vvEPA
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
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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.
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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
in
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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
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FOREWORD
The purpose of this Toxicological Review is to provide scientific support and rationale for
the hazard and dose-response assessment in 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.
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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
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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.
vn
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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 |ig/L drinking water or risk per
|ig/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
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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 at25°C
Water solubility: miscible
LogKow: 0.81
Henry's Law constant: 2.08 x 10'7 - 2.08 x 10'8 atms/nrVmole (25°C)
Flash point: 62 °C (closed cup); 70 °C (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. TOXICOKTNETICS 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
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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. Jf 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.
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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 37.5°C 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).
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Figure 1. Proposed Metabolic Scheme of EGBE In Rats and Humans
[Adapted From Medinsfcy et al,, 1990 and Corley et al., 1997]
CO2
cartoligas© oxidase dehyd rogenase
CH3CH2CH2CH2OH
(Butanol)
+ HOCH2CH2OH
(EthyleneGlycol)
CH 3CH 2CH 2CH 2OCH 2CH 2O-G luc
(EGBE - Glucuronide)
(Rats On ry?)
(Rats On ly?)
CH3CH 2CH2CH2OCH 2CH2O-SO3H
, ... (EG BE - Sulfate)
dealkylase x s
(Rats On ly?)
CH 3CH2CH2CH 2OCH2CH 2OH
(EGBE)
(Rats and Human)
a bo hoi dehydrogenase
CH 3CH 2CH 2CH 2OCH 2CHO
(BAL)
CH 3CH 2CH 2CH 2OCH 2CO2G lu
(BAA- Glutamine)
(Human Only)
aldehyde dehydrogena.se
CH 3CH 2CH 2CH 2OCH 2CO 2-G ly
(BAA- Glycine)
(Human Only)
CH3CH2CH2CH2OCH2CO2H
(BAA)
dealkyl carte ligase
1
CO2
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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 jig 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 EGBE-
induced hematotoxicity and resulted in a 10-fold lower ratio of BAA to conjugated EGBE excreted in
urine. Cyanamide treatment significantly reduced the hematotoxic response in a manner similar to that
of pyrazole, 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.
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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 C^ 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
Cj,^, 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 C^, 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
jiM BAA (range 36-57 jiM) was reached 2-4 hours postexposure. The average T1/2 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
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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.
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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.
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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
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(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.
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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
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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 > 3 67 mg/kg-day. Significant hematologic
effects in female rats at week 13 included reduced RBC counts and Hgb concentration at >82 mg/kg-
day 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/kg-
day. 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 "ground-
glass" 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.
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Werner, HW; Nawrocki, CZ; Mitchell, JL; et al. (1943 a) 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, Nffl, Research Triangle Park, NC. NTP TR 484. Nffl 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-to-
body 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
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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
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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 high-
dose 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 (pO.OOl). 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
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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 well-
conducted 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
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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-I 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.
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A final phase of this study assessed the fertility and reproductive effects of EGBE in second
generation (Fj) 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).
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Prenatal and postnatal developmental toxicity tests were conducted in CD-I 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-I 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
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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
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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/kg-
day. 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
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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 treatment-
related 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
WP2wvrA. 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
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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.
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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 |ig/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.
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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
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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 C^, 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
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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, 198la). 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
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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-yet-
undetermined 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 & 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
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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 carcinogen! city. 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
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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).
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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 C^, 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
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(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 CO2 and a diminished ability to excrete BAA in the urine.
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
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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 C^ 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
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Table 1. Subchronic 91-day drinking water studies in rats and mice
Reference
NTP (1993)
NTP (1993)
Species (strain)
Rat (F344)
Mouse (B6C3F1)
Sex
M
F
M
F
# Animals/
dose
10
10
10
10
Effect levels (mg/kg-day)
NOAEL LOAEL
54.9a
58.6a
223 553b
370 676b
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 dose-
response 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).
