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) 

October 1999 

U.S. Environmental Protection Agency 

Washington, DC 
DISCLAIMER 

This document has been reviewed in accordance with U.S. Environmental Protection 

Agency policy. Mention of trade names or commercial products does not constitute endorsement 

or recommendation for use. 

Note: This document may undergo revisions in the future. The most up-to-date version will be 

made available electronically via the IRIS Home Page at http://www.epa.gov/iris. 

ii 
CONTENTS—TOXICOLOGICAL REVIEW FOR 

ETHYLENE GLYCOL MONOBUTYL ETHER (CAS No. 111-76-2)

FOREWORD .............................................................. v

AUTHORS, CONTRIBUTORS, AND REVIEWERS ............................... vi

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

2. CHEMICAL AND PHYSICAL INFORMATION RELEVANT TO ASSESSMENTS ..... 2

3. TOXICOKINETICS RELEVANT TO ASSESSMENTS ........................... 2

4. HAZARD IDENTIFICATION ............................................... 9

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

CONTROLS ................................................... 9

4.2. PRECHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN

ANIMALS—ORAL AND INHALATION ............................ 12

4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND INHALATION

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

4.4. OTHER TOXICOLOGICALLY RELEVANT STUDIES ..................... 21

4.4.1. Single Exposure Studies .......................................... 21

4.4.2. Dermal Exposure Studies ......................................... 22

4.4.3. Ocular Exposure Studies .......................................... 23

4.4.4. Genotoxicity ................................................... 23

4.4.5. Immunotoxicity ................................................. 25

4.4.6. Other In Vitro Studies ............................................ 25

4.5. SYNTHESIS AND EVALUATION OF MAJOR NONCANCER EFFECTS AND

MODE OF ACTION (IF KNOWN)—ORAL AND INHALATION ............ 26

4.6. WEIGHT-OF-EVIDENCE EVALUATION AND CANCER

CHARACTERIZATION— SYNTHESIS OF HUMAN, ANIMAL, AND OTHER

SUPPORTING EVIDENCE, CONCLUSIONS ABOUT HUMAN

CARCINOGENICITY, AND LIKELY MODE OF ACTION ................ 29

4.7. SUSCEPTIBLE POPULATIONS ....................................... 31

4.7.1. Possible Childhood Susceptibility ................................... 32

4.7.2. Possible Gender Differences ....................................... 33

5. DOSE-RESPONSE ASSESSMENTS ........................................ 34

5.1. ORAL REFERENCE DOSE (RfD) ...................................... 34

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

............................................................... 34

5.1.2. Methods of Analysis—Including Models (PBPK, BMD, etc.) .............. 36

5.1.3. RfD Derivation—Including Application of Uncertainty Factors and Modifying

Factors ...................................................... 39

iii 
CONTENTS (continued) 

5.2. INHALATION REFERENCE CONCENTRATION (RfC) .................... 42

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

............................................................... 42

5.2.2. Methods of Analysis—Including Models (PBPK, BMD, etc.) .............. 44

5.2.3. RfC Derivation—Including Application of Uncertainty Factors and Modifying

Factors ....................................................... 47

5.3. CANCER ASSESSMENT ............................................. 49

6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF 

HAZARD AND DOSE RESPONSE ......................................... 50

6.1. HUMAN HAZARD POTENTIAL ..................................... 50

6.2. DOSE RESPONSE .................................................. 51

7. REFERENCES ......................................................... 53

APPENDIX A. EXTERNAL PEER REVIEW— 

SUMMARY OF COMMENTS AND DISPOSITION ............................ 61

APPENDIX B. CORLEY ET AL. (1994, 1997) PBPK MODEL ...................... 69

APPENDIX C. TEXT OUTPUT FROM BENCHMARK DOSE SOFTWARE

RUNS USED IN THE DERIVATION OF RfD AND RfC VALUES ................ 71

iv 
FOREWORD 

The purpose of this Toxicological Review is to provide scientific support and rationale for 

the hazard and dose-response assessment in the Integrated Risk Information System (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. 

In Section 6, EPA has characterized its overall confidence in the quantitative and 

qualitative aspects of hazard and dose response. Matters considered in this characterization 

include knowledge gaps, uncertainties, quality of data, and scientific controversies. This 

characterization is presented 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. 

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

the reader is referred to EPA’s Risk Information Hotline at 202-566-1676. 

v 
AUTHORS, CONTRIBUTORS, AND REVIEWERS 

Chemical Manager/Author 

Jeffrey S. Gift, Ph.D.

National Center for Environmental Assessment

U.S. Environmental Protection Agency

Research Triangle Park, NC

Reviewers 

In preparing the original draft of the U.S. EPA EGBE IRIS support document, EPA 

obtained valuable information and research contributions from the Chemical Manufacturers 

Association (CMA) Ethylene Glycol Ethers Panel, which collaborated with EPA scientists Jeffrey 

S. Gift, Annie M. Jarabek, and Vicki L. Dellarco in a workshop effort to produce an extensive 

EGBE health assessment review and support document (U.S. EPA, 1997). The current document 

and summary information on IRIS have received peer review both by EPA scientists and by 

independent scientists external to EPA (U.S. EPA, 1994c). Subsequent to external review and 

incorporation of comments, this assessment has undergone an Agency-wide review process 

whereby the IRIS Program Manager has achieved a consensus approval among the Office of 

Research and Development; Office of Air and Radiation; Office of Prevention, Pesticides, and 

Toxic Substances; Office of Solid Waste and Emergency Response; Office of Water; Office of 

Policy, Planning, and Evaluation; and the Regional Offices. 

Internal EPA Reviewers 

Elaina M. Kenyon, Ph.D.

National Health and Environmental Effects Research Laboratory

U.S. Environmental Protection Agency

Ralph J. Smialowicz, Ph.D.

National Health and Environmental Effects Research Laboratory

U.S. Environmental Protection Agency

Roy L. Smith, Ph.D.

Office of Air Quality Planning and Standards

U.S. Environmental Protection Agency

Judy A. Strickland, Ph.D., DABT

National Center for Environmental Assessment

U.S. Environmental Protection Agency

vi 
AUTHORS, CONTRIBUTORS, AND REVIEWERS (continued) 

External Peer Reviewers 

James Cholakis, Ph.D. 

JMC Associates

Prairie Village, KS 

Burhan Ghanayem, Ph.D.

National Institute of Environmental Health Sciences

Research Triangle Park, NC 

Kannan Krishnan, Ph.D.

Dollard-des-Ormeaux

Quebec, Canada

Mark Udden, M.D.

Houston, TX 

Summaries of the external peer reviewers’ comments and the disposition of their 

recommendations are in Appendix A. 

vii 
1. INTRODUCTION 

This document presents background and justification for the hazard and dose-response 

assessment summaries in EPA’s Integrated Risk Information System (IRIS). IRIS summaries may 

include an oral reference dose (RfD), inhalation reference concentration (RfC), and a 

carcinogenicity assessment. 

The RfD and RfC provide quantitative information for noncancer dose-response 

assessments. The RfD is based on the assumption that thresholds exist for certain toxic effects 

such as cellular necrosis, but may not exist for other toxic effects, such as some carcinogenic 

responses. It is expressed in units of mg/kg-day. In general, the RfD is 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 

noncancer effects during a lifetime. The inhalation RfC is analogous to the oral RfD, but it 

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). It is generally expressed in units of mg/m3

. 

The carcinogenicity assessment provides information on the carcinogenic hazard potential 

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

exposure. 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 are presented in three ways. The slope factor is the result 

of application of a low-dose extrapolation procedure and is presented as the risk per mg/kg-day. 

The unit risk is the quantitative estimate in terms of either risk per μg/L drinking water or risk per 

μg/m3

 air breathed. Another form in which risk is presented is a drinking water or air 

concentration providing cancer risks of 1 in 10,000; 1 in 100,000; or 1 in 1,000,000. 

Development of these hazard identification and dose-response assessments for ethylene 

glycol monobutyl ether (EGBE) has followed the general guidelines for risk assessment as set 

forth by the National Research Council (1983). EPA guidelines that were used in the 

development of this assessment may include the following: the Guidelines for Carcinogen Risk 

Assessment (U.S. EPA, 1986a), Guidelines for the Health Risk Assessment of Chemical Mixtures 

(U.S. EPA, 1986b), Guidelines for Mutagenicity Risk Assessment (U.S. EPA, 1986c), Guidelines 

for Developmental Toxicity Risk Assessment (U.S. EPA, 1991), Proposed Guidelines for 

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

Assessment (U.S. EPA, 1996b), Guidelines for Neurotoxicity Risk Assessment (U.S. EPA, 

1998a), and Proposed Guidelines for Carcinogen Risk Assessment (1996a); Recommendations 

for and Documentation of Biological Values for Use in Risk Assessment (U.S. EPA, 1988); 

(proposed) Interim Policy for Particle Size and Limit Concentration Issues in Inhalation Toxicity 

(U.S. EPA, 1994a); Methods for Derivation of Inhalation Reference Concentrations and 

Application of Inhalation Dosimetry (U.S. EPA, 1994b); Peer Review and Peer Involvement at 

the U.S. Environmental Protection Agency (U.S. EPA, 1994c); Use of the Benchmark Dose 

Approach in Health Risk Assessment (U.S. EPA, 1995b); Science Policy Council Handbook: 

Peer Review (U.S. EPA, 1998b); and memorandum from EPA Administrator, Carol Browner, 

dated March 21, 1995, Subject: Guidance on Risk Characterization. 

1
Literature search strategies employed for this compound were based on the CASRN and 

at least one common name. At a minimum, the following databases were searched: RTECS, 

HSDB, TSCATS, CCRIS, GENETOX, EMIC, EMICBACK, DART, ETICBACK, TOXLINE, 

CANCERLINE, MEDLINE, and MEDLINE backfiles. Any pertinent scientific information 

submitted by the public to the IRIS Submission Desk was also considered in the development of 

this document. 

2. CHEMICAL AND PHYSICAL INFORMATION RELEVANT TO ASSESSMENTS 

EGBE is also known as 2-butoxyethanol. Some relevant physical and chemical properties 

of EGBE are listed below. 

CASRN: 111-76-2

Empirical formula: C4H9-O-CH2CH2-OH

Molecular weight: 118.2

Vapor pressure: .0.88 mm Hg at 25EC

Water solubility: miscible

Log KOW: 0.81

Henry’s Law constant: 2.08 × 10-7 ! 2.08 × 10-8 atms/m3

/mole (25EC)

Flash point: 62EC (closed cup); 70EC (open cup)

Conversion factor: 1 ppm = 4.83 mg/m3

, 1.0 mg/m3 = 0.207 ppm

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

rate, relative to butyl acetate, is 0.08, and EGBE is therefore 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, it is unlikely that EGBE bioaccumulates. Based 

upon the magnitude of Henry’s Law constant, it is anticipated that partitioning of EGBE between 

water and air greatly favors the water phase. 

3. TOXICOKINETICS RELEVANT TO ASSESSMENTS 

In laboratory animals, EGBE is absorbed following inhalation, oral (gavage), or percutaneous 

administration, and it is distributed rapidly to all tissues via the blood stream. The uptake and 

metabolism of EGBE is essentially linear following a 6-hour inhalation exposure of up to 438 ppm, a 

concentration that caused mortality (Dill et al., 1998; Sabourin et al., 1992b). 2-Butoxyacetic acid 

(BAA) is the primary metabolite in rats following drinking water (Medinsky et al., 1990) and 

inhalation (Dill et al., 1998) exposures to EGBE. 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; Ghanayem et al., 1987a), but not humans (Corley et al., 1997). No 

significant differences in the urinary levels of BAA were found following administration of equivalent 

doses of EGBE either dermally or in the drinking water (Medinsky et al., 1990; Sabourin et al., 1992a; 

Shyr et al., 1993). Corley et al. (1997) report that elimination kinetics of EGBE and BAA appear to be 

independent of the route of exposure. Elimination of EGBE and BAA following repeated inhalation 

2
exposure appears to be dependent on species, sex, age, time of exposure, and exposure concentration 

(NTP, 1998; Dill et al., 1998). 

In a human study, Johanson and Boman (1991) attempted to define the relative importance of 

the skin to the total absorption of EGBE vapors in whole-body exposures. Four volunteers were 

exposed mouth-only to 50 ppm EGBE for 2 hours, followed by 1 hour of no exposure, followed by 2 

hours, of body-only exposure (exposed in a chamber while breathing fresh air via respirator) to 50 

ppm. 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 for the 

concentration of EGBE in the subjects’ blood samples following skin-only exposures were threefold to 

fourfold greater than following mouth-only exposure, Johanson and Boman (1991) concluded that the 

skin accounted for approximately 75% of the total uptake of EGBE in a whole-body exposure. 

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

Contrary to the conclusions of Johanson and Boman, these simulations resulted in dermal uptake 

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

temperatures and humidities (skin permeability coefficient of 3 cm/hour), assuming no clothing is worn 

that would hinder absorption. 

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

was conducted by Corley et al. (1997) in which human volunteers exposed one arm only to 50 ppm 

13C-EGBE for 2 hours. Catheters installed in the antecubital vein of the unexposed arm served as the 

primary site for collecting blood, which was analyzed for both EGBE and BAA. Finger prick blood 

samples were collected only from the exposed arm at the end of the 2-hour exposure. If Johanson and 

Boman’s (1991) 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, as 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 effects with the finger prick sampling 

technique. Corley et al. (1997) reported that the skin permeability coefficients that provided the best 

simulation of three human data sets (Johanson et al., 1988; Johanson and Boman, 1991; Corley et al., 

1997) ranged from 2 to 4 cm/hour, which covers low-high temperatures and relative humidities. Using 

these permeability coefficients, the relative contribution of the skin to the total uptake of humans 

exposed to the American Conference of Governmental Industrial Hygienists Threshold Limit Value 

(ACGIH TLV) concentration of 25 ppm EGBE for 8 hours ranged from (low to high 

temperature/humidity) 16% to 27.5% under resting conditions (normal ventilation and cardiac output) 

and 4.6% to 8.7% under working (50 W light exercise) conditions, assuming no clothing is worn that 

would hinder skin contact with EGBE. If protective clothing is worn, then only that surface area 

exposed would be available for the absorption of EGBE. 

3
The metabolism of EGBE has been studied extensively, particularly in rats, and the extensive 

literature on this subject has been thoroughly reviewed (ECETOC, 1994; Commonwealth of Australia, 

1996). Carpenter and co-workers (1956) first identified BAA as the metabolite responsible for the 

hemolytic toxicity of EGBE by incubating the acid with blood from a variety of species. Blood from 

rats, mice, and rabbits was more rapidly hemolyzed than blood from monkeys, dogs, humans, or guinea 

pigs when incubated at 37.5EC with a saline solution of 0.1% sodium butoxyacetate. These results 

correlated well with osmotic fragility studies using blood from these same species following inhalation 

exposures to EGBE. In contrast, a much higher concentration (2.5%) of EGBE was required to 

produce hemolysis in vivo. Subsequent investigations have shown that hemolytic blood concentrations 

of the acid may be produced following either oral, inhalation, or dermal administration of EGBE. 

Proposed pathways for the metabolism of EGBE in rats and humans are presented in Figure 1 (from 

Medinsky et al., 1990, and Corley et al., 1997). 

4
5
The two main oxidative pathways of EGBE metabolism observed in rats are alcohol 

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

al., 1990). EGBE may also form conjugates with glucuronide and sulfate to some extent. Primarily 

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

suggested that the former pathway, which involves production of BAA through formation of 

butoxyacetaldehyde (BAL) by ADH, would be applicable to both rats and humans (Medinsky et al., 

1990; Corley et al., 1997). However, the other three proposed metabolic pathways of EGBE may be 

applicable only to rats, as the metabolites of these pathways—ethylene glycol (EG), EGBE 

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

Ghanayem et al., 1987a) and not in the urine of humans (Corley et al., 1997). In addition, Corley et al. 

(1997) confirmed a recent observation of Rettenmeier et al. (1993) that approximately two-thirds of 

the BAA formed by humans is conjugated with glutamine and, to a lesser extent, glycine. These BAA 

glutamine and BAA glycine conjugation pathways have not been detected in the rat. 

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

acid sufficient to produce hemolysis (Bartnik et al., 1987). Metabolism, disposition, and 

pharmacokinetic studies in male F344 rats conducted by Corley et al. (1994) produced hemolytic 

blood concentrations of the acid (0.5 mM) following a single oral dose of 126 mg/kg. Using their 

physiologically based pharmacokinetic (PBPK) model, they predicted that such hemolytic blood 

concentrations would also be produced in rats following a single 6-hour inhalation exposure in excess 

of 200 ppm. A recent report on the NTP (1998) inhalation bioassay suggests that BAA blood 

concentrations in rats exceeded 0.5 mM (approximately 67 μg BAA/g blood) following exposure to 

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

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

pyrazole and cyanamide as metabolic inhibitors of alcohol and aldehyde dehydrogenases, respectively 

(Ghanayem et al., 1987b). Male F344 rats (9-13 weeks) were pretreated with pyrazole or cyanamide 

followed by administration of 500 mg/kg EGBE by gavage. Pyrazole protected rats from EGBEinduced 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, but it also resulted in a high mortality rate in rats given cyanamide and EGBE, an effect 

not observed in animals treated with cyanamide or EGBE alone. Pyrazole completely blocked the 

increase in spleen weight/body weight ratios seen in EGBE-treated animals. Gavage administration of 

either BAL or BAA at doses molar equivalent to 125 mg/kg EGBE produced identical increased 

spleen weight/body weight ratios and identical increases in free hemoglobin (Hgb) levels in plasma. 

Pretreatment of rats with cyanamide prior to administration of BAL provided significant protection 

against BAL-induced hematotoxicity. These studies confirm the central role of BAA in the 

hematotoxic response elicited in rats. 

Haufroid et al. (1997) conducted a human study on workers exposed to EGBE to test the 

possible influence of genetic polymorphism for CYP 2E1 on urinary BAA excretion rate. One 

exposed individual exhibited a mutant allele with increased cytochrome P450 oxidative activity that 

coincided with a very low urinary BAA excretion. However, the researchers did not measure BAA 

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

on the influence of genetic polymorphism for CYP 2E1 on urinary BAA excretion rate are needed 

before any firm conclusions can be drawn. 

6
The effect of age, dose, and metabolic inhibitors on the toxicokinetics of EGBE were studied 

in male F344 rats (Ghanayem et al., 1990). Rats of either 3-4 months of age or 12-13 months of age 

were dosed by gavage at 31.2, 62.5, or 125 mg/kg. Pretreatments included pyrazole, cyanamide, or 

probenecid (an inhibitor of renal anion transport). Toxicokinetic parameters for EGBE, including area 

under the curve (AUC), maximum plasma concentration (Cmax), and clearance rate (Cls) were dose 

dependent, with AUC and Cmax increasing and Cls decreasing at increasing dose levels. Other 

measured parameters were unaltered by dose. Age had no effect on half-life (T1/2), volume of 

distribution (Vs), or Cls of EGBE, but Cmax and AUC increased with increasing age. As expected from 

previous studies, inhibition of EGBE metabolism with either pyrazole or cyanamide resulted in 

significantly increased T1/2 and AUC and decreased Cls. BAA toxicokinetics were also altered by dose 

and age and by administration of metabolic inhibitors. Slight but statistically significant increases in 

Cmax, AUC, and T1/2 were seen at higher doses and 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 but produced twofold to threefold increases in AUC and twofold 

to sixfold increases in T1/2 for BAA. The results of these studies indicate that renal organic acid 

transport is vital to the renal elimination mechanism. 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 discussed previously, or they may be due to compromised renal clearance. 

Several blood and urine samples from the previously discussed human kinetic study by 

Johanson and Johnsson (1991) were analyzed for BAA. An average peak blood concentration of 44 

μM BAA (range 36-57 μM) was reached 2-4 hours postexposure. The average T 1/2 for elimination of 

BAA from blood was 4.3 hours (range 1.7-9.6 hours), suggesting little chance of accumulation of 

BAA following repeated occupational exposures to concentrations at or below existing occupational 

exposure limits of 20-25 ppm. The average renal clearance of BAA was 23-39 mL/minute, which was 

only about one-third of the glomerular filtration rate. Johanson and Johnsson (1991) suggested that the 

low clearance of BAA relative to the glomerular filtration rate 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 Johanson and Johnsson) indicates that tubular reabsorption was unlikely since more than 

99% of the BAA in normal human urine (pH ~6) is ionized. The volume of distribution (Vd) averaged 

15 L (range 6.5-25 L) based on whole blood measurements, which was approximately equal to the 

volume of extracellular water (13-16 L), a further indication of binding of BAA to blood proteins. 

BAA was measured in male workers exposed to low levels of EGBE (average airborne 

concentration of 2.91 ± 1.30 mg/m3

 [0.59 ppm]) in a beverage package production plant (Haufroid et 

al., 1997). Postshift urine samples showed average BAA concentrations of 10.4 mg/g creatinine in 

these individuals. 

