distribution seen in the glandular portions of the rodent species examined. These observations 

suggest that human stomach tissues would be less capable of accumulating and localizing BAA 

than rat tissues and, thus, would be less likely to be exposed to the irritating effects of BAA. 

4.6.3.2.1. Temporal association. All of the steps in the proposed MOA have been observed to 

occur in female mice prior to tumor formation. NTP (2000, 196293) reported that female mice 

experienced epithelial hyperplasia (1/10, 5/10, 9/10, and 10/10) after just 13 weeks of exposure 

at the same exposure levels used in the chronic study, 0, 62.5, 125, and 250 ppm. The reported 

incidence of a forestomach papilloma or carcinoma in female mice was 731, 731, and 582 days 

in the 62.5, 125, and 250 ppm exposure groups, respectively. This is consistent with the findings 

of Ghanayem et al. (1986, 011728; 1993, 597328; 1994, 042760), who investigated the temporal 

relationship between the induction of this type of forestomach lesion by another nonmutagenic 

irritant, ethyl acrylate (EA), and the development of squamous cell papillomas and carcinomas. 

These researchers observed cell proliferation/hyperplasia in the forestomach of all rats that 

received EA by gavage (200 mg/kg, 5 days/week) for 6 or 12 months. All these potentially 

precancerous forestomach lesions regressed in animals treated with EA for 6 months and allowed 

2 or 15 months of recovery, and no forestomach neoplasms were observed. Although EA, an 

unsaturated aldehyde, is not a metabolite of EGBE, it is an analog of BAL and a much more 

potent carcinogen than either EGBE or BAL (Gold et al., 1993, 056382). 

For EGBE, the high incidence of forestomach hyperplasia, the relatively lower incidence 

of papillomas, and the late occurrence of a single carcinoma in the high 250 ppm exposure group 

are suggestive of a temporal relationship and tumor progression following EGBE exposure to 

female mice. Male mice may show the beginnings of tumorigenic effects as the incidence of 

papillomas increases, but such findings have not been statistically significant compared to 

concurrent or historical controls. No hyperplasia and no tumors were observed in inhalation 

studies of rats (NTP, 2000, 196293) or in drinking water studies of mice (NTP, 1993, 042063), 

supporting the need for these steps prior to tumor formation. 

4.6.3.2.2. Dose-response relationships. The incidences of epithelial hyperplasia (6/50, 27/50, 

42/49, 44/50) and ulceration (1/50, 7/50, 13/49, 22/50) in EGBE-exposed female mice were 

dose-related and significantly increased over both concurrent and historical controls at lower 

dose levels than the forestomach tumors. The hyperplasia was often associated with ulceration, 

particularly in the female mice. Forestomach tumors observed by NTP (2000, 196293) 

(incidence 0/50, 1/50, 2/50, 6/50) increased over control animals only at exposure levels above 

those that caused significant hyperplasia. The increased incidence of the forestomach neoplasms 

 69 
occurred in groups with ulceration and hyperplasia, suggesting a dose-dependent relationship 

between the nonneoplastic and the neoplastic lesions. 

4.6.3.2.3. Biological plausibility and coherence of the database. Both mutagenic and 

nonmutagenic chemicals have been shown to induce forestomach tumors in rodents (Ghanayem, 

1986, 011728; Ghanayem et al., 1993, 597328; 1994, 042760; Kroes and Wester, 1986, 041626; 

NTP, 2000, 196293). Some nonmutagenic substances that cause such tumors appear to require 

long-term contact with the forestomach epithelium, leading to irritation, cell proliferation, and 

neoplasia. The overstimulation of repair processes and enhancement of growth-promoting 

factors are believed to be involved (Harrison et al., 1991, 041622). Promotion and other 

activities associated with the stimulation of cell proliferation have been observed for many of 

these compounds (Clayson et al., 1991, 006215; Ghanayem et al., 1994, 042760). High 

concentrations of EGBE and its BAA metabolite sequestered in the forestomach are assumed to 

cause chronic irritation and the more serious damage observed in the forestomach lining of 

female mice. The incidence of ulcers was significantly increased in all exposed groups of 

females. NTP (2000, 196293) suggests that EGBE exposure-induced irritation caused 

inflammatory and hyperplastic effects in the forestomach and that the neoplasias (papillomas and 

one carcinoma) were associated with a continuation of the injury/degeneration process. 

Other substances that induce forestomach hyperplasia in male and female mice following 

inhalation exposure include acetonitrile, 1,3-butadiene, and chloroprene (U.S. EPA, 2005, 

594432). Both propionic and butyric acid have been shown to induce proliferative responses in 

forestomach epithelium after only 7 days, and long-term propionic acid exposure has produced 

papillomas in the rat forestomach (Kroes and Wester, 1986, 041626). Since high levels of EGBE 

and BAA have been observed in the stomachs of mice following i.v., i.p., gavage, and inhalation 

exposures, it is apparent that the chemical partitions to the forestomach via multiple routes, 

including grooming of fur, systemic blood circulation, ingestion of salivary excretions and 

respiratory tract mucus, and possibly repartitioning of the stomach contents (Green et al., 2002, 

041610; Poet et al., 2003, 100190). Because the forestomach functions as a storage organ, there 

is a reduced requirement for vascularization. The planar capillary network within the epithelial 

layers of the rodent forestomach contrasts strongly with the thick mucosal network of capillaries 

in the glandular stomach of rodents (Browning et al., 1983, 041615). The cells of the 

forestomach epithelium, especially the more superficial squamous cells, are separated from 

capillaries by substantial diffusion distances (Browning et al., 1983, 041615; Bueld and Netter, 

1993, 041617). In addition, the glandular stomach contains a complex mucosal protection and 

buffering system necessary to withstand the high acidity of the digestion process. As a result, 

irritant substances that concentrate in the forestomach may produce hyperplasia in the 

forestomach, but not in the glandular stomach or other gastrointestinal tissue (Kroes and Wester, 

1986, 041626). 

 70 
4.6.3.2.4. Relevance of the hypothesized MOA to humans. While this proposed MOA is 

thought to be of qualitative relevance to humans, the EGBE exposure concentrations that would 

be necessary to cause hyperplastic effects and tumors in humans, if attainable at all, are likely to 

be much higher than the concentrations necessary to cause forestomach effects in mice for 

several reasons: 

(1) The mouse forestomach serves a food storage function and the slow rate of emptying 

provides a sink for EGBE where it is metabolized to BAA and remains in contact with 

squamous epithelium long after EGBE has been cleared from the rest of the body. While 

the human esophagus is histologically similar to the murine forestomach, the contact time 

with food and other ingested substances is short, because this organ does not have a 

storage function. Thus, the risk for esophageal tumors is low (Kroes and Wester, 1986, 

041626). The human stomach also has a faster rate of emptying than the rodent 

forestomach, and is further protected from irritant compounds by a mucous layer that is 

not present in the rodent forestomach; 

(2) The localization of the enzymes needed for acid production in the human stomach tissue 

is not the same as it is in the target, the rodent forestomach (Green et al., 2002, 041610); 

(3) A benchmark dose (BMD) analysis (see Appendix C) indicates that the exposure 

concentrations necessary to cause hyperplastic effects in humans would be much higher 

than the existing RfD and RfC for EGBE. 

4.6.3.2.5. Other possible MOAs for forestomach tumor development in female mice. 

Though the evidence favors the hypothesis that BAA is the principal toxic metabolite of EGBE, 

roles for BAL (Ghanayem et al., 1987, 041608) and butyric acid (Harrison et al., 1991) have 

been suggested. It is not likely that butyric acid plays a significant role in the toxicity of EGBE, 

particularly at environmentally relevant concentrations. High concentrations of butyric acid have 

caused ulceration and other preneoplastic lesions in mice (Harrison, 1992, 041621). However, 

low concentrations of butyric acid do not appear to be harmful, since it naturally occurs in the 

diet through the fermentation of fiber and starch and as a significant portion (up to 10 mol%) of 

total bovine milk fatty acid (Smith and German, 1995, 042103). 

Another possible alternative MOA could exist if EGBE or one of its metabolites were to 

have the capability of damaging a cell through direct interaction with its DNA. As has been 

discussed in Section 4.4.4, there is very limited evidence that EGBE or BAA is genotoxic. BAL, 

a short-lived metabolite of EGBE, has been found to be clastogenic in in vitro assays without 

enzyme activation at concentrations ranging from 0.2 to 1 mM (Elliott and Ashby, 1997, 

100111). However, as has been discussed, in vivo and in vitro genotoxicity assays do not 

suggest that BAL would have any significant genotoxicity in vivo. In addition, chemicals for 

which mutagenesis/genotoxic effects play a significant role generally induce more tumors at 

 71 
earlier time points than near the end of the conducted bioassays due to their ability to both 

initiate and promote tumor pathogenesis. The mutagenic compound ethylene dibromide, for 

instance, was reported to induce forestomach tumors in all dose groups 168–280 days from the 

start of exposure (U.S. EPA, 2004, 594429). EGBE is consistent with other forestomach 

carcinogens that are not mutagenic, such as EA, in that observed tumors generally did not 

progress to carcinoma and were not observed until well into the study (i.e., after long periods of 

forestomach cell damage and repair). The first reported incidence of forestomach papilloma or 

carcinoma in female mice was 731, 731, and 582 days in the 62.5, 125, and 250 ppm EGBE 

exposure groups, respectively (NTP, 2000, 196293). 

It does not appear that EGBE, BAL, or BAA preferentially binds to stomach tissue 

macromolecules (Green et al., 2002, 041610; Poet et al., 2003, 100190). Poet et al. (2003, 

100190) found that high levels of EGBE concentrate in the food content of the forestomach 

following i.p. exposure (Poet et al., 2003, 100190), indicating that the observed sequestering of 

EGBE in the forestomach is related to its retention in the food that remains there, not to 

preferential binding to proteins within forestomach tissue. 

4.6.3.3. Conclusions about the Hypothesized MOAs

Inhalation exposure of B6C3F1 mice to EGBE gave rise to tumors in the liver and 

forestomach. The liver tumors, hemangiosarcomas, and hepatocellular carcinomas occurred in 

males only and were significantly elevated over controls with a positive trend test for the 

hemangiosarcomas. The forestomach tumors occurred in females only, had a positive trend test 

result, and were significantly increased over controls only at the highest dose. 

The MOAs that have been developed for these tumors reflect the evidence for the 

nonmutagenic nature of EGBE and its metabolites. For the liver tumors, the hypothesized key 

steps of the MOA are metabolism of EGBE to BAA, hemolysis of RBCs with release of Hb and 

hepatic hemosiderin accumulation, followed by oxidative stress, modulation of gene expression, 

cell proliferation, promotion, and neoplasm, leading to the formation of liver tumors. For the 

forestomach tumors, the hypothesized steps are metabolism to BAA, followed by tissue irritation 

and subsequent cytotoxicity, compensatory proliferation, and the induction of forestomach 

tumors. No other viable MOAs have been identified that adequately explain the existing 

laboratory animal and human observations. 

Both of these MOAs have some degree of qualitative significance for humans since the 

principal biological components supporting them are all present and the processes can occur in 

humans. Collectively, however, the evidence presented in this assessment for these MOAs 

suggests that both MOAs have only limited quantitative significance to humans, principally due 

to kinetic/dynamic differences from the rodents. In the case of the liver tumors, in vitro data 

suggest there is a 40- to 150-fold difference in the dose that produces hemolytic changes in the 

RBCs of humans as compared to rodents. This difference is supported by the Carpenter et al. 

 72 
(1956, 066464) study in which no changes in erythrocyte fragility were measured in humans at 

the highest tested concentration, 195 ppm, but increased erythrocyte fragility was measured in 

coexposed rats. Further, PBPK model simulations performed by Corley et al. (2005, 100100) 

predict that given the vapor pressure of EGBE, the maximum blood level of BAA that can be 

obtained from inhalation exposure would be lower than the predicted concentrations from bolus 

exposures that have not resulted in hemolytic effects, and lower than concentrations that have 

been shown to produce an effect on human RBCs in vitro (Udden, 2002, 042111). In the case of 

the forestomach tumors, the primary difference between mice and humans is in the degree of 

kinetics in the metabolizing enzymes involved in the production and clearance of BAA. Thus, 

the hypothesized key events in the MOAs for the animal tumors (liver and forestomach) are not 

likely to occur in humans, especially at low doses. 

Based on the preceding analysis, EGBE is deemed not likely to be carcinogenic to 

humans at the calculated RfC and RfD values presented in this document when examining it on 

its physical-chemical properties, toxicokinetic and dynamic factors, and MOA information. 

4.7. SUSCEPTIBLE POPULATIONS 

The hemolytic effect of EGBE is presumed to be caused by its primary metabolite, BAA, 

interacting with the RBC membrane. Potentially susceptible subpopulations would include 

individuals with enhanced metabolism or decreased excretion of BAA. As discussed in 

Section 4.7.1, older rats have reduced ability to metabolize the toxic metabolite BAA to CO2 and 

a diminished ability to excrete BAA in the urine (Ghanayem et al., 1987, 066470; Ghanayem et 

al., 1990, 042016). However, the relevance of this finding to the possible susceptibility of 

elderly humans is uncertain; as discussed in Section 3, humans may have conjugation pathways 

for the excretion of BAA, such as BAA-glutamine and BAA-glycine, which are not present in 

rats. 

It would also be expected that individuals whose RBC membranes are more susceptible 

to the lysis caused by BAA would be more sensitive to effects from EGBE exposure. However, 

RBCs from normal, aged, sickle-cell anemia, and hereditary spherocytosis patients were no more 

sensitive to the hemolytic effects of BAA than RBCs from healthy volunteers when tested in 

vitro (Udden, 1994, 042109). As work in this area continues, further information on the 

metabolic or structural differences that result in the lower sensitivity of human RBCs compared 

to rat RBCs may eventually identify characteristics in humans that may indicate increased 

susceptibility. For instance, it is unknown if a genetic predisposition to hemolytic anemia from 

other causes, such as G6PD deficiency, would lead to increased susceptibility to EGBE-induced 

hemolysis. G6PD deficiency appears in approximately 400 variants, thus describing a 

genetically heterogeneous disorder. The label has been applied to all types of moderate to severe 

enzyme deficiency with intermittent, induced hemolytic episodes with chronic hemolytic anemia. 

To date, complete deficiency of this enzyme has not been identified. The clinical result is the 

 73 
reduced ability to produce nicotinamide adenine dinucleotide phosphate, an enzyme required for 

reactions of various biosynthetic pathways, as well as for the stability of catalase and the 

maintenance of GSH levels. Catalase and glutathione peroxidase are the primary enzymes in the 

detoxification of hydrogen peroxide. Thus, cells are dependent on G6PD for this pathway; 

without it, they are vulnerable to oxidative damage. RBCs are sensitive to this loss of enzyme: 

they rely on this system for their antioxidant defenses. Other human risk factors for anemia 

include ingestion of certain therapeutic drugs, infections, family history, diet, and systemic 

illnesses (Berliner et al., 1995, 001232). 

Individuals with hereditary hemochromatosis (HH) represent a population potentially 

susceptible to increased release of iron from any source. It is reported that 5/1,000 persons of 

northern European descent are homozygous for the gene or genes that cause hemochromatosis, 

although it is unknown what proportion of this population will go on to develop the HH 

phenotype (Pietrangelo, 2004, 100189). Individuals with this disorder are not able to reduce 

their absorption of iron in response to increasing iron levels in the body. Iron stores in the body 

continue to increase. The iron stores normally start out as ferritin, then eventually become 

aggregates of a breakdown product of ferritin called hemosiderin. HH is a condition 

characterized by excessive iron deposition in the form of hemosiderin, found in the liver, heart, 

skin, joints, pancreas, and other endocrine organs. It is unknown whether individuals with this 

condition would be susceptible to the effects of EGBE exposure. There is no indication in the 

literature, however, that RBCs in individuals with HH are more fragile, and it is therefore 

unlikely that HH would increase the risk of hemolysis or additional hemosiderin deposition from 

EGBE exposure. Studies have shown differences in the localization of iron between HH patients 

and rodents. In HH patients, iron appears to accumulate preferentially in the parenchymal cells 

of the liver, early on as ferritin and later as hemosiderin, due to increased iron absorption from 

the duodenum; late in the disease, iron storage is seen in Kupffer cells and reticular endothelial 

cells of the bone marrow (Knutson and Wessling-Resnick, 2003, 094393; Valberg et al., 1975, 

100217). In contrast, EGBE-induced toxicity in mice and rats results in initial and preferential 

accumulation of hemosiderin in Kupffer cells by phagocytosing senescent RBCs (NTP, 2000, 

196293). While it is clear that macrophages and other cells can in fact contain hemosiderin, the 

relative level compared to hepatocytes is much less; staining in these cells is typically seen in 

late stages of the disease (Kwittken and Tartow, 1966, 597334). The human course of 

developing hepatocellular carcinomas as a consequence of HH is also quite different compared 

to the development in the mouse model of hemangiosarcoma. Human cases of HH-induced 

hepatocellular carcinoma are typically observed in the presence of cirrhosis of the liver, another 

long-term process that reflects the chronic nature of the disease progression in humans (Harrison 

and Bacon, 2005, 100144). Hemangiosarcomas, the tumor type of concern in the male mice, 

have not been associated with HH in the literature. 

 74 
4.7.1. Possible Childhood Susceptibility 

A number of factors may differentially affect children’s responses to toxicants. The only 

human toxicity information available on the toxicity of EGBE to children is from the case study 

by Dean and Krenzelok (1991, 597279). They observed 24 children, aged 7 months to 9 years, 

after oral ingestion of at least 5 mL of glass window cleaner containing EGBE in the 0.5–9.9% 

range (potentially 25–1,500 mg EGBE exposures). Two children who had taken >15 mL did 

well after gastric emptying or lavage and observation in the hospital. The remainder were 

watched at home after receiving diluting oral fluids. No symptoms of EGBE poisoning or 

hemolysis were observed. While the effects reported in adult poisonings have been more severe 

than those reported in these children, the adults tended to consume larger volumes and different 

concentrations of EGBE, making it extremely difficult to assess the correlation of toxic effects 

with age sensitivity. 

As discussed above, there are numerous risk factors for anemia that might predispose an 

individual to, or compound the effects of, EGBE-induced hemolysis. It is generally recognized, 

however, that children do not share the same risk factors for anemia as adults for a number of 

reasons, including: (1) a higher rate of RBC turnover; (2) lower incidence of neoplastic disease 

in childhood as either a direct or indirect cause of anemia (<7,000 of the 1,000,000 new cases of 

cancer each year in the U.S. occur in individuals <15 years of age); (3) the fact that iron 

deficiency is almost always secondary to nutritional factors in children; (4) the relative rarity of 

alcoholism and its related liver disease; (5) a much lower incidence of anemia associated with 

thyroid disease; and (6) a rarity of cardiovascular disease other than congenital heart diseases, 

with the result that valve replacement, malignant hypertension, and the use of certain drugs are 

not usually a factor (Berliner et al., 1995, 001232; Hord and Lukens, 1999, 594445). 

Anemia in children is usually associated with an abnormality of the hematopoietic system 

(Berliner et al., 1995, 001232; Hord and Lukens, 1999, 594445). Studies of the osmotic fragility 

and deformability of RBCs exposed to BAA, the toxic metabolite of EGBE (Udden, 1994, 

042109), suggest that certain patients with abnormal hematopoietic systems, such as sickle-cell 

anemia or hereditary spherocytosis, are not more sensitive to the hemolytic effects of EGBE than 

normal adults. Other studies suggest that the RBCs of children may be pharmacodynamically 

less sensitive to hemolysis than those of adults. RBCs of neonates and children up to 6 months 

of age differ from normal adult RBCs in that they are larger and have higher levels of Hb F 

versus adult Hb A (Lewis, 1970, 594441). Frei et al. (1963, 100118) showed that the larger calf 

erythrocytes containing Hb F were osmotically more resistant than smaller, adult erythrocytes 

containing Hb A and suggested that, as fetal erythrocytes are replaced by postnatal erythrocytes, 

the total population of RBCs becomes more susceptible to lysis. 

The effect of age on EGBE-induced hematotoxicity was studied in male F344 rats by 

Ghanayem et al. (1987, 066470; 1990, 042016). These studies also demonstrated the time course 

for the onset and resolution of the hematological and histopathologic changes accompanying 

 75 
hemolysis. Adult male F344 rats were significantly more sensitive to the hemolytic effects of 

EGBE than were young (4–5 week) male rats following administration of a single gavage dose 

of EGBE. Concurrent metabolism studies found increased blood retention of EGBE metabolite 

BAA (as measured by increased Cmax, AUC, and t1⁄2) in young rats and that these rats eliminated a 

significantly greater proportion of the administered EGBE dose as exhaled CO2 or as urinary 

metabolites. The rats also excreted a greater proportion of the EGBE conjugates, glucuronide 

and sulfate, in the urine. These researchers suggested that a reduced ability of older rats to 

metabolize the toxic metabolite BAA to CO2 along with a diminished ability to excrete BAA in 

the urine may explain the age-dependent toxicity of EGBE. 

NTP (2000, 196293) also found that young mice eliminated BAA 10 times faster than 

aged mice following a 1-day exposure to EGBE. This difference was not as apparent after 3 

weeks of exposure, suggesting that factors other than age may be involved (Dill et al., 1998, 

041981). 

Available in vitro information suggests that children are no more and are possibly less 

sensitive to the hemolytic effects of BAA than adults. Udden (2002, 042111) compared the in 

vitro responses of erythrocytes (percent hemolysis and MCV alterations) obtained from 

hospitalized adults (n = 29–40) and hospitalized children (n = 25–46) to 0 or 10 mM BAA for 4 

hours. BAA (10 mM) produced comparable significant increases in MCV in both adults (from 

87.1 to 89.2 μm3

; 2%) and children (92.8–95.2 μm3; 3%). In the case of hemolysis in response to 

BAA, the response was noted as being significantly increased for hospitalized adults (0.54–

0.69%; 28%) but not for hospitalized children (0.68–0.75%; 10%). 

Relatively minor developmental effects due to maternal toxicity related to hematologic 

effects of EGBE exposure were found in studies using rats, mice, and rabbits dosed orally, by 

inhalation, or dermally (Hardin et al., 1984, 042030; Heindel et al., 1990, 042042; NTP, 1993, 

042063; Sleet et al., 1989, 042089; Tyl et al., 1984, 100209; Wier et al., 1987, 042123). No 

teratogenicity was noted in any studies. It can be concluded that EGBE is not significantly toxic 

to developing fetuses of laboratory animals. 

4.7.2. Possible Gender Differences 

Gender differences have been noted in a number of animal and human studies: females 

were more susceptible to effects from EGBE exposure. In the NTP (1993, 042063) 2-week 

drinking water studies with EGBE, the absolute and relative thymus weights in female F344 rats 

at the highest exposure level (265 mg/kg-day) were slightly reduced. In the 13-week studies, 

male rats in the highest three dose groups and females in all dose groups suffered mild (males) to 

moderate (females) anemia. 

Gender differences have also been noted in some studies that observed the hemotoxic 

effects of dermal administration of EGBE. Repeated application of EGBE either neat or as a 

dilute aqueous solution (occluded) to male or female NZW rabbits at exposure levels of 18, 90, 

 76 
180, or 360 mg/kg (6 hours/day, nine applications) produced hemoglobinuria in males at 

360 mg/kg and in females at 180 or 360 mg/kg (Tyler, 1984, 006769). Only female rabbits 

showed decreased RBC counts, Hb concentrations, and MCHC along with increased MCH at the 

highest treatment level. Recovery was noted following a 14-day observation period. 

A number of secondary effects resulting from the hemolytic toxicity of EGBE, such as 

effects on the rat liver, kidneys, spleen, bone marrow, and, to a lesser extent, the thymus, were 

more pronounced in females. In the NTP drinking water studies (1993, 042063), liver lesions in 

females, but not males, included cytoplasmic alterations, hepatocellular degeneration, and 

pigmentation. These effects were most pronounced in the three highest dose groups. 

Carpenter et al. (1956, 066464) reported female rats to be more sensitive than males to 

the hemolytic effects of EGBE. In dogs, slight increases in erythrocyte osmotic fragility in both 

the male and female basenji hybrids were seen, but RBC counts and Hb concentrations were 

slightly decreased in the female. Overall, the effects were seen in both genders, but appeared to 

develop more slowly in the males. In monkeys, occasional rises in erythrocyte osmotic fragility 

were recorded during the exposure period more frequently in the female than in the male. 

In the process of studying and comparing the metabolic and cellular basis of EGBEinduced hemolysis of rat erythrocytes in vitro with human erythrocytes, Ghanayem (1989, 

042014) observed that the blood from male and female human volunteers was unaffected by 4-

hour incubations with BAA at concentrations of up to 4.0 mM. At 8 mM, only slight but 

significant hemolysis of human blood was observed, with blood from females showing a slightly 

greater sensitivity. 

The NTP 2-year inhalation bioassay (Dill et al., 1998, 041981; NTP, 2000, 196293) also 

reported evidence of gender specificity in mice and rats, particularly with respect to the 

elimination of BAA in rats. Female rats eliminated BAA more slowly from the blood, as 

indicated by a smaller elimination rate constant, longer elimination t1⁄2, and larger AUC. In 

addition, the Cmax of BAA was greater for females at each concentration and time point. It has 

been suggested because a smaller amount of BAA was excreted in the urine of female rats, that 

higher blood concentrations of BAA accumulated in the females (Dill et al., 1998, 041981). 

Mouse data from the NTP (2000, 196293) study also suggest a slightly increased hematologic 

effect among female mice; however, while female mice tended to have higher blood 

concentrations of BAA, they excreted more BAA in urine than male mice. 

 77 
5. DOSE-RESPONSE ASSESSMENTS 

5.1. INHALATION REFERENCE CONCENTRATION (RFC) 

In general, the RfC is an estimate, with uncertainty spanning perhaps an order of 

magnitude, of a daily exposure to the human population—including susceptible subgroups—that 

is likely to be without an appreciable risk of adverse health effects over a lifetime. It is derived 

from a lower confidence limit on the BMD, a NOAEL, a LOAEL, or another suitable point of 

departure (POD), with uncertainty/variability factors applied to reflect limitations of the data 

used. The RfC is expressed in terms of mg/m3

 of exposure to an agent and is derived by a 

methodology similar to the RfD. Ideally, studies with the greatest duration of exposure and 

conducted via the inhalation route of exposure give the most confidence for derivation of an RfC. 

5.1.1. Choice of Principal Study and Critical Effect—with Rationale and Justification 

Only one human occupational exposure study to low levels of EGBE is available, which 

did not observe changes outside the normal clinical ranges in hepatic, renal, or hematologic 

parameters (Haufroid et al., 1997, 042040) (Section 4.1). The animal studies considered for 

selection as principal studies include the 14-week and 2-year inhalation studies by NTP (2000, 

196293) in rats and mice, the developmental toxicity study by Tyl et al. (1984, 100209) in rats 

and rabbits, the developmental toxicity study by Nelson et al. (1984, 031878) in rats, and the 

subchronic study by Dodd et al. (1983, 066465) in rats. The NTP (2000, 196293) study was 

selected as the principal study because it was conducted in two species and provides data for 

different durations and for more dose groups than the other studies. The developmental toxicity 

studies identified effects at doses higher than the doses associated with the critical effects 

identified in the NTP (2000, 196293) study and were not used for quantitative purposes. While 

the subchronic study by Dodd et al. (1983, 066465) was well-conducted, the NTP (2000, 

196293) study contained more dose groups, more animals per group, and a longer duration of 

exposure. Thus, Dodd et al. (1983, 066465) was not used for quantitative purposes. Two 

endpoints from the NTP (2000, 196293) study—the hemolytic endpoint from the 14-week 

inhalation study and the hemosiderin deposition endpoint from the 2-year inhalation study—

were used for the critical effect. The hemolytic endpoints in the 1999 EGBE Toxicological 

Review were used to derive the reference values (see Section 5.1.5), but were not used to derive 

the values in this updated assessment. New MOA information published since the 1999 EGBE 

Toxicological Review is included in this document, and this information supports the 

 

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). 

 78 
hemosiderin deposition endpoint as an important key event in the proposed MOA. A 

comparison of the NOAELs and LOAELs for the candidate studies are summarized in Table 5-1. 

Table 5-1. Results of candidate studies 

Species Effect levels (ppm) 

(strain) Gender 

Number/

Reference dose group Duration/effect NOAEL LOAEL 

9–10 14 wk, hematologic 31 62.5 NTP (2000, 

M 

50 2 yr, hematologic, 

hemosiderin (liver) – 31 

9–10 14 wk, hematologic – 31 

Rat (F344) 

F 

50 2 yr, hematologic, 

hemosiderin (liver) 

196293) 

– 31 

50 2 yr, histopathology of the 

forestomach – 62.5 NTP (2000, 

M 

50 2 yr, hematologic, 

hemosiderin (liver) 62.5 125 

50 2 yr, histopathology of the 

forestomach – 62.5 

Mouse 

(B6C3F1) 

F 

50 2 yr, hematologic, 

hemosiderin (liver) 

196293) 

– 62.5 

Tyl et al. (1984, 

100209) 

Rat (F344) F 36 GD 6–15, hematologic 50 100 

Nelson et al. 

(1984, 031878) 

Rat (SpragueDawley) F 15 GD 7–15, hematologic 150 200 

Dodd et al. 