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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. PBPKMethods
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
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Table 2. Summary of PBPK models
Model
Johanson (1986)
Shyr et al. (1993)
Corley et al. (1994, 1997)
Lee et al. (1998)
Species
Human
Rat
Rat and human
Rat and mouse
Routes of exposure
Inhalation
Inhalation, oral,
dermal
Inhalation, oral,
dermal
Inhalation
Comments
BAA not addressed
BAA excretion
BAA distribution and
excretion; male rats only
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 RED 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:
• Cj^—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 C^ 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 C^ 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
(LOAELjjgjj): (1) calculate the internal dose surrogate (C^ BAA in blood) corresponding to the
female rat LOAEL, assuming that the drinking water was consumed only during a 12-hour awake
37
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cycle on a 7 day/week schedule in model simulations; (2) verify that steady state was achieved (e.g., no
change in BAA C^ as a result of prolonging the exposure regimen); (3) simulate the internal dose
surrogate (C^ 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 (C^ 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)
CmaxBAA=103nM
Step 2: Verify steady state.
There were no changes in the C^ 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 C^ for BAA in blood for humans continuously exposed to varying
concentrations of EGBE.
Water concentration
(ppm)
24
48
94
188
375
750
Calculated dose of EGBE
from drinking water (mg/kg-day)
0.7
1.4
2.7
5.4
10.7
21.4
C^ BAA in
(MM)
9
18
36
73
147
299
blood
Step 4: Calculate the LOAELj^u 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 C^ for BAA in blood at LOAEL = 103 \M
LOAELjjED continuous exposure = 7.6 mg/kg-day (calculated by regression of the internal
dose vs. the dose of EGBE from Step 3).
The LOAELjjgo calculated using the PBPK model is likely a conservative estimate of the FED
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 (LOAELjjgo) that are lower than if female rat kinetic data had been used. In addition, the internal
dose surrogate, C^ 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
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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. Ib) are provided in Figure 2 for EGBE-
exposed 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
Cj^ 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. Ib) assessment of MCV responses in
female rats (NTP, 1998) versus corresponding PBPK estimates of C^ 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 jiM, using the 95% lower confidence limit of the dose-response curve
expressed in terms of the C^ 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 x 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 x 101/2 x lo1). Uncertainty factors applied for this RfD assessment and the
justification for their use are as follows.
39
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0)
I/)
c
o
Q.
I/)
0)
c
TO
0)
S
O
70
65
60
55
Power Model Best Fit
95% Lower Confidence Limit
50 100 150 200 250 300
Dose (mg/kg-day)
350
400
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
cone, (ppm)
750
1,500
3,000
4,500
6000
Water
intake (L/day)
0.0147
0.0155
0.0125
0.0101
00101
Female Body
weight (g)
188
185
180
164
150
BAA in
Dose (mg/kg-day)
59
125
208
277
404
Blood
CmaY(uM)
103
253
495
738
1 355
40
-------
0)
I/)
c
o
Q.
I/)
0)
c
TO
0)
S
O
70
65
60
55
Power Model Best Fit
95% Lower Confidence Limit
200 400 600 800 1000
Cmax BAA (uM)
1200
1400
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 C^, uM).
A value of 10 was selected to account for variation in sensitivity within the human population
(UFjj). 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 (UFJ 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
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pharmacodynamics x 3 for pharmacokinetics) was used. For the PBPK and the combined
PBPK/BMC (benchmark concentration) approaches, an overall UFA of 1 (1 for pharmacodynamics
x 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 dose-
response 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
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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
UFH
UFA
UFS
UFL
UFD
UF(Total)
RfD
(mg/kg-day)
Standard
10
3
1
3
1
100
55/100 = 0.6
PBPK
10
1
1
3
1
30
7.6/30 = 0.3
BMD
10
3
1
1
1
30
49/30 = 2
PBPK&
10
1
1
1
1
10
5.1/10 =
BMD
= 0.5
Table 5. Results of candidate studies
Hematologic effect
levels (ppm)
Reference
NTP (1998)
NTP (1998)
Doddetal. (1983)
Species (strain)
Rat (Fischer)
Mouse (B6C3F1)
Rat (F344)
Sex
M
F
M
F
MF
#/Dose
group
9-10
9-10
9-10
9-10
16
Duration
(months)
3, 9, 12;
14 weeks
3, 9, 12;
14 weeks
3, 9, 12;
14 weeks
3, 9, 12;
14 weeks
9
NOAEL
31
—
31
31
25
LOAEL
62.5
31
62.5
62.5
77
43
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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 (LOAELjjgc) from a LOAEL identified in an animal
study: (1) convert units to mg/m3 (3 1 ppm x 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 x 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 LOAELj^c of 150 mg/m3 was calculated for EGBE by
this standard default method.