The elimination kinetics of EGBE and BAA following repeated inhalation exposure (NTP, 

1998) appear to be dependent on species, sex, age, time of exposure, and exposure concentration (Dill 

et al., 1998). Postexposure blood samples were collected from rats and mice 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 (mice only) ppm by whole-body inhalation for 6 hours/day, 5 days/week. Urine and blood 

samples were also obtained from a separate set of aged mice (19 months) exposed to EGBE for 3 

weeks. While the systemic half-life of EGBE (<10 minutes in rats and <5 minutes in mice after 1-day 

exposure) was independent of exposure concentration and blood concentrations of EGBE (AUCEGBE) 

7
increased proportionally with exposure concentration, the rate of BAA elimination from blood 

decreased as exposure concentration increased. Female rats were significantly less efficient in clearing 

BAA from their blood than males, possibly a result of the reduced renal clearance observed in the 

female rats. EGBE clearance profiles of the 19-month-old mice exposed to 125 ppm EGBE were 

similar to young mice, but the aged mice eliminated BAA more than 10 times slower than young mice 

after a 1-day exposure. This difference was not as apparent after 3 weeks of exposure, suggesting that 

factors other than age may be involved. 

The elimination kinetics of EGBE and BAA appear to be independent of the route of 

exposure. The half-lives for the elimination of EGBE and BAA averaged 0.66 hour and 3.27 hours, 

respectively. For whole-body exposures under exercise conditions, the elimination half-lives for EGBE 

and BAA were 0.66 hour and 4 hours, respectively (Johanson, 1986; Johanson and Johnsson, 1991). 

For dermal exposure to neat liquids, the half-lives for elimination of EGBE and BAA were 1.3 hours 

and 3.1 hours, respectively (Johanson et al., 1988). For dermal exposure to vapors, the elimination 

half-life for EGBE was 0.53-0.6 hour. 

8
4. HAZARD IDENTIFICATION 

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

CONTROLS 

Bauer, P; Weber, M; Mur, JM; et al. (1992) Transient non-cardiogenic pulmonary edema following 

massive ingestion of ethylene glycol butyl ether. Inten Care Med 18:250-251. 

The effects of an acute ingestion of 500 mL of window cleaner containing 9.1% EGBE and 

2.5% ethanol by a 53-year-old male who was a chronic alcoholic were reported by Bauer et al. (1992). 

The man was admitted to a hospital comatose with metabolic acidosis, shock, and noncardiogenic 

pulmonary edema approximately 10 hours after ingestion of the dose. Heart rate was increased, blood 

pressure was decreased, and there was transient polyuria and hypoxemia. Hypochromic anemia was 

evident with a Hgb concentration of 9.1 g/100 mL, hematocrit (Hct) was 25%, and thrombocytopenia 

was noted. The man was discharged from the hospital after 15 days. 

Carpenter, CP; Pozzani, UC; Wiel, CS; et al. (1956) The toxicity of butyl cellosolve solvent. AMA 

Arch Ind Health 14:114-131. 

Three controlled studies using inhalation exposure were conducted by Carpenter et al. (1956). 

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 ft room. Effects observed in humans included nasal 

and ocular irritation, a metallic taste in the mouth, and belching. Erythrocyte osmotic fragility did not 

change for the men; however, it rose appreciably for the rats. In a second study, a group of two men, 

one woman, and three rats was exposed to 195 ppm EGBE for two 4-hour periods, separated by a 30-

minute recess, in a 6.5 cubic ft room. There was no change in the blood pressure, erythrocyte fragility, 

or pulse rate of the human subjects. Irritation of the nose and throat followed by ocular irritation and 

disturbed taste was noted, as well as one subject reporting a headache. In the rats, an increase in 

erythrocyte fragility values was noted during exposure. In the third study, a group of two men and two 

women were exposed for an 8-hour period to an EGBE concentration of 100 ppm. No changes in 

blood pressure, erythrocyte fragility, or pulse rate were observed. Irritation of the nose and throat 

followed by ocular irritation and a disturbing metallic taste were mentioned. Two of the subjects 

reported headaches. 

Dean, BS; Krenzelok, EP. (1991) Critical evaluation of pediatric ethylene glycol monobutyl ether 

poisonings. Vet Hum Toxicol 33:362. 

Twenty-four children, aged 7 months to 9 years, were observed subsequent to oral ingestion of 

at least 5 mL (two children drank more than 15 mL) of glass window cleaner containing EGBE in the 

0.5%-9.9% range. The two children consuming 15 mL were treated by gastric lavage. No symptoms 

of EGBE poisoning and no hemolysis were observed in any of the children. 

Gijsenbergh, FP; Jenco, M; Veulemans, H; et al. (1989) Acute butylglycol intoxication: a case report. 

Hum Toxicol 8:243-245. 

9
A 23-year-old woman weighing 64 kg ingested approximately 25-30 g of EGBE (~400-500 

mg/kg) and ethanol (~ 4 to 1 ratio) as a window cleaner in an apparent suicide attempt. She was 

admitted comatose to the hospital, exhibiting dilated pupils, obstructive respiration, and metabolic 

acidosis, including depression of blood Hgb 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. Hgb concentration fell from 11.9 g Hgb/100 mL blood 

on admission to 8.9 g Hgb/100 mL. The individual was discharged after 8 days. 

Gualtieri, JF; Harris, CR; Corley, RA; et al. (1995) Multiple 2-butoxyethanol intoxications in the same 

patient: clinical findings, pharmacokinetics, and therapy. Rochester, NY: North American Congress 

of Clinical Toxicology. 

A case of an intentional suicide attempt with an industrial-strength window cleaner was 

reported by Gualtieri et al. (1995). An 18-year-old male weighing 71 kg consumed between 360 and 

480 mL of a concentrated glass cleaner containing 22% EGBE (dose 1,131-1,509 mg/kg). The 

patient was admitted to the hospital within 3 hours postingestion with no abnormalities other than 

epigastric discomfort. Approximately 10 hours postadmission, the patient was noticeably more 

lethargic, weak, and hyperventilating, consistent with the onset of metabolic acidosis. BAA and EGBE 

levels were measured. The patient was transferred to a tertiary care hospital where hemodialysis was 

initiated (approximately 24 hours postingestion) and ethanol therapy was started 30 minutes later. 

Treatment also consisted of intravenous 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 following discharge, the patient was readmitted following a second 

ingestion of 480 mL of the same cleaner (EGBE dose 1,509 mg/kg). Treatment, including ethanol 

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

acidosis. Since treatment was initiated soon after ingestion, ethanol therapy did have an impact on the 

disposition of EGBE (higher concentrations were detected than following the first ingestion) and BAA 

(lower levels were detected). As with the first episode, clinical manifestations of high-dose oral 

ingestion of nearly 1.1-1.5 g/kg body weight consisted of metabolic acidosis. No evidence of 

hemolysis or renal abnormalities was detected. 

Haufroid, V; Thirion, F; Mertens, P; et al. (1997) Biological monitoring of workers exposed to low 

levels of 2-butoxyethanol. Int Arch Occup Environ Health 70:232-236. 

A cross-section of 31 male workers (22-45 years old, employed for 1-6 years) exposed to low 

levels of EGBE in a beverage packing production plant were monitored by Haufroid et al. (1997). The 

effect of external EGBE and internal BAA exposure on erythrocyte lineage (red blood cell [RBC] 

numeration, Hgb, Hct, mean corpuscular volume [MCV], mean corpuscular hemoglobin [MCH], 

mean corpuscular hemoglobin concentration [MCHC], haptoglobin [Hp], reticulocyte numeration 

[Ret], and osmotic resistance [OR]), as well as hepatic and renal creatinine and urinary retinol binding 

protein parameters was investigated. The average airborne concentration of EGBE was 2.91 mg/m3 

(0.6 ppm) (SD ± 1.30 mg/m3 or 0.27 ppm). Single determinations of BAA in post-shift urine samples 

were used to assess exposure to low levels of EGBE. No difference between exposed and control 

workers was observed for RBC count, Hgb, MCV, MCH, Hp, Ret, and OR (a measure of osmotic 

fragility). 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 

10
(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 adverse effects 

observed in laboratory animals. No significant differences were observed in hepatic and renal 

biomarkers. 

Rambourg-Schepens, MO; Buffet, M; Bertault, R; et al. (1988) Severe ethylene glycol butyl ether 

poisoning. Kinetics and metabolic pattern. Hum Toxicol 7:187-189. 

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

12% EGBE (~30-60 mL of EGBE) in an apparent suicide attempt. The woman was diagnosed with 

metabolic acidosis, hypokalemia, a rise in serum creatinine level, and a markedly increased urinary 

excretion of oxalate crystals. Moderate hemoglobinuria appeared on the third day postexposure and a 

progressive erythropenia was noted. Without more complete hematologic details from this and other 

similar case studies, it is not possible to determine whether these effects are due to hemolysis or other 

factors related to the profound changes in blood chemistry observed in these patients. The clinical 

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

11
4.2. PRECHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN 

ANIMALS—ORAL AND INHALATION 

Carpenter, CP; Pozzani, UC; Wiel, CS; et al. (1956) The toxicity of butyl cellosolve solvent. AMA 

Arch Ind Health 14:114-131. 

Carpenter et al. (1956) studied the hemolytic effects in various animal species following 

inhalation of EGBE vapors. An unspecified strain of rats (15 animals/sex) 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 or higher. 

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 

lowest-observed-adverse-effect levels (LOAELs) for these rat and mouse studies were apparently 54 

and 100 ppm, respectively. No no-observed-adverse-effect levels (NOAELs) were reported. 

Dodd, DE; Snelling, WM; Maronpot, RR; et al. (1983) Ethylene glycol monobutyl ether: acute, 9-day, 

and 90-day vapor inhalation studies in Fischer 344 rats. Toxicol Appl Pharmacol 68:405-414. 

A 90-day subchronic inhalation study was performed using F344 rats (16 rats/sex) exposed to 

EGBE for 6 hours/day, 5 days/week at concentrations of 0, 5, 25, and 77 ppm. During the course of 

the study, the 77 ppm males exhibited slight (5%) but statistically significant decreases in RBC counts 

and Hgb levels that were accompanied by increases in MCH. At the end of the study (66 exposures), 

these effects had either decreased or returned to the range of the control values. The NOAEL was 

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

Krasavage, WJ. (1986) Subchronic oral toxicity of ethylene glycol monobutyl ether in male rats. 

Fundam Appl Toxicol 6:349-355. 

A toxicity study was conducted using groups of 10 COBS CD(SD)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 consumption, clinical signs, and survival. 

Hematology and serum clinical chemistry parameters were determined following the last treatment. 

Dose-related changes were observed in the RBC counts of all treatment groups, including significantly 

decreased RBC count, decreased Hgb concentration, and increased MCH. Hematologic changes 

occurring at 443 and 885 mg/kg-day were increased MCV and decreased MCHC. The decrease in 

RBC count at a lower dose (222 mg/kg-day) seems to be inconsistent with the predominant theory 

that erythrocyte swelling (which is indicated by the increased MCV) precedes lysis of the cell (see 

discussion in Section 5.1.1). While such swelling has been documented in vitro (Ghanayem, 1989), the 

associate increases in MCV may not be detectable in vivo, given the sensitivity of the equipment used. 

Thus, the increased MCV at higher doses is more likely due to an increase in the number of larger 

reticulocytes (RTCs) in the circulation following this erythropoietic response, as has been suggested 

recently (NTP, 1998). Based on decreased RBC count and trends in Hgb and other hematologic 

12
endpoints, the LOAEL was determined to be 222 mg/kg-day, the lowest dose tested. A NOAEL was 

not identified. 

Nagano, K; Nakayama, E; Koyano, M.; et al. (1979) Testicular atrophy of mice induced by ethylene 

glycol mono alkyl ethers. Jpn J Indust Health 21:29-35. 

Nagano et al. (1979) performed a toxicity study in male mice 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 WBC counts, MCV and Hgb), absolute and relative weights of 

testes, and testicular histology. Mean RBC counts were significantly lower than the control value in 

the 357 and 714 mg/kg-day groups. WBC counts were not affected. All of the animals in the 

1430 mg/kg-day group died before examinations were performed; mortality was not observed in the 

lower dose groups, and no difference in testes weight or histology was found. The LOAEL for this 

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

National Toxicology Program (NTP). (1993) Technical report on toxicity studies of ethylene glycol 

ethers 2-methoxyethanol, 2-ethoxyethanol, 2-butoxyethanol administered in drinking water to F344/N 

rats and B6C3F1 mice. U.S. Department of Health and Human Services, Public Health Service, 

National Institutes of Health, Research Triangle Park, NC. NTP No. 26. NIH Publ. No. 93-3349. 

NTP (1993) performed a 13-week toxicity study in F344 rats and B6C3F1 mice using EGBE. 

Groups of 10/sex/species received EGBE in drinking water at doses of 0, 69, 129, 281, 367, or 

452 mg/kg-day in male rats; 0, 82, 151, 304, 363, or 470 mg/kg-day 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. 

Complete histologic 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 sexes persisting until or developing by 13 weeks 

included dose-related indications of mild to moderate anemia. Male rats evaluated at 13 weeks 

showed significantly reduced RBC counts at $281 mg/kg-day and reduced Hgb concentration, 

reduced platelets, and increased bone marrow cellularity at $367 mg/kg-day. Significant hematologic 

effects in female rats at week 13 included reduced RBC counts and Hgb concentration at $82 mg/kgday and increased RTCs, decreased platelets, and increased bone marrow cellularity at approximately 

304 mg/kg-day. There were no histopathologic changes in the testes and epididymis at $129 mg/kgday. Liver lesions, including cytoplasmic alterations, hepatocellular degeneration, and pigmentation 

were observed in the mid- and high-dose groups. 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 low-dose groups (69 

mg/kg-day for males and 82 mg/kg-day for females). The lack of cytoplasmic granularity or “groundglass” appearance of the hepatocytes suggests that this response was not due to enzyme induction 

(Greaves, 1990). The hematologic (decreased RBC count and Hgb) and hepatic changes were dose 

related and were associated with more severe blood and liver effects at higher doses; 69-82 mg/kg-day 

was considered a LOAEL. A NOAEL was not identified. Fewer effects were observed in male and 

female mice exposed to EGBE. Mean final body weight and body weight gain were essentially the 

same as control values at the two lower dose levels, but they were slightly reduced at the three highest 

dose levels. 

13
Werner, HW; Nawrocki, CZ; Mitchell, JL; et al. (1943a) Effects of repeated exposure of rats to 

vapours of monoalkyl ethers of ethylene glycol. J Ind Hyg Toxicol 25:374-379. 

Werner, HW; Mitchell, JL; Miller, JW; et al. (1943b) Effects of repeated exposure of dogs to 

monoalkyl ethylene glycol ether vapors. J Ind Hyg Toxicol 25:409-414. 

Subchronic inhalation studies were conducted using Wistar-derived rats (23 animals/group) by 

exposing them to 0, 135, or 320 ppm EGBE for 7 hours/day, 5 days/week for 5 weeks (Werner et al., 

1943a). Hematologic endpoints were evaluated (RBC, white blood cell [WBC], differential, and RTC 

counts, and Hgb estimations). The authors concluded that exposure to 320 ppm EGBE resulted in an 

increased percentage of circulating immature granulocytes, a decrease in Hgb concentration and RBC 

count, and an increase in RTC count. These hematologic changes were not severe and reversed 

3 weeks after discontinuing exposures. No effect on the WBC count was observed. In another study, 

the same researchers (Werner et al., 1943b) conducted a subchronic inhalation study using groups of 2 

dogs (of unspecified strain) and exposing them to 0 or 415 ppm EGBE for 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 Hgb concentration and RBC 

count, and increased hypochromia, polychromatophilia, and microcytosis. These hematologic effects 

were not severe and were reversed 5 weeks after the end of exposure. 

National Toxicology Program (NTP). (1998) NTP technical report on the toxicology and 

carcinogenesis studies of 2-butoxyethanol (CAS No. 111-76-2) in F344/N rats and B6C3F1 mice 

(inhalation studies). U.S. Department of Health and Human Services, Public Health Service, National 

Institutes of Health, NIH, Research Triangle Park, NC. NTP TR 484. NIH Draft Publ. No. 98-3974. 

In the subchronic portion of this study, both F344 rats and B6C3F1 mice (10/sex) were 

exposed via inhalation to concentrations of 0, 31, 62.5, 125, 250, and 500 ppm of EGBE 6 hours/day, 

5 days/week for 14 weeks (NTP, 1998). Both sexes of rats exhibited clinical signs consistent with 

hemolytic effects of EGBE at the three highest doses. Hematologic evaluation showed a mild to 

moderate regenerative anemia at all concentrations in females and at the highest three concentrations in 

males. Exposure-related trends were noted for RTCs, RBC count, MCV, Hgb, and Hct. Liver-tobody weight ratios were significantly increased in males at the two highest concentrations and in 

females at the highest concentration. Histopathologic effects 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 hemoglobin and 

hemosiderin deposition, and bone marrow hyperplasia at concentrations in excess of 62.5 ppm for male 

rats and 31 ppm for females. Also, five female rats were sacrificed moribund from the highest 

concentrations and one from the 250 ppm group. The LOAEL for hematologic 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. The mice exposed via the inhalation route of exposure exhibited clinical signs 

consistent with the hemolytic effects of EGBE at the two highest concentrations for both sexes. 

Hematologic evaluation indicated a moderate regenerative anemia with an increase in platelets at the 

three higher concentrations in both sexes. Histopathologic effects consisted of excessive 

extramedullary splenic hematopoiesis and hemosiderosis, hemosiderin accumulation in Kupffer cells, 

renal tubular degeneration and hemosiderin deposition, and testicular degeneration. Forestomach 

14
necrosis, ulceration, inflammation, and epithelial hyperplasia were observed at concentrations greater 

than 31 ppm for females and 62.5 ppm for males. Also, 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 in mice was 62.5 ppm, based on histopathologic changes in the forestomach. 

NTP also completed a two-species, 2-year inhalation study on EGBE (NTP, 1998). In the 

chronic study, exposure concentrations of EGBE 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. The highest exposure 

was selected to produce a 10% to 15% depression in hematologic indices. Survival was significantly 

decreased in male mice at 125 and 250 ppm (54.0% and 53.1%, respectively), but no effect on survival 

was observed in rats. 

Mean body weights of all groups of male and female rats exposed to 31 and 62.5 ppm were 

similar to controls. 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. Mean body weights of the exposed male and female 

mice were generally less than for controls, with females experiencing greater and earlier reductions. 

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). The severity of 

the nasal lesion was not affected by exposure and was deemed to be, in general, an adaptive rather than 

adverse response to exposure (NTP, 1998). The Kupffer cell pigmentation results from hemosiderin 

accumulation and is a recognized secondary effect of the hemolytic activity of EGBE (NTP, 1998). 

Nonneoplastic effects in mice included forestomach ulcers and epithelium hyperplasia, 

hematopoietic cell proliferation and hemosiderin pigmentation in the spleen, Kupffer cell pigmentation 

in the livers, hyaline degeneration of the olfactory epithelium (females only), and bone marrow 

hyperplasia (males only). As in the rats, the nasal lesion is deemed an adaptive rather than adverse 

response to exposure, and the Kupffer cell pigmentation is considered a secondary effect of the 

hemolytic activity of EGBE. Bone marrow hyperplasia and hematopoietic cell proliferation and 

hemosiderin pigmentation in the spleen are also attributed to the primary hemolytic effect, which is 

followed by regenerative hyperplasia of the hematopoietic tissue. The forestomach lesions do not 

appear to be related to the hemolytic effect of EGBE. Incidences of ulcer were significantly increased 

in males exposed to 125 ppm and in all exposed female groups. Ulcer consisted of a defect in the 

forestomach wall that penetrated the full thickness of the forestomach epithelium, and frequently 

contained accumulations of inflammatory cells and debris. 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. 

Using the same exposure groups described above, additional groups of rats (27/sex/exposure 

group) and mice (30/sex/exposure group) in the 2-year study were examined at 3, 6, and 12 months (8-

10 animals/duration) for hematologic effects. Rats in the 31 ppm exposure group were not examined 

at 12 months, and only hematology was examined at 3 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 

15
automated and manual Hct values and Hgb and erythrocyte counts, occurred at 3, 6, and 12 months in 

the 62.5 ppm females and the 125 ppm males and females. Statistically significant decreases in these 

same endpoints were also observed in 31 ppm females exposed for 3 and 6 months, and in 62.5 ppm 

males exposed for 12 months. At 3 months, MCV was increased following 31 ppm and higher 

exposures in both males and females. In vitro studies by Ghanayem (1989) have shown that the 

hemolysis caused by EGBE metabolite BAA is preceded by erythrocyte swelling. If the observed 

increase in MCV is in reponse 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 mean cell hemoglobin at higher exposure levels to the erythropoietic response subsequent to 

hemolysis and the corresponding increase in the number of larger RTCs in circulation (NTP, 1998). 

RTC count was increased significantly 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 RTC count was not observed at 

any duration in males or females exposed to 31 ppm, nor in males exposed to 62.5 ppm, it appears that 

RTC count alone cannot account for the increase in MCV at these levels of exposure. The observed 

increases in MCV may be a combined result of both erythrocyte swelling prior to and an increased 

number of RTCs, subsequent to hemolysis, with the former being more influential at lower exposure 

levels and the latter having more relative impact at higher exposure levels. 