(1983, 066465) 

Rat (F344) M, F 16 13 wk, hematologic 25 77 

The primary effects of EGBE exposure were hematological effects and were observed in 

both species and genders tested. Female rats (NTP, 2000, 196293) appeared to be most sensitive 

among animals studied. A mild-to-moderate regenerative anemia was observed in females 

exposed to all concentrations, with a LOAEL of 31 ppm identified for hematological effects in 

male and female rats and no NOAEL. Exposure-related trends were noted for reticulocyte count, 

RBC count, MCV, Hb concentration, and Hct. The hematological endpoints were considered for 

the derivation of the RfC; however, they presented a number of difficulties. It was not clear 

which of the hematological endpoints (changes in RBC count, reticulocyte count, MCV, Hb 

concentration, and Hct) observed in EGBE-exposed animals should be used to derive an RfC. In 

the case of BMD analysis, the proper benchmark response (BMR) level for the BMD derivation 

was uncertain. In addition, while these hematologic effects were observed in both the subchronic 

and chronic studies and persisted with exposure duration, they did not progress in severity in the 

subchronic-to-chronic study (see Tables 4-3, 4-6, and 5-2). Further, better model fits were 

obtained from the BMD analysis of the subchronic study, which used two more exposure 

 79 
concentrations than the chronic study. For these reasons, the hematologic responses from the 

14-week subchronic study were chosen for use in the BMD analyses of this endpoint (see 

Appendix C). Selection of the most appropriate hematologic endpoints for use in the BMD 

analysis also required consideration of EGBE’s MOA for hemolysis. 

Table 5-2. Female and male rat and mouse liver hemosiderin staining 

incidence and RBC counts from subchronic and chronic EGBE inhalation 

studies 

Effect/time Control 31 ppm 62.5 ppm 125 ppm 250 ppm 500 ppm 

Female rat 

Hemosiderin 

14 wk 

2 yr 

0/10 

15/50 

0/10 

19/50 

10/10c

36/50b

10/10c

47/50c

9/9c

NT 

5/5c

NT 

RBC counta

14 wk 

1 yr 

8.48 ± 0.05 

7.81 ± 0.05 

7.70 ± 0.08c 8.08 ± 0.07 (91) c

 (95) 

NT 7.42 ± 0.06c

 (95) 

6.91 ± 0.05c

 (82)

6.75 ± 0.05c

 (86)

6.07 ± 0.04c

 (72) 

NT 

4.77 ± 0.15c

 (56)

NT 

Male rat 

Hemosiderin 

14 wk 

2 yr 

0/10 

23/50 

0/10 

30/50 

0/10 

34/50b

7/10c

42/50b

10/10c

NT 

10/10c

NT 

RBC counta

14 wk 

1 yr 

9.05 ± 0.08 

8.88 ± 0.08 

8.71 ± 0.14b

 (96) 

NT 

8.91 ± 0.06 (94) 8.01 ± 0.08c

 (89)

8.39 ± 0.15c

 (94) 7.43 ± 0.20c (84)

7.10 ± 0.07c (78) 

NT 

5.97 ± 0.05c

 (66)

NT 

Female mouse 

Hemosiderin 

14 wk 

2 yr 

0/10 

0/50 

0/10 

NT 

0/10 

5/50c 

0/10 

25/49a 

10/10a

44/50a

6/6a

NT 

RBC counta

14 wk 

1 yr 

9.72 ± 0.05 

9.32 ± 0.09 

9.55 ± 0.06b

 (98) 

NT 

9.51 ± 0.06b

 (98) 9.18 ± 0.05c (94)

9.14 ± 0.08 (98) 8.50 ± 0.12c

 (91)

8.57 ± 0.06c

 (88) 

8.08 ± 0.09c

 (87) 

7.35 ± 0.07c

 (76)

NT 

Male mouse 

Hemosiderin 

14 wk 

2 yr 

0/10 

0/50 

0/10 

NT 

0/10 

0/50 

0/10 

8/49a

0/10 

30/49a

6/6a

NT 

RBC counta

14 wk 

1 yr 

9.71 ± 0.22 

9.58 ± 0.07 

10.04 ± 0.08 

(103) 

NT 

9.77 ± 0.10 (101) 

9.73 ± 0.49 (102) 

9.47 ± 0.06b

 (98)

9.36 ± 0.32b

 (98)

8.90 ± 0.07c

 (92) 

8.33 ± 0.10c

 (87) 

7.21 ± 0.23c

 (74)

NT 

a

Mean ± standard error with RBC counts expressed as106/μL; percent of control in parentheses. 

b

Statistically significant difference, p ≤ 0.05. c

Statistically significant difference, p ≤ 0.01. 

NT = not tested 

Source: NTP (2000, 196293). 

The suggested MOA of EGBE hemolysis is based on data indicating that BAA, an 

oxidative metabolite of EGBE and the first hypothesized event in the MOA, is likely to be the 

 80 
causative agent in hemolysis (Carpenter et al., 1956, 066464; Ghanayem et al., 1987, 041608; 

Ghanayem et al., 1990, 042016). The second event in the MOA is erythrocyte swelling and cell 

lysis, which is believed to be preceded by an increase in the osmotic fragility and a loss of 

deformability of the erythrocyte (Ghanayem, 1989, 042014; Udden, 1994, 042109; Udden, 1996, 

042209; Udden and Patton, 1994, 056374). This results in decreased values for RBC count, Hb, 

and Hct and in response, an increase in the production of immature RBCs (reticulocytes) by the 

bone marrow. 

Although changes in reticulocyte and nucleated erythrocyte counts sometimes represent 

the largest measurable differences between exposed animals and unexposed control animals, this 

parameter is highly variable and does not always exhibit a dose-dependent trend (NTP, 1993, 

042063; 2000, 196293). While these endpoints can be indirect markers of RBC lysis, they are 

governed by multiple feedback control processes that can be both very sensitive and variable. 

Therefore, a change in reticulocyte or nucleated erythrocyte count is not considered a suitable 

endpoint for deriving the RfC or RfD. 

Until more is known about the molecular interaction between BAA and specific cellular 

molecules, it is reasonable to assume that changes in MCV and RBC count are measurements of 

precursor events in response to both oral and inhalation EGBE exposure. Therefore, doseresponse information on MCV and RBC count are key endpoints used in the BMD analyses and 

were considered for derivation of the RfC and RfD for EGBE. 

While the toxicokinetic data suggest that MCV should theoretically be the earlier 

indicator of hemolytic effects from EGBE exposure, recent studies suggest that the relationship 

between the rate of MCV increase and RBC count decrease may not be consistent across 

exposure protocols. In the gavage studies of Ghanayem et al. (1987, 066470) and the inhalation 

studies of NTP (2000, 196293), Hct, a measure of RBC volume relative to blood volume, tended 

to decrease along with RBC count and Hb at all exposure levels for which a hematologic effect 

was observed. However, Hct did not change as RBC count and Hb decreased following drinking 

water exposures (NTP, 1993, 042063). Thus, the loss of erythrocytes in the drinking water 

studies (reduced RBC count) may have been offset by a concurrent increase in the size of the 

individual cells (increased MCV). This was not the case in the gavage and inhalation studies. 

For these reasons, greater weight is given to reduced RBC count, as opposed to increased MCV. 

While the hemolytic effects appeared to be among the earliest effects from EGBE 

exposure, the hemosiderin deposition endpoint was selected as the critical effect. This effect was 

found to occur in both species and genders of animals tested, with rats being the more sensitive 

species; the effect also occurred in the 14-week subchronic NTP inhalation study. The suggested 

MOA of EGBE-induced liver effects is based on the observation that the hemolytic effects led to 

compensatory erythropoiesis and significant increases in blood degradation products, including 

an increased accumulation of hemosiderin in the liver Kupffer cells of EGBE-exposed animals. 

The hemosiderin accumulation seen in the Kupffer cells was found to increase in severity with 

 81 
increasing dose and exposure duration (Table 5-2), unlike the hemolytic endpoints, such as 

decreased Hct, which did not progress from 3 to 12 months (Table 4-6). Thus, hemosiderin 

deposition in Kupffer cells in the rat liver is believed to be a sequela to the hematologic effects. 

Because of the progression of this effect with chronic exposure, hemosiderin is deemed to be the 

most sensitive effect. A NOAEL was not identified, while a LOAEL of 31 ppm was identified in 

both male and female rats. 

The 2-year chronic inhalation study by the NTP (2000, 196293) observed forestomach 

ulcers in female mice at all exposure levels, but this effect has not been observed in any other 

species, including mice exposed orally to EGBE (NTP, 1993, 042063). Though the incidence of 

this lesion increased with exposure, severity of the lesion did not increase with increasing dose. 

While this effect was not considered a critical effect for the derivation of an RfC, Appendix C 

contains the BMD analysis for this endpoint for comparison purposes. 

5.1.2. Methods of Analysis—Including Models (PBPK, BMD, etc.) 

PODs for the RfC derivation in terms of the human equivalent concentrations (HECs) 

have been calculated via the application of PBPK modeling and the use of internal dose metrics 

published by Dill et al. (1998, 041981) to obtain NOAEL and benchmark concentration, 95% 

lower bound (BMCL) estimates. Details of the various POD derivation approaches that were 

considered are provided in Appendix C. The selected approach is described in Section 5.1.2.1 

below. 

The PBPK models developed for EGBE are briefly summarized in Table 5-3. Shyr et al. 

(1993, 006766) and Johanson (1986, 100159) do not address BAA distribution and are only 

parameterized for humans and rats, respectively. In the 1999 EGBE Toxicological Review, the 

model described by Lee et al. (1998, 041983) was considered to be the most appropriate model 

for the estimation of rat and mouse internal doses following inhalation exposure. Since the 1999 

Toxicological Review (U.S. EPA, 1999, 597365), Corley et al. (2005, 100100) published a 

revision to the Lee et al. (1998, 041983) model for rats and mice where several assumptions used 

by Lee et al. (1998, 041983) were replaced with measured values (e.g., protein binding, partition 

coefficients, metabolism rate constants for multiple pathways, and renal clearance) as a function 

of species, gender, and age. As is described in Appendix C, that model was used to estimate the 

Cmax of BAA in rat blood for the derivation of reference values from hematologic endpoints, and 

the human PBPK model of Corley et al. (1994, 041977; 1997, 041984) was used to obtain 

estimates of human inhalation exposure concentrations associated with the rat BAA Cmax

estimates. In the analysis of the hemosiderin effect in rats and mice described in Section 5.1.2.1 

below, the human PBPK model of Corley et al. (1994, 041977; 1997, 041984) was used to obtain 

estimates of human inhalation exposure concentrations associated with the BMDs derived from 

 82 
rat BAA AUC levels reported by Dill et al. (1998, 041981).1

 Established U.S. EPA (2006, 

194568) methods and procedures were used to review, select and apply these chosen PBPK 

models.2

Table 5-3. Summary of PBPK models 

Model Species Routes of exposure Comments 

Johanson (1986, 006758) Human Inhalation BAA not addressed 

Shyr et al. (1993, 006766) Rat Inhalation, oral, dermal BAA excretion 

Corley et al. (1994, 041977; 

1997, 041984) 

Rat and human Inhalation, oral, dermal BAA distribution and excretion; male 

rats only 

Lee et al. (1998, 041983) Rat and mouse Inhalation BAA distribution and excretion; males 

and females 

Corley et al. (2005, 100100) Rat and mouse Inhalation, oral, dermal, 

i.p., i.v. 

Age-dependent BAA distribution, 

metabolism and excretion, males and 

females 

Franks et al. (2006, 100117) Human Inhalation and dermal Extended Corley et al. (1997, 041984) 

model to include bladder compartment 

for human biomonitoring studies 

5.1.2.1. BMD Approach Applied to Hemosiderin Staining Data 

For the purposes of deriving an RfC for EGBE, hemosiderin staining data were evaluated 

in male and female rats from the 2-year chronic study by NTP (2000, 196293). The current 

BMD technical guidelines (U.S. EPA, 2000, 052150) suggest the use of 10% extra risk as a 

BMR level for quantal data, as this is at or near the limit of sensitivity in most cancer bioassays 

and in some noncancer bioassays as well. Because the hemosiderin staining endpoint was 

observed in control animals and a 10% increase in incidence was within the observable range of 

the data, 10% extra risk was considered an appropriate BMR and a BMCL10 an appropriate POD 

for derivation of the RfC (1995, 005992; 2000, 052150). All BMD assessments in this review 

were performed using U.S. EPA benchmark dose software (BMDS) version 1.4.1. Graphical 

figures and text output files for selected benchmark concentration (BMC) analyses are provided 

in Appendix C. 

The AUC was selected as the appropriate dose metric due to the nature of the endpoint, 

hemosiderin deposition. This endpoint increased in severity with increased duration (subchronic 

to chronic) and is believed to be the result of the cumulative exposure to EGBE as opposed to a 

peak event. Table 5-4 reports AUC BAA blood concentrations measured at 12 months3

published by Dill et al. (1998, 041981) in both genders of B6C3F1 mice and F344 rats exposed to 

the same concentrations used in the NTP (2000, 196293) chronic studies of these test animals. 

 

1

The basic components of the Corley model are summarized in Appendix B. 

2

EPA notes that a review of the PBPK models was conducted prior to their use in the 1999 EGBE toxicological review. 

3

Dill et al. (1998, 041981) also reported 18 month data, but due to the smaller number of animals and higher variability in this data, the 12 

month data were used for the purposes of this analysis. 

 83 
Table 5-4. AUC BAA blood concentrations measured at 12 months in both 

genders of B6C3F1 mice and F344 rats 

Rats AUCBAA (μmol-hr/L)a

Exposure concentration (ppm) Gender n Mean SE 

Male 7 358.3 16.6 31.2 

Female 5 638.8 18.7 

Male 6 973.0 86.2 62.5 

Female 9 1,128.9 50.9 

Male 9 2,225.6 71.1 125 

Female 12 3,461.8 154.8 

Mice AUCBAA (μmol-hr/L)a

Exposure concentration (ppm) Gender n Mean SE 

Male 10 1,206.6 205.6 62.5 

Female 12 1,863.6 112.4 

Male 9 2,819.8 685.1 125 

Female 6 5,451.6 508.9 

Male 10 17,951.5 1,770.4 250 

Female 11 18,297.1 609.7 

a

Authors reported AUC values in terms of μg/min and g, which were converted to units consistent with the PBPK 

model of μmol-hour/L by dividing by 60 minute/hour and 132.16 g/mol and multiplying by 1,060 g/L. 

Source: Dill et al. (1998, 041981). 

The fit statistics and BMC information derived from the dichotomous models available in 

the BMD software as applied to the male and female rat hemosiderin staining data versus AUC 

BAA are shown in Table 5-5. All models were fit using restrictions and option settings suggested 

in the U.S. EPA BMD technical guidance document (U.S. EPA, 2000, 052150). The best model 

fit to these data, as determined by visual inspection, examination of low dose model fit 

(i.e., scaled residual for the dose group closest to the BMD), and comparison of overall fit 

(i.e., Akaike information criterion [AIC] values), was obtained using a multistage model (1st 

degree) for the male response data and a Log-Logistic model for the female response data. The 

male rat BMC10 was 196 μmol-hour/L and the BMCL10 was determined to be 133 μmol-hour/L, 

using the 95% lower confidence limit of the dose-response curve expressed in terms of the AUC 

for BAA in blood. The BMC10 and BMCL10 values for the female rat were determined to be 425 

and 244 μmol-hour/L, respectively. Assuming continuous exposure (24 hour/day), the Corley 

et al. (1997, 041984) PBPK model was used to back-calculate HECs of 3.4 ppm (16 mg/m3

) 

from the male rat data and 4.9 ppm (24 mg/m3

) from the female rat data. 

 84 
Table 5-5. Comparison of BMC/BMCL values for male and female rat liver 

hemosiderin staining data from inhalation chronic study using measured 

blood AUC (12 months) of the EGBE metabolite BAA as a common dose 

metric 

Model BMC10 (μmol-hr/L) BMCL10 (μmol-hr/L) p-Value AICa Scaled residualb

Male rats 

Multistage-1st degreec 196.252 133.141 0.8680 247.234 0.441 

Gammac 196.253 133.141 0.8680 247.234 0.441 

Logistic 259.296 192.773 0.7692 247.476 0.526 

Log-logistic 166.376 69.3279 0.5623 249.283 0.313 

Probit 271.525 205.882 0.7450 247.54 0.517 

Log-probit 368.336 241.992 0.6309 247.876 0.765 

Weibullc 196.253 133.141 0.8680 247.234 0.441 

Female rats 

Multistage-1st degree 122.166 214.555 0.0698 218.868 -1.945 

Gamma 316.635 134.02 0.0554 219.229 -1.238 

Logistic 273.693 221.689 0.0993 218.188 -1.294 

Log-logisticc 424.527 243.69 0.1533 217.526 -0.896 

Probit 291.017 241.206 0.0683 218.985 -1.260 

Log-probit 427.728 248.683 0.1238 217.884 -0.965 

Weibull 266.515 130.801 0.0454 219.58 -1.377 

a

AIC = -2L + 2P, where L is the log-likelihood at the maximum likelihood estimates for the parameters and P is the 

number of model degrees of freedom (usually the number of parameters estimated). 

b

χ2 residual (measure of how model predicted response deviates from the actual data) for the dose group closest to 

the BMC scaled by an estimate of its SD. Provides a comparative measure of model fit near the BMC. Residuals 

that exceed 2 in absolute value should cause one to question model fit in this region. 

c

Model choice based on adequate p-value (>0.1), visual inspection, low AIC, and low (absolute) scaled residual. 

The Multistage (1st degree) is referred to as the chosen model for male rats, though equivalent fit was obtained by 

the restricted Gamma and Weibull models. 

Likewise, the fit statistics and BMC information for male and female mouse hemosiderin 

staining data versus AUC BAA are shown in Table 5-6. All models were fit using restrictions 

and option settings suggested in the U.S. EPA BMD technical guidance document (U.S. EPA, 

2000, 052150). The best model fit to these data, as determined by visual inspection, examination 

of low dose model fit (i.e., scaled residual for the dose group closest to the BMD), and 

comparison of overall fit (i.e., AIC values), was obtained using a log-probit model for both the 

male and female response data. The male mouse BMC10 was 3,077 μmol-hour/L and the 

BMCL10 was determined to be 2,448 μmol-hour/L using the 95% lower confidence limit of the 

dose-response curve expressed in terms of the AUC for BAA in blood. The BMC10 and BMCL10

values for the female mouse were determined to be 1,735 and 1,322 μmol-hour/L, respectively. 

Assuming continuous exposure (24 hour/day), the Corley et al. (1997, 041984) PBPK model 

described in Appendix B was used to back-calculate HECs of 36 ppm (174 mg/m3) from the 

male mouse data and 20 ppm (97 mg/m3

) from the female mouse data. 

 85 
Table 5-6. Comparison of BMC/BMCL values for male and female mouse 

liver hemosiderin staining data from inhalation chronic study using 

measured blood AUC (12 months) of the EGBE metabolite BAA as a 

common dose metric 

BMC10 

(μmol-hr/L) 

BMCL10 

(μmol-hr/L) p-Value AICa Scaled residual Model b

Male mice 

Multistage-1st 

degree 

2,100.07 1,613.9 0.3067 117.571 -1.766 

Gamma 2,725.35 1,702.27 0.1452 118.559 1.358 

Logistic 6,605.45 5,333.72 0.0022 127.326 2.789 

Log-logistic 2,616.51 1,628.48 0.1882 118.02 1.193 

Probit 5,917.06 4,825.09 0.0031 126.405 2.734 

Log-probitc 3,076.8 2,448.3 0.1290 116.614 1.946 

Weibull 2,689.76 1,687.09 0.1445 118.712 -1.448 

Female mice 

Multistage-1st 

degree 

946.491 769.879 0.3680 142.669 -1.583 

Gamma 1,402.92 818.367 0.3420 143.288 -0.817 

Logistic 2,897.15 2,341.03 0.0002 162.338 -0.942 

Log-logistic 1,705.75 1,121.43 0.8223 141.501 -0.343 

Probit 2,860.03 2,364.52 0.0002 161.681 -0.829 

Log-probitc 1,734.53 1,322.06 0.8237 141.498 -0.315 

Weibull 1,282.82 804.234 0.2958 143.631 -0.988 

a

AIC = -2L + 2P, where L is the log-likelihood at the maximum likelihood estimates for the parameters and P is the 

number of model degrees of freedom (usually the number of parameters estimated). 

b

χ2 residual (measure of how model predicted response deviates from the actual data) for the dose group closest to 

the BMC scaled by an estimate of its SD. Provides a comparative measure of model fit near the BMC. Residuals 

that exceed 2 in absolute value should cause one to question model fit in this region. 

c

Model choice based on adequate p-value (>0.1), visual inspection, low AIC, and low (absolute) scaled residual. 

The Log-probit model provides a slightly better fit than other models for both genders. 

5.1.2.2. Selection of the POD 

Consideration of the available data has led to the selection of the 2-year inhalation study 

(NTP, 2000, 196293) and increased hemosiderin staining in the liver of male F344 rats as the 

principal study and critical effect for deriving the chronic RfC for EGBE. This is a high-quality 

study and, when coupled with information on the MOA, U.S. EPA concluded that this is a 

precursor to an adverse effect and is appropriate for use in deriving the RfC. A BMCL10 of 133 

μmol-hour/L for hemosiderin staining in liver of male rats chronically exposed to EGBE (NTP, 

2000, 196293) was used as the POD to calculate the RfC. A human PBPK model (Corley et al., 

1997, 041984) was used to back-calculate to a HEC of 16 mg/m3

 (3.4 ppm) for the BMCLHEC. 

 86 
5.1.3. RfC Derivation—Including Application of Uncertainty Factors 

Uncertainty factors (UFs) are applied to account for recognized uncertainties in 

extrapolation from experimental conditions to the assumed human scenario (e.g., chronic 

exposure over a lifetime when subchronic studies are used for deriving a toxicity value). The 

application of UFs may include the use of a partial UF of 101/2 (3.2) based on the assumption that 

the actual values for the UFs are log-normally distributed. Application of these factors in the 

assessments is such that, when a single partial UF is applied, the factor is rounded to 3—for 

instance, the total factor for individual UFs of 3 and 10 would be 30 (3 × 10). When two partial 

UFs are evoked, however, they are not rounded, such that a UF of 3, 3, and 10 would result in a 

total uncertainty factor of 100 (actually 101/2 × 101/2 × 101) (U.S. EPA, 2002, 088824). UFs 

applied for this RfC assessment and the justification for their use are as follows. 

A factor of 10 was selected to account for the uncertainty associated with the variability 

of the human response (UFH) to the effects of EGBE. Potentially susceptible subpopulations 

include individuals with enhanced metabolism or decreased excretion of BAA and individuals 

whose RBC membranes are more susceptible to the lysis caused by BAA, the precursor step to 

developing hemosiderin staining in the liver. Human in vitro studies suggest that the elderly and 

patients with fragile RBCs would not be more sensitive to the hemolytic effects of EGBE than 

normal adults. Laboratory animal studies suggest that older animals are more sensitive than 

neonates and that females are more sensitive than males. While developmental studies do not 

reveal increased susceptibility in infants, none of the developmental studies examined fetal or 

infant blood for signs of effects from prenatal exposure to EGBE. Additionally, human 

responses to EGBE have not been observed under a broad range of exposure conditions (e.g., 

repeated or long-term exposures) and potentially sensitive subjects (e.g., individuals predisposed 

to hemolytic anemia or infants). 

A factor of 1 was selected to account for the uncertainty associated with interspecies 

variability resulting from toxicodynamic and toxicokinetic differences between animals and 

humans (UFA). Traditionally, these components (toxicodynamic and toxicokinetic) are 

individually represented by partial UFs of 3 for a total UF of 10 in the absence of chemicalspecific information; thus, application of a full UF of 10 would depend on two areas of 

uncertainty (i.e., toxicokinetic and toxicodynamic uncertainties). In this assessment, the 

toxicokinetic uncertainty is addressed by the determination of an HEC, using a combination of 

measured internal blood levels in the test animals and PBPK modeling. Thus, a value of 1 was 

selected for the toxicokinetic portion of the UFA. Regarding toxicodynamics, in vivo (Carpenter 

et al., 1956, 066464) and in vitro (Ghanayem and Sullivan, 1993, 041609; Udden, 2002, 042111; 

Udden and Patton, 1994, 056374) studies indicate that humans may be significantly less sensitive 

than rats to the hematological effects of EGBE. For this reason, a value of 1 was selected for the 

toxicodynamic portion of the UFA. 

 87 
A factor to account for extrapolation from subchronic to chronic exposure (UFS) was not 

needed because the RfC was derived from a chronic inhalation study.

A factor to account for the extrapolation from a LOAEL to a NOAEL (UFL) was not 

applied because the current approach is to address this extrapolation as one of the considerations 

in selecting a benchmark response (BMR) for BMD modeling. In this case, EPA concluded a 

10% increase in hemosiderin staining, indicating a precursor to an adverse effect, is appropriate 

for use in deriving the RfC.

A factor of 1 was selected to account for deficiencies in the database (UFD). Studies that 

are available include chronic and subchronic studies for two species (rats and mice), and several 

reproductive and developmental studies, including a two-generation reproductive toxicity study. 

There are also limited human studies available following short-term inhalation exposure.

A total UF of 10 (10 for UFH, 1 for UFA, and 1 for UFD) was used in the derivation of the 

RfC. The combined PBPK and BMC modeling method using hemosiderin as an endpoint was 

used to derive the RfC. In addition, MOA information was used to inform the choice of the 

critical effect. The RfC for EGBE based on hemosiderin deposition in the liver was calculated as 

follows: 

RfC = BMCLHEC ÷ UF 

 = 16 mg/m3

 ÷ 10 

 = 1.6 mg/m3

Thus, the RfC is 1.6 mg/m3. 

 88 
5.1.4. RfC Comparison Information 

For comparison purposes, Figure 5-1 presents the POD, applied UFs, and derived 

reference values, including the RfC, for the effect endpoints discussed. BMC modeling was 

done using U.S. EPA BMDS version 1.4.1 (U.S. EPA, 2000, 052150), and results are provided in 

Appendix C. This comparison is intended to provide information on alternative endpoints 

associated with EGBE exposure. The selected RfC value is circled; BMC analyses are provided 

in Appendix C. 

0.1

1

10

100

1000

Concentration (mg/m3

)

PODHE UFH UFL RfC

NTP (2000); 2 yr 

male and female 

rat study; 

hematological 

effects;LOAEL 

NTP (2000); 

3 mo female rat 

study; RBC 

effects; 

BMCL05(HEC) 

NTP (2000); 2 yr 

female rat study; 

hemosiderin 

staining; 

BMCL10(HEC) 

NTP (2000); 2 yr 

male rat study; 

hemosiderin 

staining; 

BMCL10(HEC) 

NTP (2000); 2 yr 

female mouse 

study; 

hemosiderin 

staining; 

BMCL10(HEC) 

NTP (2000); 2 yr 

male mouse 

study; 

hemosiderin 

staining; 

BMCL10(HEC) 

NTP (2000); 2 yr 

female mouse 

study; 

forestomach 

hyperplasia; 

BMCL10(HEC) 

UFA

Figure 5-1. PODs for selected endpoints with corresponding applied UFs 

and derived RfC. 

Figure 5-1 shows PODs and comparison reference values (including the RfC) that could 

be derived from the various endpoints to allow a comparison with the chosen critical effect and 

the resultant RfC for the critical effect. Hemolytic effects and effects related to hemolysis (i.e., 

hemosiderin deposition) are the most sensitive endpoints for identification of a NOAEL or a 

BMCL in the subchronic and chronic studies available; these endpoints have been considered as 

 89 
the critical endpoint for derivation of the RfC. The BMCL05 for RBC count changes in female 

rats was 133 μM, using Cmax at 3 months, and was converted to an inhalation HEC (BMCLHEC) 

of 225 mg/m3 using the U.S. EPA model. Though adequate model fit per U.S. EPA BMD 

technical guidance (U.S. EPA, 2000, 052150) could not be obtained for the NTP (2000, 196293) 

3-month male rat RBC response, BMCs derived for hemolytic endpoints in male rats of this 

study were approximately twofold higher than for female rats (data not shown). For the 

hemosiderin endpoint, both male and female data from the chronic study were considered. The 

BMCL10 for hemosiderin staining in male rats was 133 μM-hour/L using the AUC for BAA in 

arterial blood at 12 months and was converted to a BMCLHEC of 16 mg/m3 using the Corley et al. 

(1994, 041977; 1997, 041984) human PBPK model. The BMCL10 for hemosiderin staining in 

female rats was 244 μM-hour/L using the AUC for BAA in arterial blood at 12 months and was 

converted to a BMCLHEC of 24 mg/m3

 using the Corley et al. (1994, 041977; 1997, 041984) 

human PBPK model. 

5.1.5. Previous Inhalation Assessment 

The previous IRIS assessment for EGBE was entered into the database on December 31, 

1999; it contains an inhalation RfC of 13 mg/m3

. The RfC was based on the BMC05(HEC) of 

380 mg/m3 for changes in RBC count in female F344 rats following a 14-week inhalation 

exposure (NTP, 1998, 594421). A total UF of 30 was used to account for human variability and 

extrapolation from an adverse effect level. 

5.2. ORAL REFERENCE DOSE (RfD) 

In general, the RfD is an estimate, with uncertainty spanning perhaps an order of 

magnitude, of a daily exposure to the human population—including susceptible subgroups—that 

is likely to be without an appreciable risk of adverse health effects over a lifetime. It is derived 

from a statistical benchmark dose, 95% lower bound (BMDL), a NOAEL, a LOAEL, or another 

suitable POD, with uncertainty/variability factors applied to reflect limitations of the data used. 