5.2.2.2. PBPKMethods
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), C^ 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 C^ concentrations
from the female rat data. The same procedure was used to calculate the HEC corresponding to the
LOAEL identified in the animal study (LOAELjjgc): (1) calculate the internal dose surrogate (C^
44
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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 C^ as a result of prolonging the exposure regimen); (3) simulate the internal
dose surrogate (C^ 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 (C^ 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
CmaxBAA= 285 pM
Step 2: Verify steady state.
There were no changes in the C^ 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)
1
5
10
20
50
100
C^ BAA in blood
(MM)
2.6
13.0
26.1
52.9
137.1
295.0
Step 4: Calculate LOAELj^c of EGBE for continuous human exposures producing the same
Cmax of BAA in blood calculated for the animal study in Step 1.
Female rat C^ BAA = 285 \M
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.
LOAELjjEc (mg/m3) = Conversion factor x LOAELj^ (ppm)
= 4.84 (mg/m3)/(ppm) x 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
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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 concentration-
response 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. Ib. 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
CO
LU
O
0)
(A
C
O
Q.
(A
Q)
O
CD
7.5
Polynomial Model Best Fit
95% Lower Confidence Limit
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).
46
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5.2.2.4. PBPK and BMC Methods Combined
Cma!i 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 C^ 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 l.lb. The
BMD05 was determined to be 225 jiM, using the 95% lower confidence limit of the dose-response
curve expressed in terms of the C^ 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 x 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 x 101/2 x 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
concentration (ppm)
31
61.5
125
250
500
Female rat body
weight (g)
216
211
214
210
201
BAA in arterial blood
Cmax (uM) in female rats
285
603
1243
1959
4227
47
-------
CD
LU
O
_><_
0)
O
Q.
or
£=
TO
O
m
or
8.5
7.5
Power model best fit
95% Lower Confidence Limit
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
EGBE (NTP, 1998) using internal dosimetry (BAA, C^, uM).
A value of 10 was selected to account for variation in sensitivity within the human population
). 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 (UFJ 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
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models or EPA default methods. Thus, an overall UFA of 1 (1 for pharmacodynamics x 1 for
pharmacokinetics) was used for all of the RfC derivation approaches.
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 RED (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 dose-
response 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.
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Table 7. Summary of application of UFs and MF for RFC calculation
Approach
Factor
UFH
UFA
UFS
UFL
UFD
^^ (Total)
RfC
mg/m3
Standard
10
1
1
3
1
30
150/30 = 5
PBPK
10
1
1
3
1
30
474/30 = 16
BMC
10
1
1
3
1
30
130/30 = 4
PBPK&
10
1
1
3
1
30
380/30
BMC
= 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 lexicological 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
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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
carcinogen!city of EGBE. A draft report of the results of a 2-year inhalation bioassay performed using
rats and mice has recently become available (NIP, 1998). NIP (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 C^ for BAA in blood as the dose metric, calculating a BMD05
of 64 jiM, 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
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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
(UFjj). 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 theRfD 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). Fligh 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 (UF^). 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.
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Tyl, RW; Millicovsky, G; Dodd, DE; et al. (1984) Teratologic evaluation of ethylene glycol monobutyl
ether in Fischer 344 rats and New Zealand white rabbits following inhalation exposure. Environ Health
Perspect 57:47-68.
Tyler, TR. (1984) Acute and subchronic toxicity of ethylene glycol monobutyl ether. Environ Health
Perspect 57:185-191.
Udden, MM. (1994) Hemolysis and decreased deformability of erythrocytes exposed to butoxyacetic
acid, a metabolite of 2-butoxyethanol. II. Resistance in red blood cells from humans with potential
susceptibility. J Appl Toxicol 14:97-102.
Udden, MM. (1995a) Effects of butoxyacetic acid on human red cells. Occup Hyg 2:283-292.