Similar effects indicating anemia were also observed in mice, with females being the more 

sensitive of the species. However, the anemia response was observed at higher doses and changed 

somewhat with duration of exposure. Statistically significant (p<0.05) decreases in automated and 

manual Hct values, Hgb, and erythrocyte 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 females at the highest duration (12 months) and exposure (250 

ppm) levels. RTC count was increased significantly in 125 ppm females at 3 and 6 months and in 125 

ppm males at 6 months. 

At the end of the 2-year chronic bioassay, neoplastic effects were observed in female rats and 

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 highdose group (16%) does not represent a statistically significant increase over the chamber control 

group, but exceeded the historical control (6.4% ± 3.5%; range 2%-13%) for this type of study. 

The low survival rate in male mice exposed to 125 and 250 ppm EGBE may have been due to 

carcinogenic effects in the liver as a high rate of hepatocellular carcinomas was found in these 

exposure groups (10/50, 11/50, 16/50, 21/50), the increase at the high exposure level being 

statistically significant (p<0.001). 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%). NTP (1998) noted that no organisms consistent with Helicobacter hepaticus 

were found in any of 14 mice evaluated. It was concluded that H. hepaticus was not a factor in the 

development of liver neoplasms in this study. No significant increase in benign or malignant 

hepatocellular tumors or hemangiosarcomas was noted in the female mice. In fact, incidence of 

16
hepatocellular adenomas actually decreased significantly (p<0.05) in relation to the control chamber 

group (16/50, 8/50, 7/49, 8/49). However, 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 = 0.017) in female mice relative to the chamber controls (0/50, 1/50, 

2/50, 6/50). The incidence of these tumor types at the highest exposure level (12%) exceeds 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, indicating a decreased latency period in the highest exposure group. While 

the incidence of these types of forestomach tumors was not significantly increased in male mice over 

controls (1/50, 1/50, 2/50, 2/50), the incidence of squamous cell papillomas in the two highest 

exposure groups (4%) exceeded the range for historical controls (0.5% ± 0.9%; range 0-2%). 

Furthermore, the increased incidences of forestomach neoplasms in males, as in females, occurred in 

groups with ulceration and hyperplasia, suggesting a relation between these nonneoplastic and 

neoplastic lesions. 

A discussion of the cancer data from this study is provided in Section 4.6. With respect to the 

noncancer findings of this study, a NOAEL could not be determined, and a LOAEL of 62.5 ppm was 

determined for nonneoplastic lesions in mice. In rats, a NOAEL of 31 ppm and a LOAEL of 62.5 ppm 

were determined for noncancer effects. 

4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND INHALATION 

Due to the known reproductive toxicity (i.e., toxicity 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 has been studied in a variety of wellconducted oral (Nagano et al., 1979, 1984; Grant et al., 1985; Foster et al., 1987; Heindel et al., 1990; 

Exon et al., 1991; NTP, 1993) and inhalation (Dodd et al., 1983; NTP, 1998) studies 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 (Wier et al., 

1987; Sleet et al., 1989), inhalation (Nelson et al., 1984; Tyl et al., 1984), and dermal (Hardin et al., 

1984) exposures to 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 adverse 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 and are 

discussed below. No teratogenic toxicities 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 

parents, nor to the developing fetuses of laboratory animals. 

As discussed in Section 4.2, Nagano et al. (1979) performed a toxicity study in male mice 

using gavage doses of 0, 357, 714, or 1,430 mg/kg-day EGBE, 5 days/week for 5 weeks. A LOAEL 

17
of 357 mg/kg-day was identified, but no changes in testes weight or histology were observed. In 

another study, Nagano et al. (1984) used the same dosing regimen to test EGBE and other glycol 

ethers to up to 2,000 mg/kg-day. Testicular atrophy was observed for EGEE and EGME, but not for 

EGBE. 

Grant et al. (1985) exposed male F344 rats 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 for rats fed up to 1,000 mg/kg-day EGBE. 

As discussed in Section 4.2, Krasavage (1986) conducted a toxicity study using groups of 

10 COBS CD(SD)BR adult male rats treated by gavage with 222, 443, or 885 mg/kg-day undiluted 

EGBE 5 days/week for 6 weeks. The researchers found no effects on testicular weight and no 

histopathologic lesions in the testes, seminal vesicles, epididymides, or prostate at any exposure level. 

Foster et al. (1987) 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 and seminal vessels 

were observed, but the decreases were not time or dose related. No treatment-related lesions were 

noted following histologic examination of the testes, epididymides, and 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 by 

exposing them to EGBE in drinking water at doses of 0, 700, 1,300, and 2,000 mg/kg-day for 7 days 

premating and 98 days as breeding pairs (Heindel et al., 1990). In the 2,000 mg/kg-day dose group, 

13/20 females died, and in the 1,300 mg/kg-day dose group, 6/20 females died during the study. Toxic 

effects in adult mice in the 1,300 and 2,000 mg/kg-day dose groups included decreased body weight 

gain, increased kidney and liver weights, and dose-related decreases in water consumption. Decreased 

pup weight and a decrease in the number of litters produced per pair and in the size of each litter were 

observed in the 1,300 and 2,000 mg/kg-day dose groups. A significant reduction (5%) of live pup 

weight was also observed in the 700 mg/kg-day dose group. No adverse effect on fertility was 

observed in the 700 mg/kg-day dose group. 

At the completion of the continuous breeding phase, first-generation (F0) breeding pairs were 

separated and housed individually and exposure to EGBE continued. When the last litter was weaned, 

a 1-week crossover mating trial was performed to determine which sex was more affected by 

treatment. 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 controls. In the only histopathologic examination carried out 

on treated females, no kidney lesions were observed. The proportion of successful copulation was the 

same in all groups, and no developmental effects were observed in the offspring of any group. 

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. 

18
A final phase of this study assessed the fertility and reproductive effects of EGBE in second 

generation (F1) pups. There were insufficient numbers of offsprings to assess the two highest dose 

groups, and no adverse effect on fertility was noted when offspring of the low, 700 mg/kg-day dose 

group were mated. 

In summary, 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 as only a very slight decrease in pup weight was observed at 

this dose. 

In an immunotoxicity study discussed in more detail in section 4.4.5 (Exon et al., 1991), 

groups of six Sprague-Dawley rats were exposed to EGBE in drinking water at doses of 0, 180, or 

506 mg/kg-day (males) or 0, 204, or 444 mg/kg-day (females) for 21 days. While testicular atrophy 

and necrosis and reduced number of spermatogenic cells were observed in males exposed to EGME, 

no adverse effect on fertility parameters was seen in males exposed to 506 mg/kg-day EGBE. 

NTP (1993) evaluated the effects of EGBE on the reproductive systems of male and female 

B6C3F1 mice by exposing them to doses of 93, 148, 210, 370, or 627 mg/kg-day EGBE for males and 

150, 237, 406, 673, or 1,364 mg/kg-day EGBE for females in drinking water for 2 weeks. No deaths 

were reported, and there were no effects on body weight. Water consumption was decreased at all 

dosages except the highest in females. Thymus weights were decreased in the highest male dose 

group. There were no treatment-related gross lesions in any of the reproductive organs, and 

histopathologic examinations were not performed. NTP (1993) also exposed male and female F344 

rats to EGBE in drinking water for 2 weeks. 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 lowered in the highest dose group in both sexes, and there were no treatment-related 

gross lesions of reproductive organs reported. 

As discussed in Section 4.2, Dodd et al. (1983) and NTP (1998) performed 90-day subchronic 

inhalation studies on F344 rats. NTP (1998) also performed a subchronic study of B6C3F1 mice and 

chronic inhalation studies of F344 rats and B6C3F1 mice. Dodd et al. (1983) exposed male and 

female rats (16/sex) to EGBE for 6 hours/day, 5 days/week at concentrations of 0, 5, 25, and 77 ppm. 

The authors reported no changes in testicular weight or in the pathology of the epididymides and testes 

of male rats at any exposure level, but reproductive organs of the female rats were not examined 

histologically. In the subchronic portion of the NTP (1998) studies, no effects were noted in 

reproductive organs of rats and mice (10/sex) exposed to concentrations of 0, 31, 62.5, 125, 250, and 

500 ppm of EGBE 6 hours/day, 5 days/week for 14 weeks, although testicular degeneration was 

reported in 2 of 4 mice from the 500 ppm group that died or were killed moribund. In the NTP 

(1998) chronic study, exposure concentrations of EGBE 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; however, survival was significantly decreased in male mice 

at 125 and 250 ppm (54.0% and 53.1%, respectively). 

19
Prenatal and postnatal developmental toxicity tests were conducted in CD-1 mice by Wier et al. 

(1987). Animals received 0, 350, 650, 1,000, 1,500, or 2,000 mg/kg-day via gavage on days 8-14 of 

gestation. Maternal toxicity included mortality of 3/6 animals in the 1,000 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 and above). Based on 

clinical signs in the prenatal study, a LOAEL for maternal effects was 350 mg/kg-day. A LOAEL for 

developmental toxicity was determined to be 1,000 mg/kg-day based on an increased number of 

resorptions and a reduced number of viable fetuses. The corresponding NOAEL for prenatal effects 

was 650 mg/kg-day. In the postnatal study, reproductive effects were evaluated in CD-1 mice 

administered EGBE via gavage at 0, 650, or 1,000 mg/kg-day on days 8-14 of gestation. Maternal 

body weight was lowered at 1,000 mg/kg-day. Survival and body weight gain of offspring were 

unaffected by treatment. No adverse reproductive or 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 gavage to groups of 28-35 pregnant F344 rats at doses of 0, 30, 100, or 200 mg/kg-day on 

gestation days 9-11, or doses of 0, 30, 100, or 300 mg/kg-day on gestation days 11-13 (Sleet et al., 

1989). Gestation days 9-13 were chosen for investigation because they are the most critical periods of 

fetal cardiovascular development. Food and water measurements, body and organ weights, clinical 

signs, hematologic analyses (dams) and number of corpora lutea, uterine contents, and dead and live 

fetuses were monitored. Maternal effects of EGBE given in either dosing sequence included marked 

reductions in body weight and/or weight gain; increases in kidney and spleen weights; severe 

hematotoxicity as evidenced by a decrease in HCT, Hgb, and RBC count; and an increase in RTCs at 

doses greater than or equal to 100 mg/kg-day. These effects were dose related. 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, but not at 300 mg/kg-day. A decreased platelet count 

was noted in the fetuses at 300 mg/kg-day. No fetal malformations, including cardiovascular 

malformations, were noted at any dose. The LOAEL for maternal toxicity was 100 mg/kg-day with a 

NOAEL established at 30 mg/kg-day. The LOAEL for developmental toxicity was 200 mg/kg-day, 

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 for days 7-15 of gestation (Nelson et al., 1984). Rats exposed to 200 ppm showed some 

evidence of hematuria on the first day of exposure; however, no adverse effects were noted thereafter. 

No adverse effects attributable to EGBE exposure were seen in offspring. The LOAEL was 200 ppm 

for slight maternal toxicity, and a NOAEL was identified at 100 ppm. The NOAEL for developmental 

toxicity was 200 ppm. Simultaneous testing revealed that 50 ppm exposures to EGME was toxic at all 

levels of embryonic and fetal development. 

Pregnant F344 rats (36/group) and New Zealand white rabbits (24/group) were exposed to 0, 

25, 50, 100, or 200 ppm EGBE via inhalation for 6 hours/day on gestational days 6-15 for rats or days 

6-18 for rabbits (Tyl et al., 1984). Fetuses were weighed and evaluated for viability, body weight, and 

morphological development, including external, visceral, and skeletal malformations. In rats, 

fetotoxicity was observed in the form of retarded skeletal ossification of vertebral arches or centra, 

sternebrae, or phalanges at 100 and 200 ppm. Maternal toxicity was also evident at 100 and 200 ppm 

20
as an increased incidence of hematuria, reduced RBC count, decreased weight gain, and reduced food 

consumption. The NOAEL and LOAEL for maternal and developmental toxicity in the rat were 50 

and 100 ppm, respectively. In rabbits, fetal skeletal ossification of sternebrae and rudimentary rib 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. The NOAEL and LOAEL for 

maternal and developmental effects in the rabbit were 100 and 200 ppm, respectively. 

Reproductive toxicity tests were performed on female Sprague-Dawley rats via dermal 

administration during days 6-15 of gestation, four times per day at 1,800 and 5,400 mg/kg-day (Hardin 

et al., 1984). 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 and there was no evidence of embryo- or 

fetotoxicity, nor were any gross malformations or variations noted. 

4.4. OTHER TOXICOLOGICALLY RELEVANT STUDIES 

4.4.1. Single Exposure Studies 

Ghanayem et al. (1987c) conducted acute toxicity studies in male F344 rats using single 

gavage doses of 0, 32, 63, 125, 250, or 500 mg/kg-day of EGBE (purity 99%) in water. These studies 

were designed to assess the effect of age on toxicity by comparing effects in treated young rats (4-5 

weeks old) and adult rats (9-13 weeks, 5-6 months, and/or 16 months). Evaluations included 

hematology (total RBC and WBC counts), urine Hgb concentration, organ weights, and histology 

(liver, spleen, bladder, kidney, and testes). Focal necrosis of the liver was observed in adult rats 

exposed at either 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 

Hgb and increases in free plasma Hgb occurred at 125 mg/kg-day in both adult and young rats, with 

the younger rats exhibiting significantly less pronounced responses. Incidence of hemoglobinuria was 

also dose- and age-dependent. Concentrations of free Hgb in urine also were significantly higher in 

older rats than in younger rats at all doses. These researchers suggested that the metabolic basis of the 

age-dependent toxicity of EGBE may be due to a reduced ability by older rats to metabolize the toxic 

metabolite BAA to CO2 and a diminished ability to excrete BAA in the urine. Based on increased Hgb 

in the urine and associated hemolytic effects at higher doses, a LOAEL for this study was determined 

to be 32 mg/kg-day for adult rats. A NOAEL was not identified. 

Ghanayem and Sullivan (1993) performed acute oral toxicity studies in rats by administering 

EGBE in single gavage doses of 250 mg/kg-day in tap water. MCV and HCT values were raised 

immediately after treatment and decreased with time following exposure. Hemolysis and decreases in 

Hgb concentrations and RBC counts occurred. 

Grant et al. (1985) 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 weights, and/or histology. Hematology evaluations showed marked dose-related effects on 

circulating RBCs and WBCs. Changes at 500 and 1,000 mg/kg-day on post-dosing day 1 included 

21
significant dose-related decreases in Hgb, total WBC, and lymphocytes, and increases in MCV, RTC, 

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 post-treatment period at 1,000 mg/kg-day. Changes in relative 

organ weights were evident on post-treatment day 1, including increased liver weight and spleen 

weight at 500 and 1,000 mg/kg-day and increased kidney and reduced thymus weights at 1,000 mg/kgday. These changes returned to normal by post-treatment day 22 except for liver and spleen weights at 

1,000 mg/kg-day, which remained somewhat increased (~5% and ~20%, respectively). 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) also administered EGBE to F344 rats via gavage for a 12-day period 

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, which became more pronounced up 

to the third day of dosing. Gradual recovery was observed up to day 12. MCV, ATP concentration, 

RTC numbers, and body weight-relative spleen weights increased up to the sixth day of dosing and 

declined thereafter. Body weight-relative liver weight ratios were slightly lowered on days 3 and 6 and 

slightly raised on day 12. 

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, 1994a), but only a 20% mortality of either at a dose 

of 1,000 mg/kg. Clinical signs and gross necropsy indicated toxicity was due to irritation of the 

stomach. There was no evidence of hemolytic toxicity. 

4.4.2. Dermal Exposure Studies 

EGBE appears to be readily absorbed after contact with the skin of animals. Rats and rabbits 

have been shown to exhibit varying degrees of hematotoxicity following dermal application of EGBE 

(Allen, 1993a,b,c,d; Bartnik et al., 1987; Tyler, 1984). Bartnik et al. (1987) performed acute dermal 

toxicity tests using rats (unspecified species). 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. 

However, in the case of rabbits, repeated application of EGBE either neat or as a dilute 

aqueous (occluded) to male or female New Zealand rabbits at exposures of 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). Only female rabbits showed decreased RBC counts, Hgb 

concentrations, and MCHC and increased MCH at the highest treatment level. Recovery was noted 

following a 14-day observation period. In a separate 13-week study, occluded dermal administration 

of EGBE to male and female New Zealand rabbits (6 hours/day, 5 days/week) at exposure levels of 10, 

50, or 150 mg/kg produced no observable hematologic effects (Tyler, 1984). 

In addition to differences in effects based on dose and duration, whether the site of EGBE 

administration was occluded or semioccluded was also a determining factor. For example, some 

22
studies have shown no clinical signs of hematotoxicity in male or female Sprague-Dawley rats 

administered EGBE dermally at 2,000 mg/kg (24-hour exposure) either semioccluded or occluded 

(Allen, 1993a,b). However, clinical signs of systemic toxicity were notable in the occluded study. In a 

similar set of studies in rabbits, red-stained urine was reported at 2,000 mg/kg EGBE (semioccluded) 

along with other clinical signs of systemic toxicity (Allen, 1993c,d). Similar effects occurred at applied 

doses of 500, 707, and 1,000 mg/kg and occluded in this species; deaths occurred at the 500 and 1,000 

mg/kg dose levels. Thus, hematotoxicity varied from nonexistent to severely affected. In guinea pigs, 

dermal administration of EGBE at 2,000 mg/kg produced no clinical signs of toxicity or treatmentrelated signs of organ toxicity (Shepard, 1994b). 

4.4.3. Ocular Exposure Studies 

EGBE has also been found to be an irritant when instilled in the eyes of rabbits in several 

studies (Jacobs and Marten, 1989; Kennah et al., 1989). Kennah et al. (1989) performed the Draize 

test to determine the effects of EGBE on eye irritation in rabbits. Scores for different concentrations 

tested at 24 hours postinstillation were 100%/66, 70%/49, 30%/39, 20%/2, and 10%/1 by the Texaco 

single-digit toxicity classification system. In an assessment that measured corneal thickness, the 

highest concentration was still classified as severely irritating, the 70% concentration was moderately 

irritating, and the others were mildly irritating. Jacobs and Marten (1989) also conducted ocular tests 

(no method specified) on rabbits (no concentrations given) to determine the effects of EGBE on eye 

irritation. Mean erythema scores and percent corneal thickening indicated that the substance should be 

classified as an irritant. 

4.4.4. Genotoxicity 

Although weakly genotoxic responses have been obtained in one laboratory, on the basis of the 

available data, EGBE is not expected to be mutagenic or clastogenic. The NTP has reported negative 

responses for mutagenicity when EGBE was tested in Salmonella typhimurium strains TA97, TA98, 

TA100, TA1535, and TA1537 up to 10 mg/plate with or without metabolic activation (Zeiger et al., 

1992). However, Hoflack et al. (1995) reported that at a high concentration (38 mmole/plate or 4.5 

mg/plate), EGBE induced a weak mutagenic response in Salmonella tester strain TA97a with or 

without S9 mix (Hoflack et al., 1995). The Corning Hazleton Laboratory (Gollapudi et al., 1996) 

conducted testing in Salmonella to confirm the positive result reported by Hoflack et al. (1995). 

Testing was conducted in Salmonella strains TA97a and TA100, as well as Escherichia coli 

WP2uvrA. 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) is unexplained. A plausible 

explanation put forth by Gollapudi et al. (1996) is that given the sensitivity of the Ames test, perhaps 

the weak positive result reported by Hoflack et al. (1995) is attributed to an impurity in their test 

material. 

In an in vitro study, EGBE was reported to induce cell-cycle delay, but neither sister chromatid 

exchanges (SCEs) nor chromosomal aberrations were observed in Chinese hamster ovary cells with or 

without liver S9 mix (NTP, 1993); a weak response for the induction of chromosomal aberrations 

23
without S9 mix was observed in a second test, but the response was not reproducible. Elias et al. 

(1996) reported that EGBE did not induce chromosomal aberrations in Chinese hamster V79 fibroblast 

cells, but indicated that EGBE at high treatment concentrations (8.5mM and higher) weakly induced 

SCEs and micronuclei and potentiated the clastogenicity induced by methyl methanesulfonate. Elias et 

al. (1996) also reported that EGBE weakly induced aneuploidy (numerical chromosomal anomalies) in 

V79 cells, but this response was also found at very high concentrations (8.4 and 16.8 mM EGBE). 

When tested at up to toxic doses, EGBE or its metabolite BAL were not found to be 

mutagenic in an in vitro gene mutation assay using Chinese hamster ovary cells (CHO-AS52) 

(Chiewchanwit and Au, 1995). In contrast, Elias et al. (1996) reported that both EGBE and BAL 

weakly induced gene mutations in Chinese hamster V79 cells, but at high treatment concentrations (7.5 

mg/mL and higher). It should be noted that Chiewchanwit and Au (1995) reported high toxicity at 

38.1 mM EGBE (4.5 mg/mL). The gene mutation data presented by Elias et al. (1996) is in graphic 

form and cannot be critically evaluated given that only mean values are displayed with no standard 

deviations. Furthermore, survival data are not reported. 

It was also determined that EGBE did not increase the incidence of micronuclei in the bone 

marrow cells of male mice or rats (NTP, 1998). Animals were given three intraperitoneal 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. 