The RfD is expressed in terms of mg/kg-day of exposure to an agent and is derived by a similar 

methodology to the RfC. Ideally, studies with the greatest duration of exposure and conducted 

via the oral route of exposure give the most confidence for derivation of an RfD. The database 

of oral studies for EGBE is more limited than the database of inhalation studies. For this reason, 

a PBPK model for EGBE has been applied to the inhalation data for derivation of an RfD. 

5.2.1. Choice of Principal Study and Critical Effect—with Rationale and Justification 

No studies have been reported in which humans have been exposed subchronically or 

chronically to EGBE by the oral route of exposure, and thus would be suitable for derivation of 

an oral RfD. No chronic oral laboratory animal studies are currently available for EGBE. The 

 90 
results of the only two available subchronic 91-day drinking water studies in rats and mice (NTP, 

1993, 042063) are summarized in Table 5-7. 

Table 5-7. Subchronic 91-day drinking water studies in rats and mice 

Effect levels 

Species (mg/kg-d) 

Reference (strain) Gender Animals/dose Effect NOAEL LOAEL 

M 10 Hepatocellular changes – 54.9a NTP (1993, Rat 

(F344) F 10 Hematological – 042063) 58.6a

M 10 Body weight 223 553b NTP (1993, Mouse 

042063) (B6C3F1) F 10 Body weight 370 676b

a

Doses were calculated using water consumption rates and body weights measured during the last week of exposure 

and, therefore, differ slightly from those reported by the authors and presented in Section 4.2. 

b

The LOAEL in mice was based on reduced body weight and body weight gain. 

Based on a comparison of NOAELs and LOAELs for hematological and liver effects, rats 

are clearly more sensitive to the noncancer effects of EGBE than mice. As discussed in 

Section 4.2, hematologic and hepatocellular changes were noted in both rat genders. In females, 

both hematologic and hepatocellular changes were noted at the low-dose level (58.6 mg/kg-day, 

using water consumption rates and body weights measured during the last week of exposure). 

Only hepatocellular cytoplasmic changes were observed in low-dose male rats (54.9 mg/kg-day, 

using water consumption rates and body weights measured during the last week of exposure). 

In the female rat study (NTP, 1993, 042063), groups of 10 F344 rats were exposed to 0, 

82, 151, 304, 363, and 470 mg/kg-day EGBE via drinking water for 13 weeks. Body and organ 

weights were measured, and clinical, hematological, gross, and histopathologic examinations 

were conducted. Decreases in body weight were observed in female rats exposed to the two 

highest dose levels. Hematologic changes were observed at all dose levels after 13 weeks and 

were indicative of mild-to-moderate anemia. These changes included reduced RBC count, Hb, 

and Hct, as well as increased reticulocyte count and MCV. Liver hemosiderin pigmentation was 

noted in the cytoplasm of Kupffer cells in both genders. In females it was noted in 0/10 controls 

in 82 mg/kg-day treated animals, 2/10 with a severity grade of 1 (minimal) at 151 mg/kg-day, 

and 10/10 in the three highest dose levels, with the severities noted as increasing from a 

numerical grade of 1.2 in the 304 mg/kg-day group to 1.9 in both of the upper two dose groups. 

In males the pigmentation was noted in animals exposed to the highest dose only (452 mg/kgday) at an incidence of 7/10 and a severity rating of 1 (minimal). No hepatic pigmentation was 

reported in the mice exposed for 13 weeks. 

Hematological endpoints indicative of hemolysis do not progress with increasing duration 

of inhalation exposure, whereas the incidence of hemosiderin pigmentation did progress 

considerably with chronic exposure (Table 5-2). A primary consideration with respect to the 

 91 
NTP (1993, 042063) study was that it did not provide a sufficient duration of exposure to EGBE 

to allow for maximal accumulation of hemosiderin deposition. In contrast, hemosiderin 

accumulation was one of the most sensitive effects observed in the NTP (2000, 196293) chronic 

inhalation study of EGBE. Furthermore, uncertainty regarding the mechanism of action of 

EGBE does not allow for a biologically-informed determination regarding which hematological 

endpoints—changes in RBC count, reticulocyte count, MCV, Hct, and Hb—observed in EGBEexposed animals should be used to derive an RfC and, in the case of BMD analysis, what a 

proper BMR level should be for the BMD derivation. Finally, hematologic effects signified by 

changes in RBC count, reticulocyte count, MCV, HCT, and Hb are considered precursor effects 

to the pathological finding of hemosiderin deposition (Section 4.5). For these reasons and 

because the hemolytic effects of EGBE appear to be consistent between oral and inhalation 

routes of exposure, an RfD has been derived via the application of a PBPK model to perform a 

route-to-route extrapolation (Section 5.2.2) from the BMD analysis (Section 5.1.2) of 

hemosiderin pigmentation observed in the NTP (2000, 196293) chronic inhalation study of 

EGBE. BMD/NOAEL analyses of hematologic endpoints and hemosiderin pigmentation 

observed in the oral NTP (1993, 042063) subchronic study are provided below for comparison 

purposes. 

Another issue that needs to be addressed with respect to the NTP (1993, 042063) study is 

the lack of reported forestomach lesions at even the highest drinking water doses administered in 

this study relative to the considerable incidence of forestomach hyperplasia and ulceration 

observed at all exposure levels in the NTP (2000, 196293) subchronic and chronic inhalation 

studies of mice. This is difficult to explain, considering the lowest dose in the subchronic 

drinking water study is predicted to result in similar, if not higher, Cmax blood levels of the 

EGBE metabolite BAA (the presumed irritant) compared to the lowest exposure concentration in 

the NTP (2000, 196293) subchronic inhalation study. There is no clear explanation. It has been 

suggested that oral nonbolus dosing of EGBE does not result in high enough local concentrations 

of EGBE and BAA (Poet et al., 2003, 100190). Studies with other forestomach carcinogens that 

are not mutagenic have demonstrated that forestomach effects are dependent not only on the dose 

but also on the chemical concentration in the dosing solution (Ghanayem et al., 1985, 042768), 

and other effects of EGBE appear to be highly dependent on the concentration attained 

(Ghanayem et al., 2000, 042748; Ghanayem et al., 2001, 042755; Long et al., 2000, 042753; 

Nyska et al., 1999, 042747; Nyska et al., 1999, 042746). In addition, first-pass liver metabolism 

of orally administered EGBE may affect the extent to which EGBE reaches the forestomach via 

the route that has been proposed following i.v. injection: distribution to salivary glands followed 

by the swallowing of EGBE-laden saliva (Green et al., 2002, 041610; Poet et al., 2003, 100190). 

In any case, since forestomach irritation was not reported in rats or mice in the NTP (1993, 

042063) drinking water study, this is not considered a sensitive endpoint, and route-to-route 

 92 
extrapolation of this endpoint from inhalation data is not considered appropriate for use in the 

RfD derivation. 

5.2.2. Methods of Analysis—Including Models (PBPK, BMD, etc.) 

PODs for the RfD derivation in terms of the human equivalent doses (HEDs) have been 

calculated via the application of PBPK modeling to NOAEL and BMDL estimates. Details of 

the various POD derivation approaches that were considered are provided in Appendix C. The 

selected approach is described below. 

5.2.2.1. BMD Approach Applied to Hemosiderin Endpoint 

Due to the limited oral database, U.S. EPA concluded that a route-to-route extrapolation 

will increase the confidence in the determination of the RfD POD. Inhalation studies considered 

for derivation of the RfC are used to supplement the oral database using the route-to-route 

extrapolation as described below. 

5.2.2.2. Route-to-Route Extrapolation from Inhalation Data 

A route-to-route extrapolation was performed on the data used in the derivation of the 

RfC from the NTP (2000, 196293) chronic inhalation study because of the lack of a chronic oral 

study for EGBE. As with the animal to human extrapolation used in the development of the 

RfC, the dose metric used for interspecies (rat to human) and route-to-route (inhalation to oral) 

extrapolation was the AUC of BAA at 12 months in arterial blood. This dose metric was used 

for dose-response modeling of chronic inhalation data (Section 5.1.2) to derive the POD of 133 

μmol-hour/L, expressed as a BMDL. The BMDL was then back-calculated using the human 

PBPK model (Corley et al., 1994, 041977; 1997, 041984) to obtain an equivalent human oral 

drinking water dose (BMDLHED) of 1.4 mg/kg-day. As for the alternative HED estimations 

described in Section C.2 of Appendix C, a simplifying assumption was used that the entire dose 

of drinking water EGBE was consumed over a 12-hour period each day. 

5.2.2.3. Selection of the POD 

The BMCL chosen for the RfC is used to determine the POD for the RfD. This value is 

based on a more comprehensive chronic data set and is below the range of estimates from 

available oral data of shorter duration of exposure. Hemosiderin deposition in male rat liver is 

the critical effect chosen for derivation of the RfC. New MOA information (see Section 4.6.3.1) 

supports the hemosiderin deposition endpoint as an important key event in the proposed MOA. 

The BMCL for the RfC (AUC of 133 μM-hour/L BAA in arterial blood at 12 months) is 

converted using the Corley et al. (1994, 041977; 1997, 041984) model to an oral HED 

(BMDLHED) of 1.4 mg/kg-day. This extrapolated oral value is consistent with and slightly lower 

than the LOAELHED of 18 mg/kg-day and the BMDLHED of 6.8 mg/kg-day estimated from the 

subchronic oral (NTP, 1993, 042063) study (see Appendix C). 

 93 
5.2.3. RfD Derivation—Including Application of UFs 

UFs are applied to account for recognized uncertainties in extrapolation from 

experimental conditions to the assumed human scenario (e.g., chronic exposure over a lifetime 

when subchronic studies are used for deriving a toxicity value). The application of UFs may 

include the use of a partial UF of 101/2 (3.2) based on the assumption that the actual values for 

the UFs are log-normally distributed. Application of these factors in the assessment is such that, 

when a single partial UF is applied, the factor is rounded to 3 (e.g., the total factor for individual 

UFs of 3 and 10 would be 30 [3 × 10]). When two partial UFs are evoked, however, they are not 

rounded, such that a UF of 3, 3, and 10 would result in a total uncertainty of 100 (actually 

101/2 × 101/2 × 101). UFs applied for this RfD assessment and the justification for their use 

follow. 

A factor of 10 was selected to account for the uncertainty associated with the variability 

of the human response (UFH) to the effects of EGBE. Potentially susceptible subpopulations 

include individuals with enhanced metabolism or decreased excretion of BAA and individuals 

whose RBC membranes are more susceptible to the lysis caused by BAA, the precursor step to 

developing hemosiderin staining in the liver. Human in vitro studies suggest that the elderly and 

patients with fragile RBCs would not be more sensitive to the hemolytic effects of EGBE than 

normal adults. Laboratory animal studies suggest that older animals are more sensitive than 

neonates and that females are more sensitive than males. While developmental studies do not 

reveal increased susceptibility in infants, none of the developmental studies examined fetal or 

infant blood for signs of effects from prenatal exposure to EGBE. Additionally, human 

responses to EGBE have not been observed under a broad range of exposure conditions (e.g., 

repeated or long-term exposures) and potentially sensitive subjects (e.g., individuals predisposed 

to hemolytic anemia or infants). 

A factor of 1 was selected to account for the uncertainty associated with interspecies 

variability resulting from toxicodynamic and toxicokinetic differences between animals and 

humans (UFA). Traditionally, these components (toxicodynamic and toxicokinetic) are 

individually represented by partial UFs of 3 for a total UF of 10 in the absence of chemicalspecific information; thus, application of a full UF of 10 would depend on two areas of 

uncertainty (i.e., toxicokinetic and toxicodynamic uncertainties). In this assessment, the 

toxicokinetic uncertainty is addressed by the determination of an HEC, using a combination of 

measured internal blood levels in the test animals and PBPK modeling. Thus, a value of 1 was 

selected for the toxicokinetic portion of the UFA. Regarding toxicodynamics, in vivo (Carpenter 

et al., 1956, 066464) and in vitro (Ghanayem and Sullivan, 1993, 041609; Udden, 2002, 042111; 

Udden and Patton, 1994, 056374) studies indicate that humans may be significantly less sensitive 

than rats to the hematological effects of EGBE. For this reason, a value of 1 was selected for the 

toxicodynamic portion of the UFA.

 94 
A factor to account for extrapolation from subchronic to chronic exposure (UFS) was not 

needed because the RfD was derived from a chronic inhalation study.

A factor to account for the extrapolation from a LOAEL to a NOAEL (UFL) was not 

applied because the current approach is to address this extrapolation as one of the considerations 

in selecting a benchmark response (BMR) for BMD modeling. In this case, EPA concluded a 

10% increase in hemosiderin staining, indicating a precursor to an adverse effect, is appropriate 

for use in deriving the RfD.

A factor of 1 was selected to account for deficiencies in the database (UFD). While no 

chronic oral studies or adequate human data are available for EGBE, PBPK models allow for 

deriving a BMDL from the chronic inhalation study using measured internal dose metrics and 

then extrapolating it back to an equivalent human oral dose. The database for inhalation exposure 

includes chronic and subchronic studies in two species (rats and mice), and several reproductive 

and developmental studies, including a two-generation reproductive toxicity study.

A total UF of 10 (10 for UFH, 1 for UFA, and 1 for UFD) was used in the derivation of the 

RfD. The RfD for EGBE based on hemosiderin deposition in the liver was calculated as follows: 

RfD = BMDLHED ÷ UF 

 = 1.4 mg/kg-day ÷ 10 

 = 0.1 mg/kg-day 

Thus, the RfD is 0.1 mg/kg-day. 

5.2.4. RfD Comparison Information 

For comparison purposes, Figure 5-2 presents the POD, applied UFs, and derived 

reference values, including the RfD, for the effect endpoints discussed. BMC modeling was 

done using U.S. EPA BMDS version 1.4.1 (U.S. EPA, 2000, 052150), and results are provided in 

Appendix C. This comparison is intended to provide information on alternative endpoints 

associated with EGBE exposure. The selected RfD value is circled; BMD analyses are provided 

in Appendix C. 

 95 
NTP 1993; 91 d 

oral study; 

hematological 

changes; 

LOAEL(HED) 

NTP 1993; 91 d 

oral study; 

RBC changes; 

BMDL05(HED) 

NTP 2000; 2 yr 

Inhalation study; 

hemosiderin 

staining; 

BMDL10(HED) 

UFL

0.01

0.1

1

10

100

Concentration (mg/kg-d) 

PODHED

(mg/kg-d) 

UFH RfD 

(mg/kg-d) 

Figure 5-2. PODs for selected endpoints with corresponding applied UFs 

and derived RfD. 

Figure 5-2 shows PODs and comparison reference values (including the RfD) that could 

be derived from the various endpoints to allow a comparison with the chosen critical effect and 

the resultant RfD for the critical effect. Hematological effects and effects related to hemolysis 

(i.e., hemosiderin deposition) are the most sensitive endpoints for identification of a NOAEL or a 

BMDL in the subchronic and chronic studies available; these endpoints have been considered as 

the critical endpoint for derivation of the RfD. 

5.2.5. Previous Oral Assessment 

The previous IRIS assessment for EGBE was entered into the database on December 30, 

1999 and contains an oral RfD of 0.5 mg/kg-day. The RfD was based on the BMD05(HED) of 

5.1 mg/kg-day for changes in MCV in female F344 rats following a 91-day drinking water 

exposure (NTP, 1993, 042063). A total UF of 10 was used to account for human variability. 

This assessment was conducted prior to the adoption of the current BMD technical guidance 

document (U.S. EPA, 2000, 052150). 

 96 
5.3. UNCERTAINTIES IN THE DERIVATION OF THE INHALATION REFERENCE 

CONCENTRATION (RfC) AND ORAL REFERENCE DOSE (RfD) 

The following is a more extensive discussion of uncertainties associated with the RfC and 

RfD for EGBE beyond the quantitative discussion in Sections 5.1.2, 5.1.3, 5.2.2, and 5.2.3. A 

summary of these uncertainties, along with uncertainties specific to the Section 5.4 cancer 

analysis, is presented in Table 5-8. 

Table 5-8. Summary of uncertainty in the EGBE noncancer and cancer risk 

assessments 

Consideration Potential impact Decision Justification 

Choice of lowdose 

extrapolation 

method 

A linear low-dose 

extrapolation would likely 

drive up the risk 

estimation when 

combined with sitespecific exposure data. 

Nonlinear approach; RfC 

and RfD are considered 

protective of the key 

events leading to cancer.

Proposed key events in the two MOAs 

(forestomach irritation and hemolysis 

leading to increased liver hemosiderin) are 

not likely to occur in humans at the RfC or 

RfD. 

Choice of 

endpoint 

Use of forestomach 

endpoint could increase 

RfC by up to 18-fold (see 

Section 5.3). 

RfC is based on the most 

sensitive endpoint, 

increased liver 

hemosiderin. 

Chosen endpoint is considered most 

relevant due to forestomach toxicokinetic 

and exposure issues. 

Choice of dose 

metric 

Alternatives could 

increase or decrease 

RfC/RfD (e.g., use of Cmax

BAA increases RfC by 

two to threefold). 

AUC for BAA in arterial 

blood. 

Evidence for a causal association between 

the proposed key steps of BAA and 

hemolysis leading to increased liver 

hemosiderin and increased liver tumors. 

AUC chosen because hemosiderin 

increased with cumulative exposure to 

EGBE/BAA. 

RfC/RfD threefold lower 

than for NOAEL. 

BMD method used. Advantages include capacity to account for 

sample size and to provide confidence 

bounds on dose. 

POD derivation 

method 

Choice of model 

for BMCL 

derivation 

Alternative models could 

increase RfC up to 

threefold (see 

Section 5.3). 

Multistage (1st degree) 

model chosen. 

The best-fitting model was chosen based 

on U.S. EPA (2000, 052150) BMD 

technical guidance. 

Choice of animal 

to human 

extrapolation 

method 

Alternatives could 

increase or decrease 

RfC/RfD (e.g., default 

would increase RfC by 

twofold) (see Section 5.3).

A PBPK model was used 

to extrapolate animal to 

human concentrations. 

Use of a PBPK model reduces uncertainty 

associated with the animal to human 

extrapolation. AUC blood levels of BAA 

are an appropriate dose metric, and a peerreviewed and verified PBPK model exists 

that estimates this metric. 

Statistical 

uncertainty at 

POD 

POD would be ~40% 

higher if BMD were used.

BMDL used per U.S. 

EPA BMD guidance 

(U.S. EPA, 2000, 

052150). 

Limited size of bioassay results in 

sampling variability; lower bound is 95% 

CI on administered exposure. 

Choice of 

bioassay 

Alternatives could 

increase or decrease 

RfC/RfD. 

NTP (2000, 196293) 

study. 

Alternative bioassays were inadequate. 

 97 
Consideration Potential impact Decision Justification 

Choice of species/ 

gender 

RfC would be increased if 

based on another 

species/gender. 

RfC is based on the most 

sensitive endpoint, 

increased liver 

hemosiderin, in the most 

sensitive species and 

gender, male rats. 

Male mice are the only species/gender to 

experience an increase in liver tumors 

from EGBE exposure. If one relies on the 

MOA, however, liver hemosiderin increase 

in male rats is the appropriate key event 

and species to utilize for the RfC. Female 

mice exhibit forestomach papilloma but at 

higher doses. 

Human relevance 

of rodent tumor 

data 

If MOAs for tumors from 

EGBE were deemed not 

relevant, cancer descriptor 

would be “suggestive of 

human carcinogenic 

potential.” 

MOAs for liver and 

forestomach tumors are 

used. 

Although EGBE has some evidence for 

liver tumors in male mice and forestomach 

tumors in female mice, the MOAs describe 

rationales as to why humans are unlikely to 

experience appreciable risk at 

environmental concentrations. 

Human 

population 

variability 

Low-dose toxicity would 

increase to an unknown 

extent. 

10-fold UF applied to 

derive the RfD and RfC 

values. 

10-fold UF is applied principally because 

of limited data on human variability or 

potential susceptible subpopulations. 

5.3.1. Choice of Endpoint 

Comparison RfC values were calculated (see Section 5.1.4) and are intended to provide 

information on alternative health effects associated with EGBE exposure. The comparison RfCs 

ranged from 1 to 23 mg/m3

, depending on whether irritation (forestomach), hematologic effects, 

or hemosiderin deposition data were used to derive the POD, with the latter endpoint 

representing the lower end of the RfC range. 

5.3.2. Choice of Dose Metric 

The AUC for BAA in arterial blood was selected as the appropriate measure of dose due 

to evidence for a causal association between BAA and hemolysis, between hemolysis and the 

accumulation of hemosiderin in the liver, and between hemosiderin accumulation in the liver and 

increased incidence of liver hemangiosarcoma. AUC is considered to be a more appropriate 

response measure because hemosiderin pigmentation increases in incidence and severity with 

increased duration (subchronic to chronic) and is believed to be the result of the cumulative 

exposure to EGBE/BAA as opposed to a peak exposure. The corresponding aldehyde of BAA, 

BAL, was also considered as a choice for an internal dose measure. As discussed in Section 4.6, 

BAL is the EGBE metabolite considered to have the greatest potential to interact directly with 

DNA. However, high ADH activity in the liver and forestomach is expected to result in very 

short residence time and in very low tissue concentrations of BAL; this scenario has been 

demonstrated in simulations using the Corley et al. (2005, 100098) PBPK model. Also, the 

Corley et al. (2005, 100098) PBPK model along with the gavage study of Deisinger and 

Boatman (2004, 594446) suggest that the conditions of in vitro assays showing BAL to be 

clastogenic (e.g., no metabolic activation; high cytotoxic concentrations of BAL) are considered 

to be of little relevance to the expected target organ (liver) environment (e.g., high metabolic 

 98 
activity; low concentrations of BAL). Use of an alternate measure of internal dose, for instance, 

a parent compound or Cmax, would be more difficult to justify based on available empirical 

information. However, for comparative purposes, a BMD analysis was done on the critical 

endpoint to determine the impact that choosing Cmax of BAA in blood rather than AUC of BAA 

in blood would have had on the BMCL10(HEC) derivation. If Cmax had been used as the dose 

measure, the BMCL10(HEC) value would have been 109 mg/m3

, approximately 6.8-fold higher 

than the 16 mg/m3 BMCL10(HEC) value derived using AUC as the dose measure. 

5.3.3. Use of BMC Approach 

Utilization of the BMC approach has advantages over other approaches to dose-response 

analysis, such as the NOAEL/LOAEL approach. These advantages include the capacity of the 

BMC approach to accommodate study sample size and reflect this in providing confidence 

bounds to the lower limit on dose. As shown in Figures 5-1 and 5-2, use of the BMC approach 

on the incidence of chronic hemosiderin deposition resulted in RfC and RfD values lower than 

RfC and RfD values that would have been derived via the NOAEL/LOAEL approach. 

5.3.4. Choice of Model for BMCL Derivations 

BMCL10 estimates used in the derivation of the RfC, which formed the basis for both the 

noncancer and cancer quantitative assessments, spanned a twofold range (60–124 μmol-hour/L) 

for female rats and threefold range (40–126 μmol-hour/L) for male rats. All of the models fit 

reasonably well (p-values above 0.1). Hence, this range of results can be considered a reflection, 

in part, of model uncertainty. 

5.3.5. Choice of Animal to Human Extrapolation Method 

A PBPK model (Corley et al., 1997, 041984) was used to extrapolate animal to human 

concentration. An AUC blood level of BAA associated with a 10% increase in male mice with 

hemosiderin pigmentation of 69.6 μmol-hour/L was estimated using the mouse PBPK model; the 

human PBPK model was used to convert back to a human equivalent exposure concentration, or 

a BMCL10(HEC), of 12 mg/m3

. If no PBPK models were available, the BMCL10(HEC) would have 

been derived by dividing the BMCL10 for external exposure concentration of 75 mg/m3

 by the 

threefold pharmacokinetic portion of the animal to human default adjustment factor (U.S. EPA, 

1994, 006488), resulting in a BMCL10(HEC) of 25 mg/m3

. This default value would have been 

twofold higher than the value derived using the PBPK model. 

5.3.6. Route-to-Route Extrapolation 

To estimate an oral dose POD for chronic hemosiderin deposition, a route-to-route 

extrapolation was performed on the inhalation exposure POD used to derive the RfC using a 

PBPK model and assuming, as discussed above, that the metric most closely associated with the 

effect seen is the AUC measure of BAA in blood. One way of characterizing the uncertainty 

 99 
associated with this approach is to compare dose levels (BMCL values) using this metric for 

hemolytic effects—assumed to be associated with chronic hemosiderin deposition—derived 

from (1) an existing oral subchronic NTP (1993, 042063) study; or (2) from a model estimating 

this metric from an existing inhalation subchronic NTP (1998, 594421; 2000, 196293) study. 

This analysis was performed (data not shown) and the values derived using the second procedure 

were slightly but consistently lower than those derived using the former, suggesting that 

estimates using the procedure employed for route-to-route extrapolation would tend to 

overestimate the toxicity value and result in lower RfD estimates. 

5.3.7. Statistical Uncertainty at the POD 

Parameter uncertainty can be assessed through CIs. Each description of parameter 

uncertainty assumes that the underlying model and associated assumptions are valid. For the 

linear multistage model applied to the male mouse hemosiderin data, there is a reasonably small 

degree of uncertainty at the 10% excess incidence level (the POD for derivation of the RfC), 

with the 95%, single-sided lower confidence limit (BMCL) being about 30% below the 

maximum likelihood estimate of the BMC. 

5.3.8. Choice of Bioassay 

The NTP (2000, 196293) inhalation study was used for development of the RfC and RfD. 

This was a well-designed study, conducted in both genders in two species with an adequate 

number of animals per dose group and with examination of appropriate toxicological endpoints 

in both genders of rats and mice. Alternative comparable bioassays were unavailable. 

5.3.9. Choice of Species/Gender 

The RfC was based on the incidence of liver hemosiderin pigmentation in male rats, the 

species and gender most sensitive to this effect (NTP, 2000, 196293). This event also occurs in 

female rats and in mice, and is thought to be a precursor to the observed increase in liver tumors 

in male mice. The cancer assessment was based on the more sensitive hemosiderin response in 

the male rat, not the male mouse, because there is no evidence to suggest that the proposed MOA 

for tumor formation would not be relevant to rats, and because the lack of an observed tumor 

response in rats may have been due to the fact that rats were exposed to lower doses for a shorter 

portion of their average lifespan (see further discussion in Sections 4.6 and 4.7.2). 

If the RfC had been based on increased incidence of liver hemosiderin in another 

species/gender, such as male mice, a higher RfC value would have been derived. Similarly, the 

RfC would also have been higher had it been based on forestomach irritation, an effect less 

sensitive than hemosiderin deposition but considered to be a precursor event leading to the 

increase in incidence of forestomach squamous cell papillomas and one high-dose carcinoma 

observed by NTP (2000, 196293) in female mice. 

 100 
5.3.10. Human Relevance of Noncancer Responses Observed in Mice 

The effects of hemosiderin deposition and forestomach irritation may both have 

qualitative relevance to humans. However, for reasons discussed in Section 4.5, the exposure 

concentrations that would be necessary to cause these effects in humans, if attainable at all, are 

likely to be much higher than the RfC/RfD and well above concentrations necessary to cause 

these effects in mice. 

5.3.11. Human Population Variability 

The extent of interindividual variability associated with EGBE metabolism is not well 

characterized in humans. As is discussed in Section 4.7, the hemolytic effect of EGBE is 

presumed to be caused by the interaction of its primary metabolite, BAA, with the RBC 

membrane. Potentially susceptible subpopulations or life stages would include individuals with 

enhanced metabolism or decreased excretion of BAA. In addition, individuals whose RBC 

membranes are more susceptible to the lysis caused by BAA could be more sensitive to EGBE. 

However, RBCs from normal, aged, sickle-cell anemia, and hereditary spherocytosis patients 

were no more sensitive to the hemolytic effects of BAA than RBCs from healthy volunteers 

(Udden, 1994, 042109). As is discussed further in Section 4.7, some potentially susceptible 

subpopulations or life stages have not been tested, and, when combined with the lack of 

understanding about EGBE’s mechanism of hemolytic action, this represents a considerable 

source of uncertainty and forms the principal basis for the 10-fold UF applied to derive the RfD 

and RfC values. 

5.4. CANCER ASSESSMENT 

In accordance with the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005, 

086237), the method used to characterize and quantify cancer risk from a chemical depends on 

what is known about the MOA of carcinogenicity and the shape of the cancer dose-response 

curve for that chemical. An assumption of linearity is appropriate when evidence supports an 

MOA of gene mutation due to DNA reactivity or an MOA that is anticipated to be linear. The 

linear approach is used as a default option if the MOA of carcinogenicity is not known. A 

nonlinear approach “can be used for cases with sufficient data to ascertain the MOA and to 

conclude that it is not linear at low doses...” (U.S. EPA, 2005, 086237). Alternatively, the MOA 

may theoretically have a threshold; that is, the carcinogenicity may be a secondary effect of 

toxicity that is itself a threshold phenomenon. 

In the case of EGBE, the MOA of carcinogenicity for hepatic hemangiosarcoma, 

hepatocellular adenoma and carcinoma, and forestomach tumor formation in animals is 

reasonably well understood. An RfC and RfD approach has been used for EGBE because 

“When adequate data on MOA provide sufficient evidence to support a nonlinear MOA for the 

general population and/or any subpopulations of concern, a different approach—a RfD/RfC that 

 101 
assumes nonlinearity—is used” (U.S. EPA, 2005, 086237). It is recognized, however, that while 

this approach fits this case, other nonlinear approaches may be appropriate in other settings. 