Udden, MM. (1995b) Effects of butoxyacetic acid on rat and human erythrocytes. Abstract, 37th
annual meeting, American Society of Hematology, Dec. 1-5, Seattle, WA.
Udden, MM; Patton, CS. (1994) Hemolysis and decreased deformability of erythrocytes exposed to
butoxyacetic acid, a metabolite of 2-butoxyethanol. I. Sensitivity in rats and resistance in normal
humans. J Appl Toxicol 14:91-96.
U.S. Environmental Protection Agency (U.S. EPA). (1986a) Guidelines for carcinogen risk
assessment. Federal Register 51(185):33992-34003.
U.S. EPA. (1986b) Guidelines for the health risk assessment of chemical mixtures. Federal Register
51(185):34014-34025.
U.S. EPA. (1986c) Guidelines for mutagenicity risk assessment. Federal Register 51(185):34006-
34012.
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U.S. EPA. (1988) Recommendations for and documentation of biological values for use in risk
assessment. Environmental Criteria and Assessment Office, Office of Health and Environmental
Assessment, U.S. Environmental Protection Agency, Cincinnati, OH. February 1988. EPA/600/6-
87/008, NTIS PB88-179874/AS.
U.S. EPA. (1991) Guidelines for developmental toxicity risk assessment. Federal Register
56(234):63798-63826.
U.S. EPA. (1994a) Interim policy for particle size and limit concentration issues in inhalation toxicity:
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U.S. EPA. (1994b) Methods for derivation of inhalation reference concentrations and application of
inhalation dosimetry. National Center for Environmental Assessment, U.S. Environmental Protection
Agency. Research Triangle Park, NC. EPA/600/8-90/066F.
U.S. EPA. (1994c) Peer review and peer involvement at the U.S. Environmental Protection Agency.
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Forum, Office of Research and Development. EPA/630/R-94-007.
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1996. Federal Register 61(212):56274-56322.
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96-002A.
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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. (1943 a) Effects of repeated exposure of rats to
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Wier, PJ; Lewis, SC; Traul, KA. (1987) A comparison of developmental toxicity evident at term to
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and ethanol. Teratog Carcinog Mutag 7:55-64.
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MM, ed. Philadelphia: Lea & Febiger, pp. 677-697.
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induced mouse liver neoplasia. Abstract, annual meeting, Society of Toxicology, March 14-18, New
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the testing of 311 chemicals. Environ Mol Mutagen 19 (Suppl 21):2-141.
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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.
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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
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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.
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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 ofC^ as the appropriate dose surrogate may be
added" to the PBPK method discussions in Sections 5.1.2 and 5.2.2.
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,
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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 (UFjj)—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
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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 C,^ 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-to-
day attainment of steady state with respect to the C^ 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.
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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 RED 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 UF^ 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 x 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
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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 RED 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.
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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-l). 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.
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m
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or Gavage
D—
Metabolis
J
usion
r
1 8
g m
S S