Furthermore, NTP (1998) reported high mortality (two mice out of five survived) to mice injected with 

1,000 mg/kg doses of EGBE. The protocol and results of Elias et al. (1996) appear to be adequate 

and consistent with the NTP results for this assay. 

In conclusion, EGBE has adequately been tested in conventional genotoxicity tests for its 

potential to induce gene mutations in in vitro systems and cytogenetic damage in both in vitro and in 

vivo systems. The available data do not support a mutagenic or clastogenic potential for EGBE. One 

laboratory has reported weak genotoxicity responses at toxic doses (Elias et al., 1996; Hoflack et al., 

1995). These data, however, are questionable given the limited information reported on results. 

24
4.4.5. Immunotoxicity 

Based upon the results of the Exon et al. (1991) study, it appears that the immune system is 

not a sensitive target of EGBE. In this immunotoxicity study, groups of six Sprague-Dawley rats were 

exposed to EGBE in drinking water at doses of 0, 180, or 506 mg/kg-day (males) or 0, 204, or 

444 mg/kg-day (females) for 21 days. All rats were injected subcutaneously with heat-aggregated 

aqueous keyhole limpet hemocyanin (KLH) 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 natural killer (NK) cell cytotoxicity, serum anti-KLH IgG antibody 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 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 response was significantly enhanced in males at 

180 mg/kg-day and females at 204 mg/kg-day, but not at the high dose in either sex. 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 adversity. No significant alterations in other immune parameters 

were noted. 

Smialowicz et al. (1992) reported that EGBE may potentiate the lethality of low-level 

exposure to lipopolysaccharide (LPS). They immunized F344 rats with a single intravenous injection 

of 0.5 mL of 40 μg/mL trinitrophenyl-LPS (TNP-LPS), then dosed them (six per dose group) by 

gavage with 50 to 400 mg/kg-day of various glycol ethers, including EGBE, 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. However, EGBE was not found to be immunosuppressive, as indicated by the fact 

that it did not suppress the primary plaque-forming cell response to TNP-LPS. 

4.4.6. Other In Vitro Studies 

Ghanayem (1989) has studied the metabolic and cellular basis of EGBE-induced hemolysis of 

rat erythrocytes in vitro and has compared this with human erythrocytes. EGBE is not metabolized 

when incubated with blood from male F344 rats and causes no hemolysis or metabolic alterations at 

concentrations 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 of no 

relevance physiologically. 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 (increased 

HCT) followed by hemolysis. This response was more pronounced for BAA, with nearly complete 

hemolysis observed following a 4-hour incubation at 2.0 mM. The aldehyde produced only slight 

hemolysis under the same conditions. The addition of aldehyde dehydrogenase and its cofactors to rat 

blood followed by BAL produced a potentiation of the hemolytic effects. Addition of cyanamide, an 

aldehyde dehydrogenase inhibitor, significantly decreased the effects either with or without added 

aldehyde dehydrogenase. 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. 

25
Addition of exogenous ATP failed to reverse the hemolytic effects. Neither EGBE, BAL, nor BAA 

caused any detectable changes in the concentrations of glutathione or glucose-6-phosphate 

dehydrogenase in rat erythrocytes. Blood from male and female human volunteers was unaffected by 

4-hour incubations with BAA at concentrations up to 4.0 mM. At 8 mM, only slight but significant 

hemolysis of human blood was observed, with blood from female volunteers showing a slightly greater 

sensitivity. It was concluded from these studies that the erythrocyte membrane is the likely target for 

the hemolysin BAA. Loss of osmotic homeostasis results in cell swelling and lysis, and humans of both 

sexes are relatively insensitive to the hemolytic effects of BAA. 

The relative insensitivity of human erythrocytes to the hemolytic effects of BAA has been 

demonstrated in vitro. However, the possibility exists that certain human subpopulations, including the 

aged and those predisposed to hemolytic disorders, might be at an increased risk from exposure to 

EGBE. Udden (1994, 1995a) has investigated this possibility using blood from the elderly (mean age 

71.9 years; range 64-79 years; five men and four women) or from patients with sickle cell disease 

(seven patients) or hereditary spherocytosis (three subjects; all were studied following splenectomy; 

one was studied presplenectomy). Using a sensitive assay for erythrocyte deformability (Udden, 1994; 

Udden and Patton, 1994), it was shown that blood from all of these potentially sensitive groups was 

unaffected by incubations for up to 4 hours with 2.0 mM concentrations of BAA. In more recent work 

(Udden, 1995b), the deformability of human erythrocytes incubated at BAA concentrations of 7.5-10 

mM displayed a slight but significant decrease that was accompanied by slight increases in osmotic 

fragility and MCV. These effects were judged prehemolytic and corresponded to similar changes 

reported in rat erythrocytes but at approximately 15-fold lower concentrations (0.5 mM). 

4.5. SYNTHESIS AND EVALUATION OF MAJOR NONCANCER EFFECTS AND MODE 

OF ACTION (IF KNOWN)—ORAL AND INHALATION 

Intravascular hemolysis is the primary response elicited in sensitive species following 

inhalation, oral, or dermal administration of EGBE. Humans are less sensitive to the hemolytic effects 

of EGBE than are typical laboratory species. Effects generally thought to be secondary to hemolysis 

are observed in the liver, kidneys, bone marrow, spleen, and thymus and may result from increased 

hematopoiesis. The acid metabolite of EGBE, BAA, has been shown to be the causative agent in this 

hemolysis. Hemolysis can be induced in vivo following administration of EGBE or in vitro following 

addition of BAA to either whole blood or to washed erythrocytes. In vitro tests have shown that BAA 

produces a concentration- and time-dependent swelling of rat erythrocytes, and changes in the normal 

erythrocyte morphology from the typical discocyte form to a spherocytic form prior to lysis. This 

response appears to be mediated by the erythrocyte membrane and results in an increase in osmotic 

fragility and a loss of deformability of the erythrocyte. Older erythrocytes are apparently more sensitive 

to the hemolytic effects of BAA than are younger cells or newly formed RTCs. Macrocytosis and 

increased MCV have been observed in sensitive species (rat) and are attributed to the increased number 

of larger RTCs in the circulation following this erythropoietic response (NTP, 1998). The following 

issues relate to the relevance of this hemolytic effect to humans and to EGBE’s mode of action. 

The weight of evidence obtained from a variety of studies in animals and humans would 

suggest that certain species are more susceptible to the hemolytic effects of EGBE. The sensitivities 

range from that of the guinea pig, which displays no hemolytic effects from EGBE at exposure levels 

26
as high as 1,000 mg/kg when given orally or at 2,000 mg/kg when given dermally, to the rat, which 

displays increased osmotic fragility of erythrocytes at single-inhalation exposures below 100 ppm and 

single oral exposures below 100 mg/kg EGBE. No hemolysis has been observed in controlled 

laboratory acute inhalation exposures of human volunteers to up to 195 ppm EGBE; reversible 

hemolytic effects have been observed in cases where humans consumed single oral doses of 400 to 

1,500 mg/kg EGBE (see Section 4.1). Effects in humans from chronic exposure have not been 

studied. 

With respect to gender sensitivity, it has been consistently noted (Carpenter et al., 1956; Dodd 

et al., 1983; NTP, 1993, 1998) that female rats are more sensitive to EGBE-induced hemolysis than 

male rats. This gender difference is consistent with toxicokinetic data for male and female rats 

reported for the NTP 2-year study (NTP, 1998). Female rats eliminated BAA, the toxic metabolite of 

EGBE, more slowly from the blood, resulting in a larger area under the blood concentration versus 

time curve (Appendix K of NTP, 1998). This may be a result of the reduced renal excretion observed 

in female versus male rats. NTP (1998) 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 anemia found by NTP in female over male mice. However, unlike 

female rats, female mice excrete slightly more BAA than male mice, and no significant difference 

between female and male mice has been noted in the overall rate of elimination or the half-life of BAA. 

Several studies (Ghanayem et al., 1987c, 1990) 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) and hematologic effects (Ghanayem et al., 

1987c) were found to be dose- and age-dependent, with older rats retaining more of the EGBE 

metabolite BAA in their blood and being more sensitive than the younger rats. The increased blood 

retention (as measured by increased Cmax, AUC, and T1/2) in older versus younger rats may be due to 

metabolic differences or compromised renal clearance. These researchers suggested that the 

pharmacokinetic basis of the age-dependent toxicity of EGBE may be due to a reduced ability by older 

rats to metabolize the toxic metabolite BAA to CO2 and a diminished ability to excrete BAA in the 

urine. 

While older rats appear to be more severely impacted by acute doses of EGBE, chronic 

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 (Krasavage, 1986; Grant et 

al., 1985) and chronic (NTP, 1998) studies. Ghanayem and co-workers (1990, 1992) investigated this 

adaptive effect in the male F344 rat. Daily gavage administration of EGBE at 125 mg/kg (12 days) 

resulted initially in hemolytic anemia, which was more pronounced following the third day, but rats 

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 and co-workers proposed from the results of these studies that the tolerance to hemolysis 

following repetitive dosing is not due to changes in EGBE metabolism but is due to the replacement of 

older and more susceptible erythrocytes with less susceptible, younger cells. However, chronic studies 

in rats and mice (NTP, 1998) 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 final blood 

27
collection at 12 months of age. Apparently, there is a balance in these rodents between the release of 

immature erythrocytes (RTCs) to the circulation and the aging process, so that the level of susceptible 

cells and severity of anemia remain 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 the 

liver, kidneys, spleen, bone marrow, and, to a lesser extent, the thymus. Typically, increased liver and 

kidney weights are observed with corresponding decreases in body weights at doses that produce a 

hematotoxic response. Accompanying this are hepatocellular degeneration, pigmentation of the liver, 

and congested spleens. Renal damage is often reported, accompanied by hemosiderin accumulation, 

renal tubular degeneration, and intracytoplasmic hemoglobin. Often these effects are more 

pronounced in females. Changes noted in the liver and kidneys generally return to normal following 

recovery periods of from 2 to 3 weeks. Hematopoiesis in bone marrow and spleen, increased 

cellularity of bone marrow, and splenic congestion are all secondary to the hematotoxicity of EGBE 

and result 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, suggesting an increased rate of removal of damaged erythrocytes in EGBE-treated rats 

(Ghanayem et al., 1987c). Mild lymphopenia and neutrophilia were observed at hemolytic doses of 

EGBE (Ghanayem et al., 1987c) and were reported to be consistent with a “stress” leukogram 

produced by the release of endogenous corticosteroids (Wintrobe, 1981a). Neutrophilia, commonly 

associated with acute hemolysis or hemorrhage (Wintrobe, 1981b), was also observed. 

All of the liver effects, predominantly hemosiderin pigmentation of Kupffer’s cells, noted in the 

NTP (1998) report of subchronic and chronic inhalation studies in rats and mice are discernible as 

secondary effects of the hemolytic activity caused by EGBE exposure. However, in an NTP (1993) 

subchronic drinking water study, hepatocellular cytoplasmic changes were observed in male rats at an 

exposure level (750 ppm) below the level at which hematologic changes were recorded (1,500 ppm), 

raising possibility of a direct, primary hepatic toxicity of either EGBE or a metabolite. Similar liver 

effects observed in female rats at the 750 ppm exposure level were accompanied by hematologic 

effects. In the same NTP (1993) 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. 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), consisted of hepatocytes staining more eosinophilic and lacked the 

amphophilic to basophilic granularity of the cytoplasm present in hepatocytes from control animals. 

Greaves (1990) has suggested that the lack of cytoplasmic granularity or “ground-glass” appearance of 

the hepatocytes is an indication that this response was not due to a mechanism involving enzyme 

induction. The hepatocellular degeneration and pigmentation observed at the higher exposure levels in 

both sexes was centrilobular, which is consistent with Kupffer’s cell pigmentation of the NTP (1998) 

inhalation studies. These facts, along with the fact that all other rat and mouse oral and inhalation 

studies of EGBE report hemolysis at or below exposure levels that result in liver effects, suggest that 

the hepatocellular changes in male rats reported in the NTP (1993) drinking water study may reflect 

adaptation to a subclinical level of hemolysis. However, other data indicate that there is reason for 

caution. Focal necrosis of the liver observed in male rats following gavage administration of 250 and 

500 mg/kg EGBE (Ghanayem et al., 1987b) has been judged to be inconsistent with typical anoxic 

centrilobular necrosis associated with anemia (Edmonson and Peters, 1985). The effects observed in 

28
this study may be associated with the high bolus exposures employed. However, these results and the 

results of the NTP (1993) drinking water study in male rats indicate that an assessment of human risk 

from EGBE exposure should allow for the possibility of direct liver effects by some as-yetundetermined mechanism. 

In conclusion, humans are significantly less sensitive to the hemolytic toxicity of EGBE than 

are typical laboratory species such as mice, rats, or rabbits. This 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 minimal prehemolytic 

changes to occur. Comparable effects in rat blood occur at in vitro concentrations approximately 15-

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 

BAA. Based on the results of PBPK modeling, 6-hour exposures of humans (whole body) to 

saturated atmospheres of EGBE will result in maximum blood concentrations of BAA below those 

needed to produce hemolysis (Corley et al., 1994). Although liver effects observed in rats and mice 

may be secondary to hemolysis, the possibility of a direct liver effect through a mechanism more 

relevant to humans has not been ruled out. 

4.6. WEIGHT-OF-EVIDENCE EVALUATION AND CANCER CHARACTERIZATION— 

SYNTHESIS OF HUMAN, ANIMAL, AND OTHER SUPPORTING EVIDENCE, 

CONCLUSIONS ABOUT HUMAN CARCINOGENICITY, AND LIKELY MODE OF 

ACTION 

Under the existing Guidelines for Carcinogen Risk Assessment (U.S. EPA, 1986a), EGBE is 

deemed to be a possible human carcinogen based on limited laboratory animal evidence and a lack of 

human studies. Under the Proposed Guidelines for Carcinogen Risk Assessment (U.S. EPA, 1996a), 

it is concluded that the human carcinogenic potential of EGBE cannot be determined at this time, but 

suggestive evidence exists from rodent studies. As was discussed in Section 4.2, 2-year inhalation 

bioassays conducted in mice and rats with EGBE indicate significant increases in several tumor types 

(NTP, 1998). NTP (1998) 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. They 

also reported some evidence of carcinogenic activity in male B6C3F1 mice based on increased 

incidences of hemangiosarcoma of the liver, and some evidence of carcinogenic activity in female 

B6C3F1 mice based on increased incidences of forestomach squamous cell papilloma or carcinoma 

(mainly papilloma). As discussed below, there are questions regarding the relevance of these tumors to 

an assessment of the carcinogenicity of this compound to humans. No reliable human epidemiologic 

studies are available that address the potential carcinogenicity of EGBE. 

With respect to the pheochromocytomas reported in female rats, the NTP (1998) tables 

indicate a marginally significant trend (p=0.044), and high-dose findings (16%) are only slightly 

different from the upper range of historical controls (13%). Further, pheochromocytomas can be 

difficult to distinguish from nonneoplastic adrenal medullary hyperplasia, and according to the NTP 

29
report, most of these tumors were “small and not substantially larger than the more severe grades of 

adrenal medullary hyperplasia.” Thus, these tumors must be interpreted with caution. 

The hemangiosarcomas in livers of male mice appear to be exposure related. However, the 

fact that the incidence of hemangiosarcomas was only slightly higher than the upper end of the range 

for historical controls (8% vs. 4%), was not increased in other organs (bone, bone marrow), and was 

not noted in either rats or female mice raises the question of whether this effect is related to 

accumulation of hemosiderin from hemolytic effects in the liver and related oxidative stress in male 

mice. Mice are known to be more susceptible to oxidative stress than are rats because of their lower 

antioxidant capability (Bachowski et al., 1997). On page 118 of its draft report, NTP (1998) states 

that a review of past NTP studies found no association between hemosiderin deposition in the liver and 

liver neoplasms in 79 male mice and 103 female mice from 2-year NTP studies in which liver was a site 

of chemical-related neoplasms. NTP (1998) goes on to state: “At least for mice, it does not appear 

that an accumulation of hemosiderin and possible oxidative stress alone were the cause of liver 

neoplasm in male mice.” However, recent work dose suggest that iron accumulation from the 

hemolytic effects of EGBE occurs in the livers of mice and may lead to oxidative stress (Xue et al., 

1999). Humans have been shown to be much less sensitive to the hemolytic effects of EGBE. Thus, if 

the slight increase in the incidence of hemangiosarcomas in male mice observed in the NTP study is 

related to the hemolytic effects of EGBE, they are unlikely to be relevant to human risk. Ongoing 

research on the effects of hemosiderin accumulation in male mice could help to resolve this issue. 

The increased incidence of forestomach squamous cell papillomas or carcinomas was another 

effect observed in mice but not in rats. Increased incidences of forestomach neoplasms in the male and 

female mice occurred in groups in which ulceration and hyperplasia were also noted. NTP (1998) 

notes (p. 115): “A direct association of neoplasia with ulceration and hyperplasia was not shown in 

this study although it is hypothesized that 2-butoxyethanol exposure-induced irritation caused the 

inflammatory and hyperplastic effects in the forestomach, and that the neoplasia was associated with a 

continuation of the injury/degeneration process.” The mechanism for forestomach accumulation of 

EGBE or a metabolite following inhalation exposure is not known. However, Ghanayem et al. 

(1987a) found that the levels of EGBE in the forestomach of rats 48 hours after gavage exposure were 

three times the levels in the glandular stomach, suggesting a different reactivity and/or absorption in the 

two parts of the stomach. 

In addition to the 2-year bioassay data, data from short-term tests and subchronic studies were 

evaluated along with EGBE’s chemical and physical properties to gain some insight into EGBE’s 

potential carcinogenicity. From what is known of the metabolic pathways of EGBE in animals, 

metabolic production of a species capable of significant reactivity with DNA is not anticipated. 

Available data on EGBE derived from conventional genotoxicity tests do not support a mutagenic or 

clastogenic (chromosomal breaking) potential of the compound. Further details on these genotoxicity 

tests can be found in Section 4.4. Not all carcinogens, however, are DNA reactive (Ashby and 

Tennant, 1991). A paucity of information was available on other potential modes of action for EGBE. 

Some information was available on gap-junctional intercellular communication (GJIC), which is widely 

believed to play a role in tissue and organ development and in the maintenance of a normal cellular 

phenotype with tissues. Thus, interference of GJIC may be a contributing factor in tumor 

development. Elias et al. (1996) reported that EGBE inhibited intercellular communication in Chinese 

hamster V79 fibroblast cells. They reported negative results for cell transformation in Syrian Chinese 

30
hamster embryo. This cell transformation assay is capable of detecting genotoxic or nongenotoxic 

carcinogens; however, the gene mutation data presented by Elias et al. (1996) are in graphic form and 

cannot be critically evaluated given that only mean values are displayed with no standard deviations. 

Furthermore, survival data are not reported. 

Structure-activity relationship (SAR) analyses were conducted to provide some insight into 

EGBE's potential carcinogenicity. SAR analysis is useful mostly for agents that are believed to initiate 

carcinogenesis through DNA reactive mechanisms. Based on chemical structure, EGBE does not 

resemble any known chemical carcinogens and is not expected to have electrophilic or DNA reactive 

activity. As discussed in Section 4.4.4, this is supported by genotoxicity data on EGBE, which were 

predominantly negative with the exception of one laboratory reporting weak mutagenic activity in 

some in vitro tests at toxic concentrations (Elias et al., 1996; Hoflack et al., 1995). Many of the 

conclusions reached in the Elias et al. (1996) paper cannot be evaluated because of the lack of data 

reported, and the weak Salmonella response in the Hoflack et al. (1995) study could not be repeated 

(Gollapudi et al., 1996). Thus, considering the weight of evidence on EGBE, it is not expected to be 

mutagenic or clastogenic. 

4.7. SUSCEPTIBLE POPULATIONS 

The hemolytic effect of EGBE is caused by its primary metabolite, BAA, presumably on the 

RBC membrane. Potentially susceptible subpopulations would include individuals with enhanced 

metabolism or decreased excretion of BAA. As discussed in Section 4.7.1 below, older rats have a 

reduced ability to metabolize the toxic metabolite BAA to CO2 and a diminished ability to excrete 

BAA in the urine (Ghanayem et al., 1987c, 1990). However, the relevance of this finding to the 

possible susceptibility of elderly humans is uncertain due to the fact that, as discussed in section 3, 

humans may have conjugation pathways for the excretion of BAA (BAA-glutamine and BAA-glycine) 

that are not available to the rat. 

In addition, it is expected that individuals whose RBC walls are less resistant to the lysis caused 

by BAA would be more sensitive to EGBE. However, RBCs from normal, aged, sickle-cell anemia, 

and hereditary spherocytosis patients were all resistant to the hemolytic effects of BAA (Udden, 1994). 

As work in this area continues, further information on the metabolic or structural differences that result 

in the lower sensitivity of human RBCs compared with rat RBCs may eventually illuminate 

characteristics in the human population that may indicate increased susceptibility. It is unknown at this 

time, for instance, whether people with a genetic predisposition to hemolytic anemia from other causes 

(e.g., glucose-6-phosphate dehydrogenase deficiency) would be more susceptible to EGBE-induced 

hemolysis. Other human risk factors for anemia include ingestion of certain therapeutic drugs 

(hydralazine, dilantin, chloramphenicol and others, sulfa, etc.); infections (malaria, parasites, syphilis, 

herpes, rubella, etc.); family history (e.g., of gallstones, cholestectomy, jaundice, Rh or ABO 

isoimmunization); diet (e.g., iron deficiency); and systemic illnesses such as cardiac, gastrointestinal, 

liver, renal diseases, and hypothyroidism (Berliner et al., 1999). 