Available data indicate that EGBE is not likely to be mutagenic and that it is not expected to 

produce rodent tumors via a mutagenic MOA. Rather, there is evidence that carcinogenic 

responses observed in animals are associated with erythrocyte hemolysis (leading to oxidative 

damage, increased hepatocyte and endothelial cell proliferation, and initiation for the liver 

tumors) and with the formation of BAA, an acidic metabolite, that leads to regenerative 

hyperplasia in response to irritation for the forestomach tumors. Because cytolethality occurs 

only at exposure levels above some critical dose, a nonlinear (threshold) approach is considered 

to be the most appropriate method for characterizing the cancer risk from EGBE. 

As discussed in Sections 4.1 and 4.6, there are currently no human studies addressing the 

potential carcinogenicity of EGBE. A 2-year inhalation bioassay in mice and rats (NTP, 2000, 

196293) reported some evidence of carcinogenic activity in male B6C3F1 mice based on 

increased incidences of hemangiosarcoma of the liver and an increase in the incidence of 

hepatocellular carcinoma, as well as some evidence of carcinogenic activity in female B6C3F1

mice based on increased incidences of forestomach squamous cell papilloma or carcinoma 

(mainly papilloma). The study also reported no evidence of carcinogenic activity in male 

F344/N rats and equivocal evidence of carcinogenic activity in female F344/N rats based on 

increased combined incidences of benign and malignant pheochromocytoma (mainly benign) of 

the adrenal medulla. The hypothesized MOA for the induction of hepatic hemangiosarcomas 

and hepatocellular carcinomas reported in male mice exposed to EGBE may comprise a 

sequence of events that are dose-dependent. First, EGBE is metabolized to the carboxylic acid, 

which then hemolyzes RBCs. This event leads to the release of excess iron that in turn could 

result in iron-induced formation of ROS and subsequent oxidative damage to target tissues 

within the liver. Induction of cell proliferation and neoplasm formation follows. This sequence 

of events is considered necessary for the formation of the observed neoplasms. Strategies 

intended to control or omit any of these key events, including the initial hemolytic event, would 

interrupt the process and prevent formation of neoplasms. For these reasons, formation of 

neoplasms in humans would likely be prevented by establishing levels of EGBE exposure in the 

dose range that does not result in the initial hemolytic events and subsequent ROS-mediated 

cellular damage. Thus, for the assessment of human cancer risk associated with the formation of 

hemangiosarcomas and hepatocellular adenomas and carcinomas in animals, the RfD and RfC 

derived in Sections 5.1 and 5.2 should be considered protective, as the carcinogenicity may be a 

secondary effect of toxicity that is itself a threshold phenomenon. 

Available data suggest that the MOA for the induction of forestomach tumors reported in 

female mice exposed to EGBE is dependent on the initial formation of the irritating metabolite 

BAA. This acidic metabolite produces chronic cytotoxicity, which results in compensatory 

epithelial cell regeneration. Chronic cell proliferation in preneoplastic cells is in turn associated 

 102 
with the formation of forestomach papillomas and carcinomas. In addition, the available data 

indicate that BAA may be preferentially formed in the forestomach due to the presence of levels 

of ADH that are higher than those found in the human stomach or esophagus. BMD modeling of 

the dose response for epithelial hyperplasia in female mice forestomachs was performed as a 

comparison to reference values derived for precursor effects in the liver as discussed above and 

is presented in Appendix C. The analysis in Appendix C shows that, had hyperplastic effects in 

female mice been used as a POD, the resultant RfD and RfC values would have been much 

higher than the RfD and RfC values derived in Sections 5.1 and 5.2 using hemosiderin 

deposition as the critical effect for EGBE exposure. Thus, the analysis indicates that the RfD 

and RfC based on liver effects would also be protective of forestomach toxicity. The exposure 

concentrations that would be necessary to cause these effects in humans, if attainable at all, are 

likely to be much higher than the RfC and RfD. 

Female rats reported a marginally significant trend for pheochromocytomas, and the high 

dose frequencies reported in the rats were only slightly different from the upper range of 

historical controls (see Section 4.6). In addition, the histopathologic data indicated that there 

was difficulty distinguishing pheochromocytomas from nonneoplastic adrenal medullary 

hyperplasia. Thus, these lesions are interpreted with caution as tumors. Given the marginal dose 

response, lack of tumor evidence in any other organ system of the rats, and reported difficulties 

in distinguishing pheochromocytomas from nonneoplastic adrenal medullary hyperplasia, this 

tumor was not given significant weight in the qualitative or quantitative assessment of EGBE 

cancer potential. 

5.4.1. Quantification for Oral and Inhalation Cancer Risk 

Following the U.S. EPA (2005, 088823) Guidelines for Carcinogen Risk Assessment, a 

nonlinear approach to dose-response assessment is taken for agents, such as EGBE, for which the 

most plausible mode of action at low doses is consistent with nonlinearity. The RfC of 1.6 

mg/m3

 and RfD of 0.1 mg/kg-day derived in Sections 5.1 and 5.2 represent the outcome of 

nonlinear assessments based on hemolytic effects (i.e., hemosiderin deposition)associated with 

both oral and inhalation exposures to EGBE. Doses (or concentrations) of EGBE below the RfC 

(or RfD) would not be expected to produce hemolytic effects (i.e., hemosiderin deposition) and 

therefore are not expected to produce any increase in cancer risk. 

5.4.2. Uncertainties in Cancer Risk Assessment 

The cancer assessment of EGBE is based largely on the premise that key events in the 

MOAs proposed for mice (forestomach irritation and hemolysis leading to increased hemosiderin 

deposition) are not likely to occur in humans at concentrations at or below the RfC and RfD 

values. Uncertainties in the RfC and RfD derivations are addressed in part in Sections 5.1.2, 

5.1.3, 5.2.2, 5.2.3, and 5.3. This section will discuss additional uncertainties relative to the 

 103 
human relevance of cancer responses observed in mice and the extrapolation method employed 

for the estimation of low-dose cancer risk. All of these uncertainties are summarized in 

Table 5-8. 

5.4.2.1. Choice of Low-Dose Extrapolation Method 

The MOA is a consideration in estimating risks. A linear low-dose extrapolation 

approach was not considered to be optimal for the estimation of human carcinogenic risk 

associated with EGBE exposure because of the physicochemical properties of EGBE, 

toxicokinetic limitations, specific MOAs articulated earlier, and limitations in data to 

parameterize appropriate models. It should be noted that the demonstration that a chemical is not 

mutagenic is insufficient, alone, to postulate a nonlinear dose response. Key events in the MOAs 

proposed for mice (forestomach irritation and hemolysis leading to increased hemosiderin 

deposition) are not likely to occur in humans at concentrations at or below the RfC and RfD 

values. This assumes that the proposed MOAs in mice are reasonably correct. If, for instance, 

hemosiderin accumulation in the mouse liver is not the result of increased hemolysis but of 

EGBE interaction with other cell types, a more direct, linear MOA for the observed increase in 

male mouse hemangiosarcomas might be hypothesized. In order to illustrate the predicted 

cancer risk under such a scenario, the cancer risks associated with the tumor types that were 

increased following EGBE exposure were calculated using the default approach of low-dose 

linear extrapolation outlined in the cancer guidelines (U.S. EPA, 2005, 086237). The results of 

this linear analysis are presented in Table 5-9. 

Table 5-9. Illustrative potency estimates for tumors in mice, using a linear 

analysis approach 

BMCL10HEC

(mg/m3

)

a

Inhalation unit risk 

0.1/BMCL10HEC

( per mg/m3

) 

Hepatocellular carcinoma (males) 208 4.8 × 10-4

Hemangiosarcomas (males) 575 1.7 × 10-4

Papilloma or carcinoma of the forestomach (females) 544 1.8 × 10-4

a

BMDL10HEC values were calculated using AUC as the dose metric. 

For illustrative purposes, an estimate of the increased cancer risk at the RfC (1.6 mg/m3) 

was calculated, using the inhalation unit risk for hepatocellular tumors in male mice, that was 

derived using a default linear low-dose extrapolation approach. The estimated increased cancer 

risk at the RfC in this comparison exercise would be 1.6 mg/m3 × 4.8 × 10-4 = 7.6 × 10-4. This 

value is only for illustrative purposes and indicates the differences in the two approaches. It 

should not be misconstrued as an estimate of the cancer risk at the RfC since the default linear 

approach is not recommended. 

 104 
5.4.2.2. Human Relevance of Cancer Responses Observed in Mice 

The hypothesized MOAs for EGBE-induced liver tumors observed in male mice and 

EGBE-induced forestomach tumors observed in female mice may both have qualitative 

relevance to humans. However, for reasons discussed in Section 4.6, the exposure 

concentrations that would be necessary to cause these effects in humans, if attainable at all, are 

likely to be much higher than the RfC and RfD. 

5.5. POTENTIAL IMPACT OF SELECT UNCERTAINTIES ON THE RFC 

In this assessment, the RfC forms the basis for the RfD. The range of possible results 

associated with some of the areas of uncertainty in the RfC derivation can be estimated (see 

Sections 5.3 and 5.4). Figure 5-3 graphically illustrates the change in the RfC that would result 

had particular choices, other than those presented in this assessment, been made (see summary in 

Table 5-8). These specific areas were presented in this illustration because there are data 

available that could be used to quantify their contribution to the uncertainty in the noncancer and 

cancer assessments. The “Cancer approach” illustrates the dose that would represent a 10-6

increased cancer risk if a default linear low-dose extrapolation approach were used. This dose is 

expressed as X-fold change in the RfC. 

 105 
Figure 5-3. Potential impact of select uncertainties on the RfC for EGBE. 

The area of uncertainty that has the potential to have the greatest impact on the EGBE 

assessment is that which is associated with the approach used to extrapolate human cancer risk 

from the available rodent studies; that is, if human cancer risk had been estimated from a linear 

extrapolation of responses observed in rodent studies. Figure 5-3 provides a graphic of the areas 

of uncertainty, described in Table 5-8, for which there is quantitative information and impact on 

the RfC can be estimated. The “Cancer Approach” value in Figure 5-3 represents the difference 

in the dose that was selected for the RfC and the dose that would represent a 10-5 increased 

cancer risk if a default linear low-dose extrapolation approach were to be used. In this instance, 

the comparison indicated that the dose associated with a 10-6 cancer risk, estimated via the linear 

dose-response method using the inhalation unit risk for hepatocellular tumors in male mice as 

indicated in Table 5-9, is 56-fold lower than the RfC. Note that the default linear approach is not 

recommended. 

These discussions and illustrative tools should not be viewed as a comprehensive analysis 

of all possible uncertainty considerations; rather, they exemplify what are believed to be 

important areas of uncertainties for which data are available. This characterization is presented 

 106 
in an effort to make apparent the limitations of the assessment and to aid and guide the risk 

assessor in the ensuing steps of the risk assessment process. 

 107 
6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HAZARD AND 

DOSE RESPONSE 

6.1. HUMAN HAZARD POTENTIAL 

EGBE is a clear, miscible solvent used in formulating cleaning products and protective 

coatings. It is metabolized primarily to the carboxylic acid, BAA, the proximate toxicant, in 

both humans and animals. BAA and its conjugates are readily excreted in the urine. 

Hemosiderin deposition in the liver of male rats, as a consequence of hemolysis, is 

identified as the critical endpoint of concern in toxicological studies on EGBE. Toxicologically, 

this effect increases with both exposure concentration and duration, is not compensated for (as 

with hemolysis), and is considered to be a pathological finding. Mechanistically, this effect is at 

the interface between noncancer and cancer effects from exposure to EGBE. 

Observations regarding the potential relevance of EGBE toxicity to humans include the 

insensitivity of human RBCs to the hemolytic effects of EGBE and its metabolite, BAA. While 

it is established that humans can experience hemosiderin deposition in the liver as a consequence 

of excessive hemolysis (Iancu et al., 1976, 100149), the relative insensitivity of human blood to 

the effects of EGBE have been demonstrated in numerous in vitro studies through the use of 

either whole blood or washed erythrocytes (e.g., Ghanayem and Sullivan, 1993, 041609; Udden, 

2002, 042111). Humans appear significantly less sensitive to the hemolytic toxicity of EGBE 

than are typical laboratory species, such as mice, rats, or rabbits, with reports from analyses of 

isolated RBCs demonstrating that human RBCs are 40- to 150-fold less sensitive than rat RBCs 

(Udden, 2002, 042111). These observations are inclusive of human RBCs from individuals with 

hereditary spherocytosis and sickle cell anemia, conditions characterized by RBC sensitivity to 

hemolysis. Available in vivo information with human exposure supports this species disparity in 

sensitivity to the hemolytic effects of EGBE. Male rats in one study (NTP, 1993, 042063) 

experienced mild liver effects at a drinking-water dose lower than that which caused observable 

hemolytic effects. Available human toxicity data show that the primary effects after acute oral 

ingestion of large doses of EGBE (most often combined with other solvents) are reversible 

metabolic acidosis from the production of BAA and some hematological changes. Occupational 

exposure to low levels of EGBE were not reported to cause changes outside the normal clinical 

ranges in hepatic, renal, or hematologic parameters (Haufroid et al., 1997, 042040). 

Due to the known reproductive toxicity (i.e., toxicity to male testes and sperm) of two 

other glycol ethers, EGME and EGEE, the reproductive toxicity of EGBE has been studied in a 

 

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). 

 108 
variety of well-conducted oral studies (Exon et al., 1991, 100112; Foster et al., 1987, 100116; 

Grant et al., 1985, 006770; Heindel et al., 1990, 042042; Nagano et al., 1979, 100179; Nagano et 

al., 1984, 100180; NTP, 1993, 042063) and inhalation studies (Dodd et al., 1983, 066465; Doe, 

1984, 006754; Nachreiner, 1994, 081825; NTP, 2000, 196293) using rats, mice, and rabbits. In 

addition, several developmental studies have addressed EGBE’s toxicity from conception to 

sexual maturity, including toxicity to the embryo and fetus, following oral exposures (Sleet et al., 

1989, 042089; Wier et al., 1987, 042123), inhalation exposures (Nelson et al., 1984, 031878; Tyl 

et al., 1984, 100209), and dermal exposures (Hardin et al., 1984, 042030) to rats, mice, and 

rabbits. EGBE did not cause significant effects in any reproductive organ, including testes, in 

any study. In a two-generation reproductive toxicity study, fertility was reduced in mice only at 

very high maternally toxic doses (>1,000 mg/kg). Maternal toxicity related to the hematologic 

effects of EGBE and relatively minor developmental effects have been reported in 

developmental studies. No teratogenic effects were noted in any of the studies. It can be 

concluded from these studies that EGBE is not significantly toxic to the reproductive organs 

(male or female) of either parents or to the developing fetuses of laboratory animals. 

No reliable human epidemiological studies are available that address the potential 

carcinogenicity of EGBE. The NTP (2000, 196293) performed a 2-year inhalation bioassay with 

rats and mice and found no evidence of carcinogenic activity in male F344/N rats and equivocal 

evidence of carcinogenic activity in female F344/N rats based on increased combined incidences 

of benign and malignant pheochromocytoma (mainly benign) of the adrenal medulla. The 

researchers reported some evidence of carcinogenic activity in male B6C3F1 mice based on an 

increased incidence of hemangiosarcoma of the liver and an increase in the incidence of 

hepatocellular carcinoma that may have been exposure related. They also reported some 

evidence of carcinogenic activity in female B6C3F1 mice based on an increased incidence of 

forestomach squamous cell papilloma or carcinoma (mainly papilloma). Based on its physicalchemical properties, toxicokinetic and dynamic factors, and MOA information, under existing 

U.S. EPA guidelines (U.S. EPA, 2005, 086237), EGBE is judged not likely to be carcinogenic to 

humans at expected environmental concentrations (see Section 4.6). The MOAs presented for 

the animal tumors indicate that both high doses and sustained periods of exposure are necessary 

for the carcinogenic response. The available human exposure/response information indicates 

that these conditions are unlikely to occur because the primary response of humans to high oral 

doses of EGBE, as shown in the case studies in Section 4.1 is metabolic acidosis, which, if not 

treated, can lead to shock and eventually death. Further, based on simulations from PBPK 

modeling, the maximum blood concentrations of BAA that could be produced in humans 

following exposure to a saturated atmosphere of EGBE would be below those needed to produce 

hemolysis (Corley et al., 2005, 100100). Evidence from the only human inhalation exposure 

study available showed that, while nasal and ocular irritation were reported in research subjects 

exposed to up to 195 ppm, no changes in erythrocyte fragility were observed (Carpenter et al., 

 109 
1956, 066464). For a more complete discussion of the carcinogenic potential of EGBE, see 

Section 4.6. 

6.2. DOSE RESPONSE 

6.2.1. Noncancer—Inhalation 

Studies have not been reported in which humans were exposed subchronically or 

chronically via inhalation to EGBE. After consideration of the available animal inhalation 

studies with EGBE, the NTP (2000, 196293) study was selected as the principal study because it 

was conducted in two species and provides data for different durations and for multiple dose 

groups compared to other available studies. 

For derivation of the RfC, the most sensitive endpoint for dose-response assessment is the 

effect of inhalation exposure on hemosiderin staining in Kupffer cells, as reported in the study by 

NTP (2000, 196293). The concentration of BAA in the blood was used as an internal dose 

metric for EGBE exposure. The human PBPK model of Corley et al. (1994, 041977; 1997, 

041984) was used to obtain estimates of human inhalation exposure concentrations associated 

with the BMCs derived from rat BAA AUC levels reported by Dill et al. (1998, 041981). The 

RfC is based on the human equivalent BMCL10 of 16 mg/m3, which was back-calculated from 

rat data using the BMD and PBPK approach. 

The use of uncertainty factors used to derive the RfC are as follows: a factor of 10 was 

selected to account for the uncertainty associated with the variability of the human response 

(UFH) to the effects of EGBE, a factor of 1 was selected to account for the uncertainty associated 

with interspecies variability resulting from toxicodynamic and toxicokinetic differences between 

animals and humans (UFA), and a factor of 1 was selected to account for deficiencies in the 

database. A total UF of 10 (10 for UFH, 1 for UFA, and 1 for UFD) was used in the derivation of 

the RfC. Thus, the RfC is 16 mg/m3 ÷ 10 = 1.6 mg/m3

. 

The overall confidence in the RfC is medium to high. A high degree of confidence is 

placed in the RfC because it was derived from internal dose measures (PBPK method and 

combined PBPK/BMC method) which account for pharmacokinetic differences between rats and 

humans (Corley et al., 1997, 041984; Corley et al., 2005, 100100; Lee et al., 1998, 041983) and 

it is based on actual measurements of internal blood concentrations in test animals of interest 

(Dill et al., 1998, 041981). There is a high level of confidence in the NTP (2000, 196293) study 

because it was a chronic study, it employed both male and female rats and mice, it had a wide 

range of exposure levels, and animals were observed twice daily. There is medium-to-high 

confidence. While the database lacks long-term human studies, the available short-term human 

controlled studies and case reports, as well as laboratory animal and in vitro studies provide 

evidence to suggest that, with respect to the hemolytic effects of EGBE, long-term human 

exposures would be no more adverse than long-term rat exposures. Additionally, the selection of 

 110 
medium-high confidence is supported because the potential for effects in humans from repeated, 

long-term exposures has not been investigated. 

6.2.2. Noncancer—Oral 

Studies have not been reported in which humans have been exposed subchronically or 

chronically to EGBE by the oral route of exposure, and thus would be suitable for derivation of 

an oral RfD. No chronic oral laboratory animal studies are currently available for EGBE. 

Hematological effects leading to RBC lysis and organ accumulation of iron in the form of 

hemosiderin accumulation appear to be the most sensitive of the effects caused by EGBE in 

laboratory animals. Based on the limited oral database and because the critical endpoint, 

hemosiderin pigmentation, was more pronounced in the chronic inhalation study (NTP, 2000, 

196293), EPA concluded that the use of a route to route extrapolation from the NTP, 2000 

(196293) study would increase the confidence in the derived RfD (see Section 5.2.1). An RfD 

has been derived via the application of a PBPK model using hemosiderin pigmentation observed 

in the NTP (2000, 196293) chronic inhalation study of EGBE. As with the animal to human 

extrapolation used in the development of the RfC, the dose metric used for interspecies (rat to 

human) and route-to-route (inhalation to oral) extrapolation was the AUC of BAA at 12 months 

in arterial blood. This dose metric was used for dose-response modeling of chronic inhalation 

data (Section 5.1.2) to derive the POD of 133 μmol-hour/L, expressed as a BMDL10. The 

BMDL10 was then back-calculated using the human PBPK model (Corley et al., 1994, 041977; 

Corley et al., 1997, 041984) to obtain an equivalent human oral drinking water dose (BMDLHED) 

of 1.4 mg/kg-day. 

The use of uncertainty factors used to derive the RfD are as follows: a factor of 10 was 

selected to account for the uncertainty associated with the variability of the human response 

(UFH) to the effects of EGBE, a factor of 1 was selected to account for the uncertainty associated 

with interspecies variability resulting from toxicodynamic and toxicokinetic differences between 

animals and humans (UFA), and a factor of 1 was selected to account for deficiencies in the 

database. A total UF of 10 (10 for UFH, 1 for UFA, and 1 for UFD) was used in the derivation of 

the RfD. Thus, the RfD is 1.4 mg/kg-day ÷ 10 = 0.1 mg/kg-day. 

The overall confidence in the RfD is medium to high. The RfD has been calculated for 

EGBE using a route-to-route extrapolation from the inhalation PBPK/BMC method used to 

derive the RfC. High confidence is placed in the RfD since pharmacokinetic differences 

between rats and humans were accounted for using a validated PBPK model (Corley et al., 1994, 

041977; Corley et al., 1997, 041984). There is high confidence in the NTP (2000, 196293) study 

because it was a chronic study, it employed both male and female rats and mice, it had a wide 

range of exposure levels, and animals were observed twice daily. There is medium-to-high 

confidence in the database, because data are available for a variety of animal species, including 

humans. While the database lacks long-term human studies, the available short-term human 

 111 
controlled studies, case reports, laboratory animal, and in vitro studies provide evidence to 

suggest that, with respect to the hemolytic effects of EGBE, long-term human exposures would 

be no more adverse than long-term rat exposures. Additionally, the selection of medium-high 

confidence is supported because the potential for effects in humans from repeated, long-term 

exposures has not been investigated. 

6.2.3. Cancer—Oral and Inhalation 

Information regarding the reported liver and forestomach tumors observed in laboratory 

animals exposed to EGBE indicates that the MOAs underlying these lesions are nonmutagenic 

and include intermediate processes that have nonlinear dose-response characteristics. Control or 

omission of these intermediate events would likely be sufficient to prevent the occurrence of 

such tumors in humans, including potentially sensitive subpopulations such as children. 

Application of nonlinear quantitative assessment methods indicate that the noncancer 

RfD (0.1 mg/kg-day) and RfC (1.6 mg/m3) values developed for EGBE are considered protective 

of these key events and would serve to prevent the occurrence of carcinogenic effects in 

humans.1

 The exposure concentrations that would be necessary to cause these effects in humans, 

if attainable at all, are likely to be much higher than the RfC and RfD. 

The estimation of uncertainty in this analysis is based on the alternative approaches for 

estimating the dose response that are discussed and shown in Section 5.1.4 and Appendix C. 

These alternatives include using the NOAEL approach combined with the measure of the 

internal dose estimated with the PBPK model and the BMC approach combined with the same 

PBPK model. Sections 5.1.3 and 5.2.3 summarize the uncertainty associated with each of these 

approaches for the RfC and RfD, respectively. Other uncertainties associated with the noncancer 

and cancer assessments presented in this Toxicological Review are summarized in Table 5-8 and 

in Sections 5.4.1 and 5.5. 

 

1

 These analyses are consistent with the nonlinear assessment approach described in the 2005 cancer guidelines (U.S. EPA, 2005a)

 112 
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APPENDIX A. SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC 

COMMENTS AND DISPOSITION 

The Toxicological Review of Ethylene Glycol Monobutyl Ether (U.S. EPA, 2008, 

597575) has undergone formal external peer review performed by scientists in accordance with 

U.S. EPA guidance on peer review (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 U.S. 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. Public 

comments that were received are summarized and addressed following the peer-reviewers 

comments and disposition. 

On April 10, 2008, U.S. EPA introduced revisions to the IRIS process for developing 

chemical assessments. As part of the revised process, the disposition of peer reviewer and public 

comments, as found in this Appendix, and the revised IRIS Toxicological Review was provided 

to the external peer review panel members for their comment on May 15, 2009. Any additional 

peer review panel comments received as part of this second review and U.S. EPA’s responses are 

included at the end of this Appendix. 

A.1. GENERAL CHARGE QUESTIONS: 

1. Is the Toxicological Review logical and clear? Has EPA accurately, clearly and 

objectively represented and synthesized the scientific evidence for noncancer and cancer 

hazard? 

Comments: 

In general, reviewers found the Toxicological Review to be logical and clear. In addition, 

they found that the review accurately, clearly, and objectively represented and synthesized the 

scientific evidence for noncancer and cancer hazard. 

More specifically, several reviewers provided suggestions to improve the clarity of the 

document and one reviewer questioned the quality of analytical procedures used in the critical 

study. 

 

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-1 
Two reviewers stated that the PBPK modeling of EGBE and BAA was not well 

illustrated or adequately described in the document and one reviewer felt that Figure 4-2 was not 

very informative. 

One reviewer commented that the low renal clearance of BAA relative to GFR needs to 

be discussed in more depth. The same reviewer posed several questions: is there data on plasma 

albumin binding of BAA and does it vary between rodents and humans? If the binding is less 

than ≈95%, it is unlikely to be the explanation of the restricted renal elimination. The alternate 

explanation of active reuptake by a carrier mechanism is amenable to study: is there relevant 

information in the literature? Resolution of this question is deemed crucial in understanding the 

factors that determine both the Cmax and AUC of BAA in the various species/sexes of rodents as 

well as the determination of HECs. 

One reviewer commented that the toxicokinetic discussion (Section 3.2) is, perhaps of 

necessity, somewhat diffuse and suggested a table incorporating some of the relevant kinetic 

information. 

Response: 

The suggestions for document improvement were incorporated into the Toxicological 

Review wherever possible and appropriate. Additional text has been added in Section 4.5 to 

clarify the effects of EGBE on RBCs and the possible pathophysiological mechanisms involved. 

Comments concerning the quality of the critical study were considered, but do not negate the 

positive findings nor preclude the use of study dose-response data. 

A brief summary of the existing PBPK models and rationale for use has been included in 

Section 3.2 as well as in Appendix B. We have eliminated Figure 4-2 as PBPK simulations for 

peak (Cmax) concentrations of EGBE, BAL, and BAA in the liver of male mice following 6-hour 

inhalation exposures to 250 ppm (the highest concentration used in the NTP bioassay) were 

adequately described in the text. 

The PBPK modeling paper by Corley et al. (1994, 041977) describes the basis for 

inclusion of plasma protein binding and renal clearance of BAA for male F344 rats and humans. 

The parameters for protein binding were assumed to be similar to phenolsulfophthalein and renal 

clearance optimized from in vivo pharmacokinetic studies that indicated that the elimination of 

BAA in urine by male F344 rats was saturable, leading to nonlinear increases in blood BAA Cmax

and AUC values at high dose levels. Parameters developed from rats were successfully scaled to 

humans as demonstrated by Franks et al. (2006, 100117), Jones et al. (2003, 100161), and Corley 

et al. (1994, 041977; 1997, 041984), for ‘low-dose’ inhalation and dermal exposures, and 

Gualtieri et al. (1995, 594443; 2003, 100140) for ‘high dose’ oral exposures (suicide attempt). 

In their 1998 publication, Lee et al. (1998, 041983) updated the model parameters using the in 

vivo toxicokinetic data for male and female, young and old, rats and mice. As identified by the 

reviewer, these processes are experimentally accessible. Thus, Corley et al. (2005, 100100) 

 A-2 
conducted a series of studies to determine the species, gender, and age-dependent tissue:blood 

partitition coefficients for BAA, plasma protein binding of BAA, metabolism of EGBE and 

BAA, and renal active transport of BAA. These parameters were used to update the PBPK 

model of Lee et al. (1998, 041983). The toxicokinetic summary (Section 3.2) has been revised to 

include additional information from Corley et al. (2005, 100100) who determined plasma protein 

binding, partition coefficients, and renal elimination of BAA in young vs. aged male and female 

rats and mice. 

A table of relevant toxicokinetic information has been included in Section 3.2 to 

summarize the information presented in the text. 

2. Please identify any additional studies that should be considered in the assessment of the 

noncancer and cancer health effects of EGBE. 

Comments: 

Most reviewers did not identify any additional studies that should be considered in the 

assessment of the health effects. However, a few reviewers did provide suggestions for 

supporting references and expanded discussions that are listed below. 

a. References on the subjects of thrombosis and infarction. 

b. References on the subjects of hematology instrument methodology differences and iron 

overload. 

c. References on the subject of the role of Kupffer cells in hepatotoxicity and 

carcinogenicity. 

d. While no specific references were supplied, one reviewer suggested an increased 

discussion on the MOA of EGBE on the RBC and its fate. 

e. An increased discussion on the subject of olfactory hyaline membrane degeneration. 