Butoxy acetic Acid
^lodel for 2-Butoxyacetic Acid
^ fTun-nn-l lil-riil \_
l^ Blood JT
^ Raoidlv Perfused 1
|^ Organs J
_ [ Slowly Perfused |
^ (^ Organs J
*( r 1
1 J
["^
Skm J-
.^ 1 Gastrointestinal 1 ""^
^\ Tract J '
^S
er -^ ^>
L
fcf I", V ^
» ^ K,dney ^
1
§
""^^ Urine
(Butoxy acetic Acid)
Figure A-l. 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
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APPENDIX C. TEXT OUTPUT FROM BENCHMARK DOSE SOFTWARE
RUNS USED IN THE DERIVATION OF RfD AND RfC VALUES
Power Model, Version Number: 1.1. Ib
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 * doseApower
Dependent variable = MEAN
Independent variable = DOSE
The power is not restricted
The variance is to be modeled as Var(i) = alpha*mean(i)Arho
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
alpha
rho
control
slope
power
Estimate Standard Error
0.0008464 0.00159172
1.95327 0.449801
54.7753 0.330267
0.0520655 0.00218389
0.946046 0.069827
Asymptotic Correlation Matrix of Parameter Estimates
alpha
alpha -1
rho 1
control -0.0078
slope 0.012
power 9.5e-005
rho control slope power
1 -0.0078 0.012 9.52-005
-1 0.0057 -0.012 -0.00011
0.0057 1 -0.77 -0.02
-0.012 -0.77 1 -0.029
-0.00011 -0.02 -0.029 1
Table of Data and Estimated Values of Interest
Dose N Obs Mean Obs Std Dev Est Mean Est Std Dev
0
59
125
208
277
404
10
10
10
10
10
10
54.8
57
60.5
62.4
65.3
70.1
0.92
1.25
1.27
1.78
1.83
2.76
54.8
57.2
59.8
62.9
65.4
70
1.45
1.52
1.58
1.66
1.73
184
Model Descriptions for Likelihoods Calculated
72
-------
Model Al: Yij = Mu(i) + e(ij)
Var{e(ij)} = SigmaA2
Model A2: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma(i)A2
Model A3: Yij = Mu(i) + e(ij)
Var{e(ij)} = alpha* (Mu(i))Arho
Model R: Yi = Mu + e(i)
Var{e(i)} = SigmaA2
Likelihoods of Interest
Model
Al
A2
A3
fitted
R
Log(likelihood)
-60.057590
-52.588573
-57.206449
-58.818246
-131.090823
DF
7
12
8
5
2
AIC
-67.057590
-64.588573
-65.206449
-63.818246
-133.090823
Explanation of Tests
Test 1: Do response and/or variances differ among dose levels? (A2 vs. R)
Test 2: Are variances homogeneous? (Al 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-va\ue
Test 1 157.005 10 O.00001
Test 2 14.938 5 0.01063
Test3 9.23575 4 0.05547
Test4 3.22359 3 0.3584
The/>-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/>-value for Test 2 is less than 0.05. A nonhomogeneous variance model appears to be
appropriate.
The/>-value for Test 3 is greater than 0.05. The modeled variance appears to be appropriate here.
The/7-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
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Power Model, Version Number: 1.1. Ib
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 * doseApower
Dependent variable = MEAN
Independent variable = CMAX
The power is not restricted
The variance is to be modeled as Var(i) = alpha*mean(i)Arho
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 =
rho =
control =
slope =
power =
3.02612
0
54.8
0.0828926
0.731677
75
-------
Parameter Estimates
Variable
alpha
rho
control
slope
power
Estimate
0.0008464
1.95428
54.6662
0.136613
0.656823
Standard Error
0.00161642
0.456969
0.335163
0.00575923
0.0430356
Asymptotic Correlation Matrix of Parameter
alpha
rho
control
slope
power
Alpha
-1
1
-0.015
0.02
Rho Control
1 -0.015
-1 0.013
0.013 1
-0.021 -0.78
-0.00069 0.00077 -0.023
Estimates
Slope
0.02
-0.021
-0.78
1
-0.039
Power
-0.00069
0.00077
-0.023
-0.039
1
Table of Data and Estimated Values of Interest
Dose
0
103
253
495
738
1355
N Obs Mean
10 54.8
10 57
10 60.5
10 62.4
10 65.3
10 70.1
Obs Std Dev
0.92
1.25
1.27
1.78
1.83
2.76
Est Mean
54.7
57.5
59.8
62.7
65.1
70.2
Est Std Dev
1.45
1.53
1.59
1.66
1.72
1.85
76
-------
Model Descriptions for Likelihoods Calculated
Model Al: Yij = Mu(i) + e(ij)
Var{e(ij)} = SigmaA2
Model A2: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma(i)A2
Model A3: Yij = Mu(i) + e(ij)
Var{e(ij)} = alpha* (Mu(i))Arho
Model R: Yi = Mu + e(i)
Var{e(i)} = SigmaA2
Likelihoods of Interest
Model
Al
A2
A3
fitted
R
Log(likelihood)
-60.057590
-52.588573
-57.206449
-58.950987
-131.090823
DF
7
12
8
5
2
AIC
-67.057590
-64.588573
-65.206449
-63.950987
-133.090823
Explanation of Tests
Test 1: Do response and/or variances differ among dose levels? (A2 vs. R)
Test 2: Are variances homogeneous? (Al 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-va\ue
Test 1 157.005 10 O.OOOOl
Test 2 14.938 5 0.01063
Test3 9.23575 4 0.05547
Test4 3.48907 3 0.3222
The/>-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/>-value for Test 2 is less than 0.05. A nonhomogeneous variance model appears to be
appropriate.