31
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), who observed 24 children, age 7 months to 9 years, subsequent to oral 

ingestion of at least 5 mL of glass window cleaner containing EGBE in the 0.5% to 9.9% range 

(potentially 25 to 1,500 mg EGBE exposures). The two children who had taken greater than 15 mL 

amounts of the cleaner 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 a comparison of toxic effects observed to age sensitivity of the 

human extremely difficult. 

As discussed above, numerous risk factors for anemia might predispose an individual to or 

compound the adverse effects of EGBE-induced hemolysis. It is generally recognized, however, that 

children have fewer risk factors for anemia than are present for adults due to (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 United States 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 so that valve replacement, malignant hypertension, and the use of certain 

drugs are not usually a factor (Berliner et al., 1999; Hord and Lukens, 1999). 

The primary cause for anemia in children is usually associated with an abnormality of the 

hematopoietic system (Berliner et al., 1999; Hord and Lukens, 1999). Studies of the osmotic fragility 

and deformability of RBCs exposed to EGBE’s toxic metabolite BAA (Udden, 1994) suggest that 

certain patients with abnormal hematopoietic systems (sickle-cell anemia and hereditary spherocytosis 

patients) 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 adults. 

RBCs of neonates and children (up to 6 months) differ from normal adult RBCs in that they are larger 

and have higher levels of Hgb F versus adult Hgb A (Lewis, 1970). Frei et al. (1963) showed that the 

larger calf erythrocytes containing Hgb F were osmotically more resistant than smaller, adult 

erythrocytes containing Hgb A. Frei et al. (1963) 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 and co-workers (1987c, 1990). These studies also demonstrated the time course for the 

onset and resolution of hematologic and histopathologic changes accompanying hemolysis. Adult (9-

13 weeks) male F344 rats were significantly more sensitive to the hemolytic effects of EGBE than 

were young (4-5 weeks) male rats following administration of a single gavage dose of EGBE at 32, 63, 

125, 250, or 500 mg/kg. In concurrent metabolism studies, it was also found that there was increased 

blood retention of EGBE metabolite BAA (as measured by increased Cmax, AUC, and T1/2), and that 

young rats eliminated a significantly greater proportion of the administered EGBE dose as exhaled CO2 

or as urinary metabolites as well as excreting a greater proportion of the EGBE conjugates 

32
(glucuronide and sulfate) in the urine. These researchers suggested that the pharmacokinetic basis of 

the age-dependent toxicity of EGBE may be due to a reduced ability by older rats to metabolize the 

toxic metabolite BAA to CO and a diminished ability to excrete BAA in the urine. 2

NTP (1998) also found that young mice (6-7 weeks) eliminated BAA 10 times faster than aged 

mice (19 months) following a 1-day exposure to 125 ppm 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). 

Developmental studies, which may also be of possible relevance to this issue, have been 

conducted using rats, mice, and rabbits dosed orally, by inhalation, or, in one study, dermally (Hardin et 

al., 1984; Heindel et al., 1990; Nelson et al., 1984; NTP, 1993; Sleet et al., 1989; Tyl et al., 1984; Wier 

et al., 1987). Maternal toxicity related to the hematologic effects of EGBE and relatively minor 

developmental effects were reported in most studies. No teratogenic toxicities were noted in any of 

the studies. It can be concluded from these studies 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, with the female 

gender being more susceptible. In the NTP (1993) 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 following a 2-week exposure. 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. 

In addition, female rats displayed significantly increased urea nitrogen creatine. 

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 New Zealand rabbits at exposure levels of 18, 90, 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). Only female rabbits showed decreased erythrocyte counts, Hgb 

concentrations, and MCHC and 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, are more 

pronounced in females. In drinking water studies conducted by NTP (1993), liver lesions included 

cytoplasmic alterations, hepatocellular degeneration, and pigmentation. These effects were most 

pronounced in the three highest dose groups. 

Carpenter et al. (1956) reported female rats to be more sensitive than male rats to the 

hemolytic effects of EGBE. In studies on dogs, 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). Erythrocyte counts and Hgb concentrations were slightly decreased in the female. 

Erythrocyte permeabilities (determined by radioiodine uptake) were increased in both sexes but were 

not statistically different from control values. A female dog succumbed after 8 days of inhalation 

33
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 

leukocyte count. Autopsy 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 in one adrenal 

was found. A male dog survived 28 days of inhalation exposure to 385 ppm of EGBE (7 hours/day). 

Toxic manifestations in the male were similar to the female but developed more slowly. At autopsy, 

congestion of the kidneys was not observed for the male animal. Also, two monkeys (male and 

female) were exposed to 100 ppm EGBE by inhalation (exposure period not specified but presumed to 

be 7 hours) for 90 days. Occasional rises in erythrocyte osmotic fragility were recorded during the 

exposure period and were more frequent in the female monkey. 

In the process of studying and comparing the metabolic and cellular basis of EGBE-induced 

hemolysis of rat erythrocytes in vitro with human erythrocytes, Ghanayem (1989) observed that the 

blood from male and female human volunteers was unaffected by 4-hour incubations with BAA at 

concentrations up to 4.0 mM. At 8 mM, only slight but significant hemolysis of human blood was 

observed, with blood from female volunteers showing a slightly greater sensitivity. 

A recently completed 2-year inhalation bioassay (NTP, 1998; Dill et al., 1998) also reports 

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 half-life, and larger area under the blood concentration-versus-time 

curve. In addition, the Cmax of BAA were greater for females at each concentration and time point. It 

has been suggested that higher blood concentrations of BAA accumulate in female rats because a 

smaller amount is excreted in their urine relative to male rats (Dill et. al., 1998). Mouse data from the 

NTP (1998) study also suggest a slightly increased hematologic effect among female mice, but while 

female mice tended to have higher blood concentrations of BAA, they actually excreted more BAA in 

urine than male mice. 

5. DOSE-RESPONSE ASSESSMENTS 

5.1. ORAL REFERENCE DOSE (RfD) 

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

No chronic oral studies are currently available for EGBE. The results of the only two 

subchronic 91-day drinking water studies in rats and mice (NTP, 1993) are summarized in Table 1. 

Based on a comparison of NOAELs and LOAELs for hematologic and liver effects, rats are 

clearly more sensitive than mice. As discussed in Section 4.2, hematologic and hepatocellular changes 

were noted in both sexes of rats. In female rats, 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 

34
--

--

Table 1. Subchronic 91-day drinking water studies in rats and mice 

Effect levels (mg/kg-day) 

Reference Species (strain) Sex 

# Animals/ 

dose NOAEL LOAEL 

NTP (1993) Rat (F344) M 10 54.9a 

F 10 58.6a 

NTP (1993) Mouse (B6C3F1) M 10 223 553b 

F 10 370 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 presented in Section 4.2.

b The LOAEL in mice was based on reduced body weight and body weight gain.

in low-dose male rats (54.9 mg/kg-day using water consumption rates and body weights measured 

during the last week of exposure). However, as discussed in Section 4.5, these hepatocellular changes 

probably represent adaptation to a subclinical level of hemolysis produced at this dose. Although a 

lower LOAEL was reported in male rats, this value gives no indication of the relative slope of the doseresponse curve for males and females. Because this is an important factor for benchmark dose(BMD) 

analyses (U.S. EPA, 1995b, 1996c), a comparison of the MCV and RBC count results for both male 

and female rats was performed, which demonstrates that female rats are more sensitive to the effects of 

EGBE than are males. For this reason, dose-response information on the hematologic effects in female 

rats was selected as the basis for the oral RfD BMD analyses discussed below. 

In the female rat study (NTP, 1993), groups of 10 female F344 rats were exposed to 0, 750, 

1,500, 3,000, 4,500, and 6,000 ppm EGBE via the drinking water for 13 weeks. Body and organ 

weights were measured. In addition, clinical, hematologic, 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, Hgb, and Hct, and 

increased RTC and MCV. 

Hematologic effects appear to be the most sensitive of the adverse effects caused by EGBE in 

laboratory animals. Less clear, however, is the decision as to which of the hematologic endpoints 

(changes in RBC count, RTC, MCV, Hct, and Hgb) observed in EGBE-exposed animals is the most 

appropriate basis for an RfC/RfD. The suggested mechanism of action of EGBE is based on the fact 

that BAA, an oxidative metabolite of EGBE, appears to be the causative agent in hemolysis 

(Carpenter et al., 1956; Ghanayem et al., 1987b, 1990). The first event in this mechanism of action is 

the interaction between BAA and cellular molecule(s) in erythrocytes. The second event is erythrocyte 

swelling. The third event is cell lysis mediated by the increase in osmotic fragility, and a loss of 

deformability of the erythrocyte (Ghanayem, 1989; Udden, 1994, 1995a; Udden and Patton, 1994), 

which results in decreased values for RBC count, Hgb, and Hct. The last event is compensatory 

erythropoiesis; that is, in response to the loss of erythrocytes, the bone marrow responds by increasing 

the production of young RBCs (RTCs). 

35
Although changes in RTC sometimes represent the largest measurable differences between 

exposed animals and unexposed controls, this parameter is highly variable (covariance = 30%-60%) 

and does not always exhibit a clear dose-dependent trend (NTP, 1993, 1998). The use of RTC as the 

critical endpoint is analogous to the use of cell proliferation versus quantification of cell death 

(histopathologic). While cell death has a direct relationship to chemical exposure, cell proliferation has 

multiple feedback control processes that can be both very sensitive and variable. Therefore, changes in 

RTC are not considered a suitable endpoint for deriving the RfC or RfD. As discussed in Section 4.2, 

both cell swelling (second event of the proposed mechanism of action) and an increased number of 

larger RTCs (last event) can result in increased MCV, and decreased RBC count suggests cell lysis 

(third event). Until more is known about the molecular interaction between BAA and specific cellular 

molecules, changes in MCV and RBC count must serve as the earliest measurable responses for both 

oral and inhalation exposures to EGBE. For this reason, dose-response information on MCV and 

RBC count is considered for derivation of an RfC and an RfD for EGBE. 

While the toxicokinetic mechanism proposed above may 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 gavage studies of Ghanayem et al. (1987c) and NTP (1998) inhalation 

studies, Hct, a measure of erythrocyte volume relative to blood volume, tended to decrease along with 

RBC count and Hgb at all exposure levels for which a hematologic effect was observed. However, 

Hct did not change as RBC count and Hgb decreased following drinking water exposures (NTP, 

1993). Thus, the loss of erythrocytes (reduced RBC count) was apparently offset by a concurrent 

increase in the size of the individual cells (increased MCV) in the drinking water studies. This was not 

the case in the gavage and inhalation studies. Until the reason for this difference is known, EPA has 

chosen to make use of the empirically more sensitive endpoint (the endpoint that results in the steepest 

dose response curve) in the following RfD/RfC derivations. 

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

The human equivalent doses (HEDs) have been calculated via four methods summarized 

below. 

5.1.2.1. Standard Default Methods 

Because animals were exposed continuously to EGBE in the critical study (NTP, 1993), 

adjustments to the male rat LOAEL of 55 mg/kg-day were not required to obtain an HED. The male 

rat LOAEL was chosen for this method because it provides the more conservative RfD. The 

preceding paragraphs discuss why female rat data were used for the BMD analysis used in two of the 

other three RfD derivation methods discussed below. The female rat data were also used for the 

following PBPK method because this method was not applicable to the male rat endpoint. 

5.1.2.2. PBPK Methods 

Several PBPK models have been developed for EGBE, all of which are capable of estimating 

internal doses. These models are summarized briefly in Table 2. 

36
Table 2. Summary of PBPK models 

Model Species Routes of exposure Comments 

Johanson (1986) Human Inhalation BAA not addressed 

Shyr et al. (1993) Rat Inhalation, oral, 

dermal 

BAA excretion 

Corley et al. (1994, 1997) Rat and human Inhalation, oral, 

dermal 

BAA distribution and 

excretion; male rats only 

Lee et al. (1998) Rat and mouse Inhalation BAA distribution and 

excretion; males and females 

Of these models, the Corley et al. (1994, 1997) model is considered the most complete and 

appropriate for use in the derivation of the oral RfD because it has been experimentally validated, 

covers all routes of exposure, and addresses both the distribution and excretion of the toxic metabolite, 

BAA, via the oral route of exposure. This model is summarized in Appendix B. In 

addition to selecting a PBPK model, it is also important to determine what estimate of internal dose 

(i.e., dose metric) can serve as the most appropriate for adverse health effects. 

The PBPK model of Corley et al. (1994, 1997) is capable of calculating several measures of 

dose for both EGBE and BAA, including the following: 

• Cmax—this represents the peak concentration of EGBE or BAA in the blood during the 

exposure period. 

• AUC—On the other hand, this represents the cumulative product of concentration and 

time for EGBE and BAA in the blood. 

Two important pieces of information were used to select Cmax for BAA in the blood as the 

more appropriate dose metric. 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; Ghanayem et al., 1987b, 1990). 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. (1987c) found that gavage doses to F344 male rats of 125 mg/kg 

EGBE resulted in hemolytic effects, including reduced RBC count, Hgb and Hct, and kidney 

pathology (Hgb casts and intracytoplasmid Hgb). However, hemolytic effects were not reported at a 

similar acute drinking water dose of 140 mg/kg (Medinsky et al., 1990). While a slight drop in RBC 

count and Hgb (9% and 7%, respectively) was noted in F344 male rats after 1 week of drinking water 

exposure to 129 mg/kg-day EGBE, dose-related kidney pathology was not observed in these rats, 

even after 13 weeks of drinking water exposure to up to 452 mg/kg-day EGBE (NTP, 1993). Finally, 

Corley et al. (1994) have also suggested that Cmax may be a better dose metric than AUC. 

Four steps were involved in using the Corley et al. (1994, 1997) PBPK model as modified by 

Corley et al. (1997) to calculate the HED corresponding to the LOAEL identified in the animal study 

(LOAELHED): (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 

37
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 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 liters of water during a 12-hour awake cycle; and (4) calculate the 

HED (mg/kg-day) for the amount of EGBE consumed in 2 liters of water that resulted in the same 

internal dose (Cmax BAA) simulated for the animal in Step 1 as shown below. 

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 hematologic determination) 

Cmax BAA = 103 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 hours/day, 7 days/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. 

Water concentration 

(ppm) 

Calculated dose of EGBE 

from drinking water (mg/kg-day) 

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 LOAELHED for a 70 kg human consuming EGBE in 2 liters 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 = 103 μM 

LOAELHED continuous exposure = 7.6 mg/kg-day (calculated by regression of the internal 

dose vs. the dose of EGBE from Step 3). 

The LOAELHED calculated using the PBPK model is likely a conservative estimate of the HED 

since the model is based on male rat kinetic data and female rats have been observed to have slightly 

higher concentrations of BAA in blood than male rats at similar exposure levels. In other words, use of 

male rat kinetic data results in estimates of the BAA concentrations in human blood associated with an 

effect (LOAELHED) that are lower than if female rat kinetic data had been used. In addition, the internal 

dose surrogate, Cmax for BAA in blood, is highly dependent upon the rate of water ingestion. Since 

drinking water exposures are highly complex and variable, a simplifying assumption was used in all 

simulations that the entire dose of EGBE in drinking water was consumed over a 12-hour period each 

38
day corresponding to the awake cycle for both rats and humans. This assumption resulted in higher 

peak blood concentrations of BAA in both rats and humans than would have been calculated using the 

original Corley et al. (1994) structure that assumed that drinking water uptake occurred over a 24 

hours/day dosing period. 

5.1.2.3. BMD Method 

All BMD assessments in this review were performed using EPA Benchmark Dose Software 

(BMDS). A copy of the latest version of BMDS can be obtained from the Internet at 

www.epa.gov/ncea/bmds.htm. For the purposes of deriving an RfD for EGBE, hematologic 

endpoints (see discussion in Section 5.1.1) were evaluated as continuous data. For this reason, the 

BMD05 was considered a more appropriate basis than the BMD10 (U.S. EPA, 1995b, 1996c). MCV 

was the most sensitive hematologic endpoint in the NTP (1993) study. Observed versus predicted 

MCV responses using the BMDS Power model (version 1.1.1b) are provided in Figure 2 for EGBEexposed female rats. A textual description of these results is provided in Appendix C. The BMD05 

was determined to be 49 mg/kg-day, using the 95% lower confidence limit of the dose-response curve 

expressed in terms of administered dose. This value is considered to be an HED under this method of 

analysis because animals were exposed continuously, negating the need for a duration adjustment. 

5.1.2.4. PBPK and BMD Methods Combined 

Cmax for BAA in arterial blood was determined using the PBPK model of Corley et al. (1994) 

as modified by Corley et al. (1997). The results of this modeling effort are summarized in Table 3. 

Graphic results of the BMDS Power model (version 1.1.1b) assessment of MCV responses in 

female rats (NTP, 1998) versus corresponding PBPK estimates of Cmax for BAA in female rat blood 

are provided in Figure 3. A textual description of these results is provided in Appendix C. The BMD05 

was determined to be 64 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. (1994, 1997) PBPK model was 

used to “back-calculate” an HED of 5.1 mg/kg-day, assuming that rats and humans receive their entire 

dose of EGBE from drinking water over a 12-hour period each day. 

5.1.3. RfD Derivation—Including Application of Uncertainty Factors and Modifying Factors 

Uncertainty factors (UFs) are applied to account for recognized uncertainties in extrapolation 

from experimental conditions to the assumed human scenario (i.e., chronic exposure over a lifetime). 

Historically, UFs are applied as values of 10 in a multiplicative fashion (Dourson and Stara, 1983). 

Recent EPA practice, however, also includes use of a partial UF of 101/2 (3.162; U.S. EPA, 1994b) on 

the assumption that the actual values for the UFs are log-normally distributed. Application of these 

factors in the assessments is that, when a single partial UF is applied, the factor is rounded to 3; the 

total factor for a UF of 3 and 10, for example, would be 30 (3 × 10). When two partial UFs are 

evoked, however, they are not rounded, so UFs of 3, 3, and 10 would result in a total uncertainty of 

100 (actually 101/2 × 101/2 × 101). Uncertainty factors applied for this RfD assessment and the 

justification for their use are as follows. 

39
70 

BMD05 = 58 

Power Model Best Fit 

95% Lower Confidence Limit 

MCV Mean Response (fL) 

65 

60 

55 

0 50 100 150 200 250 300 350 400 

Dose (mg/kg-day) 

Figure 2. BMD plot of MCV data (expressed in terms of femtoliters, fL) in female rats 

following oral exposure to EGBE (NTP, 1993) using external dose (mg/kg-day). 

Table 3. Corley et al. (1994, 1997) model estimates of BAA blood levels in female rats 

following oral exposures 

Water Water Female Body BAA in Blood 

conc. (ppm) intake (L/day) weight (g) Dose (mg/kg-day) Cmax (uM) 

750 0.0147 188 59 103 

1,500 0.0155 185 125 253 

3,000 0.0125 180 208 495 

4,500 0.0101 164 277 738 

6,000 0.0101 150 404 1,355 

40
70 

BMD05 = 64 

Power Model Best Fit 

95% Lower Confidence Limit 

MCV Mean Response (fL) 

65 

60 

55 

0 200 400 600 800 1000 1200 1400 

Cmax BAA (uM) 

Figure 3. BMD plot of MCV data (expressed in terms of fL) in female rats following oral 

exposure to EGBE (NTP, 1993) using internal dosimetry (BAA Cmax, μM). 

A value of 10 was selected to account for variation in sensitivity within the human population 

(UFH). Potentially susceptible subpopulations include individuals with enhanced metabolism or 

decreased excretion of BAA and individuals whose RBC walls are less resistant to the lysis caused by 

BAA. A UF of 10 was retained to account for the uncertainty associated with the variability of the 

human response to the effects of EGBE. 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, 

and laboratory animal (rats, calves, and mice) studies suggest that older animals are more sensitive than 

neonates and that females are more sensitive than males (see other details in Section 4.7). However, 

actual human responses to EGBE have not been observed in a broad enough range of exposure 

conditions (e.g., repeat/long-term exposures) and potentially sensitive subjects (e.g., individuals 

predisposed to hemolytic anemia, infants) to warrant the reduction of the UFH below the default value 

of 10. 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. 

The UF for interspecies variation (UFA) accounts for pharmacodynamic and pharmacokinetic 

differences between animals and humans. There is in vivo (Carpenter et al., 1956) and in vitro 

(Ghanayem and Sullivan, 1993; Udden and Patton, 1994; Udden, 1995b) information indicating that, 

pharmacodynamically, humans are less sensitive than rats to the hematologic effects of EGBE. For this 

reason, a fractional component of the UFA was considered. However, the in vivo relative insensitivity 

of humans cannot be quantified at this time. Thus, for all RfD derivation approaches discussed above, 

a value of 1 was used to account for pharmacodynamic differences between rats and humans. Under 

the standard default and BMD approaches described above, an overall UFA of 3 (1 for 

41 
pharmacodynamics × 3 for pharmacokinetics) was used. For the PBPK and the combined 

PBPK/BMC (benchmark concentration) approaches, an overall UFA of 1 (1 for pharmacodynamics 

× 1 for pharmacokinetics) was used because pharmacokinetic differences between rats and humans are 

adequately accounted for by a PBPK model. 