Response: 

a. The subject of thrombosis and infarction is briefly discussed in Section 4.4.1. Additional 

text has been added in Section 4.5 of the Toxicological Review including the suggested 

reference as well as others. The added references are as follows: 

Bevers, EM; Comfurius, P; van Rijn, JL; et al. (1982) Generation of prothrombinconverting activity and the exposure of phosphatidylserine at the outer surface of 

platelets. Eur J Biochem 122(2):429–436. (1982, 100088) 

Connor, J; Bucana, C; Fidler, IJ; et al. (1989) Differentiation-dependent expression of 

phosphatidylserine in mammalian plasma membranes: quantitative assessment of 

outer-leaflet lipid by prothrombinase complex formation. Proc Natl Acad Sci USA. 

86(9):3184–3188. (1989, 100097) 

Ezov, N; Levin-Harrus, T; Mittelman, M; et al. (2002) A chemically induced rat 

model of hemolysis with disseminated thrombosis. Cardiovasc Toxicol 2:181–194. 

(2002, 100113) 

 A-3 
Nyska, A; Maronpot, RR; Long, PH; et al. (1999b) Disseminated thrombosis and 

bone infarction in female rats following inhalation exposure to 2-butoxyethanol. 

Toxicol Pathol 27(3):287–294.(1999, 042746) 

Ramot, Y; Lewis, DA; Ortel, TL; et al. (2007) Age and dose sensitivities in the 

2-butoxyethanol F344 rat model of hemolytic anemia and disseminated thrombosis. 

Exp Toxicol Pathol 58(5):311–322. (2007, 100192) 

Yoshizawa, K; Kissling, GE; Johnson, JA; et al. (2005) Chemical-induced atrial 

thrombosis in NTP rodent studies. Toxicol Pathol 33(5):517–532. (2005, 100224) 

b. All of the references cited in the Toxicological Review pertaining to MCV used the 

impedance type analyzers (the more sensitive of the two types commented on). 

Therefore, no changes were made to the document. The references suggested on iron 

overload were reviewed. It was determined that the information in the papers would not 

add significantly to the outcome of the health assessment. 

c. Additional text and the suggested reference have been added to the Toxicological Review

on the role of Kupffer cells in hepatotoxicity and carcinogenicity in Section 4.5. The 

following reference was added: 

Roberts, RA; Ganey, PE; Ju, C; et al. (2007) Role of the Kupffer cell in mediating 

hepatic toxicity and carcinogenesis. Toxicol Sci 96(1):2–15. (2007, 100196) 

d. Additional text has been added to the Toxicological Review discussing the MOA of 

EGBE on the RBC and its fate in Section 4.5. 

e. Additional text and references have been added to the Toxicological Review discussing 

the effect of olfactory hyaline membrane degradation and the U.S. EPA’s rationale for its 

exclusion as a critical effect. The references added are as follows: 

Buckley, LA; Morgan, KT; Swenberg, JA; et al. (1985) The toxicity of 

dimethylamine in F344 rats and B6C3F1 mice following a 1-year inhalation 

exposure. Fundam Appl Toxicol 5(2):341–352. (1985, 062668) 

Nikula, KJ; Novak, RF; Chang, IY; et al. (1995) Induction of nasal carboxylesterase 

in F344 rats following inhalation exposure to pyridine. Drug Metab Dispos 

23(5):529–535. (1995, 100182) 

St. Clair, MBG; Morgan, KT. (1992) Changes in the upper respiratory tract. In Mohr, 

U; Dungworth, DL; Capen, CC; eds. Pathobiology of the Aging Rat, Vol. 1, ILSI 

Press; Washington, DC: pp. 111–127. (1992, 594426) 

3. Please discuss research that you think would be likely to increase confidence in the 

database for future assessments of EGBE. 

Comments: 

All reviewers provided research suggestions that may increase confidence in the database 

for future EGBE assessments. The following is a brief summary of these comments and 

suggestions: 

• Measure iron levels in the liver; 

 A-4 
• Do dose/response for oxidant-induced liver cell initiation and promotion; 

• Define the age of erythrocyte susceptibility to BAA; 

• Definition of the MOA of the toxic insult to the erythrocyte with definition of dose 

metrics; 

• Determine the loss of sidedness in the phospholipid composition of the outer leaflet of the 

red cell membrane to the premature loss of red cells in the circulation and the possibility 

that this may induce microvascular thrombosis and infarction; 

• Determine the role of the spleen vs. the liver in the removal of damaged red cells and 

effects on Hp and hemopexin levels and saturation during chronic exposure to EGBE; 

• Determine the time and dose/response relationships for hemosiderin deposition in the 

liver as a whole and in specific cell types; 

• Determine the MOA and dose/response relationships for hemosiderin in 

hemangiosarcoma development; 

• Experiments designed to determine the fate of BAA damaged RBCs and the role of 

spleen and liver macrophages; 

• Measure the phosphatidylserine levels on RBCs following exposure to BAA; 

• Experiments designed to better define the cellular effects of BAA exposure and their role 

in hemosiderin deposition and ROS toxicity; 

• Determination of the dose and temporal relationships between EGBE-induced 

hemosiderin and its induction of hemolysis; 

• Experiments designed to define the nature and dose response of effects seen in humans 

following moderate exposures; 

• Experiments designed to characterize the species differences in RBC membrane 

physiology; 

• Long term human inhalation and oral studies of EGBE; 

• Experiments designed to address the mechanism(s) by which BAA induces RBC 

hemolysis in various species; 

• Experiments designed to investigate the role of ROS in modulation of endothelial and 

hepatic cell gene expression; 

• In vivo studies designed to quantify the internal threshold doses that must be met to 

progress from one key precursor event in the mechanistic sequence to another; and 

 A-5 
• Experiments designed to determine the relative susceptibility of endothelial cells and 

hepatocytes to oxidative damage. 

Response: 

A number of research suggestions were received, and U.S. EPA agrees further research 

may enhance future risk assessments of EGBE. No additional changes to the Toxicological

Review are indicated at this time. 

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? 

Comments: 

All of the reviewers found Sections 5 and 6 to be well written and complete. A number 

of reviewers had comments on the selection of the factors that account for uncertainty (UFs). 

Additional suggestions included the following: (1) an alternative endpoint of olfactory 

epithelial degeneration; (2) Sections 5.1.3 and 5.2.3 be expanded for clarity; and (3) further 

analyses (Hazard Quotients and Hazard Indices) be performed for Section 6. 

Response: 

Specific comments on the values of the UFs are addressed in the response to charge 

question A5. 

The potential use of olfactory epithelial degeneration as an endpoint is discussed in 

Section 5. Additional references have been added that support the position that olfactory 

epithelial degeneration is not an adverse effect but an adaptive response. 

Substantial improvements were made to Section 5 that address the suggestion for 

improved clarity. Supporting text for alternative derivations has been moved to Appendix C to 

improve the readability of the document. 

Computing Hazard Quotients and Hazard Indices would require an exposure analysis, 

and while an portent component of risk assessment, is beyond the scope of this human health risk 

assessment. 

 A-6 
A.2. CHEMICAL-SPECIFIC CHARGE QUESTIONS: 

A.2.1. (A) Inhalation reference concentration (RfC) for EGBE 

A1. The 2-year inhalation study by the National Toxicology Program (NTP, 2000, 196293) 

was selected as the basis for the chronic inhalation RfC. Please comment on whether the 

selection of this study as the principal study has been scientifically justified. Has this study 

been 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. 

Comments: 

All reviewers agreed with the selection of the NTP (2000, 196293). No additional studies 

were identified for use as the principal study. A number of editorial comments were provided to 

improve the document. One reviewer commented that since humans are substantially less 

sensitive than rodents to the EGBE-induced hemolysis, they could be prone to other effects 

which are masked in the rodent study. He concludes with the statement that there does not seem 

to be any adequate alternative to the approach taken.

Response: 

Based on the favorable comments from the reviewers, no substantial changes were made 

to the Toxicological Review with respect to the selection of the principal study for the RfC. 

Suggested editorial comments have been reviewed and revisions were made to the Toxicological 

Review where appropriate. 

A2. The incidence of hemosiderin staining in the liver of male rats was selected as the 

critical effect because it is considered by EPA to be a precursor to an adverse effect. Please 

comment on whether 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 discussion. Please identify and provide the rationale 

for any other endpoints that should be considered in the selection of the critical effect. 

Comments: 

Five of the seven reviewers agreed with the conclusion that hemosiderin staining in the 

liver of male rats was the most appropriate choice for the critical effect. However, two of those 

five reviewers communicated reservations regarding this conclusion and discussed the 

deficiencies of its use. It was suggested that both hemosiderin and Hct, as parameters for POD 

purposes, be discussed and illustrated in the review. 

Two reviewers thought that alternate endpoints were more appropriate, such as RBC 

counts, Hct levels, MCV, or hyaline degeneration. 

 A-7 
Response: 

RfC and RfD derivations from all endpoints were considered and are now illustrated in 

Appendix C as well as summarized in Sections 5.1.4 and 5.2.4 of the assessment. The doseresponse assessments used in the derivation of the RfC and RfD are reviewed in Sections 5.1.2 

and 5.1.4, respectively. Based on the most recent information on the MOA for EGBE, 

hemosiderin deposition was selected as the critical effect. While the U.S. EPA recognizes that 

other hematological endpoints are biologically relevant, hemosiderin provides the best overall 

choice based on pathological considerations, dose-response relationships, and model fit. The 

support and rationale for the choice of the critical effect can be found in Sections 5.1, 5.2, 6.1, 

and 6.2. 

A3. Benchmark dose (BMD) modeling was applied to incidence data for hemosiderin 

staining in male rat liver to derive the point of departure (POD) for the RfC. Please 

provide comments with regard to whether BMD modeling is the best approach for 

determining the POD. Has the BMD modeling been appropriately conducted and 

objectively and transparently described? Has the benchmark response (BMR) selected for 

use in deriving the POD (i.e., 10% extra risk of hemosiderin staining in the liver) been 

scientifically justified, and transparently and objectively described? Please identify and 

provide the rationale for any alternative approaches for the determination of the POD and 

discuss whether such approaches are preferred to EPA’s approach. 

Comments: 

All reviewers agreed that BMD modeling was the best approach for determining the 

POD. However, alternatives were suggested for the choice of species and gender. One reviewer 

commented that male mice should have been used since they have shown correlation between the 

critical effect and the adverse effect (tumor). Two other reviewers commented that female rats 

should have been used since they have been shown to be more sensitive to the hemolytic effects 

and were used in the previous IRIS assessment. Lastly, one reviewer commented on the choice 

of 10% response level. This reviewer felt that a 5% response level is scientifically supported 

because it approximates the NOAEL. 

Response: 

Based on the majority of comments agreeing with the choice of gender and endpoint to 

model, no substantial changes to the Toxicological Review are indicated. Rationale for the 

selection of the species and gender are discussed in Section 5.3.9. Briefly, the male rat was 

chosen based on the NTP (2000, 196293) report showing male rats were the most sensitive 

species and gender with respect to the critical effect (hemosiderin deposition). In addition, the 

 A-8 
modeling of the data for male rats provided a much better fit than the modeling for the female rat 

data. As for the selection of the response rate, utilizing U.S. EPA guidance on BMD modeling 

of qualitative data 10% was chosen because it was within the range of experimental responses 

and appropriate for the power of the study. Additional justification has been added regarding the 

fact that this tumor response may not have been observed in the rats because the rats were 

exposed to lower doses and for a shorter duration of their average lifespan than the mice. 

A4. PBPK modeling was used to extrapolate the POD from rats to humans. Please 

comment on whether the PBPK modeling for interspecies extrapolation is scientifically 

justified, and transparently and objectively described in the document. Does the model 

properly represent the toxicokinetics of the species under consideration? Was the model 

applied properly? Are the model assumptions, parameter values, and selection of dose 

metrics clearly presented and scientifically supported? 

Comments: 

All reviewers that provided comments agreed that the PBPK modeling was the 

appropriate method for extrapolating the POD from rats to humans. Two reviewers commented 

that a comparison between the 1999 assessment and this one would be helpful. 

Response: 

Based on the comments, no change in the Toxicological Review is indicated. 

Comparisons to the previous assessment are made throughout the document in the appropriate 

sections. 

A5. Please comment on the selection of all of the uncertainty factors applied to the POD 

for the derivation of the chronic RfC. For instance, are they scientifically justified, and 

transparently and objectively described in the document? An UF of 10 for extrapolation 

from animals to humans (UFA) is generally applied when data are not available to inform 

potential pharmacokinetic (PK-UF) and pharmacodynamic (PD-UF) differences. In this 

assessment, an UFA of 1 was applied. 

• A PBPK model was used to inform pharmacokinetic differences and a PK-UF of 1 was 

selected. Please comment on whether this selection is scientifically justified. Is the 

rationale transparently and objectively described? Please comment on whether there are 

sufficient scientific data and support for the use of this PBPK model to estimate 

interspecies toxicokinetic differences and to replace the default interspecies factor for 

toxicokinetic differences (i.e., 101/2). 

• Evidence from human and animal in vitro and in vivo studies were used to inform 

pharmacodynamic differences and a PD-UF of 1 was selected. Please comment on 

 A-9 
whether this selection is scientifically justified. Is the rationale transparently and 

objectively described? Please comment on whether a higher value for the PD-UF should 

be used (e.g., to account for the limited information available on the potential for effects 

in human cell types other than red blood cells) or alternatively, should a lower (i.e., 

fractional) PD-UF be used (e.g., to account for the 40–150-fold difference in the 

concentrations that cause prehemolytic effects in human red blood cells (RBCs), 

including RBCs from potential susceptible populations such as the elderly, and patients 

suffering from anemia and RBC disorders that weaken the cellular membrane such as 

hereditary spherocytosis). 

Please identify and provide the rationale for any alternative approaches for the selection 

of the uncertainty factors. 

Comments: 

Several reviewers commented that the selection of factors to account for uncertainty 

(UFs) was not supported by the available science. 

a. Two reviewers concluded that the UFA should be <1. 

b. Two reviewers commented that the UFH should be <10. 

c. One reviewer concluded that the UFD was inconsistent with the confidence level. 

d. Most reviewers agreed that the selection of the UF values should be more clearly 

justified. 

Response: 

The current UFA value in the Toxicological Review is 1. The UFA accounts for a 

reduction from 3 for the toxicokinetic differences between animals and humans through the use 

of a PBPK model for extrapolation of doses. The toxicodynamic portion, likewise was reduced 

from 3 to 1. The toxicological effect in question being the deposition of hemosiderin is 

identified as a key event in the MOA for the development of liver hepatomas in mice. To 

implement a further reduction in the UFA (i.e., fractional) would logically indicate that a 

preponderance of data are available to describe this key event (toxicodynamics) in both animals 

and humans to the extent of describing why humans are less sensitive to the hemolytic effects 

leading to hemosiderin deposition. This is not the case, although in vitro data (Ghanayem, 1989, 

042014; Udden, 2000, 042110; Udden, 2002, 042111; Udden and Patton, 2005, 100213) do 

suggest humans are less sensitive than rodents to the hemolytic effects of EGBE. Likewise, the 

few human studies (Carpenter et al., 1956, 066464; Haufroid et al., 1997, 042040) indicate the 

same finding. However, these studies (Carpenter et al., 1956, 066464; Ghanayem, 1989, 

042014; Haufroid et al., 1997, 042040; Udden, 2000, 042110; Udden, 2002, 042111; Udden and 

 A-10 
Patton, 2005, 100213) do not characterize hemosiderin deposition, the key event for the MOA In 

addition, little is known of the long-term or repeated exposure responses in humans to EGBE. 

a. There is very limited information on the sensitivity of various human subpopulations to 

the hemolytic effects of EGBE. In addition, the long-term and repeated exposure effects 

in potentially sensitive populations remain to be determined. Therefore, the default UFH

of 10 is appropriate. This subject is discussed in Section 5.2.3. 

b. Response to this comment can be found in question A6 below. 

c. Additional text has been added to Sections 5.1.3 and 5.2.3 for improved justification of 

the chosen UFs; the sections were reformatted for improved clarity. 

A6. Please comment specifically on the database uncertainty factor of 1 applied in the RfC 

derivation. Are the criteria and rationale for the selection of the database uncertainty 

factor transparently and objectively described in the document? Please comment on the 

body of information regarding the hemato and hepatic toxicity of EGBE and the use of the 

toxicokinetic data in the determination of the database uncertainty factor. Please comment 

on whether the selection of the database uncertainty factor for the RfC has been 

scientifically justified. Has this selection been transparently and objectively described in 

the document? 

Comments: 

The majority of reviewers commented that the UFD value was appropriate. One reviewer 

commented that the value was inconsistent with the U.S. EPA’s confidence level of medium to 

high. 

Response: 

Based on the reviewers comments, no change in the document is indicated. The value 

assigned for the database UF is based on the completeness of the database in terms of 

toxicological studies assessing the range of likely potential effects including reproductive and 

developmental effects as well as information from more than one species. 

A.2.2. (B) Oral reference dose (RfD) for EGBE 

B1. A conclusion was reached that the available oral toxicity data are inadequate to 

support derivation of a chronic oral RfD value. Is the rationale for not developing an RfD 

from the available database of oral studies transparently and objectively described? If 

other oral studies are identified that would be suitable for the derivation of the RfD, please 

identify and provide the rationale for their use. 

 A-11 
Comments: 

All reviewers commented that the 91-day drinking water study should be considered in 

the RfD development and that the justification for not using it to at least derive comparative RfD 

estimates was inadequate. 

Response: 

Currently, there are no chronic oral studies of EGBE and very limited sub-chronic 

studies. The larger database of inhalation studies, including 2 year studies that identify chronic 

endpoints (e.g. hemosiderin deposition), supports the use of a route-to-route extrapolation using 

the chronic inhalation study and PBPK modeling to produce an RfD estimate. The 91-day 

drinking water study has been included in Chapter 5 for comparison purposes and Section 5.2.1 

was amended to include a more concise rationale for the exclusion of the oral study in 

determining the RfD. Alternative endpoints including the endpoint selected for the previous 

assessment are presented graphically and discussed in a new section (5.2.4). 

B2. A route-to-route extrapolation was performed to derive the chronic RfD, using the 

chronic inhalation study and PBPK modeling. The Human Equivalent Concentration 

(HEC) was based on a continuous oral exposure to EGBE in drinking water that would 

yield the same AUC for the metabolite BAA (in the arterial blood over three months) as 

that estimated for the rat following an external inhalation exposure to EGBE at the level of 

the proposed POD (i.e., the BMCL10). Please comment on whether the PBPK model is 

adequate for use to conduct a route-to-route extrapolation for EGBE to derive an RfD in 

the absence of adequate oral animal or human dose-response data to derive the RfD 

directly. Was the extrapolation correctly performed and objectively and transparently 

documented? 

Comments: 

Five of seven reviewers agreed with the extrapolation. Two reviewers abstained from 

commenting citing their lack of expertise in this area. Additionally, it was noted by two 

reviewers that while the extrapolation was correctly performed, it would not be necessary if the 

drinking water study were used. 

Response: 

Based on the reviewer’s comments, no change in the Toxicological Review is indicated. 

 A-12 
B3. Please comment specifically on the database uncertainty factor of 1 applied in the RfD 

derivation. Are the criteria and rationale for the selection of the database uncertainty 

factor transparently and objectively described in the document? Measured internal doses 

in rats and a human PBPK model were used to perform a route-to-route extrapolation to 

derive the RfD. Please comment on the use of the PBPK model and the inhalation database 

in the determination of the database uncertainty factor for the RfD. Please comment on 

whether the selection of the database uncertainty factor for the RfD has been scientifically 

justified. Has this selection been transparently and objectively described in the document? 

Comments: 

There was general agreement among the reviewers with the selection of the database UF 

for the RfD. One reviewer commented that the explanation of the rationale for the UF selection 

should be improved. 

Response: 

Based on the reviewer’s comments, the explanation of the rationale for the UF selection 

has been revised for improved transparency in the Toxicological Review where indicated. 

A.2.3. (C) Carcinogenicity of EGBE 

C1. Under the EPA’s 2005 Guidelines for Carcinogen Risk Assessment 

http://www.epa.gov/cancerguidelines/, the Agency concluded that EGBE is not likely to be 

carcinogenic to humans at expected exposure concentrations. Please comment on the 

scientific justification for the cancer weight of evidence characterization and describe the 

basis for your view. Has the scientific justification for the weight of evidence descriptor 

been sufficiently, transparently and objectively described? 

Comments: 

Four of seven reviewers indicated that they agreed with the Agency’s conclusion 

regarding the cancer descriptor for EGBE. One reviewer abstained from commenting while 

another reviewer only commented on the wording of the cancer descriptor. One reviewer 

commented that the cancer descriptor was carefully explained. Two reviewers suggested the 

phrase “at expected exposure concentrations” should either be deleted or better defined with 

exposure concentrations encountered. These reviewers provided a possible revision as follows: 

“at the calculated RfC and RfD values presented in this document.” 

 A-13 
Response: 

Because all reviewers that commented agreed with the Agency conclusion that EGBE is 

not likely to be carcinogenic to humans at expected exposure concentrations, qualitatively this 

conclusion was not revised. As suggested by two of the reviewers on the External Peer Review 

Panel, the specific text to the weight of evidence descriptor was revised to account for their 

suggested language. Lastly, all reviewers that commented, responded favorably concerning the 

scientific justification for the cancer weight of evidence descriptor, thus, no revisions were 

indicated for the document. 

C2. EPA has proposed a mode of action (MOA) for male mouse liver cancer involving 

metabolism, hemolysis of RBCs, hemosiderin deposition in the liver, oxidative damage and 

proliferation leading to tumor induction as key events best supported by the data. Please 

provide detailed comments on whether this analysis regarding the MOA for liver cancer is 

scientifically sound, and transparently and objectively described in the Toxicological 

Review. Considerations include the scientific support regarding the plausibility for the 

hypothesized MOA and the characterization of uncertainty regarding this MOA. 

Comments: 

In general, the reviewers agreed with the conclusions of the analysis regarding the MOA 

for liver cancer, although several reviewers provided additional scientific considerations 

regarding the MOA. Most notably, remarks regarding hemosiderin deposition include: (1) Why 

does hemosiderin deposition not occur in the spleen?; (2) Tumor induction from initiated cells is 

speculation; (3) If male mice represent the most sensitive species for tumor formation due to 

exposure to EGBE, why is a more marked dose response for hemosiderin deposition observed in 

female mice?; and (4) Two reviewers commented that hemosiderin deposition was a biomarker 

of exposure, not effect. 

Response: 

Responses to this charge question will be addressed in the order in which they were 

presented in the comment overview (above): (1) One reviewer inquired as to why hemosiderin 

deposition does not occur in the spleen. NTP (2000, 196293) reported hemosiderin deposition 

does occur in the spleen for which these data are presented in Table 4-5; (2) Tumor induction 

from initiated cells is not speculated within the Toxicological Review. With respect to the 

hypothesized MOA for forestomach tumors, the MOA is the same as that proposed for 

hepatocellular tumors observed in male mice (2000, 196293). Within this MOA, the occurrence 

of initiated cells is not specifically addressed but could occur from two of the key events 

including oxidative stress and cytotoxicity. Although data do not currently exist for EGBE, the 

possibilities also include epigenetic pathways that could lead to the formation of initiated cells. 

 A-14 
A key point regarding the hypothesized MOA is that the key events represent obligatory (rate 

limiting) steps or biomarkers of such events leading to the formation of tumors, but do not 

represent a complete description of the biologic mechanisms that lead to tumor development; 

(3) Hemosiderin deposition observed was indeed greater in female compared to male mice (NTP, 

2000, 196293; see table 4-5). NTP has observed liver hemangiosarcomas in 105/4,183 (2.51%) 

male versus just 35/4,177 (0.84%) female historical controls (Klaunig and Kamendulis, 2005, 

100165; NTP, 2000, 196293). In addition, other chemicals reported by NTP to cause both early 

onset hemosiderin buildup and liver tumors have also exhibited this male specificity (U.S. EPA, 

2005, 594432). While the reason for the sex difference in liver tumor susceptibility between 

male and female mice is not clear, it has been shown that estrogens can be protective through 

their antioxidant capacities and through their modulation of the activities of other antioxidants 

(Nyska et al., 2004, 056379; Section 4.6.3). Given the significant mortality of the male mice in 

the study, the doses received by the female mice may not have represented a sufficient dose to 

induce tumors (i.e., maximum tolerated dose); and (4) Hemosiderin deposition is a key event in 

the MOA for the formation of liver tumors in male mice. As stated previously, a key event 

represents an obligatory (rate limiting) step or a biomarker of such an event. This would not 

preclude the use of hemosiderin deposition as a biomarker of such an event, as long as a doseresponse and temporal relationship are observed to indicate it as an obligatory step in the MOA. 

C3. EPA has proposed a MOA for female mouse forestomach tumors involving 

metabolism, irritation and regenerative proliferation leading to tumor induction as key 

events best supported by the data. Please provide detailed comments on whether this 

analysis regarding the MOA for forestomach tumors is scientifically sound, and 

transparently and objectively described in the Toxicological Review. Considerations 

include the scientific support regarding the plausibility for the hypothesized MOA and the 

characterization of uncertainty regarding this MOA. 

Comments: 

Five of seven reviewers generally agreed with the conclusions of the analysis regarding 

the MOA for forestomach tumors. One reviewer abstained from commenting while another 

focused their comments on editorial aspects stating that the discussion on the MOA was too 

speculative and that steps 5 and 6 should be deleted. A few reviewers provided comments to 

refine the supporting text and conclusions for the MOA. 

Response: 

The overall conclusions regarding the MOA for forestomach tumors were not modified. 

Editorial and clarification changes to refine the supporting text were made to the document. 

 A-15 
While step 5 of the proposed sequence of events leading to forestomach tumors seems 

redundant, it is necessary to illustrate that the development of tumors is not a single exposure 

scenario but requires a repeated cycle of injury and repair. Regarding step 6, EGBE-specific 

evidence of ROS modulation in endothelial cells and hepatocytes is a biologically plausible step 

within the hypothesized MOA, even though it is not definitively supported. It is important to 

note that key events within the MOA may have different levels of supporting scientific evidence. 

C4. EPA has not proposed a MOA for the female rat pheochromocytomas of the adrenal 

medulla. NTP rated the female rat pheochromocytomas as providing equivocal evidence of 

carcinogenic activity and the pathology report expressed concern as to whether the 

observed tumors met the criteria used to diagnose pheochromocytomas. For these reasons, 

this tumor was not given significant weight in the qualitative or quantitative assessment of 

EGBE cancer potential. Please provide detailed comments on whether this analysis 

regarding the female rat pheochromocytomas is scientifically sound, and transparently and 

objectively described in the Toxicological Review. Please comment on whether and the 

extent to which the female rat pheochromocytomas are adequate to support alternative 

analyses of qualitative and quantitative cancer risks to humans and discuss approaches to 

consider if such analyses are warranted. 

Comments: 

Six of seven reviewers generally agreed with the conclusions of the analysis regarding 

female rat pheochromocytomas. No dissenting scientific opinions were submitted that would 

warrant alternative analysis. One reviewer did suggest a possible extension of the NTP 

conclusion that pheochromocytomas represented equivocal findings. This reviewer also 

suggested the possibility of including a linear low-dose extrapolation for this tumor type as an 

academic exercise to explore the range of possibilities but did not recommend its use for 

determining risk. One reviewer abstained from commenting. 

Response: 

As stated in the charge question, U.S. EPA has not proposed a MOA for the female rat 

pheochromocytomas of the adrenal medulla. NTP concluded that the data for the female rat 

pheochromocytomas provided equivocal evidence of carcinogenic activity and the pathology 

report expressed concern as to whether the observed tumors met the criteria used to diagnose 

pheochromocytomas. For these reasons, this tumor was not given significant weight in the 

qualitative or quantitative assessment of EGBE cancer potential. 

Thus, cancer quantification using a linear low-dose extrapolation for pheochromocytomas 

was not included in the Toxicological Review. 

 A-16 
C5. Please comment on the choice of the nonlinear threshold approach for the quantitative 

assessment of the carcinogenic potential of EGBE. Please comment on whether this 

approach is scientifically sound, and transparently and objectively described. Please 

comment on whether the example calculations using linear low-dose extrapolation for 

cancer as discussed in Section 5.4.1 represent useful characterizations of the potential 

quantitative uncertainty associated with exposure to EGBE. Please comment on whether 

the linear analysis should be presented as an alternative to the threshold approach 

considering the Agency conclusion that EGBE is not likely to be carcinogenic to humans at 

expected exposure concentrations. 

Comments: 

Six of seven reviewers that commented supported the choice of the nonlinear threshold 

approach. Most reviewers found the presentation of the linear low-dose extrapolation to be 

informative for comparative purposes, but two reviewers felt it should not be presented as an 

alternative to the threshold approach. 

Response: 

Based on the majority of comments, no change in the Toxicological Review is indicated. 

A.3. PUBLIC COMMENTS 

Comment: 

The use of hemosiderin staining as the critical effect for RfC and RfD development is not 

biologically appropriate and is inconsistent with the available dose-response data. 

Response: 

The use of hemosiderin staining as the critical effect for RfC and RfD development has 

been peer reviewed by a panel of experts and found to be appropriately used. While some 

increase in hemosiderin accumulation can be expected with advancing age, excessive 

accumulation beyond that contributed by age is considered pathological (Muller et al., 2006, 

597291). While the hemolytic effects appeared to be among the earliest effects from EGBE 

exposure, the hemosiderin deposition endpoint was selected as the critical effect. This effect was 

found to occur in both species and genders of animals tested, with rats being the more sensitive 

species; the effect also occurred in the 14-week subchronic NTP inhalation study. The suggested 

MOA of EGBE-induced liver effects is based on the observation that the hemolytic effects led to 

compensatory erythropoiesis and significant increases in blood degradation products, including 

an increased accumulation of hemosiderin in the liver Kupffer cells of EGBE-exposed animals. 