The/>-value for Test 3 is greater than 0.05. The modeled variance appears to be appropriate here.
The/>-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
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Polynomial Model, Version Number: 1.1 .Ob
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_l*dose + beta_2*doseA2 + ...
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)Arho
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_l = -0.0125321
beta 2 = 4.71226e-008
79
-------
Parameter Estimates
Variable Estimate Standard Error
alpha 0.000870086 0.0064509
rho 1.82359 3.61441
beta_0 8.47429 0.0626104
beta_l -0.0123721 0.0024627
beta 2 -1.16375e-006 1.78152e-005
Asymptotic Correlation Matrix of Parameter Estimates
alpha
rho
beta_0
beta_l
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
0.61
beta_l beta_2
-0.14 0.13
0.14 -0.13
-0.76 0.62
1 -0.97
-0.97 1
Table of Data and Estimated Values of Interest
Dose N Obs Mean Obs Std Dev Est Mean Est Std Dev
0.0000 10 8.480 0.160 8.474 0.043
31.0000 10 8.080 0.230 8.090 0.039
62.5000 10 7.700 0.250 7.696 0.036
125.000 10 6.910 0.150 6.909 0.030
80
-------
Model Descriptions for Likelihoods Calculated
Model Al: Yij = Mu(i) + e(ij)
Var{e(ij)} = SigmaA2
Model A2: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma(i)A2
Model A3: Yij = Mu(i) + e(ij)
Var{e(ij)} = alpha* (Mu(i))Arho
Model R: Yi = Mu + e(i)
Var{e(i)} = SigmaA2
Warning: Likelihood for model Al 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
Al
A2
A3
fitted
R
Log(likelihood)
46.051943
-0.501998
46.173884
46.159466
1.343542
DF
5
8
6
5
2
AIC
41.051943
-8.501998
40.173884
41.159466
-0.656458
Explanation of Tests
Test 1: Do response and/or variances differ among dose levels? (A2 vs. R)
Test 2: Are variances homogeneous? (Al 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-va\ue
Test 1 0 6 O.OOOOl
Test 2 0 3 O.OOOOl
Test3 0 2 O.OOOOl
Test4 0.0288374 1 0.8652
The/>-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/>-value for Test 2 is less than 0.05. A nonhomogeneous variance model appears to be
appropriate.
The/>-value for Test 3 is less than 0.05. You may want to consider a different variance model.
The/>-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
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Power Model, Version Number: 1.1. Ib
Input Data File: C:\BMDS4ME\DATA\EGBE\EGBE_F.(D)
MonJan 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 * doseApower
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 =
control =
slope =
power =
0.040875
8.48
-0.00208697
0.928193
83
-------
Parameter Estimates
Variable Estimate Standard Error
alpha 0.0368453 0.00823886
control 8.47733 0.0469956
slope -0.00181442 9.50703e-005
power 0.948686 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
1
Table of Data and Estimated Values of Interest
Dose N Obs Mean Obs Std Dev Est Mean Est Std Dev
0 10 8.48 0.16 8.48 0.192
285 10 8.08 0.23 8.09 0.192
603 10 7.7 0.25 7.69 0.192
1243 10 6.91 0.15 6.91 0.192
84
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Model Descriptions for Likelihoods Calculated
Model Al: Yij = Mu(i) + e(ij)
Var{e(ij)} = SigmaA2
Model A2: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma(i)A2
Model R: Yi = Mu + e(i)
Var{e(i)} = SigmaA2
Likelihoods of Interest
Model Log(likelihood) DF AIC
Al 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? (Al vs. A2)
Test 3: Does the model for the mean fit? (Al vs. fitted)
Tests of Interest
Test -2*log(likelihood ratio) DF p-va\ue
Test 1 89.4575 6 O.00001
Test 2 3.82397 3 0.2811
Test3 0.0628093 1 0.8021
85
-------
The/>-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/>-value for Test 2 is greater than 0.05. A homogeneous variance model appears to be
appropriate here.
The/>-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
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