For all RfD calculation approaches, a value of 1 was selected for extrapolating the results from 

a subchronic study to chronic exposures (UFS). Although no chronic oral studies are currently 

available for EGBE, there does not appear to be a significant increase in the severity of hemolytic 

effects beyond 1-3 weeks of oral (NTP, 1993) or inhalation (NTP, 1998) EGBE exposures. 

For the standard default and PBPK methods, a value of 3 was selected for extrapolating a 

LOAEL to a NOAEL (UFL). A value of less than 10 is justifiable because there is information that 

indicates the LOAEL is very near the threshold level for the hematologic effects of concern. For 

example, the effects observed in the critical study at the lowest drinking water doses were fairly mild. 

All hematologic endpoints except for RTCs were within 5% of the control value. For BMD analyses, 

a value of 1 was used because of the minimal and precursive nature of the critical lesion (cell swelling 

as measured by increased MCV) and the fact that a BMD05 for a minimally adverse effect is typically 

deemed to be equivalent to a NOAEL for continuous data sets (U.S. EPA, 1995b, 1996c). 

A value of 1 was used for the database UFD for all methods of analyses. While no chronic oral 

studies or adequate human data are available for EGBE, oral and inhalation dose-response data 

indicate that there would be little if any increase in severity of hemolytic effects beyond subchronic 

exposure durations (NTP, 1993, 1998). There are chronic and subchronic studies available in two 

species (rats and mice) and adequate reproductive and developmental studies, as well as limited studies 

in humans following short-term inhalation exposure. 

A modifying factor (MF) of 1 was used for all approaches. A summary of how the five UFs 

and one MF were applied for the four RfD calculation approaches discussed is provided in Table 4. 

The combined PBPK and BMD method was used to derive the RfD, since this approach 

incorporates much of the mechanistic information available for EGBE, best characterizes the doseresponse relationships for EGBE-induced hematologic effects, and reduces the potential uncertainties 

to the greatest extent. Thus, the total UF is 10 and the MF is 1. The RfD = 5.1 mg/kg-day ÷ 10 = 0.5 

mg/kg-day. 

5.2. INHALATION REFERENCE CONCENTRATION (RfC) 

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

Short-term studies by Dodd et al. (1983) in rats and Tyl et al. (1984) in rats and rabbits, 

although well conducted, were not considered long enough to be useful for predicting toxicity 

following chronic inhalation exposures. In addition, the blood samples evaluated by Tyl et al. (1984) 

42
--

were collected 6 days following the last exposure. For this reason, these studies are not considered 

further. The results of several candidate subchronic and chronic studies are summarized in Table 5. 

Hematologic effects from EGBE exposure are the only effects consistently observed across 

both sexes and species that have been studied. NTP (1998) observed forestomach ulcers in female 

mice at all exposure levels, but this effect has not been observed in any other species, nor in mice 

exposed orally to EGBE (NTP, 1993), and although incidence of this lesion increased with exposure, 

severity of the lesion did not increase with increasing dose. For these reasons, and because 

hematologic effects have been observed in humans acutely exposed to EGBE, hematologic effects are 

considered the critical effects of concern for the purpose of the RfC derivation. Based on a 

comparison of effect levels, female rats (NTP, 1998) appear to be more sensitive to the hematologic 

Table 4. Summary of application of uncertainty factors and 

modifying factor for RfD calculation 

Approach 

Factor Standard PBPK BMD PBPK & BMD 

UFH 10 10 10 10 

UFA 3 1 3 1 

UFS 1 1 1 1 

UFL 3 3 1 1 

UFD 1 1 1 1 

UF(Total) 100 30 30 10 

RfD 

(mg/kg-day) 

55/100 = 0.6 7.6/30 = 0.3 49/30 = 2 5.1/10 = 0.5 

Table 5. Results of candidate studies 

Hematologic effect 

levels (ppm) 

Reference Species (strain) Sex 

#/Dose 

group 

Duration 

(months) NOAEL LOAEL 

NTP (1998) Rat (Fischer) M 9-10 3, 9, 12; 

14 weeks 

31 62.5 

F 9-10 3, 9, 12; 

14 weeks 

31 

NTP (1998) Mouse (B6C3F1) M 9-10 3, 9, 12; 

14 weeks 

31 62.5 

F 9-10 3, 9, 12; 

14 weeks 

31 62.5 

Dodd et al. (1983) Rat (F344) MF 16 9 25 77 

43
effects of EGBE than the other animals. In the female rat study, NTP (1998) exposed groups of 9-10 

female F344 rats to 0, 31, 62.5, 125, 250, and 500 ppm EGBE in air for 3 to 12 months (6 hours/day, 

5 days/week). Body and organ weights were measured, and clinical, hematologic, gross, and 

histopathologic examinations were conducted. Female rats exposed to the three highest 

concentrations at all exposure durations developed clinical signs consistent with the hemolytic effects 

associated with EGBE exposures. Mild to moderate regenerative anemia was observed in females 

exposed to all concentrations. Exposure-related trends were noted for RTC, RBC count, MCV, Hgb, 

and Hct. Liver-to-body weight ratios were significantly increased in females exposed to the highest 

concentration. Histopathologic effects observed in rats included excessive extramedullary splenic 

hematopoiesis, hemosiderosis, and hemosiderin accumulation in Kupffer cells of the liver (secondary to 

hemolysis). A LOAEL of 31 ppm was identified in this study for hematologic and histopathologic 

effects in female rats. A NOAEL for these effects was not identified. Therefore, the female rat data 

from this study were used as the basis for an RfC. 

It is recognized that the NOAEL/LOAEL designations listed above for each study do not 

necessarily indicate the slope of the concentration-response curve, an important factor in BMC (used 

to assess inhalation studies in the same manner as BMDs are used to assess oral studies) analysis (U.S. 

EPA, 1995b, 1996c). For this reason, BMC analyses were also performed on the other subchronic and 

chronic studies (Dodd et al., 1983; NTP, 1998 in mice). The rationale for choice of the critical effect 

for use in the BMC analyses, changes in MCV and RBC count, is the same as for oral exposure to 

EGBE and is summarized in Section 5.1.1. 

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

The human equivalent concentrations (HECs) have been calculated via four methods. 

5.2.2.1. Standard Default Methods 

There are two steps to calculating an HEC (LOAELHEC) from a LOAEL identified in an animal 

study: (1) convert units to mg/m3 (31 ppm × 4.84 [mg/m3]/ppm = 150 mg/m3), and (2) account for the 

ratio of blood:air partitioning of the chemical for laboratory animals to humans (150 mg/m3 × 1 [default 

ratio] = 150 mg/m3). This value is considered an HED and is not adjusted for less than continuous 

exposure because, as discussed in Section 5.1.2, dose rate is considered a more important determinant 

of effects from EGBE than total dose. Thus, a LOAELHEC of 150 mg/m3

 was calculated for EGBE by 

this standard default method. 

5.2.2.2. PBPK Methods 

The model of Lee et al. (1998) was used to estimate BAA blood concentrations in female rats 

following inhalation exposure to EGBE because it is a recent extension of the Corley et al. (1994, 

1997) model for inhalation exposures and includes added parameters for female rats. As in the case of 

the RfD (see Section 5.1.2.2), Cmax is considered a more appropriate dose metric than AUC, and the 

PBPK model of Corley et al. (1994, 1997) was used to obtain estimates of human Cmax concentrations 

from the female rat data. The same procedure was used to calculate the HEC corresponding to the 

LOAEL identified in the animal study (LOAELHEC): (1) calculate the internal dose surrogate (Cmax 

44
BAA in blood) corresponding to the female rat LOAEL using the actual experimental exposure 

regimen (6 hours/day, 5 days/week) in model simulations; (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 continuously exposed (24 hours/day, 7 days/week) to 

varying concentrations of EGBE; (4) calculate the continuous HEC for EGBE in air that resulted in the 

same internal dose (Cmax BAA) simulated for the animal in Step 1; and (5) convert the EGBE exposure 

units from ppm to mg/m3

 as shown below. 

Step 1: Calculate Cmax for BAA in blood corresponding to female rat LOAEL (Lee et al., 1998). 

Female rat LOAEL = 31 ppm 

Cmax BAA = 285 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 6 hours/day, 5 days/week 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 (Corley et al., 1994, 1997). 

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 

Step 4: Calculate LOAELHEC of EGBE for continuous human exposures producing the same 

Cmax of BAA in blood calculated for the animal study in Step 1. 

Female rat Cmax BAA = 285 μM 

HEC continuous exposure = 98 ppm (calculated by regression of internal dose versus the 

concentration of EGBE in air from Step 3). 

Step 5: Unit conversion. 

LOAELHEC (mg/m3) = Conversion factor × LOAELHEC (ppm) 

= 4.84 (mg/m3)/(ppm) × 98 ppm 

= 474 mg/m3 

5.2.2.3. BMC Method 

For the purposes of deriving an RfC for EGBE, both MCV and RBC count response data 

were evaluated in female rats (see discussion in Section 5.1.1) for all exposure durations studied by 

45
NTP (1998). Because these endpoints were evaluated as continuous data, the BMC05 was considered 

a more appropriate basis than the BMC10 (U.S. EPA, 1995b, 1996c). The steepest concentrationresponse curves (and the lowest BMC05 estimate) were obtained for decreased RBC count in female 

rats. Since severity of the effect did not change with increasing exposure duration, all exposure 

durations (14 weeks, 3, 6, and 12 months) were considered. The best model fit (p=0.8652) was 

obtained from response data from the 14-week subchronic study, using the BMDS polynomial model 

and excluding the two highest exposure levels (250 and 500 ppm). Observed versus predicted RBC 

count responses using a polynomial model are provided in Figure 4. A textual description of these 

results is provided in Appendix C. As discussed in Section 5.1.2.3, all BMD assessments in this review 

were performed using EPA BMDS version 1.1b. The BMC05 was determined to be 27 ppm (130 

mg/m3

) using the 95% lower confidence limit of the dose-response curve expressed in terms of 

administered concentration. Use of response information from just the lowest four exposure groups 

(0, 31, 62.5, and 125 ppm) is justified based on both a significantly improved model fit and increased 

relevancy to any potential human exposure and effect scenario. Although poorer model fits were 

obtained, assessments using other model and duration combinations were supportive of these results 

(BMC05 estimates ranging from 15 to 58 ppm). The estimated BMC05 of 130 mg/m3

 is considered an 

HED and is not adjusted for less than continuous exposure because, as discussed in Section 5.1.2.3, 

dose rate is considered a more important determinant of effects from EGBE than total dose. 

8.5 

8 

7.5 

7 

BMD05 = 27 

Polynomial Model Best Fit 

95% Lower Confidence Limit 

0 20 40 60 80 100 120 

Exposure Concentration (ppm) 

Figure 4. BMC plot of RBC count in female rats following 14-week inhalation exposure 

to EGBE (NTP, 1998) using external dose (ppm). 

RBC Mean Response (x10E6) 

46
5.2.2.4. PBPK and BMC Methods Combined 

Cmax for BAA in arterial blood of rats was determined using the PBPK model of Lee et al. 

(1998). Dermal exposures to the EGBE vapor were not considered in the predicted blood levels. This 

is because the estimated relative contribution of the skin to the total uptake of unclothed humans 

exposed to 25 ppm EGBE for 8 hours ranged only from 4.6% to 27.5%, depending on temperature, 

humidity, and exercise level (Corley et al., 1997). Thus, dermal uptake is predicted to contribute less 

than 10%, even if 50% of an individual’s skin is exposed. The results of this modeling effort are 

summarized in Table 6. 

Graphic results of a Power model assessment of RBC count responses in female rats (NTP, 

1998) versus corresponding PBPK estimates of Cmax for BAA in female rat blood are provided in 

Figure 5. A textual description of these results is provided in Appendix C. As discussed in Section 

5.1.2.3, all BMD assessments in this review were performed using EPA BMDS version 1.1b. The 

BMD05 was determined to be 225 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) PBPK model was 

used to “back-calculate” HEC of 78 ppm (380 mg/m3) assuming continuous exposure (24 hours/day). 

5.2.3. RfC Derivation—Including Application of Uncertainty Factors and Modifying Factors 

UFs are applied to account for recognized uncertainties in extrapolation from experimental 

conditions to the assumed human scenario (i.e., chronic exposure over a lifetime). Historically, UFs 

are applied as values of 10 in a multiplicative fashion (Dourson and Stara, 1983). Recent EPA 

practice, however, also includes use of a partial UF of 101/2 (3.162; U.S. EPA, 1994b) on the 

assumption that the actual values for the UFs are log-normally distributed. Application of these factors 

in the assessments is that, when a single partial UF is applied, the factor is rounded to 3, such that the 

total factor for a UF of 3 and 10, for example, 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). Uncertainty factors applied for this RfC assessment 

and the justification for their use are as follows. 

Table 6. Lee et al. (1998) model estimates of BAA blood levels in female rats 

following inhalation exposures 

Exposure Female rat body BAA in arterial blood 

concentration (ppm) weight (g) Cmax (μM) in female rats 

31 216 285 

61.5 211 603 

125 214 1243 

250 210 1959 

500 201 4227 

47
8.5 

BMD05 = 225 uM 

Power model best fit 

95% Lower Confidence Limit 

RBC Mean Response (x10E6) 

8 

7.5 

7 

0 200 400 600 800 1000 1200 

Cmax BAA (uM) 

Figure 5. BMC plot of RBC count in female rats following 14-week inhalation exposure to 

max EGBE (NTP, 1998) using internal dosimetry (BAA, C , μM).

A value of 10 was selected to account for variation in sensitivity within the human population 

(UFH). Potentially susceptible subpopulations include individuals with enhanced metabolism or 

decreased excretion of BAA and individuals whose RBC walls are less resistant to the lysis caused by 

BAA. A UF of 10 was retained to account for the uncertainty associated with the variability of the 

human response to the effects of EGBE. 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, 

and laboratory animal (rats, calves, and mice) studies suggest that older animals are more sensitive than 

neonates and that females are more sensitive than males (see other details in Section 4.7). However, 

actual human responses to EGBE have not been observed in a broad enough range of exposure 

conditions (e.g., repeat/long-term exposures) and potentially sensitive subjects (e.g., individuals 

predisposed to hemolytic anemia, infants) to warrant the reduction of the UFH below the default value 

of 10. 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. 

The UF for interspecies variation (UFA) accounts for pharmacodynamic and pharmacokinetic 

differences between animals and humans. There is in vivo (Carpenter et al., 1956) and in vitro 

(Ghanayem and Sullivan, 1993; Udden and Patton, 1994; Udden, 1995b) information indicating that, 

pharmacodynamically, humans are less sensitive than rats to the hematologic effects of EGBE. For this 

reason, a fractional component of the UFA was considered. However, the in vivo relative insensitivity 

of humans cannot be quantified at this time. Thus, for all RfC derivation approaches discussed above, 

a value of 1 was used to account for pharmacodynamic differences between rats and humans. Further, 

each approach accounts for pharmacokinetic differences between rats and humans by either PBPK 

48 
models or EPA default methods. Thus, an overall UFA

pharmacokinetics) was used for all of the RfC derivation approaches. 

of 1 (1 for pharmacodynamics × 1 for 

For all RfC calculation approaches, a value of 1 was selected for extrapolating the results from 

a subchronic study to chronic exposures (UFS). Recent chronic studies indicate that a significant 

increase in the severity of hemolytic effects beyond 1-3 weeks of inhalation exposure time to EGBE 

(NTP, 1998) would not be expected. 

For RfC derivation methods, a value of 3 was selected for extrapolating a LOAEL or BMC 

estimate to a NOAEL (UFL). A UFL value of less than 10 is justifiable because there is information 

that indicates that both the chosen LOAEL and estimated BMC05 values are near the threshold level for 

the hematologic effects of concern. The measured hematologic effects that formed the basis for these 

values were mildly adverse and within 5% of the control value. In addition, the female rat LOAEL 

(150 mg/m3

) and BMC05 (130 mg/m3) values derived from the NTP (1998) subchronic/chronic 

inhalation study are very close to the 121 mg/m3 (25 ppm) NOAEL identified for male and female rats 

in the Dodd et al. (1983) subchronic inhalation study. In the case of the RfD (Section 5.1.3), a UFL 

value of 1 was used for the BMD analyses because the RfD BMDs were based on a minimal and 

precursive lesion (cell swelling as measured by increased MCV). A threefold UFL is retained for the 

RfC BMC analyses because the RfC BMCs are based on a more serious hematologic endpoint (RBC 

lysis as measured by a decrease in RBC count). 

A value of 1 was used for the database UFD for all methods of analyses. Subchronic and 

chronic inhalation studies suggest that there is little, if any, increase in severity of hemolytic effects 

beyond subchronic exposure durations (NTP, 1993, 1998). There are chronic and subchronic studies 

available in two species (rats and mice) and adequate reproductive and developmental studies, as well 

as limited studies in humans following short-term inhalation exposure. 

An MF of 1 was used for all approaches. A summary of how the five UFs and one MF were 

applied for the four RfC calculation approaches discussed is provided in Table 7. 

The combined PBPK and BMD/C method was used to derive the RfC, since this approach 

incorporates much of the mechanistic information available for EGBE, best characterizes the doseresponse relationships for EGBE-induced hematologic effects, and reduces the potential uncertainties 

to the greatest extent. Thus, the total UF is 30 and the MF is 1; RfC = 380 mg/m3 ÷ 30 = 13 mg/m3. 

5.3. CANCER ASSESSMENT 

As discussed above (Sections 4.2 and 4.6), there are currently no human epidemiologic, 

occupational studies addressing the potential carcinogenicity of EGBE. A 2-year inhalation bioassay 

using mice and rats has recently been completed (NTP, 1998) and reports significant increases in 

certain types of tumors in exposed mice compared with controls, but not in rats. The relevancy of 

these tumors to humans is not clear at this time, as is discussed in Section 4.6, and a quantitative 

assessment was not performed. 

49
Table 7. Summary of application of UFs and MF for RFC calculation 

Approach 

Factor Standard PBPK BMC PBPK & BMC 

UFH 10 10 10 10 

UFA 1 1 1 1 

UFS 1 1 1 1 

UFL 3 3 3 3 

UFD 1 1 1 1 

UF(Total) 30 30 30 30 

RfC 

mg/m3 

150/30 = 5 474/30 = 16 130/30 = 4 380/30 = 13 

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 BAA, the proximate toxicant, in both humans and animals. 

This acid and its conjugates are readily excreted in the urine. 

Hemolysis has been identified as the critical endpoint of concern in toxicological studies on 

EGBE. Humans are significantly less sensitive to the hemolytic toxicity of EGBE than are typical 

laboratory species such as mice, rats, or rabbits. This has been demonstrated in numerous laboratory 

studies and through the use of in vitro studies using either whole blood or washed erythrocytes. In 

addition to hemolytic effects of EGBE, other effects (e.g., liver, spleen, and kidney) have been 

observed in laboratory animals with exposure to EGBE. While male rats in one study (NTP, 1993) 

experienced mild liver effects at a drinking water dose lower than that which caused observable 

hemolytic effects, human case report and controlled study data and most laboratory animal evidence 

suggest that these other effects are secondary to hemolysis. Available human toxicity data show that 

after acute oral ingestion of large doses of EGBE combined with other solvents, hematologic changes 

and metabolic acidosis are the primary effects. Occupational exposure to low levels of EGBE did not 

cause adverse changes in hepatic, renal, or hematologic parameters (Haufroid et al., 1997). 

Due to the known reproductive toxicity (i.e., toxicity to male testes and sperm) of two other 

glycol ethers, EGME (2-methoxyethanol) and EGEE (2-ethoxyethanol), the reproductive toxicity of 

EGBE has been studied in a variety of well-conducted oral (Nagano et al., 1979, 1984; Grant et al., 

1985; Foster et al., 1987; Heindel et al., 1990; Exon, 1991; NTP, 1993) and inhalation (Dodd et al., 

1983; Doe, 1984; Nachreiner, 1994; NTP, 1998) studies 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 (Wier et al., 1987; Sleet et al., 1989), 

inhalation (Nelson et al., 1984; Tyl et al., 1984), and dermal (Hardin et al., 1984) exposures to rats, 

50
mice, and rabbits. EGBE did not cause adverse 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 toxicities 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 parents, nor to the 

developing fetuses of laboratory animals. 

No reliable human epidemiologic studies are available that address the potential 

carcinogenicity of EGBE. A draft report of the results of a 2-year inhalation bioassay performed using 

rats and mice has recently become available (NTP, 1998). NTP (1998) 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. They also reported some evidence of carcinogenic activity in 

male B6C3F1 mice based on increased incidences of hemangiosarcoma of the liver, and some evidence 

of carcinogenic activity in female B6C3F1 mice based on increased incidences of forestomach 

squamous cell papilloma or carcinoma (mainly papilloma). As discussed in more detail in Section 4.6, 

because of the uncertain relevance of these tumor increases to humans, the fact that EGBE is generally 

negative in genotoxic tests, and the lack of human data to support the findings in rodents, the human 

carcinogenic potential of EGBE, in accordance with the recent Proposed Guidelines for Carcinogen 

Risk Assessment (U.S. EPA, 1996a), cannot be determined at this time, but suggestive evidence exists 

from rodent studies. Under existing EPA guidelines (U.S. EPA, 1986a), EGBE is judged to be a 

possible human carcinogen. For a more complete discussion of the carcinogenic potential of EGBE 

see Section 4.6. 