The hemosiderin accumulation seen in the Kupffer cells was found to increase in severity with 

 A-17 
increasing dose and exposure duration (Table 5-2), unlike the hemolytic endpoints, such as 

decreased Hct, which did not progress from 3 to 12 months (Table 4-6). Thus, hemosiderin 

deposition in Kupffer cells in the rat liver is believed to be a sequela to the hematologic effects. 

Because of the progression of this effect with chronic exposure, hemosiderin is deemed to be the 

most sensitive effect (Section 5.1.1). Additional support for the use of hemosiderin, as opposed 

to the various hematological parameters, is the location of the effect. Kupffer cell accumulation 

of hemosiderin in the liver is consistent with the pathological findings of liver 

hemangiosarcomas and hepatocellular carcinomas found in EGBE-exposed male mice. 

Comment: 

The use of male rat data as the POD is inappropriate because the available data 

convincingly demonstrate that female rats are more sensitive to the hemolytic effects of EGBE. 

Response: 

U.S. EPA agrees that female rats are more sensitive to the hemolytic effects of EGBE. 

However, hemolysis is one step in a complex process leading to the critical endpoint, 

hemosiderin accumulation. Male rats have been shown to be more sensitive to hemosiderin 

accumulation than females (NTP, 2000, 196293). The fit statistics and BMC information 

derived from the dichotomous models available in the BMD software as applied to the male and 

female rat hemosiderin staining data versus AUC BAA are shown in Table 5-5. All models were 

fit using restrictions and option settings suggested in the U.S. EPA BMD technical guidance 

document (U.S. EPA, 2000, 052150). The best model fit to these data, as determined by visual 

inspection, examination of low dose model fit (i.e., scaled residual for the dose group closest to 

the BMD), and comparison of overall fit (i.e., AIC values) was obtained using a multistage 

model (1st degree) for the male response data and a Log-Logistic model for the female response 

data. The male rat BMC10 was 196 μmol-hour/L and the BMCL10 was determined to be 133 

μmol-hour/L, using the 95% lower confidence limit of the dose-response curve expressed in 

terms of the AUC for BAA in blood. The BMC10 and BMCL10 values for the female rat were 

determined to be 425 and 244 μmol-hour/L, respectively. Assuming continuous exposure (24 

hours/day), the Corley et al. (1997, 041984) PBPK model was used to back-calculate HECs of 

3.4 ppm (16 mg/m3

) from the male rat data and 4.9 ppm (24 mg/m3) from the female rat data 

(Section 5.1.2.1). If you combine the males and females for the BMC analysis, the result is 

<10% of the original BMCL10 for males alone. This indicates that there is an inconsistency with 

at least one of the data sets in the female study. Based on all the information, the use of male 

rats in the assessment is warranted. 

Comment: 

 A-18 
The choice of dose metric, and the application of PBPK modeling in the derivation of 

candidate RfCs based on hematological data should be re-examined. The selection of an internal 

dose metric to be used in the dose-response assessment using the incidence of hemosiderin 

staining is not adequately justified. 

The Lee et al. (1998, 041983) model has been incorrectly applied in the derivation of the 

Cmax BAA values reported for female rats in Table 5-4. 

Similar discrepancies (current draft vs. 1999 document) are evident in the BMD 

modeling based on hematological endpoints. 

Response: 

The AUC for BAA in arterial blood was selected as the appropriate measure of dose due 

to evidence for a causal association between BAA and hemolysis, between hemolysis and the 

accumulation of hemosiderin in the liver, and between hemosiderin accumulation in the liver and 

increased incidence of liver hemangiosarcoma. AUC is considered to be a more appropriate 

response measure because hemosiderin pigmentation increases in incidence and severity with 

increased duration (subchronic to chronic) and is believed to be the result of the cumulative 

exposure to EGBE/BAA as opposed to a peak exposure. However, for comparative purposes, a 

BMD analysis was done on the critical endpoint to determine the impact that choosing Cmax of 

BAA in blood rather than AUC of BAA in blood would have had on the BMCL10(HEC) derivation. 

If Cmax had been used as the dose measure, the BMCL10(HEC) value would have been 39 mg/m3, 

approximately 2.4-fold higher than the 16 mg/m3 BMCL10(HEC) value derived using AUC as the 

dose measure (Section 5.3.2). The choice of hemosiderin as the critical effect warrants the use of 

AUC as the dose metric. 

The reviewer is correct; the arterial blood BAA Cmax estimates for female rats reported in 

Table 5-4 were in error. Table 5-4 was revised using the most recent PBPK model of Corley et 

al. (2005, 100100), which replaced several of the assumptions utilized by Lee et al. (1998, 

041983) with measured values for protein binding, partition coefficients, metabolism, and renal 

clearance. For comparison, the Lee et al. (1998, 041983) simulations along with the simulations 

using the Corley et al. (2005, 100100) model are as follows: 

 Exposure Female rat 

 concentration body weight Cmax BAA in arterial blood (μM) 

 (ppm) (g) Lee et al. (1998, 041983) Corley et al. (2005, 100100) 

 31.25 216 285 167 

 61.5 211 603 408 

 125 214 1,243 1,091 

 250 210 1,959 2,752 

 500 201 4,227 6,483 

 A-19 
As the reviewer pointed out, these revised Cmax estimates are now consistent with the 

peak blood concentrations of BAA observed following 3 months of exposure to the three 

concentrations used in the NTP chronic inhalation bioassay (31.25, 61.5, and 125 ppm) reported 

by Dill et al. (1998, 041981). Table 5-3 and Appendix A were also revised to include a 

description of the Corley et al. (2005, 100100) PBPK model. 

The BMD calculations (Table 5-5) were revised using the Corley et al. (2005, 100100) 

estimates of arterial blood BAA Cmax values for female rats as described above. The HEC 

calculations were again based upon the Corley et al. (1997, 041984) model. 

Comment: 

The use of inhalation data to derive the RfD is inappropriate. The current IRIS Review 

of EGBE (U.S. EPA, 1999, 597365) http://www.epa.gov/ncea/iris/toxreviews/0500tr.pdf selects 

a 91-day drinking water study in rats as the principal study for the derivation of the RfD. 

Because information available from chronic inhalation studies with EGBE indicates that the 

primary effect, hematological changes, does not become more severe with prolonged exposures, 

the 91-day drinking water study in rats remains the appropriate POD for deriving the RfD. Use 

of this study would preclude the need for a route-to-route extrapolation from chronic inhalation 

data, with the substantial errors that can be associated with this type of analysis (Pauluhn, 2003, 

100188). 

In evaluating the results of the route-to-route extrapolation, a comparison to what is 

observed in the available oral studies is critical to determine whether the modeling results are 

realistic or whether, instead, the modeling results contribute additional uncertainty to the derived 

RfD. The route-to-route extrapolation in the draft IRIS Review is based on the observation of 

male rat liver hemosiderin staining from the NTP (2000, 196293) inhalation study. With that 

data set as the POD, PBPK modeling is used to derive the proposed new RfD of 0.14 mg/kg-day. 

In comparing this RfD to the results from the 13-week oral study in rats (NTP, 1993, 042063), a 

significant increase in hemosiderin staining was not observed in male rats until doses of 452 

mg/kg-day were achieved or in female rats until a dose of 281 mg/kg-day was achieved 

(Table 4-1). In addition, significant changes in hematological endpoints were observed at lower 

doses (69 mg/kg-day). The doses associated with significant increases in hemosiderin staining 

are orders of magnitude above the POD of 1.4 mg/kg-day, suggesting that the use of the route-toroute endpoint is not appropriate. Additional analyses are needed to determine why a difference 

in response by route of exposure would be observed. 

While route-to-route extrapolation is a valuable tool for chemicals for which no adequate 

study is available for a selected route of exposure, in the case of EGBE, an adequate oral study is 

available and should be used to derive the RfD. 

 A-20 
Response: 

Hematological endpoints indicative of hemolysis (e.g., RBC count) do not progress with 

increasing duration of inhalation exposure, whereas the incidence of hemosiderin pigmentation 

did progress considerably with chronic exposure (Table 5-2). A primary consideration with 

respect to the NTP (1993, 042063) study was that it did not provide a sufficient duration of 

exposure to EGBE to allow for maximal accumulation of hemosiderin deposition. In contrast, 

hemosiderin accumulation was one of the most sensitive effects observed in the NTP (2000, 

196293) chronic inhalation study of EGBE. Furthermore, uncertainty regarding the mechanism 

of action of EGBE does not allow for a biologically-informed determination regarding which 

hematological endpoints—changes in RBC count, reticulocyte count, MCV, Hct, and Hb—

observed in EGBE-exposed animals should be used to derive an RfC and, in the case of BMD 

analysis, what a proper BMR level should be for the BMD derivation. Finally, hematologic 

effects signified by changes in RBC count, reticulocyte count, MCV, HCT, and Hb are 

considered precursor effects to the pathological finding of hemosiderin deposition (Section 4.5). 

For these reasons and because the hemolytic effects of EGBE appear to be consistent between 

oral and inhalation routes of exposure, an RfD has been derived via the application of a PBPK 

model to perform a route-to-route extrapolation (Section 5.2.2) from the BMD analysis (Section 

5.1.2) of hemosiderin pigmentation observed in the NTP (2000, 196293) chronic inhalation study 

of EGBE. BMD/NOAEL analyses of hematologic endpoints and hemosiderin pigmentation 

observed in the oral NTP (1993, 042063) subchronic study are provided below for comparison 

purposes. 

The 91-day drinking water study has been included in Chapter 5 for comparison 

purposes. Alternative endpoints including the endpoint selected for the previous assessment are 

presented graphically and discussed in a new section (5.2.4). Inhalation studies considered for 

derivation of the RfC were used to supplement the oral database using the route-to-route 

extrapolation (Section 5.2.2.1). 

Comment: 

The intrahuman and interspecies UFs applied in the derivation of the RfC and RfD are 

greatly overprotective and should be reevaluated. The draft IRIS Review repeatedly (e.g., pages 

50, 59, and 109) mentions that humans are much less sensitive to the toxic effects of EGBE than 

are the rodents that provide the basis for derivation of the RfC and RfD. These differences in 

sensitivity are not simply due to pharmacokinetic differences (which are addressed in the PBPK 

modeling), but differences in inherent sensitivity (pharmacodynamics) as illustrated by the 

marked differences in sensitivity of human and rodent blood cells in vitro to EGBE and BAA. 

For example, the work of Udden (2002, 042111) demonstrates about a 150-fold greater 

sensitivity of rat blood cells than human blood cells to the effects of BAA on RBC deformability, 

osmotic fragility, and hemolysis. Even potentially hypersensitive subgroups of the human 

 A-21 
population (the elderly, and patients with sickle cell disease or hereditary spherocytosis) show 

similar resistance to these effects of BAA (Udden, 2002, 042111). 

Given the consistent, substantial difference in sensitivity between human and rats and the 

data that is the basis for the RfD and RfC, there is no scientific justification for using a 

pharmacodynamic UF as large as 1. The weight of the evidence supports the use of a fractional 

value of perhaps 0.01, or even less. 

Furthermore, the default UF of 10 for human variability is not needed. This factor is 

typically applied to account for variations in human sensitivity or to be protective of sensitive 

subpopulations. In this case, however, typical hypersensitive subgroups, such as the elderly, also 

show resistance to the hematological effects of BAA, as do individuals with disease conditions 

(patients with sickle cell disease or hereditary spherocytosis) that might be expected to make 

them more sensitive (Udden, 2002, 042111). While animal studies suggest that older animals are 

more sensitive than neonates, and females are more sensitive than males, these have been shown 

to be a reflection of differences in pharmacokinetics, not pharmacodynamics or sensitivity 

(Corley et al., 2005, 100100). Based on these findings, the proposed 10-fold intraspecies UF is 

clearly excessive. For further information, see the response for charge question A5. 

Response: 

The current evidence indicates that the human RBC response to EGBE exposure is 

considerably less than that of rodents. However, this information comes from a relatively small 

number of studies. The current UFA value in the Toxicological Review is 1 and considers the 

limited number of studies available as well as the unknown effect of EGBE on the cellular events 

leading to hemolysis in human populations. In addition, little is known of the long-term or 

repeated exposure responses in humans to EGBE. 

The UF for human variation has been assigned a value of 10. Some studies have shown 

that in vitro exposure of RBCs from proposed sensitive populations, such as the elderly and those 

with iron handling diseases, produces similar effects as in the general population. However, the 

database is small and represents only acute or short-term exposures. The effects of longer or 

repeated exposures to the general population as well as the proposed sensitive populations 

remain to be determined. Therefore, the use of the default value for UFH is justified. 

Comment: 

There are several problems with BMD modeling. While benchmark modeling is the most 

scientifically appropriate approach for determining the POD using the available noncancer data 

for EGBE, there are several problems in the implementation of the procedure. The additional 

documentation of the modeling results provided in Appendix B of the draft has several 

deficiencies. The output provided in Appendix B for the hemosiderin modeling does not use the 

AUC doses so is not an example of the output used to derive the RFC. Specifically, the 

 A-22 
multistage model output labeled “BMD Method for RFC: Hemosiderin deposition in male rats 

versus AUC BAA, 2-year inhalation study (NTP, 2000, 196293)” in Appendix B does not use 

the doses indicated in Table 5-6 as the AUC doses. The BMD and BMDL in this output are not 

the values reported in Table 5-7. The same is also true of the log-logistic output labeled “BMD 

Method for RfC: Hemosiderin deposition in female rats versus AUC BAA, 2-year inhalation 

study, (NTP, 2000, 196293)” in Appendix B. The BMD and BMDLs reported in these outputs 

are not given anywhere else in the document. In addition, in Table 5-7, the female rat multistage 

(1-stage) output has the BMDL in the BMD column and an incorrect number in the BMDL 

column (source unknown). 

Response: 

The reviewer is correct that the outputs of the BMD modeling do not match the cited 

source studies. Older BMD outputs were inadvertently placed in the document appendix while 

the correct values incorporated into the text. The BMD calculations have been revised and the 

corrected outputs placed in the appendix. 

A.4. ADDITIONAL EXTERNAL PEER REVIEW PANEL COMMENTS – SECOND 

REVIEW IN RESPONSE TO REVISIONS AS INDICATED ABOVE 

A.4.1. General Charge Questions 

Comment: 

One reviewer questioned the replacement of the term “adverse” with the terms 

“significant” and “biologically significant” throughout the text. 

Response: 

The term “adverse” was generally replaced throughout the text to provide a more 

meaningful description. Many of the instances where “biologically significant” was used in the 

document has been replaced with the words “statistically significant.” 

A.4.2. Chemical-Specific Charge Questions 

(A) Inhalation reference concentration (RfC) for EGBE 

A2. 

Comment: 

One reviewer commented that hyaline membrane degeneration could be regarded as a 

precursor lesion and, while not suggesting that this is a suitable critical endpoint in isolation, it 

might be a useful endpoint for quantitative comparison with other endpoints. 

Response: 

 A-23 
The potential use of hyaline membrane degeneration as endpoints was considered and 

discussed in chapter 5 of the Toxicological Review. Based on the available literature, U.S. EPA 

maintains the position that olfactory hyaline membrane degeneration is not suitable as a critical 

endpoint. Based on the available literature for EGBE, U.S. EPA concludes that hyaline 

membrane degeneration represents an adaptive response and not an adverse effect. Any 

quantitative comparison between hyaline membrane degeneration and hemosiderin deposition 

would therefore not be appropriate for comparative purposes. 

Comment: 

One reviewer felt the discussion on hemolytic anemia in Section 4.5 still required 

revisions and provided alternative text. Specifically, the discussion on osmotic fragility and its 

relationship to EGBE-induced hemolytic anemia required clarification. 

Response: 

The alternative text suggested by the reviewer has been incorporated into the discussion 

on hemolytic anemia in Section 4.5 and the Toxicological Review was reviewed for other 

occurrences of the phrase “osmotic fragility”. The alternative text clearly discusses the available 

information and limitation of what is known about the mechanism of EGBE-induced hemolytic 

anemia. 

A3. 

Comment: 

One reviewer questioned the selection of male rats as the most sensitive gender when the 

data demonstrate that females are more sensitive to the hematological effects of EGBE and 

display an increased incidence of hemosiderin deposition. 

Response: 

It is agreed that female rats are more sensitive to the hemolytic effects of EGBE. 

However, hemolysis is one step in a complex process leading to the critical endpoint,, 

hemosiderin accumulation. While the data from the subchronic study show females as more 

sensitive, it is the 2-year chronic study that is the focus of our analysis. In that study, male rats 

were shown to be more sensitive to hemosiderin accumulation than females (NTP, 2000, 

196293). In addition, the cumulative blood concentration of the active metabolite BAA was 

chosen as the internal dose metric, as opposed to the exposure concentration of EBGE. The fit 

statistics and BMC information derived from the dichotomous models available in the BMD 

software as applied to the male and female rat hemosiderin staining data versus AUC BAA are 

shown in Table 5-5. All models were fit using restrictions and option settings suggested in the 

U.S. EPA BMD technical guidance document (U.S. EPA, 2000, 052150). The best model fit to 

 A-24 
these data, as determined by visual inspection, examination of low dose model fit (i.e., scaled 

residual for the dose group closest to the BMD), and comparison of overall fit (i.e., AIC values) 

was obtained using a multistage model (1st degree) for the male response data and a LogLogistic model for the female response data. The male rat BMC10 was 196 μmol-hour/L and the 

BMCL10 was determined to be 133 μmol-hour/L, using the 95% lower confidence limit of the 

dose-response curve expressed in terms of the AUC for BAA in blood. The BMC10 and BMCL10

values for the female rat were determined to be 425 and 244 μmol-hour/L, respectively. 

Assuming continuous exposure (24 hours/day), the Corley et al. (1994, 041977) PBPK model 

was used to back-calculate concentrations for the HECs of 3.4 ppm (16 mg/m3) from the male rat 

data and 4.9 ppm (24 mg/m3) from the female rat data (Section 5.1.2.1). If the data for males and 

females are combined for the BMC analysis, the result is <10% of the original BMCL10 for 

males alone. This indicates that there is a inconsistency with at least one of the data sets in the 

female study. Based on all the information, the use of male rats in the assessment is warranted. 

A5. 

Comment: 

One reviewer continued to question the use of a UFA of 1 when in their view the 

scientific data support a UFA <1. 

Response: 

The basis for selection of a UFA of 1 is presented in the EPA response to the comment on 

charge question A5. A new rationale has not been provided by the reviewer to alter the U.S. 

EPA’s selection of this value. The current UFA value in the Toxicological Review is 1. The UFA

accounts for a reduction from 3 for the toxicokinetic differences between animals and humans 

through the use of a PBPK model for extrapolation of doses. Likewise, the toxicodynamic 

portion was reduced from 3 to 1. The toxicological effect in question being the deposition of 

hemosiderin is a key event of the MOA for the development of liver hepatomas in mice. To 

implement a further reduction in the UFA (i.e., fractional) would logically indicate that a 

preponderance of data are available to fully describe this key event (toxicodynamics) in both 

animals and humans to the extent of describing why humans are less sensitive to the hemolytic 

effects leading to hemosiderin deposition. This is not the case, although in vitro data 

(Ghanayem, 1989, 042014; Udden, 2000, 042110; Udden, 2002, 042111; Udden and Patton, 

2005, 100213) do suggest humans are less sensitive than rodents to the hemolytic effects of 

EGBE. Likewise, the few human studies (Carpenter et al., 1956, 066464; Haufroid et al., 1997, 

042040) indicate the same finding. However, these studies (Carpenter et al., 1956, 066464; 

Ghanayem, 1989, 042014; Haufroid et al., 1997, 042040; Udden, 2000, 042110; Udden, 2002, 

042111; Udden and Patton, 2005, 100213) do not characterize hemosiderin deposition, the key 

event for the MOA. The current UFA value in the Toxicological Review is 1 and considers the 

 A-25 
limited number of studies available as well as the unknown effect of EGBE on the cellular events 

leading to hemolysis in human populations. In addition, little is known of the long-term or 

repeated exposure responses in humans to EGBE. 

A6. 

Comment: 

One reviewer commented that our interpretation of his original comment on the UFD in 

charge question A6 was inaccurate. The reviewer stated that he did not think the value of the 

UFD should be higher but that it was inconsistent with the Agency’s confidence statement. 

Response: 

The inaccuracies have been corrected in the reviewer responses for charge question A6. 

The assigning of a value of 1 for the UFD reflects that the database contains a composite of the 

basic studies needed for an overall toxicological assessment. However, U.S. EPA’s conclusion 

regarding the overall confidence for the derivation of a human health toxicity value (i.e., RfD, 

RfC) considers the quality, strength, and adequacy of the principal study as well as the database. 

The selection of a confidence level of medium – high is appropriate and also considers the 

potential for effects in humans from repeated, long-term exposures has not been investigated. 

 A-26 
APPENDIX B. PBPK MODELS (CORLEY ET AL.[1994;1997;2005]) 

Corley et al. (1994, 041977) developed PBPK models for rats and humans with the 

primary objective of describing the concentration of BAA in the target tissue (blood) of rats and 

humans for use in risk assessment (Figure B-1). The models incorporate allometrically scalable 

physiological and biochemical parameters (e.g., blood flows, tissue volumes, and metabolic 

capacity) in place of the standard values for a 70-kg human. These parameters normalize 

standard values to the actual body weights of the subjects in several human kinetic studies. The 

physiology of humans under exercise conditions initially used by Johanson (1986, 006758) to 

describe the kinetics of EGBE following inhalation exposures while exercising was maintained 

in the model. The rat was included to expand the database for model validation and to assist in 

interspecies comparisons of target tissue doses (BAA in blood). The formation of BAA from 

EGBE was assumed to occur only in the liver and was simulated in a second model linked via 

the formation of BAA. 

 

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-1 
Rapidly Perfused

Organs

Slowly Perfused

Organs

Fat

Skin

Muscle

Liver

Lungs and 

Arterial Blood

Gastrointestinal 

Tract

Rapidly Perfused

Organs

Slowly Perfused

Organs

Fat

Skin

Muscle

Liver

Lungs and 

Arterial Blood

Gastrointestinal 

Tract

Kidney

Model for 2-Butoxyethanol Model for 2-Butoxyacetic Acid

Inhalation Exhalation

Dermal; Vapor / Liquid

Urine 

(Butoxyacetic acid)

Arterial Blood

Other 

metabolites

iv infusion

Drinking water

or gavage

Metabolism

to BAA

Arterial Blood

Venous BloodVenous Blood

Source: Modified from Corley et al. (1994, 041977). 

Figure B-1. PBPK model of Corley et al. (1994, 041977). 

The Corley et al. (1994, 041977) model included additional routes of exposure such as 

gavage, drinking water, i.v. infusion, and dermal (liquids and vapor) to facilitate comparisons to 

several published toxicokinetic studies utilizing these routes of administration. The formation of 

BAA was assumed to occur only in the liver, using the rat liver perfusion data of Johanson et al. 

(1986, 006758) scaled to the human. A second model was linked to the EGBE model 

specifically to track the disposition of BAA following its formation in the liver. The kidney was 

added to the BAA model because it is the organ of elimination for BAA. All other metabolic 

routes for EGBE (formation of EG and glucuronide conjugate) were combined, as they were 

 B-2 
used only to account for the total disposition of EGBE in the rat metabolism studies and not for 

cross-species extrapolations. 

Contrary to observations in rats, Corley et al. (1997, 041984) found no evidence of 

metabolites in urine that would indicate that humans form conjugates of EGBE or EG. Thus, 

these pathways, which were lumped together in the model of Corley et al. (1994, 041977) to 

simulate rat kinetic data, were eliminated for human simulations. The human blood:air partition 

coefficient of 7,965, from Johanson and Dynesius (1988, 100157), was also used in the Corley et 

al. (1994, 041977) model. In addition, the partition coefficients for both EGBE and BAA were 

measured in human blood, rat blood, and rat tissues by using a modification of the Jepson et al. 

(1994, 006570) technique for ultrafiltration. Human tissue:blood partition coefficients were 

assumed to be equal to those of the rat. The skin:air partition coefficient, used to calculate the 

dermal uptake of vapors, was assumed to be the same as the blood:air partition coefficient. With 

the exception of the lung:blood partition coefficient for EGBE (11.3), the tissue:blood partition 

coefficients ranged from 0.64 to 4.33 for EGBE and from 0.77 to 1.58 for BAA. Protein binding 

of BAA in blood and saturable elimination of BAA by the kidneys were necessary components 

to describe the BAA kinetic data in rats and humans as discussed above. Since no direct 

measurements of protein binding were available, these parameters were arbitrarily set to the 

molar equivalent values reported for phenolsulfonphthalein as described by Russel et al. (1987, 

100197). Constants for the saturable elimination of BAA by the kidneys were then estimated by 

optimization from the data of Ghanayem et al. (1990, 042016), where rats were administered 

EGBE i.v. and the concentrations of BAA in blood were determined, following three different 

dose levels. These parameters were then held constant (protein binding) or scaled by (body 

weight)0.74 × (renal elimination) for all simulations. 

Corley et al. (1997, 041984) published the results from a human dermal exposure study 

and updated their 1994 human model. This study tested the hypothesis put forward by Johanson 

and Boman (1991, 006759) that dermal absorption can be more significant than inhalation 

absorptions by humans exposed to EGBE vapors. The results from this study verified the 

predictions from the Corley et al. (1994, 041977) model that blood samples taken via fingerprick sampling methods are confounded by locally high concentrations of EGBE on the surface 

or within the skin compartment and do not represent system blood concentrations. This study, 

and PBPK simulations have more recently been verified by Franks et al. (2006, 100117) and 

Jones et al. (2003, 100161). 

Lee et al. (1998, 041983) published an upgrade to the rat PBPK model of Corley et al. 

(1994, 041977) and included female rats, male and female rats, and the effects of aging to 

simulate the results from the kinetic studies conducted as part of the NTP 2-year bioassay (Dill et 

al., 1998, 041981). In their model, Lee et al. (1998, 041983) adjusted the rates of metabolism of 

EGBE to BAA in female rats, plasma protein binding of BAA, and the rates of renal active 

transport of BAA into urine to describe the kinetics of EGBE and BAA following short-term and 

 B-3 
long-term exposures. This model was used in the 1999 EGBE Toxicological Review to calculate 

the blood BAA Cmax values in female rats, which were used as the basis for RfC determination. 

Corley et al. (2005, 100100) continued the work of Lee et al. (1998, 041983) by 

experimentally measuring the blood and tissue partition coefficients for BAA, plasma protein 

binding of BAA, liver metabolism of EGBE, and renal clearance of BAA in young and old, male 

and female F344 rats and B6C3F1 mice to replace assumptions used in the rat and mouse PBPK 

model of Lee et al. (1998, 041983). This revised PBPK model (Figure B-2) was used as the 

basis for calculating the Cmax for BAA in the blood of female rats in the subchronic NTP 

inhalation study as the basis for the current RfC. The HEC was then calculated from BMD 

analysis of internal dose-response for hemolysis using the Corley et al. (1997, 041984) human 

PBPK model. A summary of the female rat and human model parameters are shown in Table 

B-1. 

The model was based upon the PBPK model of Lee et al. (1998, 041983) with the 

exception that (a) the spleen was combined with the rapidly perfused tissue group; (b) the i.p. 

injection route was added to the EGBE submodel; (c) salivary glands were added; (d) the skin 

compartment was separated into exposed and unexposed skin; and (e) metabolism of BAA to 

conjugates or CO2 were included. 

 B-4 
Source: Corley et al. (2005, 100100) 

Figure B-2. PBPK model of Corley et al. (2005, 100100). 

 B-5 
Table B-1. Selected parameters used in the PBPK model for EGBE 

developed by Corley et al. (1997, 041984; 2005, 100100) 

Parameter Human Young female rat Old female rat 

Weights

Body weight (kg) 70 0.20 0.34 

Liver (% BW) 3.14 3.22 2.52 

Kidney (EGBE model) 

(BAA model) (% BW) 

N/A 

0.44 

0.69 

0.69 

0.694 

0.694 

Rapidly perfused (EGBE model) 3.71 

3.27 (BAA model) (% BW) 

4.39 

4.39 

4.39 

4.39 

Slowly perfused (% BW) 9.4 24.6 14.3 

Flows 

Alveolar ventilation (L/hr) 347.9 5.47 8.10 

Cardiac output (COP) (L/hr) 347.9 5.47 8.10 

Liver (% COP) 25.0 18.3 18.3 

Kidney (% COP) 25.0 14.1 14.1 

Rapidly perfused (EGBE model) (% COP) 50.0 

25.0 (BAA model) 

23.3 

23.3 

23.3 

23.3 

Slowly perfused (% COP) 2 2 2 

Partition coefficients 

Blood/air 7,965 7,965 7,965 

Liver/blood 

(BAA model) 

1.46 

1.30 

1.48 

0.66 

1.48 

0.66 

Kidney/blood (EGBE model) 

(BAA model) 

1.83 

1.07 

1.83 

0.87 

1.83 

0.87 

Rapidly perfused/blood 

(BAA model) 

1.46 

1.30 

1.47 1.47 

0.66 0.66 

Slowly perfused/blood 

(BAA model) 

0.64 

1.31 

0.65 

0.54 

0.65 

0.54 

 B-6 
Parameter Human Young female rat Old female rat 

Metabolic constants 

EGBE to BAA 

Vmax1C (mg/hr/kg BW) 375 213 189 

Km1 (mg/L) 26.9 20.1 20.1 

EGBE to others (human only) 

Vmax2C (mg/hr/kg BW) 5 

Km2 (mg/L) 0.5 

EGBE to EG (rat only) 

Vmax2C (mg/hr/kg BW) 3.3 3.3 

Km2 (mg/L) 2.7 2.7 

EGBE to glucuronide conjugate (rat only) 

Vmax3C (mg/hr/kg BW) 30 30 

Km3 (mg/L) 55.7 55.7 

BAA to CO2 (rat only) 

Vmax4C (mg/hr/kg BW) 2.6 2.6 

Km4 (mg/L) 31.8 31.8 

Plasma protein binding of BAA

P (binding sites; mg/L) 164 n/a n/a 

Kd (dissociation constant; mg/L) 46 n/a n/a 

Bind (unitless fraction) n/a 0.298 0.433 

Renal elimination of BAA

VmaxEC (mg/hr/kg BW) 20.4 4.0 10.8 

KmEC (mg/L) 21.9 40.0 40.0 

 B-7 
APPENDIX C. RfD AND RfC DERIVATION OPTIONS 

C.1. DERIVATION OF THE RfC 

C.1.1. Potential RfC Derivations Based on Hematologic Data 

The lowest NOAEL/LOAEL observed in any subchronic or chronic study of EGBE is the 

31 ppm LOAEL for hematologic effects observed in the NTP study (2000, 196293) in rats. In 

order to estimate a corresponding human equivalent exposure, an internal dose metric associated 

with this exposure level is estimated and a PBPK model is used to estimate the human exposure 

level that would result in that internal dose. 