6.2. DOSE RESPONSE 

The quantitative estimates of human risk from lifetime exposure to EGBE are based on animal 

experiments, because no relevant human data exist. 

The human oral dose that is likely to be without an appreciable risk of deleterious noncancer 

effect during a lifetime (the RfD) is 0.5 mg/kg-day. This value was obtained by dividing the estimated 

human equivalent BMC05 of 5 mg/kg-day by a UF of 10 (see summary of UF below). The human 

equivalent BMC05 was estimated using Cmax for BAA in blood as the dose metric, calculating a BMD05 

of 64 μM, and then using the BMD approach described in the previous section and a PBPK model to 

“back-calculate” an HED, assuming that rats and humans receive their entire dose of EGBE from 

drinking water over a 12-hour period each day. 

The overall confidence in the RfD assessment is medium to high. The RfD value has been 

calculated for EGBE using the combined PBPK/BMD method. A higher confidence is placed in the 

RfD values derived from internal dose measures, since pharmacokinetic differences between rats and 

humans were accounted for using a validated PBPK model (Corley et al., 1994, 1997). Medium 

confidence is placed on the NTP (1993) study because it was not a chronic study; however, the study 

employed both male and female rats and mice, provided a wide range of exposure levels (0-6,000 ppm 

EGBE in drinking water), and observed animals twice daily. Medium to high confidence is placed on 

51
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 controlled studies and case 

reports, and laboratory animal and in vitro studies provide ample evidence to suggest that long-term 

human exposures would be no more adverse than long-term rat exposures. Confidence is not “high” 

because the potential for effects in humans from repeat, long-term exposures has not been 

investigated. 

A value of 10 was selected to account for variation in sensitivity within the human population 

(UFH). Potentially susceptible subpopulations include individuals with enhanced metabolism or 

decreased excretion of BAA and individuals whose RBC walls are less resistant to the lysis caused by 

BAA. A UF of 10 was retained to account for the uncertainty associated with the variability of the 

human response to the effects of EGBE. Human responses to EGBE have not been observed in a 

broad enough range of exposure conditions (e.g., repeat/long-term exposures) and potentially sensitive 

subjects (e.g., individuals predisposed to hemolytic anemia, infants) to warrant the reduction of the 

UFH below the default value of 10. An MF was not employed (MF = 1). For a more detailed 

discussion of the RfD UF, see Section 5.1.3. 

The daily inhalation exposure to the human population that is likely to be without an 

appreciable risk of deleterious noncancer effect during a lifetime (the RfC) is 13 mg/m3. This amount is 

1/30 the human equivalent BMC05 of 380 mg/m3

, which was “back-calculated” from rat data using the 

BMD and PBPK approach described in the previous section. 

The overall confidence in the RfC assessment is medium to high. A higher confidence is 

placed in the RfC values derived from internal dose measures (PBPK method and combined 

PBPK/BMC method) because pharmacokinetic differences between rats and humans were accounted 

for using PBPK models (Lee et al., 1998; Corley et al., 1994, 1997). High confidence is placed on the 

NTP (1998) 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. Medium to high 

confidence is placed on 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 controlled 

studies and case reports, and laboratory animal and in vitro studies, provide ample 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. Confidence is not “high” because the potential for effects in 

humans from repeat, long-term exposures has not been investigated. 

In the derivation of the RfC, a 30-fold UF was applied, which is intended to account for 

intrahuman variability and extrapolation from an adverse effect level. A value of 10 was selected to 

account for variation in sensitivity within the human population (UFH). Potentially susceptible 

subpopulations include individuals with enhanced metabolism or decreased excretion of BAA and 

individuals whose RBC walls are less resistant to the lysis caused by BAA. An uncertainty factor of 

10 was retained to account for the uncertainty associated with the variability of the human response to 

the effects of EGBE. Human responses to EGBE have not been observed in a broad enough range of 

exposure conditions (e.g., repeat/long-term exposures) and potentially sensitive subjects (e.g., 

individuals predisposed to hemolytic anemia, infants) to warrant the reduction of the UFH below the 

default value of 10. In the case of the RfC, a partial threefold LOAEL to NOAEL UF (UFL) is 

retained because the BMC used in the derivation of the RfC was based on a more serious hematologic 

endpoint (RBC lysis as measured by a decrease in RBC count) than the effect that formed the basis for 

the RfD BMD (cell swelling as measured by an increase in MCV). An MF was not employed (MF = 

1). For a more detailed discussion of the RfC UF, see Section 5.2.3. 

52
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U.S. EPA. (1996c) Benchmark dose technical guidance document (external review draft). EPA/600/P96-002A. 

U.S. EPA. (1997) IRIS support document. Ethylene glycol monobutyl ether. 

U.S. EPA. (1998a) Guidelines for neurotoxicity risk assessment, April 1998. EPA 630/R-95/001F, 

NTIS PB98-117831. 

U.S. EPA. (1998b) Science policy handbook: peer review. Office of Science Policy, Office of 

Research and Development, January 1998. EPA/600/B-98/001. 

Werner, HW; Nawrocki, CZ; Mitchell, JL; et al. (1943a) Effects of repeated exposure of rats to 

vapours of monoalkyl ethers of ethylene glycol. J Ind Hyg Toxicol 25:374-379. 

Werner, HW; Mitchell, JL; Miller, JW; et al. (1943b) Effects of repeated exposure of dogs to 

monoalkyl ethylene glycol ether vapors. J Ind Hyg Toxicol 25:409-414. 

59
Wier, PJ; Lewis, SC; Traul, KA. (1987) A comparison of developmental toxicity evident at term to 

postnatal growth and survival using ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, 

and ethanol. Teratog Carcinog Mutag 7:55-64. 

Wintrobe, MM. (1981a) Variations of leukocytes in disease. In: Clinical hematology. Wintrobe, MM, 

ed. Philadelphia: Lea & Febiger, pp. 1284-1323. 

Wintrobe, MM. (1981b) The normocytic, normochromic anemias. In: Clinical hematology. Wintrobe, 

MM, ed. Philadelphia: Lea & Febiger, pp. 677-697. 

Xue, H; Kamendulis, LM; Klaunig, JE. (1999) A potential mechanism for 2-butoxyethanol (2-BE) 

induced mouse liver neoplasia. Abstract, annual meeting, Society of Toxicology, March 14-18, New 

Orleans, LA. 

Zeiger, E; Anderson, B; Haworth, S; et al. (1992) Salmonella in mutagenicity tests. V. Results from 

the testing of 311 chemicals. Environ Mol Mutagen 19 (Suppl 21):2-141. 

60
APPENDIX A. EXTERNAL PEER REVIEW— 

SUMMARY OF COMMENTS AND DISPOSITION 

The support document and IRIS summary for ethylene glycol butyl ether (EGBE) have 

undergone both internal peer review performed by scientists within EPA and a more formal external 

peer review performed by scientists chosen by EPA’s contractor in accordance with guidance on peer 

review (U.S. EPA, 1994c). Comments made by the internal reviewers were addressed prior to 

submitting the documents for external peer review and are not part of this appendix. Public comments 

also were read and carefully considered. The four 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 comments made by the external reviewers 

and EPA’s response to these comments follows. 

(1) General Comments 

Reviewers felt that the IRIS file for EGBE was an acceptable basis for the derivation of an RfD 

and RfC, calling it “an excellent synthesis of the current state of knowledge,” “a well written, concise 

review,” and “a very thorough, credible, and lucid scientific presentation.” However, two reviewers 

felt that the cancer assessment was premature and should be postponed pending publication of a final 

report on the NTP chronic inhalation bioassay. This key issue is addressed briefly in the general 

comment review below and more extensively in Section 6, “Comments on Chemical-Specific 

Questions.” 

A. Comment: One reviewer commented extensively regarding his belief that “the NTP 2-year studies 

must be addressed more appropriately.” This and one other reviewer suggested that EPA should wait 

for the final report on the NTP 2-year cancer bioassay. 

Response to Comment: The NTP 2-year studies could not be addressed fully in the external 

peer review EGBE file because the report on its findings was not available. The only materials 

available at the time were pathology tables from the NTP Internet Web site, which did not include any 

descriptive text, nor did it include any hematologic information or blood chemistry data. The external 

peer review draft made appropriate conclusions based on the preliminary nature of the information 

available. Subsequent to the external peer review, a draft of the NTP 2-year study has been forwarded 

to EPA and considered in the preparation of the consensus review draft. The final NTP report will 

also be considered when it becomes available. 

B. Comment: One reviewer suggested that EPA consider the following additional studies: 

1. The 14-week studies performed in conjunction with the NTP 2-year study. 

2. Lee, K; et al. (in press) Physiologically-based pharmacokinetics model for chronic 

inhalation of 2-butoxyethanol. Toxicol Appl Pharmacol. 

3. Dill, J; et al. (in press) Toxicokinetics of 2-butoxyethanol and its major metabolites, 2-

butoxyacetic acid, in F344 rats and B6C3F1 mice. Toxicol Appl Pharmacol. 

4. Ghanayem, BI. (1996) An overview of the hematotoxicity of ethylene glycol ethers. 

Occup Hyg 2:253-268. 

61
Response to Comment: Like the NTP 2-year study, the draft report of the 14-week NTP 

study was not available at the time of the EGBE IRIS file’s external peer review. Much more 

information was available on this study in tabular form, however, and EPA was able to derive an RfC 

from the available preliminary information on the 14-week study. As with the 2-year study, the draft 

14-week study has since been made available to EPA and has been considered in the current EGBE 

IRIS file version. Studies 2, 3 and 4 above are now available for consideration in the consensus review 

draft as well, and have been retrieved, reviewed, and cited in the current EGBE IRIS file version. 

(2) Study Descriptions 

A. Comment: One reviewer suggested inclusion of a “developmental/repro” studies discussion in 

Section 6.1. 

Response to Comment: A paragraph has been added to Section 6.1 to summarize the 

developmental and reproductive system effect findings of the document. 

B. Comment: One reviewer suggested that the lesions observed in rats and mice in the NTP 14-week 

inhalation exposure study were not discussed. 

Response to Comment: This comment is incorrect. These lesions are discussed in Section 4.2 

in the discussion of the NTP (1998) study. 

C. Comment: One reviewer did not understand the statement in section 4.2 in the discussion of the 

Krasavage (1986) study, “hematologic changes occurring at 443 and 885 mg/kg-day were increased 

MCV and decreased MCHC. This seems to be inconsistent with the predominant theory that 

erythrocyte swelling precedes lysis of the cell.” 

Response to Comment: The sentence before this statement indicates that all doses of EGBE 

caused decreased RBC counts and decreased Hgb concentrations. The fact that RBC counts were 

decreased at doses lower than doses that caused increased MCV is what was intended to be proffered 

as inconsistent with “swelling precedes lysis.” Thus, the latter sentence above has been replaced with 

“The decrease in RBC count at a lower dose (222 mg/kg-day) seems to be inconsistent with the 

predominant theory that erythrocyte swelling (which is indicated by the increased MCV) precedes...” 

D. Comment: This same reviewer stated that “the report failed to address the effects of EGBE on 

the morphology of erythrocytes and how these changes resemble morphological changes reported in 

certain human blood disorders,” and provided a citation. 

Response to Comment: The effects of EGBE on the morphology of erythrocytes, particularly 

in rats, are discussed, to the extent that they are currently known with any degree of certainty, in 

several places in Sections 4.2 and 4.5 of the IRIS support document. Additional language has been 

added to Section 4.5 regarding erythrocyte morphological changes in rats from EGBE exposure that 

are similar to morphological changes observed in certain human blood disorders. However, no 

evidence exists that suggests that humans with these blood disorders represent sensitive 

subpopulations. In fact, experiments by Udden et al. (1994) have shown that blood from persons with 

62
sickle cell anemia is no more sensitive to the hemolytic effects of EGBE than blood from normal 

persons. 

E. Comment: One reviewer pointed out some additional reservations concerning the Haufroid et al. 

(1997) occupational EGBE exposure study, including the fact that the authors did not account for an 

important metabolic detoxification pathway (BAA conjugation with glutamine), the higher alcohol 

consumption among exposed workers, and the lack of any relation between hematologic results and 

parameters of internal exposure. 

Response to Comment: The additional caveats have been added to the EGBE IRIS file. 

F. Comment: One reviewer commented that “in the absence of a complete hematologic 

investigation, a role for hemolysis [in cases of anemia observed after human ingestion during suicide 

attempts] cannot be substantiated.” 

Response to Comment: EPA agrees that the anemia reported in human case reports, 

particularly Rambourg-Schepens et al. (1988), may have been related to more than just the hemolytic 

effect of EGBE. A statement regarding the speculative nature of any conclusions in the absence of 

more detailed hemolysis data has been added to the support document’s discussion of this case report 

and to Section 4.5, Synthesis and Evaluation of Major Noncancer Effects and Mode of Action—Oral 

and Inhalation. 

G. Comment: A reviewer suggested that the eosinophilic inclusions in rat hepatocytes observed after 

subchronic exposure to EGBE are reminiscent of Mallory bodies seen in cases of alcoholic hepatitis. 

The reviewer pointed out that “there is some evidence that Vitamin A depletion accentuates this finding 

[Mallory bodies],” and that a comparison of the rat diet and liver lesions from the subchronic versus 

chronic studies would help to resolve whether diet was a contributing factor. 

Response to Comment: An association between diet and the eosinophilic inclusions would be 

difficult, if not impossible, to determine even if the suggested dietary comparison could be performed. 

Further, since the eosinophilic inclusions were observed in a high percentage (40%-100%) in male and 

female rats of EGBE exposure groups, and not in the control rats of either sex, it is not likely that they 

would be due to a dietary deficiency. A comparison was not attempted. 

H. Comment: One reviewer did not agree that the bone marrow hyperplasia observed in rats in the 

NTP subchronic oral and inhalation studies was an indication of “bone marrow toxicity” or 

“leukemogenicity.” This reviewer argued that this hyperplasia was a secondary adaptive response to 

hemolysis and that changes in white cell and platelet counts “can be attributed to splenomegaly or 

splenic congestion.” 

Response to Comment: The section of text this reviewer was referring to has been edited. 

To account for the reviewer’s comment, the sentence that refers to bone marrow and liver toxicity has 

been supplemented with language indicating that they are possible secondary effects of hemolysis. 

New information relevant to the association of liver toxicity and hemolysis has been added to Section 

4.5 as well. 

63
I. Comment: According to one reviewer, the table in Section 5.1.1 falsely implies that hematologic 

effects were observed in mice from the NTP subchronic drinking water study. 

Response to Comment: A column header for the table has been changed and a footnote was 

added to the LOAEL for mice indicating that it was based on reduced body weight and reduced body 

weight gain. 

(3) RfD/RfC Calculation 

A. Comment: The use of a male rat LOAEL for the standard default method of deriving an oral RfD 

is “contradictory to the basis of selection of LOAEL provided in preceding paras.” 

Response to Comment: The basis for the LOAEL used in the standard default RfD derivation 

method was not the focus of the paragraphs that precede this derivation. The preceding paragraphs 

focused on the assumptions, including the use of female rats, for the benchmark dose analysis used in 

two of the other three RfD derivation methods discussed. 

B. Comment: “A sentence to justify the choice of Cmax

added” to the PBPK method discussions in Sections 5.1.2 and 5.2.2. 

as the appropriate dose surrogate may be 

Response to Comment: The PBPK methods discussion in Section 5.1.2 has been rewritten to 

include an additional page of discussion on this issue. Section 5.2.2 refers to the discussion in Section 

5.1.2. 

C. Comment: There is no need to adjust for less-than-continuous exposures for the BMC RfC 

derivation method if, as is stated earlier, “BAA blood concentrations attain steady state during any 24-

hr period.” The reviewer contends that “the fact that steady state is attained implies that regardless of 

the duration of further exposure, there will be no further change in blood concentration of the dose 

surrogate.” 

Response to Comment: The Agency agrees with this comment, and both the standard and 

BMC methods discussed in Section 5.2.2 have been revised to reflect that these values are not adjusted 

for less than continuous exposure because, as is now discussed in section 5.1.2, dose rate is considered 

a more important determinant of effects from EGBE than total dose. 

D. Comment: A reviewer argued that the selection of “mean cell volume (MCV) as the earlier 

endpoint and development of the HEC without further explanation may create conflicting and 

confusing scientific discussions,” that “using the MCV as an endpoint may have overestimated [the 

oral HED] by approximately 8 to 20 fold,” and that RBC count data should have been used to 

determine the NOAEL for hematologic parameters. 

Response to Comment: The reviewer is correct in that better dose-response curves are 

available from RBC count data in the NTP (1998) inhalation study. This was realized originally, but 

the MCV data were used because it was felt that there were mechanistic reasons to use MCV as the 

endpoint that measured an early event in the RBC hemolysis process. This thinking has changed after 

review of the recently released draft NTP (1998) report (see response to Question C, section 6, below, 

64
and current discussions of the 1998 NTP study in Toxicological Review Section 4.2 and Section 

5.1.2). Both MCV and RBC count data were considered for the current assessment, with RBC count 

data being chosen for derivation of the current RfC value. 

(4) Uncertainty Factors 

Intrahuman Uncertainty Factor (UFH)—The proposed UFH for both the RfD and RfC was 3, based 

on the fact that red blood cells from the elderly and from patients with hemolytic disorders do not 

show an increased sensitivity to the hemolytic effects of EGBE (Udden, 1994; Udden and Patton, 

1994). A UF of 1 was not considered justifiable given that not all potentially sensitive subpopulations 

have been tested in this manner. 

A. Comments on UFH: One reviewer commented that the threefold UFH should be retained based on 

known toxicodynamic differences (factor of 1) and unresolved toxicokinetic differences (factor of 3) 

between animals and humans. 

A second reviewer suggested that the UFH should be increased to 10-fold because (a) “some 

humans have exhibited signs of hemolytic anemia after ingestion of EGBE” and “some humans, for 

reasons not characterized at this time, may be more sensitive than the average population”; (b) some 

lesions observed in laboratory animals exposed to EGBE resemble lesions observed in humans with 

certain diseases such as hereditary spherocytosis and sickle cell anemia; and (c) the yet to be published 

NTP report of a 2-year bioassay “suggests that EGBE is a multisite carcinogen in mice.” 

A third reviewer supports an intrahuman UF of 1 because he felt confident in the “decrease in 

sensitivity of human red cells for hemolysis” and was “not certain at all that the liver toxicity described 

is independent of hemolysis.” This reviewer felt that it was more reasonable to attribute the 

hepatoxicity of EGBE to the secondary changes related to hemolysis and iron deposition. He cited 

evidence from the NTP drinking water study that rats experienced both liver damage and hemolysis at 

55 mg/kg-day, whereas mice experienced hemolysis at much higher doses (550-670 mg/kg-day) and 

showed no signs of liver damage. 

Like the first reviewer, the fourth reviewer commented that “since it was clearly established 

that humans are more resistant (less sensitive) to potential hemolytic effects of EGBE than the female 

rat, it is scientifically justifiable to assign an uncertainty factor of 1, not 3, for this part of the 

determination.” This reviewer felt that the Agency should apply UF to account for “potential chronic 

hepatic effects in humans and for subchronic to chronic extrapolation by applying factors ranging from 

1 to 10.” 

Response to Comments: In their comments on this issue, all four reviewers have confused the 

UFH with the interspecies UFA to some extent. The purpose of the UFH is to ensure protection of 

sensitive subpopulations. Differences between species are more appropriately accounted for by the 

UFA. Nevertheless, it has been determined that, as suggested by the second reviewer, a UFH of 10 is 

required to fully account for possible human sensitive subpopulations, including children (see expanded 

discussion in Section 4.7.1). Potentially susceptible subpopulations include individuals with enhanced 

metabolism or decreased excretion of BAA and individuals whose RBC walls are less resistant to the 

lysis caused by BAA. Human in vitro studies suggest that the elderly and patients with fragile RBCs 

65
would not be more sensitive to the hemolytic effects of EGBE than normal adults, and laboratory 

animal (rats, calves, and mice) studies suggest that older animals are more sensitive than neonates and 

that females are more sensitive than males. However, actual human responses to EGBE have not been 

observed in a broad enough range of exposure conditions (e.g., repeat/long-term exposures) and 

potentially sensitive subjects (e.g., individuals predisposed to hemolytic anemia, infants) to warrant the 

reduction of the UFH below the default value of 10. The concern over undiagnosed human liver effects 

expressed by the fourth reviewer is also a valid argument for retention of the 10-fold UFH default. 

Subchronic to Chronic Uncertainty Factor (UFS)—For all RfC and RfD calculation 

approaches proposed in the EGBE IRIS Support Document, a partial UFS of 3 was selected for 

extrapolating from a subchronic study to chronic exposures. This was based on the fact that, although 

no chronic studies are currently available (results of a 2-year bioassay have not been reported 

completely), there does not appear to be a significant increase in the severity of hemolytic effects 

beyond 1-3 weeks of exposure to EGBE (NTP, 1993). 

B. Comment on UFS: One reviewer commented that “if Cmax is chosen as the dose surrogate and 

steady state is attained during subchronic exposures, then the use of a factor of 3 [for the UFS] is not 

defensible.” He supported a UFS of 1 based on this and earlier statements in the document that 

suggested “no increase in severity of hemolytic effects beyond subchronic exposure durations.” 

Response to Comment: The Agency basically agrees with this comment. While the day-today attainment of steady state with respect to the Cmax does not necessarily ensure that the health 

effects observed at the end of a subchronic study would not progress as a result of chronic exposures, 

recent dose-response information obtained from the NTP (1998) chronic inhalation study does suggest 

that the hemolytic effects of EGBE do not progress significantly with chronic exposure. Information 

from the NTP (1993) drinking water study also suggests a lack of progression in severity beyond the 

first 1-3 weeks of exposure. For this reason, the UFS used in the derivation of the oral RfD and the 

inhalation RfC were reduced to 1. 

(5) Weight-of-Evidence/Confidence Levels 

No comments were received regarding the cancer weight-of-evidence classification or the 

noncancer confidence levels; however, two reviewers felt that the cancer assessment was premature 

and should be postponed, pending publication of a final report on the NTP chronic inhalation bioassay. 

This was done. 

(6) Comments on Chemical-Specific Questions 

A. Question: Have we gone too far in analyzing the NTP (1998) chronic inhalation study given that 

it exists only in the form of data tables on the Internet that have not been peer reviewed at this time? 

Comments: Two reviewers felt strongly that EPA should wait for the complete report of the chronic 

NTP bioassay before finalizing the IRIS support document’s cancer assessment. One reviewer did not 

address the question directly. Another reviewer had some suggestions on how to interpret the mouse 

liver and forestomach tumors, but did not feel that it was necessarily inappropriate to use the data 

tables to support a cancer assessment at this time. 

66
Response to Comments: The draft NTP (1998) report on the chronic inhalation study became 

available after this peer review and is incorporated into the IRIS file for EGBE. 

B. Question: Is adequate justification provided for basing the RfC and RfD for hematologic effects in 

rats, despite in vitro indications of human insensitivity? Are the liver effects observed by NTP (1993) 

in rats adequately addressed as likely secondary effects of hemolysis (e.g., in Section 4.5 and as they 

would impact the RfD/RfC derivations in Section 5)? 

Comments: One reviewer did not address this question. All reviewers generally agreed with the use 

of hematologic data as the basis for the RfD and RfC. One reviewer suggested “modifying factors” be 

used (or applied to the UFA) to take into account the difference in sensitivity to hematologic effects of 

EGBE between humans and rats, and the reviewer did not feel that concern over a possible direct liver 

effect of EGBE warranted maintaining a threefold intrahuman UFH. Another reviewer suggested that 

any conclusion regarding the potential for EGBE to cause liver effects that are not secondary to 

hemolysis “can only be determined after chronic oral exposure to EGBE or comparison with other 

similar chemicals that have linked findings in subchronic to observations in chronic studies.” 

Response to Comments: The use of a fractional component of the interspecies UFA, or 

“modifying factors” as suggested by the reviewer, was considered. However, due primarily to 

limitations in the database of human response information, the in vivo relative insensitivity of humans 

cannot be quantified at this time. Thus, a value of 1 was used to account for pharmacodynamic 

differences between rats and humans, and an overall UFA of 1 (1 for pharmacodynamics × 1 for 

pharmacokinetics) was used for derivation of the RfD and RfC. 

With respect to the possible direct liver effect of EGBE, the Agency concurs with the second 

reviewer in believing that this issue requires further oral animal and oral/inhalation human studies 

before it can be conclusively resolved. Thus, a threefold UFD has been retained for both the RfD and 

the RfC. 

C. Question: Is the rationale in Section 5 for the selection of MCV as the hematologic endpoint 

convincing? 

Comments: One reviewer did not address this question. Two reviewers agreed with the rationale and 

choice of MCV as the basis for the RfD and RfC. However, one indicated that he would have 

measured the MCV and RTC counts by different, more sensitive and reproducible methods. One 

commenter disagreed with the use of MCV, pointing out that MCV is not a “bench-level measured 

clinical determination.” 

Response to Comments: Data from the recently released NTP (1998) chronic inhalation 

bioassays in rats and mice caused the Agency to rethink its position on the strict use of MCV as the 

basis for both the RfD and RfC. In vitro studies by Ghanayem (1989) show that the hemolysis caused 

by EGBE metabolite BAA is preceded by erythrocyte swelling. However, increased MCV from 

EGBE exposures can also be attributed to the erythropoietic response subsequent to hemolysis and the 

corresponding increase in the number of larger RTCs in circulation (NTP, 1998). RTC count was 

increased significantly in females at 62.5 ppm (6 and 12 months) and in males at 125 ppm (3 and 6 

months) of the NTP (1998) chronic rat bioassay. On the other hand, since a statistically significant 

67
increase in RTC count was not observed in this study at any duration in males or females exposed to 

31 ppm, nor in males exposed to 62.5 ppm, it appears that RTC count alone cannot account for the 

increase in MCV at these levels of exposure. The observed increases in MCV may be a combined 

result of both erythrocyte swelling prior to and an increased number of RTCs subsequent to hemolysis, 

with the former being more influential at lower exposure levels and the latter having more relative 

impact at higher exposure levels. Thus, other endpoints, including RBC count changes, were 

considered for use in the derivation of the RfD and RfC. One reviewer commented that the Agency 

rationale for use of MCV was reasonable, but that more sensitive and reproducible methods should 

have been used to measure both MCV and RTC counts. The Agency agrees with this reviewer and 

with the reviewer who commented that the MCV as measured was not a bench-level measured clinical 

determination. Nevertheless, the Agency concurs with the former reviewer’s contention that MCV 

should be considered (along with other endpoints) because of the potential for MCV to provide an 

indication of primary events in the pathology of EGBE-induced hemolysis. 

68
APPENDIX B. CORLEY ET AL. (1994, 1997) PBPK MODEL 

Corley et al. (1994) 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 A-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 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 Corley et al. (1994) model included additional routes of exposure such as oral (gavage), 

drinking water, intravenous infusion, and dermal (liquids and vapor). The formation of BAA was 

assumed to occur only in the liver, using the rat liver perfusion data of Johanson et al. (1986) 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 since they were 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) found no evidence of metabolites in urine that would indicate that humans form 

conjugates of EGBE or ethylene glycol. Thus, these pathways, which were lumped together in the 

model of Corley et al. (1994) to simulate rat kinetic data, were eliminated for human simulations. 

The human blood:air partition coefficient of 7965, from Johanson and Dynesius (1988), was 

also used in the Corley et al. (1994) model. In addition, the partition coefficients for both EGBE and 

BAA were measured in human blood, rat blood, and rat tissues using a modification of the Jepson et al. 

(1994) 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 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). Constants for the saturable elimination of BAA by the kidneys 

were then estimated by optimization from the data of Ghanayem et al. (1990), where rats were 

administered EGBE intravenously 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. Significant increases in the concentrations 

of EGBE were observed by Corley et al. (1997) in the first postexposure blood samples. Since 

the subjects were able to freely move their arms after the exposure, Corley et al. hypothesized that 

the local blood flow to the exposed arm increased for a few minutes postexposure. By adjusting 

the blood flow to the skin by fourfold for 5 minutes postexposure, the model is able to simulate 

this change in the concentration of EGBE in blood. 

69
Dermal 

Vapor/Liquid 

Lungs and Arterial 

Blood 

Rapidly Perfused 

Organs 

Slowly Perfused 

Organs 

Fat Arterial Blood 

Venous Blood 

Rapidly Perfused 

Organs 

Slowly Perfused 

Organs 

A

Fat rterial Blood 

Venous Blood 

Inhalation 

Lungs and Arterial 

Blood 

Urine 

(Butoxyacetic Acid) 

Liver 

Gastrointestinal 

Tract 

Muscle 

Kidney 

Liver 

Gastrointestinal 

Tract 

Muscle 

Drinking Water 

or Gavage 

Metabolism to 

Butoxyacetic Acid 

Skin Skin 

IV Infusion 

Other Metabolites 

Exhalation 

Model for 2-Butoxyethanol Model for 2-Butoxyacetic Acid 

Figure A-1. PBPK model of Corley et al. (1994). 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. 

70
APPENDIX C. TEXT OUTPUT FROM BENCHMARK DOSE SOFTWARE 

RUNS USED IN THE DERIVATION OF RfD AND RfC VALUES 

==================================================================== 

Power Model, Version Number: 1.1.1b 

Input Data File: C:\BMDS4ME\DATA\EGBE\EGBEORAL.(D) 

Fri Jan 08 13:18:25 1999 

==================================================================== 

BMD Method for RfD: MCV Response in Orally Exposed Female Rats (NTP, 1993) 

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

The form of the response function is: 

Y[dose] = control + slope * dose^power

Dependent variable = MEAN

Independent variable = DOSE

The power is not restricted

The variance is to be modeled as Var(i) = alpha*mean(i)^rho

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: 2.22045e-016

Parameter convergence has been set to: 1.49012e-008

Default Initial Parameter Values

alpha = 3.02612 

rho = 0 

control = 54.8 

slope = 0.0438974 

power = 0.976715 

71
Parameter Estimates 

Variable Estimate Standard Error 

alpha 0.0008464 

rho 1.95327 

control 54.7753 

slope 0.0520655 

power 0.946046 

0.00159172 

0.449801 

0.330267 

0.00218389 

0.069827 

Asymptotic Correlation Matrix of Parameter Estimates 

alpha rho 

alpha -1 1 

rho 1 -1 

control -0.0078 0.0057 

slope 0.012 -0.012 

power 9.5e-005 -0.00011 

control slope 

-0.0078 0.012 

0.0057 -0.012 

1 -0.77 

-0.77 1 

-0.02 -0.029 

Table of Data and Estimated Values of Interest 

power 

9.52-005 

-0.00011 

-0.02 

-0.029 

1 

Est Std Dev 

1.45 

1.52 

1.58 

1.66 

1.73 

184 

Dose N Obs Mean Obs Std Dev Est Mean 

0 10 54.8 0.92 54.8 

59 10 57 1.25 57.2 

125 10 60.5 1.27 59.8 

208 10 62.4 1.78 62.9 

277 10 65.3 1.83 65.4 

404 10 70.1 2.76 70 

Model Descriptions for Likelihoods Calculated 

72
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)} = alpha*(Mu(i))^rho 

Model R: Yi = Mu + e(i) 

Var{e(i)} = Sigma^2 

Likelihoods of Interest

Model Log(likelihood) DF AIC

A1 -60.057590 7 -67.057590

A2 -52.588573 12 -64.588573

A3 -57.206449 8 -65.206449

fitted -58.818246 5 -63.818246

R -131.090823 2 -133.090823

Explanation of Tests 

Test 1: Do response 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)

Tests of Interest 

Test -2*log(likelihood ratio) DF p-value

Test 1 157.005 10 <0.00001

Test 2 14.938 5 0.01063

Test 3 9.23575 4 0.05547

Test4 3.22359 3 0.3584

The p-value for Test 1 is less than 0.05. There appears to be a difference between response

and/or variances among the dose levels. It seems appropriate to model the data.

73
The p-value for Test 2 is less than 0.05. A nonhomogeneous variance model appears to be

appropriate.

The p-value for Test 3 is greater than 0.05. The modeled variance appears to be appropriate here.

The p-value for Test 4 is greater than 0.05. The model chosen seems to adequately describe the

data.

Benchmark Dose Computation

Specified effect = 2.7388 (5% of background estimate of 54.7753)

Risk type = Added response

Confidence level = 0.950000

BMD = 65.941824

BMDL = 48.792442

74
 ==================================================================== 

Power Model, Version Number: 1.1.1b 

Input Data File: C:\BMDS4ME\DATA\EGBE\EGBEORAL.(D) 

Fri Jan 08 15:26:07 1999 

==================================================================== 

BMD + PBPK Method for RfD: MCV in Orally Exposed Female Rats (NTP, 1993) 

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

The form of the response function is: 

Y[dose] = control + slope * dose^power

Dependent variable = MEAN

Independent variable = CMAX

The power is not restricted

The variance is to be modeled as Var(i) = alpha*mean(i)^rho

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: 2.22045e-016

Parameter convergence has been set to: 1.49012e-008

Default Initial Parameter Values 

alpha = 3.02612 

rho = 0 

control = 54.8 

slope = 0.0828926 

power = 0.731677 

75
Parameter Estimates 

Variable Estimate 

alpha 0.0008464 

rho 1.95428 

control 54.6662 

slope 0.136613 

power 0.656823 

Standard Error 

0.00161642 

0.456969 

0.335163 

0.00575923 

0.0430356 

Asymptotic Correlation Matrix of Parameter Estimates 

Alpha Rho Control Slope 

alpha -1 1 -0.015 0.02 

rho 1 -1 0.013 -0.021 

control -0.015 0.013 1 -0.78 

slope 0.02 -0.021 -0.78 1 

power -0.00069 0.00077 -0.023 -0.039 

Table of Data and Estimated Values of Interest 

Dose N Obs Mean Obs Std Dev Est Mean 

0 10 54.8 0.92 54.7 

103 10 57 1.25 57.5 

253 10 60.5 1.27 59.8 

495 10 62.4 1.78 62.7 

738 10 65.3 1.83 65.1 

1355 10 70.1 2.76 70.2 

Power 

-0.00069 

0.00077 

-0.023 

-0.039 

1 

Est Std Dev 

1.45 

1.53 

1.59 

1.66 

1.72 

1.85 

76
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)} = alpha*(Mu(i))^rho 

Model R: Yi = Mu + e(i) 

Var{e(i)} = Sigma^2 

Likelihoods of Interest 

Model Log(likelihood) DF AIC 

A1 -60.057590 7 -67.057590 

A2 -52.588573 12 -64.588573 

A3 -57.206449 8 -65.206449 

fitted -58.950987 5 -63.950987 

R -131.090823 2 -133.090823 

Explanation of Tests 

Test 1: Do response 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)

77
Tests of Interest 

Test -2*log(likelihood ratio) DF p-value

Test 1 157.005 10 <0.00001

Test 2 14.938 5 0.01063

Test 3 9.23575 4 0.05547

Test4 3.48907 3 0.3222

The p-value for Test 1 is less than 0.05. 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 less than 0.05. A nonhomogeneous variance model appears to be

appropriate.

The p-value for Test 3 is greater than 0.05. The modeled variance appears to be appropriate here.

The p-value for Test 4 is greater than 0.05. The model chosen seems to adequately describe the

data.

Benchmark Dose Computation

Specified effect = 2.733000 (5% of background estimate of 54.66)

Risk type = Added response

Confidence level = 0.950000

BMD = 95.712853

BMDL = 63.695782

78
 ==================================================================== 

Polynomial Model, Version Number: 1.1.0b 

Input Data File: C:\BMDS4ME\DATA\EGBE\EGBE_F.(D) 

Tue Jan 12 08:56:59 1999 

==================================================================== 

BMD Method for RfC: RBC Count for Female Rats Exposed 14 Weeks (NTP, 1998) 

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

The form of the response function is: 

Y[dose] = beta_0 + beta_1*dose + beta_2*dose^2 + ...

Dependent variable = MEAN

Independent variable = DOSE

Signs of the polynomial coefficients are not restricted

The variance is to be modeled as Var(i) = alpha*mean(i)^rho

Total number of dose groups = 4

Total number of records with missing values = 0

Maximum number of iterations = 250

Relative function convergence has been set to: 2.22045e-016

Parameter convergence has been set to: 1.49012e-008

Default Initial Parameter Values

alpha = 0.040875

rho = 0

beta_0 = 8.47683

beta_1 = -0.0125321

beta_2 = 4.71226e-008

79
Parameter Estimates 

Variable Estimate 

alpha 0.000870086 

rho 1.82359 

beta_0 8.47429 

beta_1 -0.0123721 

beta_2 -1.16375e-006 

Standard Error 

0.0064509 

3.61441 

0.0626104 

0.0024627 

1.78152e-005 

Asymptotic Correlation Matrix of Parameter Estimates 

alpha rho beta_0 beta_1 beta_2 

alpha 1 -1 0.1 -0.14 0.13 

rho -1 1 -0.1 0.14 -0.13 

beta_0 0.1 -0.1 1 -0.76 0.62 

beta_1 -0.14 0.14 -0.76 1 -0.97 

beta_2 0.13 -0.13 0.61 -0.97 

Table of Data and Estimated Values of Interest 

Dose N Obs Mean Obs Std Dev Est Mean 

0.0000 10 8.480 0.160 8.474 

31.0000 10 8.080 0.230 8.090 

62.5000 10 7.700 0.250 7.696 

125.000 10 6.910 0.150 6.909 

1 

Est Std Dev 

0.043 

0.039 

0.036 

0.030 

80
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)} = alpha*(Mu(i))^rho 

Model R: Yi = Mu + e(i) 

Var{e(i)} = Sigma^2 

Warning: Likelihood for model A1 larger than or equal to that one for model A2. 

Warning: Likelihood for model A3 larger than or equal to that one for model A2. 

Warning: Likelihood for model R larger than or equal to that one for model A2. 

Likelihoods of Interest 

Model Log(likelihood) DF AIC 

A1 46.051943 5 41.051943 

A2 -0.501998 8 -8.501998 

A3 46.173884 6 40.173884 

fitted 46.159466 5 41.159466 

R 1.343542 2 -0.656458 

Explanation of Tests 

Test 1: Do response 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)

81
Tests of Interest 

Test -2*log(likelihood ratio) DF p-value

Test 1 0 6 <0.00001

Test 2 0 3 <0.00001

Test 3 0 2 <0.00001

Test4 0.0288374 1 0.8652

The p-value for Test 1 is less than 0.05. 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 less than 0.05. A nonhomogeneous variance model appears to be

appropriate.

The p-value for Test 3 is less than 0.05. You may want to consider a different variance model.

The p-value for Test 4 is greater than 0.05. The model chosen seems to adequately describe the

data.

Benchmark Dose Computation

Specified effect = 0.423700 (5% of background estimate of 8.474)

Risk type = Added response

Confidence level = 0.950000

BMD = 34.136806

BMDL = 26.942074

82
 ==================================================================== 

Power Model, Version Number: 1.1.1b 

Input Data File: C:\BMDS4ME\DATA\EGBE\EGBE_F.(D) 

Mon Jan 11 13:20:21 1999 

==================================================================== 

BMD + PBPK Method for RfC: RBC Count for Female Rats Exposed 14 Wks (NTP, 1998) 

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

The form of the response function is: 

Y[dose] = control + slope * dose^power

Dependent variable = MEAN

Independent variable = CMAX3

rho is set to 0

The power is not restricted

A constant variance model is fit

Total number of dose groups = 4

Total number of records with missing values = 0

Maximum number of iterations = 250

Relative function convergence has been set to: 2.22045e-016

Parameter convergence has been set to: 1.49012e-008

Default Initial Parameter Values 

alpha = 0.040875

control = 8.48

slope = -0.00208697

power = 0.928193

83
Parameter Estimates 

Variable Estimate 

alpha 0.0368453 

control 8.47733 

slope -0.00181442 

power 0.948686 

Standard Error 

0.00823886 

0.0469956 

9.50703e-005 

0.132835 

Asymptotic Correlation Matrix of Parameter Estimates 

Alpha Control Slope Power 

alpha 1 -1.6e-006 -2.7e-006 -0.00016 

control -1.6e-006 1 -0.76 0.01 

slope -2.7e-006 -0.76 1 0.017 

power -0.00016 0.01 0.017 

Table of Data and Estimated Values of Interest 

1 

Est Std Dev 

0.192 

0.192 

0.192 

0.192 

Dose N Obs Mean Obs Std Dev Est Mean 

0 10 8.48 0.16 8.48 

285 10 8.08 0.23 8.09 

603 10 7.7 0.25 7.69 

1243 10 6.91 0.15 6.91 

84
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 R: Yi = Mu + e(i) 

Var{e(i)} = Sigma^2 

Likelihoods of Interest 

Model Log(likelihood) DF AIC 

A1 46.051943 5 41.051943 

A2 47.963928 8 39.963928 

fitted 46.020539 4 42.020539 

R 1.323169 2 -0.676831 

Explanation of Tests 

Test 1: Do response and/or variances differ among dose levels? (A2 vs. R)

Test 2: Are variances homogeneous? (A1 vs. A2)

Test 3: Does the model for the mean fit? (A1 vs. fitted)

Tests of Interest 

Test -2*log(likelihood ratio) DF p-value

Test 1 89.4575 6 <0.00001

Test 2 3.82397 3 0.2811

Test 3 0.0628093 1 0.8021

85
The p-value for Test 1 is less than 0.05. 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 0.05. A homogeneous variance model appears to be

appropriate here.

The p-value for Test 3 is greater than 0.05. The model chosen appears to adequately describe the

data.

Benchmark Dose Computation

Specified effect = 0.424000 (5% of background estimate of 8.48)

Risk type = Added response

Confidence level = 0.950000

BMD = 313.866166

BMDL = 224.956831

86