Initially, it was important to determine what estimate of internal dose (i.e., dose metric) 

could serve as the most appropriate metric for the effects under consideration. PBPK models of 

Lee et al. (1998, 041983) and Corley et al. (1994, 041977; 1997, 041984) are capable of 

calculating several measures of dose for both EGBE and BAA, including: 

CmaxC The peak concentration of EGBE or BAA in the blood during the exposure 

period; 

AUCC Represents the cumulative product of concentration and time for EGBE and BAA 

in the blood. 

Dill et al. (1998, 041981) published measured AUC, but not Cmax, blood concentrations 

of EGBE and its principal metabolite BAA at various exposure durations in both genders of 

B6C3F1 mice and F344 rats exposed to the same concentrations used in the NTP (2000, 196293) 

chronic studies. Cmax values would need to be derived from a PBPK model. Two pieces of 

information were used to select Cmax for BAA in the blood as the more appropriate dose metric 

for the main hemolytic endpoint associated with this LOAEL. First, as discussed in Section 4.5, 

there is convincing evidence to indicate that an oxidative metabolite, BAA, is the causative agent 

for EGBE-induced hemolysis (Carpenter et al., 1956, 066464; Ghanayem et al., 1987, 041608; 

Ghanayem et al., 1990, 042016). With this in mind, dose metrics for BAA in blood appear to be 

more appropriate than those for EGBE in blood, since they are more closely linked 

mechanistically to the toxic response. Second, EGBE-induced hemolysis appears to be 

dependent upon the dose rate. Ghanayem et al. (1987, 066470) found that gavage doses to F344 

male rats of 125 mg/kg EGBE resulted in hemolytic effects including reduced RBC count, Hb, 

and Hct, as well as kidney pathology (Hb casts and intracytoplasmic Hb). However, it should be 

 

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-1 
noted that hemolytic effects were not reported at a similar acute drinking water dose of 140 

mg/kg (Medinsky et al., 1990, 041986). While a drop in RBC count and Hb (9 and 7%, 

respectively) was noted in F344 male rats after 1 week of drinking water exposure to 129 mg/kgday EGBE, dose-related kidney pathology was not observed in these rats, even after 13 weeks of 

drinking water exposure up to 452 mg/kg-day EGBE (NTP, 1993, 042063). Consistent with the 

hypothesis that exposure concentration plays a larger role than exposure duration for EGBEinduced hemolytic effects, hematological endpoints indicative of hemolysis do not progress with 

increased inhalation duration (Section 5.1.1). Corley et al. (1994, 041977) also suggested that 

Cmax is a more appropriate dose metric for the hemolytic effects of EGBE than AUC. 

The PBPK models developed for EGBE are briefly summarized in Table C-1. Shyr et al. 

(1993, 006766) and Johanson (1986, 006758) do not address BAA distribution, and are only 

parameterized for humans and rats, respectively. In the 1999 EGBE Toxicological Review 

(U.S. EPA, 1999, 597365), the model described by Lee et al. (1998, 041983) was determined to 

be the most appropriate model for the estimation of rat and mouse internal doses following 

inhalation exposure. Since the 1999 Toxicological Review (U.S. EPA, 1999, 597365), Corley et 

al. (2005, 100100) published a revision to the Lee et al. (1998, 041983) model for rats and mice 

where several assumptions used by Lee et al. (1998, 041983) were replaced with measured 

values (e.g., protein binding, partition coefficients, metabolism rate constants for multiple 

pathways, and renal clearance) as a function of species, gender, and age. That model is used 

here to estimate the Cmax of BAA in blood following inhalation exposure to female rats, the more 

sensitive gender. For transparency, the results from both PBPK models are presented in Table 

C-2 below. However, only the values derived from the Corley et al. (2005, 100100) model were 

utilized in the derivation of the LOAEL and BMD in Sections C.1.1.1 and C.1.1.2. The human 

PBPK model of Corley et al. (1994, 041977; 1997, 041984) was then used to obtain estimates of 

human inhalation exposure concentrations associated with the female rat BAA blood 

concentrations.1

 Established U.S. EPA (2006, 194568) methods and procedures were used to 

review, select, and apply these chosen PBPK models.2

 

1

The basic components of the Corley model are summarized in Appendix B. 

2

EPA notes that the review of the PBPK models was conducted prior to their use in the 1999 EGBE toxicological review. 

 C-2 
Table C-1. Summary of PBPK models 

Model Species Routes of exposure Comments 

Johanson (1986, 006758) Human Inhalation BAA not addressed 

Shyr et al. (1993, 

006766) 

Rat Inhalation, oral, dermal BAA excretion 

Corley et al. (1994, 

041977; 1997, 041984) 

Rat and human Inhalation, oral, 

dermal, i.v. 

BAA distribution and excretion; male rats 

only 

Lee et al. (1998, 041983) Rat and mouse Inhalation BAA distribution and excretion; males and 

females 

Corley et al. (2005, 

100100) 

Rat and mouse Inhalation, oral, 

dermal, i.p., i.v. 

Age-dependent BAA distribution, 

metabolism, and excretion, males and females

Franks et al. (2006, 

100117) 

Human Inhalation and dermal Extended Corley et al. (1997, 041984) model 

to include bladder compartment for human 

biomonitoring studies 

Table C-2. Model estimates of BAA blood levels in female rats following 

inhalation exposures 

BAA in arterial blood Cmax in 

female rats (μM) 

Lee et al. (1998, 041983) 

BAA in arterial blood Cmax

in female rats (μM) 

Corley et al. (2005, 100100)

Exposure 

concentration (ppm) Female rat body weight (g)

31 216 285 167 

61.5 211 603 408 

125 214 1,243 1,091 

250 210 1,959 2,752 

500 201 4,227 6,483 

Source: Lee et al. (1998, 041983). Model results used in the 1999 EGBE Toxicological Review are included for 

comparison to the updated model of Corley et al. (2005, 100100). 

C.1.1.1. NOAEL/LOAEL Method Applied to Hematologic Data 

A five-step procedure was used to calculate the LOAEL HEC: 

Step 1: Calculate the internal dose surrogate (Cmax for BAA in blood) corresponding to 

female rat LOAEL (Corley et al., 2005, 100100) by using the actual experimental 

exposure regimen (6 hours/day, 5 days/week) in model simulations. 

 Female rat LOAEL = 31 ppm 

 Cmax BAA = 167 μM 

Step 2: Verify that steady state was achieved (e.g., no change in BAA Cmax as a result of 

prolonging the exposure regimen). 

There were no changes in the Cmax of BAA in blood during any 24-hour simulation 

period, using a 6 hour/day, 5 day/week exposure regimen at the female rat LOAEL, 

indicating that steady state was achieved. 

 C-2 
Step 3: Simulate the internal dose surrogate by calculating the Cmax for BAA in blood for 

humans continuously exposed (24 hours/day, 7 days/week) to varying concentrations 

of EGBE. 

Table C-3. Estimated Cmax for BAA in blood for humans 

continuously exposed to varying concentrations of EGBE 

Concentration of EGBE 

in air (ppm) 

Cmax BAA in blood 

(μM) 

1 2.6 

5 13.0 

10 26.1 

20 52.9 

50 137.1 

100 295.0 

200a 732.3 

a

Steady-state concentrations are not achieved within 5 days of continuous exposure to EGBE 

concentrations >200 ppm. 

Source: Corley et al. (1994, 041977; 1997, 041984) 

Step 4: Calculate the LOAELHEC of EGBE for continuous human exposure in air that 

resulted in the same internal dose (Cmax of BAA) in blood calculated for the animal 

study in step 1. 

 Female rat Cmax BAA = 167 μM 

 HEC continuous exposure = 56 ppm (calculated by regression of internal dose 

versus the concentration of EGBE in air from step 3). 

Step 5: Convert the EGBE exposure units from ppm to mg/m3

LOAELHEC (mg/m3

) = conversion factor × LOAELHEC (ppm) 

= 4.84 (mg/m3

) ÷ (ppm) × 56 ppm 

= 271 mg/m3

C.1.1.2. BMC Method Applied to Hematologic Data 

For the purposes of deriving an RfC for EGBE from hemolytic endpoints, both MCV and 

RBC count response data were evaluated in female rats from the 14-week subchronic NTP 

(2000, 196293) study (see Section 5.1.1). BMCs derived for these same hemolytic endpoints in 

male rats of this study were approximately twofold higher than for female rats (data not shown). 

The current BMD technical guidelines (U.S. EPA, 2000, 052150) suggest the use of 1 SD 

from the control mean for the BMR level for continuous data in the absence of additional 

information. Because the hemolytic endpoints are continuous measurements that have a 

relatively small historical variance in rats, and because low-dose responses for these endpoints 

 C-3 
were generally near or within 5% of the control mean, the BMCL05 was considered to be a more 

appropriate POD for derivation of the RfC (U.S. EPA, 1995, 005992; U.S. EPA, 2000, 052150). 

The steepest concentration-response curves (and the lowest BMCL05 estimate) were obtained for 

decreased RBC count in female rats, and a 5% change was found to be statistically significant. 

Higher levels of response (e.g., ≥ 10% reductions) are in the exposure range where other more 

severe responses related to anemia occur, such as MCV increases and increased reticulocyte 

counts. Lower levels of response, for instance, 1 SD from the control mean, an approximate 2% 

reduction for these data, are relatively distant from the observable data and other responses 

related to anemia. Cmax for BAA in arterial blood of rats was determined by using the PBPK 

model of Corley et al. (2005, 100100). Dermal exposure to EGBE vapor was not considered in 

the predicted blood levels because the estimated relative contribution of the skin to the total 

uptake of unclothed humans exposed to 25 ppm EGBE for 8 hours ranged from only 4.6 to 

27.5%, depending on temperature, humidity, and exercise level (Corley et al., 1997, 041984). 

Thus, dermal uptake is predicted to contribute <10%, even if 50% of an individual’s skin is 

exposed. The results of this modeling effort are summarized in Table C-2. 

All BMD analyses were performed using models in U.S. EPA BMDS, version 1.4.1c 

(U.S. EPA, 2000, 052150). The fit statistics and BMC information derived from the continuous 

models available in the BMD software as applied to the female rat RBC count data versus Cmax

BAA are shown in Table C-4. The best model fit to these data, from visual inspection and 

comparison of AIC values, was obtained using the Hill model. The BMCL05 was determined to 

be 133 μM using the 95% lower confidence limit of the dose-response curve expressed in terms 

of the Cmax for BAA in blood. The graphic and textual output from the Hill model run is 

displayed after Table C-4. BMCLSD values are provided for comparative purposes. The Corley 

et al. (1997, 041984) PBPK model was used to back-calculate a HEC of 46.5 ppm (225 mg/m3) 

from the 133 μM BMCL05, assuming continuous exposure (24 hours/day). 

 C-4 
Table C-4. Comparison of BMC/BMCL values for female rat RBC count 

data from a 14-week subchronic inhalation studya

, using modeled blood Cmax

(3 months) of the EGBE metabolite BAA as a common dose metric 

Model BMC05 (μM) 

BMCL05

(μM) 

BMCSD

(μM)

BMCLSD

(μM) p-Value AICa

Scaled 

residualb

Polynomial (2°) 495.468 460.317 357.566 301.089 <0.0001 -66.057235 -1.51 

Powerc 119.313 82.782 29.4231 16.5251 0.022 -106.196640 1.29 

Hillc,d 189.394 133.005 72.9703 38.8183 0.621 -111.611016 -0.287 

a

To obtain adequate model fits, the high dose group data was dropped from the analysis.

b

AIC = −2L + 2P, where L is the log-likelihood at the maximum likelihood estimates for the parameters and P is the 

number of model degrees of freedom (usually the number of parameters estimated). 

c

χ2 residual (measure of how model predicted response deviates from the actual data) for the dose group closest to 

the BMC scaled by an estimate of its SD. Provides a comparative measure of model fit near the BMC. Residuals 

that exceed 2.0 in absolute value should cause questioning of the model fit in this region. 

d

Power and Hill models were run with power terms unrestricted to obtain adequate fit; estimates of the power terms 

were 0.56 and 0.97 for the Power and Hill model, respectively. 

e

Model choice based on adequate p-value (>0.1), visual inspection, low AIC, and low (absolute) scaled residual. 

 C-5 
 6

 6.5

 7

 7.5

 8

 8.5

 0 500 1000 1500 2000 2500

Mean Response

dose

Hill Model with 0.95 Confidence Level

16:26 02/27 2009

BMDL BMD

 

Hill

 

Source: NTP (2000, 196293) 

Figure C-1. Hill Model run of female rat RBC count versus CMAX BAA from 

a 14-week inhalation study. 

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

 Hill Model. (Version: 2.14; Date: 06/26/2008) 

 Input Data File: C:\Usepa\BMDS21Beta\Data\EGBE\femalerat\2Hilrbchil.(d) 

 Gnuplot Plotting File: C:\Usepa\BMDS21Beta\Data\EGBE\femalerat\2Hilrbchil.plt 

 Fri Feb 27 16:26:00 2009 

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

 BMDS Model Run 

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

 

 The form of the response function is: 

 Y[dose] = intercept + v*dose^n/(k^n + dose^n) 

 Dependent variable = RBC 

 Independent variable = Cmax

 C-6 
 rho is set to 0 

 Power parameter is not restricted 

A constant variance model is fit 

 Total number of dose groups = 5 

 Total number of records with missing values = 0 

 Maximum number of iterations = 250 

 Relative Function Convergence has been set to: 1e-008 

 Parameter Convergence has been set to: 1e-008 

 Default Initial Parameter Values 

 alpha = 0.0357308 

 rho = 0 Specified 

 intercept = 8.48 

 v = -2.41 

 n = 1.34368 

 k = 775.437 

 Asymptotic Correlation Matrix of Parameter Estimates 

 ( *** The model parameter(s) -rho 

 have been estimated at a boundary point, or have been specified by the user, 

 and do not appear in the correlation matrix ) 

 alpha intercept v n k 

 alpha 1 9.2e-008 -5.9e-008 -8.5e-008 2.5e-008 

 intercept 9.2e-008 1 -0.36 -0.47 0.23 

 v -5.9e-008 -0.36 1 0.95 -0.99 

 n -8.5e-008 -0.47 0.95 1 -0.92 

 k 2.5e-008 0.23 -0.99 -0.92 1 

 Parameter Estimates 

 C-7 
 95.0% Wald Confidence Interval 

 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

 alpha 0.0323151 0.00646302 0.0196478 0.0449824 

 intercept 8.47617 0.0566598 8.36512 8.58722 

 v -3.86075 0.809864 -5.44805 -2.27344 

 n 0.971125 0.157912 0.661623 1.28063 

 k 1634.54 734.333 195.274 3073.81 

 Table of Data and Estimated Values of Interest 

 Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res. 

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

 0 10 8.48 8.48 0.158 0.18 0.0674 

 167 10 8.08 8.1 0.221 0.18 -0.287 

 408 10 7.7 7.68 0.253 0.18 0.353 

 1091 10 6.91 6.92 0.158 0.18 -0.175 

 2752 10 6.07 6.07 0.126 0.18 0.0415 

 Model Descriptions for likelihoods calculated 

 Model A1: Yij = Mu(i) + e(ij) 

 Var{e(ij)} = Sigma^2 

 Model A2: Yij = Mu(i) + e(ij) 

 Var{e(ij)} = Sigma(i)^2 

 Model A3: Yij = Mu(i) + e(ij) 

 Var{e(ij)} = Sigma^2 

 Model A3 uses any fixed variance parameters that 

 were specified by the user 

 Model R: Yi = Mu + e(i) 

 C-8 
 Var{e(i)} = Sigma^2 

 Likelihoods of Interest 

 Model Log(likelihood) # Param's AIC 

 A1 60.927568 6 -109.855137 

 A2 64.091535 10 -108.183070 

 A3 60.927568 6 -109.855137 

 fitted 60.805508 5 -111.611016 

 R -18.641036 2 41.282072 

 Explanation of Tests 

 Test 1: Do responses and/or variances differ among Dose levels? 

 (A2 vs. R) 

 Test 2: Are Variances Homogeneous? (A1 vs A2) 

 Test 3: Are variances adequately modeled? (A2 vs. A3) 

 Test 4: Does the Model for the Mean Fit? (A3 vs. fitted) 

 (Note: When rho=0 the results of Test 3 and Test 2 will be the same.) 

 Tests of Interest 

 Test -2*log(Likelihood Ratio) Test df p-value 

 Test 1 165.465 8 <.0001 

 Test 2 6.32793 4 0.176 

 Test 3 6.32793 4 0.176 

 Test 4 0.244121 1 0.6212 

The p-value for Test 1 is less than .05. There appears to be a 

difference between response and/or variances among the dose levels 

It seems appropriate to model the data 

The p-value for Test 2 is greater than .1. A homogeneous variance 

model appears to be appropriate here 

 C-9 
The p-value for Test 3 is greater than .1. The modeled variance appears 

 to be appropriate here 

The p-value for Test 4 is greater than .1. The model chosen seems 

to adequately describe the data 

 

 Benchmark Dose Computation 

Specified effect = 0.05 

Risk Type = Relative risk 

Confidence level = 0.95 

 BMD = 189.394 

 BMDL = 133.005 

 C-10 
C.1.2. BMC Method Applied to Hemosiderin Data 

For the purposes of deriving an RfC for EGBE, hemosiderin staining data were evaluated 

in male and female rats from the 2-year chronic study by NTP (2000, 196293). The current 

BMD technical guidelines (U.S. EPA, 2000, 052150) suggest the use of 10% extra risk as a 

BMR level for quantal data as this is at or near the limit of sensitivity in most cancer bioassays 

and in some noncancer bioassays as well. Because the hemosiderin staining endpoint was 

observed in control animals and a 10% increase in incidence was within the observable range of 

the data, 10% extra risk was considered an appropriate BMR and a BMCL10 an appropriate POD 

for derivation of the RfC (U.S. EPA, 1995, 005992; U.S. EPA, 2000, 052150). All BMD 

assessments in this review were performed using U.S. EPA BMDS version 1.4.1c. 

The AUC was selected as the appropriate dose metric due to the nature of the endpoint, 

hemosiderin deposition. This endpoint increased in severity with increased duration (subchronic 

to chronic) and is believed to be the result of the cumulative exposure to EGBE as opposed to a 

peak event. Table C-5 reports AUC BAA blood concentrations measured at 12 months3

published by Dill et al. (1998, 041981) in both genders of B6C3F1 mice and F344 rats exposed to 

the same concentrations used in the NTP (2000, 196293) chronic studies of these test animals. 

 

3

Dill et al. (1998, 041981) also reported 18 month data, but due to the smaller number of animals and higher variability in this data the 12 

month data were used for the purposes of this analysis. 

 C-11 
Table C-5. AUC BAA blood concentrations measured at 12 months in both 

genders of B6C3F1 mice and F344 rats 

AUCBAA (μmol-hr/L)a

Exposure concentration (ppm) Gender n Mean SE 

Rats 

31.2 Male 7 358.3 16.6 

Female 5 638.8 18.7 

62.5 Male 6 973.0 86.2 

Female 9 1,128.9 50.9 

125 Male 9 2,225.6 71.1 

Female 12 3,461.8 154.8 

Mice 

62.5 Male 10 1,206.6 205.6 

Female 12 1,863.6 112.4 

125 Male 9 2,819.8 685.1 

Female 6 5,451.6 508.9 

250 Male 10 17,951.5 1,770.4 

Female 11 18,297.1 609.7 

a

Authors reported AUC values in terms of μg-min/g, which were converted to units consistent with the PBPK model 

of μmol-hr/L by dividing by 60 min/h and 132.16 g/mol and multiplying by 1,060 g/L. 

Source: Dill et al. (1998, 041981). 

The fit statistics and BMC information derived from the dichotomous models available in 

the BMD software as applied to the male and female rat hemosiderin staining data versus AUC 

BAA are shown in Table C-6. All models were fit using restrictions and option settings 

suggested in the U.S. EPA BMD technical guidance document (U.S. EPA, 2000, 052150). The 

best model fit to these data, as determined by visual inspection, examination of low dose model 

fit (i.e., scaled residual for the dose group closest to the BMD), and comparison of overall fit 

(i.e., AIC values) was obtained using a multistage model (1st degree) for the male response data 

and a Log-Logistic model for the female response data. The male rat BMC10 was 196 μmolhour/L and the BMCL10 was determined to be 133 μmol-hour/L, using the 95% lower confidence 

limit of the dose-response curve expressed in terms of the AUC for BAA in blood. The BMC10

and BMCL10 values for the female rat were determined to be 425 and 244 μmol-hour/L, 

respectively. The graphic and textual output from the model runs that resulted in these male and 

female rat BMC10 and BMCL10 estimates are displayed below, after Table C-7. Assuming 

continuous exposure (24 hour/day), the Corley et al. (1997, 041984) PBPK model was used to 

back-calculate HECs of 3.4 ppm (16 mg/m3) from the male rat data and 4.9 ppm (24 mg/m3) 

from the female rat data. 

 C-12 
Table C-6. Comparison of BMC/BMCL values for male and female rat liver 

hemosiderin staining data from inhalation chronic study using measured 

blood AUC (12 months) of the EGBE metabolite BAA as a common dose 

metric 

Model BMC10 (μmol-hr/L) BMCL10 (μmol-hr/L) p-Value AICa Scaled residualb

Male rats 

Multistage-1st degreec 196.252 133.141 0.8680 247.234 0.441 

Gammac 196.253 133.141 0.8680 247.234 0.441 

Logistic 259.296 192.773 0.7692 247.476 0.526 

Log-Logistic 166.376 69.3279 0.5623 249.283 0.313 

Probit 271.525 205.882 0.7450 247.54 0.517 

Log-Probit 368.336 241.992 0.6309 247.876 0.765 

Weibullc 196.253 133.141 0.8680 247.234 0.441 

Female rats 

Multistage-1st degree 122.166 214.555 0.0698 218.868 -1.945 

Gamma 316.635 134.02 0.0554 219.229 -1.238 

Logistic 273.693 221.689 0.0993 218.188 -1.294 

Log-Logisticc 424.527 243.69 0.1533 217.526 -0.896 

Probit 291.017 241.206 0.0683 218.985 -1.260 

Log-Probit 427.728 248.683 0.1238 217.884 -0.965 

Weibull 266.515 130.801 0.0454 219.58 -1.377 

a

AIC = -2L + 2P, where L is the log-likelihood at the maximum likelihood estimates for the parameters and P is the 

number of model degrees of freedom (usually the number of parameters estimated) 

b

χ2 residual (measure of how model predicted response deviates from the actual data) for the dose group closest to 

the BMC scaled by an estimate of its SD. Provides a comparative measure of model fit near the BMC. Residuals 

that exceed 2.0 in absolute value should cause one to question model fit in this region. 

c

Model choice based on adequate p-value (>0.1), visual inspection, low AIC, and low (absolute) scaled residual. 

The Multistage (1st degree) is referred to as the chosen model for male rats, though equivalent fit was obtained by 

the restricted Gamma and Weibull models. 

 C-13 
Table C-7. Comparison of BMC/BMCL values for male and female mouse 

liver hemosiderin staining data from inhalation chronic study using 

measured blood AUC (12 months) of the EGBE metabolite BAA as a 

common dose metric 

Model 

BMC10 

(μmol-hr/L) 

BMCL10 

(μmol-hr/L) p-Value AICa Scaled residualb

Male mice 

Multistage-1st 

degree 

2,100.07 1,613.9 0.3067 117.571 -1.766 

Gamma 2,725.35 1,702.27 0.1452 118.559 1.358 

Logistic 6,605.45 5,333.72 0.0022 127.326 2.789 

Log-logistic 2,616.51 1,628.48 0.1882 118.02 1.193 

Probit 5,917.06 4,825.09 0.0031 126.405 2.734 

Log-probitc 3,076.8 2,448.3 0.1290 116.614 1.946 

Weibull 2,689.76 1,687.09 0.1445 118.712 -1.448 

Female mice 

Multistage-1st 

degree 

946.491 769.879 0.3680 142.669 -1.583 

Gamma 1,402.92 818.367 0.3420 143.288 -0.817 

Logistic 2,897.15 2,341.03 0.0002 162.338 -0.942 

Log-logistic 1,705.75 1,121.43 0.8223 141.501 -0.343 

Probit 2,860.03 2,364.52 0.0002 161.681 -0.829 

Log-probitc 1,734.53 1,322.06 0.8237 141.498 -0.315 

Weibull 1,282.82 804.234 0.2958 143.631 -0.988 

a

AIC = -2L + 2P, where L is the log-likelihood at the maximum likelihood estimates for the parameters and P is the 

number of model degrees of freedom (usually the number of parameters estimated). 

b

χ2 residual (measure of how model predicted response deviates from the actual data) for the dose group closest to 

the BMC scaled by an estimate of its SD. Provides a comparative measure of model fit near the BMC. Residuals 

that exceed 2.0 in absolute value should cause one to question model fit in this region. 

c

Model choice based on adequate p-value (>0.1), visual inspection, low AIC, and low (absolute) scaled residual. 

The Log-probit model provides a slightly better fit than other models for both genders. 

Likewise, the fit statistics and BMC information for male and female mouse hemosiderin 

staining data versus AUC BAA are shown in Table C-7. All models were fit using restrictions 

and option settings suggested in the U.S. EPA BMD technical guidance document (U.S. EPA, 

2000, 052150). The best model fit to these data, as determined by visual inspection, examination 

of low dose model fit (i.e., scaled residual for the dose group closest to the BMD), and 

comparison of overall fit (i.e., AIC values) was obtained using a log-probit model for both the 

male and female response data. The male mouse BMC10 was 3,077 μmol-hour/L and the 

BMCL10 was determined to be 2,448 μmol-hour/L using the 95% lower confidence limit of the 

dose-response curve expressed in terms of the AUC for BAA in blood. The BMC10 and BMCL10

values for the female mouse were determined to be 1,735 and 1,322 μmol-hour/L, respectively. 

 C-14 
Assuming continuous exposure (24 hour/day), the Corley et al. (1997, 041984) PBPK model was 

used to back-calculate HECs of 36 ppm (174 mg/m3) from the male mouse data and 20 ppm 

(97 mg/m3) from the female mouse data. 

 0.3

 0.4

 0.5

 0.6

 0.7

 0.8

 0.9

 0 500 1000 1500 2000

Fraction Affected

dose

Multistage Model with 0.95 Confidence Level

16:50 02/27 2009

BMDL BMD

 

Multistage

 Source: NTP (2000, 196293) 

Figure C-2. Multistage Model run of hemosiderin deposition in male rats 

versus AUC BAA at 12 months from a 2-year inhalation study. 

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

 Multistage Model. (Version: 2.8; Date: 02/20/2007) 

 Input Data File: U:\BMDS\DATA\EGBE\RAT-HEMOSID-12MTH-DILLAUC\MALE-MULT1.(d) 

 Gnuplot Plotting File: U:\BMDS\DATA\EGBE\RAT-HEMOSID-12MTH-DILLAUC\MALE-MULT1.plt 

 Mon Mar 24 14:56:58 2008 

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

 C-15 
BMD Model Run

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

 

 The form of the probability function is: 

 P[response] = background + (1-background)*[1-EXP( 

 -beta1*dose^1)] 

 The parameter betas are restricted to be positive 

 Dependent variable = Hemo_M 

 Independent variable = AUC-M-Dill 

 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: 1e-008 

 Parameter Convergence has been set to: 1e-008 

 Default Initial Parameter Values 

 Background = 0.481866 

 Beta(1) = 0.000526977 

 Asymptotic Correlation Matrix of Parameter Estimates 

 Background Beta(1) 

Background 1 -0.64 

 Beta(1) -0.64 1 

 Parameter Estimates 

 95.0% Wald Confidence Interval 

 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

 Background 0.477747 * * * 

 Beta(1) 0.000536865 * * * 

 C-16 
* - Indicates that this value is not calculated. 

 Analysis of Deviance Table 

 Model Log(likelihood) # Param's Deviance Test d.f. P-value 

 Full model -121.475 4 

 Fitted model -121.617 2 0.28443 2 0.8674 

 Reduced model -130.097 1 17.2453 3 0.0006292 

 AIC: 247.234 

 Goodness of Fit 

 Scaled 

 Dose Est._Prob. Expected Observed Size Residual 

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

 0.0000 0.4777 23.887 23 50 -0.251 

 358.3000 0.5691 28.457 30 50 0.441 

 973.0000 0.6902 34.512 34 50 -0.157 

 2225.6000 0.8419 42.094 42 50 -0.037 

 Chi^2 = 0.28 d.f. = 2 P-value = 0.8680 

 Benchmark Dose Computation 

Specified effect = 0.1 

Risk Type = Extra risk 

Confidence level = 0.95 

 BMD = 196.252 

 BMDL = 133.141 

 BMDU = 342.382 

Taken together, (133.141, 342.382) is a 90 % two-sided confidence 

interval for the BMD 

 C-17 
 0.2

 0.3

 0.4

 0.5

 0.6

 0.7

 0.8

 0.9

 1

 0 500 1000 1500 2000 2500 3000 3500

Fraction Affected

dose

Log-Logistic Model with 0.95 Confidence Level

17:03 02/27 2009

BMDL BMD

 

Log-Logistic

Source: NTP (2000, 196293) 

Figure C-3. Log-Logistic Model run of hemosiderin deposition in female rats 

versus AUC BAA at 12 months from a 2-year inhalation study. 

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

 Logistic Model. (Version: 2.10; Date: 09/23/2007) 

 Input Data File: U:\BMDS\DATA\EGBE\RAT-HEMOSID-12MTH-DILLAUC\FEMALE-LOGLOG.(d) 

 Gnuplot Plotting File: U:\BMDS\DATA\EGBE\RAT-HEMOSID-12MTH-DILLAUC\FEMALE-LOGLOG.plt 

 Mon Mar 24 15:20:55 2008 

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

BMD Model Run

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

 

 The form of the probability function is: 

 P[response] = background+(1-background)/[1+EXP(-intercept-slope*Log(dose))] 

 Dependent variable = Hemo_F 

 C-18 
 Independent variable = AUC-F-Dill 

 Slope parameter is restricted as slope >= 1 

 Total number of observations = 4 

 Total number of records with missing values = 0 

 Maximum number of iterations = 250 

 Relative Function Convergence has been set to: 1e-008 

 Parameter Convergence has been set to: 1e-008 

 User has chosen the log transformed model 

 Default Initial Parameter Values 

 background = 0.3 

 intercept = -17.7354 

 slope = 2.49241 

 Asymptotic Correlation Matrix of Parameter Estimates 

 background intercept slope 

background 1 -0.24 0.19 

 intercept -0.24 1 -1 

 slope 0.19 -1 1 

 Parameter Estimates 

 95.0% Wald Confidence Interval 

 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

 background 0.280971 * * * 

 intercept -16.4459 * * * 

 slope 2.35478 * * * 

* - Indicates that this value is not calculated. 

 Analysis of Deviance Table 

 Model Log(likelihood) # Param's Deviance Test d.f. P-value 

 Full model -104.742 4 

 Fitted model -105.763 3 2.04091 1 0.1531 

 C-19 
 Reduced model -135.725 1 61.9658 3 <.0001 

 AIC: 217.526 

 Goodness of Fit 

 Scaled 

 Dose Est._Prob. Expected Observed Size Residual 

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

 0.0000 0.2810 14.049 15 50 0.299 

 638.8000 0.4430 22.149 19 50 -0.896 

 1128.9000 0.6595 32.974 36 50 0.903 

 3461.8000 0.9566 47.828 47 50 -0.575 

 Chi^2 = 2.04 d.f. = 1 P-value = 0.1533 

 Benchmark Dose Computation 

Specified effect = 0.1 

Risk Type = Extra risk 

Confidence level = 0.95 

 BMD = 424.527 

 BMDL = 243.69 

 C-20 
C.1.3. BMD Method Applied to Forestomach Lesions in Female Mice 

Cmax of blood BAA is considered a reasonable measure of internal dose for the 

forestomach lesions reported from EGBE exposure. BAA is believed to be the toxic moiety 

responsible for the forestomach effects observed following EGBE exposure, with concentration 

(rather than AUC) appearing to be determinative in the development of these effects as well 

(Corley et al., 2005, 100098). Other information supportive of the Cmax as an appropriate metric 

include the findings of NTP (1993, 042063), where no signs of forestomach irritation were 

observed in mice at very high dose levels of 1,400 mg/kg-day in 2-week and 13-week drinkingwater studies conducted by NTP (1993, 042063). It is likely that such oral nonbolus dosing of 

EGBE does not result in high enough local concentrations of EGBE and BAA (Poet et al., 2003, 

100190) to produce irritation. Consistent with this observation are results with other 

forestomach carcinogens that are not mutagenic, demonstrating that forestomach effects are 

dependent not only on the dose but also on the chemical concentration in the dosing solution 

(Ghanayem et al., 1985, 042768). 

The blood level of BAA is a valid surrogate for the local (i.e., forestomach) BAA 

concentration as blood levels of BAA follow the severity of the irritant and irritant-associated 

hyperplastic responses in the forestomach. Further, this relationship between blood BAA levels 

and irritant response holds true for routes of EGBE administration other than oral, (e.g., 

inhalation, i.p., and i.v.) (Corley et al., 1999, 042003; Green et al., 2002, 041610). The basis for 

this route-independent response may be related to the Green et al. (2002, 041610) results from 

whole-body autoradiography of mice exposed via inhalation, showing appreciable amounts of 

EGBE-associated radioactivity being present in salivary glands and ducts. 

Plausible evidence exists for considering the incidence of forestomach tumors in female 

mice, following chronic inhalation exposure to EGBE, to occur via a nonlinear, nongenotoxic 

MOA. EGBE appears to be one of a group of compounds that are not mutagenic but can 

indirectly cause forestomach tumors through the sustained cytotoxicity and cell regeneration 

brought about by irritant and irritant-associated hyperplastic effects and breakdown of the 

forestomach’s gastric mucosal barrier. This sequence of events is considered obligate for the 

formation of the observed neoplasms. Strategies intended to control or omit any of these key 

mode-of-action events, including the initial hyperplastic event, would interrupt the process and 

prevent formation of neoplasms. While this MOA may be of qualitative relevance to humans, 

the exposure concentrations that would be necessary to cause these hyperplastic effects and 

resultant tumors in humans, if attainable, are likely to be much higher than the concentrations 

necessary to cause forestomach effects in mice, primarily because humans lack a comparable 

organ for storage and long-term retention of EGBE. 

Another line of reasoning that may be used to address issues relating to the occurrence of 

these irritant/hyperplastic lesions (and potential for progression) in humans is to order the dose-

 C-21 
response among those effects noted for EGBE, most prominently the hematologic effects that 

underlie the hepatic tumors and that are the basis of the oral RfD and inhalation RfC. A BMD 

analysis of the irritant/hyperplastic response observed in the NTP (2000, 196293) follows. 

The endpoint used in this analysis was epithelial hyperplasia of the female mouse 

forestomach, since it was the most sensitive forestomach effect observed in the NTP (2000, 

196293) study. Consistent with the 1999 IRIS assessment, four steps were employed to estimate 

human equivalent oral and inhalation benchmark exposures from this endpoint: (1) estimate a 

BMDL10 value using modeled “end-of-the-week” internal dose (Cmax BAA in blood) levels; 

(2) verify that steady state was achieved (e.g., no change in BAA Cmax as a result of prolonging 

the exposure regimen); (3) simulate the internal dose surrogate (Cmax BAA in blood) for humans 

(continuous air exposure; drinking water assumption was that a 70-kg human consumes an 

average of 2 L of water during a 12-hour awake cycle); and (4) calculate the HED/concentration 

that resulted in the same internal dose (Cmax BAA) simulated for the animal in step 1. 

Step 1: Estimation of BMDL10 (Cmax) dose. 

Cmax for BAA in arterial blood was determined using the PBPK model of Lee et al. (1998, 

041983). The model results and incidence data for the endpoint of concern are summarized in 

Table C-8. 

Table C-8. PBPK model estimates of BAA Cmax blood levels and incidence 

of forestomach epithelial hyperplasia in female mice 

Air concentration (ppm) Cmax BAA (μM) 

Incidence of forestomach 

hyperplasia 

0 0 0/50 

62.5 529 6/50 

125 1,200 27/50 

250 2,620 42/49 

 BMD and BMDL10 estimates were derived using the available models in version 1.3.2 of 

the U.S. EPA BMDS. The estimates for each model, along with statistical goodness-of-fit 

information, are provided in Table C-9. 

 C-22 
Table C-9. BMDS model estimates of Cmax BMD10 and BMDL10 values for 

forestomach epithelial hyperplasia in female mice 

BMDS model BMD (μM) BMDL (μM) 

AICa

(lowest = best fit) 

p-Value 

(>0.1 = adequate fit) 

Gamma 420.56 266.87 151.16 0.5287 

Logistic 544.757 444.896 162.191 0.0067 

Log-logisticb 462.513 329.04 150.153 0.8717 

Multistage (1st degree) 177.442 145.713 156.244 0.0648 

Multistage (2nd degree) 338.483 202.437 152.681 0.0976 

Multistage (3rd degree) 338.485 197.436 152.681 0.2535 

Probit 525.521 430.612 161.304 0.0086 

Log-probit 470.876 344.412 150.163 0.8673 

Weibull 376.085 238.952 151.855 0.3807 

a

AIC = -2L + 2P, where L is the log-likelihood at the maximum likelihood estimates for the parameters and P is the 

number of model degrees of freedom (usually the number of parameters estimated). 

b

Model choice based on adequate p-value (>0.1), visual inspection, low AIC, and low (absolute) scaled residual. 

The Log-logistic model provides a slightly better fit than other models. 

Step 2: Verification of steady state. 

As can be seen from Table C-10, Cmax levels are relatively constant through 6 months 

then increase at and beyond 12 months, presumably due to clearance problems in aging animals. 

However, the earlier steady-state levels are appropriate for use in this assessment, because that is 

the more conservative approach, and because similar effects were observed during the 

subchronic portion of the NTP (2000, 196293) study at the same dose levels, indicating that the 

higher internal doses at and beyond 12 months were not required for the effects to appear. 

Table C-10. Female mouse Cmax values for various time points of the NTP 

(2000, 196293) study estimated by the Lee et al. (1998, 041983) model 

62.5 ppm 125 ppm 250 ppm 

Months on study Male Female Male Female Male Female 

1 403 529 921 1,200 2,080 2,620 

3 402 527 925 1,202 2,120 2,652 

6 399 523 914 1,184 2,071 2,582 

12 484 639 1,079 1,414 2,349 2,951 

16 643 849 1,443 1,839 2,798 3,501 

18 756 995 1,625 2,102 3,067 3,803 

Step 3: Simulation of internal human doses. 

The tables below summarize the results of model simulations of the internal dose 

surrogate (Cmax BAA in blood) for a 70-kg human who consumes an average of 2 L of drinking 

 C-23 
water during a 12-hour awake cycle (Table C-11) or is continuously exposed to air 

concentrations (Table C-12) of EGBE. 

Table C-11. Estimated Cmax for BAA in blood for humans continuously 

exposed to varying drinking water concentrations of EGBE 

EGBE concentration in water 

(ppm) 

Calculated dose of EGBE from 

drinking water (mg/kg-d) 

Cmax BAA 

in blood (μM) 

24 0.7 9 

48 1.4 18 

94 2.7 36 

188 5.4 73 

375 10.7 147 

750 21.4 299 

Sources: Corley et al. (1994, 041977; 1997, 041984). 

Table C-12. Estimated Cmax for BAA in blood for humans continuously 

exposed to varying concentrations of EGBE 

Concentration of EGBE in air (ppm) Cmax BAA in blood (μM) 

1 2.6 

5 13.0 

10 26.1 

20 52.9 

50 137.1 

100 295.0 

200 733.7 

Sources: Corley et al. (1994, 041977; 1997, 041984). 

Step 4: Calculate the HED/concentration 

The Corley et al. (1994, 041977; 1997, 041984) PBPK model was used to back-calculate 

a human equivalent oral dose of 23.6 mg/kg-day from the Cmax BMDL10 of 329 μM estimated in 

step 1, assuming that mice and humans receive their entire dose of EGBE from drinking water 

over a 12-hour period each day. The Corley et al. (1997, 041984) PBPK model was used to 

back-calculate a human equivalent air concentration of 551 mg/m3 (113 ppm) from the Cmax

BMDL10 of 320 μM estimated in step 1, assuming continuous exposure (24 hours/day). 

The PODs calculated above are significantly higher than the PODs of 1.4 mg/kg-day and 

16 mg/m3

 used to derive the RfD and RfC, respectively. Thus, these results indicate that the RfD 

and RfC values for EGBE, which were based on hemosiderin accumulation due to hemolytic 

 C-24 
effects in rats, should be adequate for the prevention of gastrointestinal hyperplastic effects as 

well. 

Concentration (μM) 

 Source: NTP (2000, 196293) 

Figure C-4. Log-Logistic Model run of forestomach epithelial hyperplasia in 

female mice versus CMAX BAA from a 2-year inhalation study. 

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

 Logistic Model $Revision: 2.1 $ $Date: 2000/02/26 03:38:20 $ 

 Input Data File: F:\BMDS\DATA\EGBE\F_MOUSE_HYP_LOG-LOGIST.(d) 

 Gnuplot Plotting File: F:\BMDS\DATA\EGBE\F_MOUSE_HYP_LOG-LOGIST.plt 

 Fri Jul 11 19:53:31 2003 

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

BMDS Model Run 

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

 

 The form of the probability function is: 

 P[response] = background+(1-background)/[1+EXP(-intercept-slope*Log(dose))] 

 Dependent variable = F_Hyperplasia 

 C-25 
 Independent variable = Dose 

 Slope parameter is restricted as slope >= 1 

 Total number of observations = 4 

 Total number of records with missing values = 0 

 Maximum number of iterations = 250 

 Relative Function Convergence has been set to: 1e-008 

 Parameter Convergence has been set to: 1e-008 

 User has chosen the log transformed model 

 Default Initial Parameter Values 

 background = 0 

 intercept = -16.768 

 slope = 2.36735 

 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 -1 

 slope -1 1 

 Parameter Estimates 

 Variable Estimate Std. Err. 

 background 0 NA 

 intercept -16.7132 2.64108 

 slope 2.36545 0.372243 

NA - Indicates that this parameter has hit a bound 

 implied by some inequality constraint and thus 

 has no standard error. 

 Analysis of Deviance Table 

 Model Log(likelihood) Deviance Test DF P-value 

 Full model -72.9391 

 Fitted model -73.0765 0.274637 2 0.8717 

 Reduced model -131.841 117.804 3 <.0001 

 AIC: 150.153 

 C-26 
 Goodness of Fit 

 Scaled 

 Dose Est._Prob. Expected Observed Size Residual 

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

 0.0000 0.0000 0.000 0 50 0 

 529.0000 0.1324 6.622 6 50 -0.2596 

 1200.0000 0.5145 25.725 27 50 0.3608 

 2620.0000 0.8705 42.653 42 49 -0.2777 

 Chi-square = 0.27 DF = 2 P-value = 0.8717 

 Benchmark Dose Computation 

Specified effect = 0.1 

Risk Type = Extra risk 

Confidence level = 0.95 

 BMD = 462.513 

 BMDL = 329.04 

C.2. DERIVATION OF THE RfD 

C.2.1. Potential RfD Derivations Based on Hematologic Data 

PODs for the RfD derivation in terms of the HEDs have been calculated via the 

application of PBPK modeling to NOAEL and BMDL estimates. 

Of the available PBPK models (Table C-1), the Corley et al. (1997, 041984; 2005, 

100100) model is considered the most complete and appropriate for use in the derivation of the 

oral RfD because it has been experimentally validated for the most sensitive species (rats) and 

humans, it covers both oral and inhalation routes of exposure, and it addresses both the 

distribution and excretion of the toxic metabolite, BAA, following oral EGBE exposure. This 

model is summarized in Appendix B. As in the case of the RfC (see Section C.1.1), Cmax is 

considered a more appropriate dose metric than AUC for the hematological effects. The PBPK 

model of Corley et al. (1997, 041984; 2005, 100100) was used to obtain estimates of human Cmax

values from the female rat drinking water study data. 

The four steps involved in using the Corley et al. (1997, 041984; 2005, 100100) PBPK 

model to calculate the HED corresponding to the LOAEL identified in the animal study 

(LOAELHED) were to: (1) calculate the internal dose surrogate (Cmax BAA in blood) 

corresponding to the female rat LOAEL, assuming that the drinking water was consumed only 

during a 12-hour awake cycle on a 7 day/week schedule in model simulations; (2) verify that 

steady state was achieved (e.g., no change in BAA Cmax as a result of prolonging the exposure 

 C-27 
regimen); (3) simulate the internal dose surrogate (Cmax BAA in blood) for humans consuming 

EGBE in drinking water, assuming that a 70-kg human consumes an average of 2 L of water 

during a 12-hour awake cycle; and (4) calculate the HED (mg/kg-day) for the amount of EGBE 

consumed in 2 L of water that resulted in the same internal dose (Cmax BAA) simulated for the 

animal in step 1 as shown below. 

C.2.1.1. NOAEL/LOAEL Method and PBPK Model Applied to Hematologic Data 

Step 1: Calculate the Cmax for BAA in blood corresponding to female rat LOAEL. 

Female rat LOAEL = 59 mg/kg-day (calculated for final week of 13-week study to 

correspond with the final hematological determination) Cmax BAA = 255 μM.

Step 2: Verify steady state. 

There were no changes in the Cmax of BAA in blood during any 24-hour simulation 

period using a 12 hour/day, 7 day/week drinking water exposure regimen at the female rat 

LOAEL, indicating that steady state was achieved.

Step 3: Calculate the Cmax for BAA in blood for humans continuously exposed to varying 

concentrations of EGBE. 

Table C-13 shows modeled estimates of BAA in blood of humans exposed continuously 

to varying concentrations of EGBE in water (Corley et al., 1994, 041977; Corley et al., 1997, 

041984). Drinking water volume is 2 L consumed over 12 hours in a day. 

Table C-13. Modeled estimates of BAA in human blood exposed to EGBE in 

water. 

EGBE concentration (ppm) in 

water 

Calculated dose of EGBE from 

drinking water (mg/kg-d) Cmax BAA in blood (μM) 

24 0.7 9 

48 1.4 18 

94 2.7 36 

188 5.4 73 

375 10.7 147 

750 21.4 299 

Step 4: Calculate the LOAEL HED for a 70-kg human consuming EGBE in 2 L of 

drinking water/day that results in the same internal dose of EGBE (Cmax of BAA in blood) 

calculated for the animal study in step 1.

Female rat Cmax for BAA in blood at LOAEL = 255 μM 

LOAELHED continuous exposure = 18 mg/kg-day (calculated by regression of the internal 

dose versus the dose of EGBE from step 3). 

The internal dose surrogate, Cmax for BAA in blood, is highly dependent on the rate of 

water ingestion. Since drinking-water exposures are highly complex and variable, a simplifying 

 C-28 
assumption was used in all simulations that the entire dose of EGBE in drinking water was 

consumed over a 12-hour period each day corresponding to the awake cycle for both rats and 

humans. This assumption resulted in higher Cmax blood concentrations of BAA in both rats and 

humans than would have been calculated using the original Corley et al. (1994, 041977) structure 

that assumed that drinking-water uptake occurred over a 24 hour/day dosing period. 

C.2.1.2. BMD Method and PBPK Model Applied to Hematologic Data 

Although a lower LOAEL was reported in male rats, this value gives no indication of the 

relative slopes of the dose-response curves for males and females. Because this is an important 

factor for BMD analyses (U.S. EPA, 1995, 005992; U.S. EPA, 2000, 052150), a comparison of 

the MCV and RBC count results for both male and female rats was performed and demonstrated 

that female rats are more sensitive to the effects of EGBE than are males. Therefore, doseresponse information on these hematological effects in female rats was selected as the basis for 

the oral RfD BMD analyses discussed below. 

As was discussed in Section 5.2.1, MCV and RBC count are continuous response 

measurements of precursor events associated with EGBE exposure and are considered the most 

appropriate hematologic endpoints for use in a BMD analysis. Cmax is considered the more 

appropriate dose metric for use in evaluating the chosen hemolytic endpoints. Cmax for BAA in 

arterial blood was determined using the PBPK model of Corley et al. (1997, 041984; 2005, 

100100). The results of this modeling effort are summarized in Table C-14. 

Table C-14. Model estimates of BAA blood levels in female rats following 

oral exposures 

EGBE concentration BAA in blood 

in water (ppm) 

Water EGBE intake 

(L/d) 

Female body 

weight (g) Dose (mg/kg-d) Cmax (μM) 

750 0.0147 188 59 255 

1,500 0.0155 185 125 914 

3,000 0.0125 180 208 2,370 

4,500 0.0101 164 277 3,231a

6,000 0.0101 150 404 5,464a

a

Steady-state not reached within 5 days. 

Source: Corley et al. (2005, 100100). 

A BMD analysis was performed using U.S. EPA BMDS version 1.4.1 

(http://www.epa.gov/ncea/bmds/about.html ). As can be seen from the results in Table C-15, 

RBC count was the more sensitive of the two hematological endpoints assessed. All models 

were fit using restrictions and option settings suggested in the U.S. EPA BMD technical 

guidance document (U.S. EPA, 2000, 052150) except for the choice of BMR. 

 C-29 
Table C-15. Comparison of female rat RBC count and MCV BMD/BMDL 

values from an oral subchronic study using modeled blood Cmax (3 months) 

of the EGBE metabolite BAA as a common dose metric 

Model 

BMD05

(μM) 

BMDL05

(μM) 

BMDSD

(μM)

BMDLSD

(μM) p-Value AICa Scaled residualb

RBC count

Polynomial (1°) 1,623.31 1,350.41 1,937.36 1,532.88 <0.0001 -27.563915 -0.85 

Power 1,623.31 1,350.41 1,937.36 1,532.88 <0.0001 -27.563915 -0.85 

Hillc 180.667 93.9053 166.258 84.5854 0.4084 -47.398016 -0.228 

MCV 

Polynomial (1°) 1,036.38 934.455 578.088 442.513 <0.0001 146.041856 3.37 

Power 1,036.38 934.455 578.088 442.513 <0.0001 146.041856 3.37 

Hill 475.072 347.356 156.553 100.308 0.001166 135.552419 0.947 

a

AIC = −2L + 2P, where L is the log-likelihood at the maximum likelihood estimates for the parameters and P is the 

number of model degrees of freedom (number of parameters estimated). 

b

χ2 residual (measure of how model predicted response deviates from the actual data) for the dose group closest to 

the BMD scaled by an estimate of its SD provides a comparative measure of model fit near the BMD. Residuals 

that exceed 2.0 in absolute value should cause questioning of model fit in this region. 

c

Model choice based on adequate p-value (>0.1), visual inspection, low AIC, and low (absolute) scaled residual. 

Source: NTP (1993, 042063). 

For continuous response data, the current BMD technical guidelines (U.S. EPA, 2000, 

052150) suggest the use of 1 SD for the BMR level in the absence of additional information, 

such as a minimal level of change in the endpoint that is generally considered to be biologically 

significant. Because the chosen hemolytic endpoints are continuous measurements that have a 

relatively small historical variance in rats, and because low-dose responses for these endpoints 

were generally near or within 5% of the control mean, the BMDL05 was considered to be a more 

appropriate POD for derivation of the RfD (U.S. EPA, 1995, 005992; U.S. EPA, 2000, 052150). 

Higher levels of response (e.g., ≥ 10% reduction) are in the exposure range where other more 

severe responses related to anemia occur (e.g., RBC decreases, increased reticulocyte counts). 

Lower levels of response (e.g., 1 SD, approximately a 3% reduction for these data) are relatively 

distant from the observable data and other responses related to anemia. 

Adequate model fit could not be obtained for the MCV data. The best model fit to the 

RBC count data (from visual inspection and comparison of AIC values and scaled residuals near 

the BMD) was obtained using a Hill model (see Table C-15). A graphical plot and textual 

description of the results of the Hill model assessment of RBC count responses in female rats 

(NTP, 2000, 196293) versus corresponding PBPK estimates of Cmax for BAA in female rat blood 

are provided below. 

 C-30 
The BMD05 was 181 μM and the BMDL05 was determined to be 94 μM using the 95% 

lower confidence limit of the dose-response curve expressed in terms of the Cmax for BAA in 

blood. The Corley et al. (1997, 041984; 2005, 100100) PBPK model was used to back-calculate 

a HED (BMDLHED) of 6.8 mg/kg-day, assuming that rats and humans receive their entire dose of 

EGBE from drinking water over a 12-hour period each day. 

 6.5

 7

 7.5

 8

 8.5

 0 1000 2000 3000 4000 5000

Mean Response

dose

Hill Model with 0.95 Confidence Level

08:27 03/04 2009

BMDL BMD

 

Hill

Source: NTP (1993, 042063) 

Figure C- 5. Hill Model run of female rat RBC count versus CMAX BAA from 

a 3-month oral study. 

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

 Hill Model. (Version: 2.14; Date: 06/26/2008) 

 Input Data File: C:\Usepa\BMDS21Beta\Data\EGBE\femaleratoral\1Hilrbchil.(d) 

 Gnuplot Plotting File: C:\Usepa\BMDS21Beta\Data\EGBE\femaleratoral\1Hilrbchil.plt 

 Tue Mar 03 15:29:39 2009 

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

BMDS Model Run 

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

 C-31 
 

 The form of the response function is: 

 Y[dose] = intercept + v*dose^n/(k^n + dose^n) 

 Dependent variable = RBC 

 Independent variable = Cmax

 rho is set to 0 

 Power parameter restricted to be greater than 1 

 A constant variance model is fit 

 Total number of dose groups = 6 

 Total number of records with missing values = 0 

 Maximum number of iterations = 250 

 Relative Function Convergence has been set to: 1e-008 

 Parameter Convergence has been set to: 1e-008 

 Default Initial Parameter Values 

 alpha = 0.154717 

 rho = 0 Specified 

 intercept = 8.15 

 v = -1.57 

 n = 0.982521 

 k = 551.55 

 Asymptotic Correlation Matrix of Parameter Estimates 

 ( *** The model parameter(s) -rho -n 

 have been estimated at a boundary point, or have been specified by the user, 

 and do not appear in the correlation matrix ) 

 alpha intercept v k 

 alpha 1 2.2e-010 1.9e-009 6.6e-009 

 intercept 2.2e-010 1 -0.55 -0.54 

 v 1.9e-009 -0.55 1 -0.29 

 k 6.6e-009 -0.54 -0.29 1 

 Parameter Estimates 

 C-32 
 95.0% Wald Confidence Interval 

 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

 alpha 0.146124 0.0266785 0.0938351 0.198413 

 intercept 8.13659 0.120911 7.89961 8.37357 

 v -1.57421 0.156637 -1.88122 -1.26721 

 n 1 NA 

 k 518.419 227.998 71.5517 965.286 

NA - Indicates that this parameter has hit a bound 

 implied by some inequality constraint and thus 

 has no standard error. 

 Table of Data and Estimated Values of Interest 

 Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res. 

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

 0 10 8.15 8.14 0.28 0.382 0.111 

 255 10 7.59 7.62 0.48 0.382 -0.228 

 914 10 7.09 7.13 0.43 0.382 -0.348 

 2370 10 7 6.84 0.37 0.382 1.28 

 3231 10 6.8 6.78 0.36 0.382 0.165 

 5464 10 6.58 6.7 0.41 0.382 -0.983 

 Model Descriptions for likelihoods calculated 

 Model A1: Yij = Mu(i) + e(ij) 

 Var{e(ij)} = Sigma^2 

 Model A2: Yij = Mu(i) + e(ij) 

 Var{e(ij)} = Sigma(i)^2 

 Model A3: Yij = Mu(i) + e(ij) 

 Var{e(ij)} = Sigma^2 

 Model A3 uses any fixed variance parameters that 

 were specified by the user 

 Model R: Yi = Mu + e(i) 

 Var{e(i)} = Sigma^2 

 Likelihoods of Interest 

 Model Log(likelihood) # Param's AIC 

 C-33 
 A1 29.145609 7 -44.291218 

 A2 30.744881 12 -37.489762 

 A3 29.145609 7 -44.291218 

 fitted 27.699008 4 -47.398016 

 R -3.574142 2 11.148285 

 Explanation of Tests 

 Test 1: Do responses and/or variances differ among Dose levels? 

 (A2 vs. R) 

 Test 2: Are Variances Homogeneous? (A1 vs A2) 

 Test 3: Are variances adequately modeled? (A2 vs. A3) 

 Test 4: Does the Model for the Mean Fit? (A3 vs. fitted) 

 (Note: When rho=0 the results of Test 3 and Test 2 will be the same.) 

 Tests of Interest 

 Test -2*log(Likelihood Ratio) Test df p-value 

 Test 1 68.638 10 <.0001 

 Test 2 3.19854 5 0.6694 

 Test 3 3.19854 5 0.6694 

 Test 4 2.8932 3 0.4084 

The p-value for Test 1 is less than .05. There appears to be a 

difference between response and/or variances among the dose levels 

It seems appropriate to model the data 

The p-value for Test 2 is greater than .1. A homogeneous variance 

model appears to be appropriate here 

The p-value for Test 3 is greater than .1. The modeled variance appears 

 to be appropriate here 

The p-value for Test 4 is greater than .1. The model chosen seems 

to adequately describe the data 

 

 Benchmark Dose Computation 

Specified effect = 0.05 

Risk Type = Relative risk 

Confidence level = 0.95 

 BMD = 180.667 

 C-34 
 BMDL = 93.9053 

 C-35