United States
Environmental Protection
1=1 m m Agency
EPA/690/R-13/012F
Final
6-12-2013
Provisional Peer-Reviewed Toxicity Values for
2-Ethoxyethanol
(CASRN 110-80-5)
Superfund Health Risk Technical Support Center
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268

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AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGER
Scott C. Wesselkamper, PhD
National Center for Environmental Assessment, Cincinnati, OH
DRAFT DOCUMENT PREPARED BY
ICF International
9300 Lee Highway
Fairfax, VA 22031
PRIMARY INTERNAL REVIEWERS
Ambuja Bale, PhD, DABT
National Center for Environmental Assessment, Washington, DC
Q. Jay Zhao, PhD, MPH, DABT
National Center for Environmental Assessment, Cincinnati, OH
This document was externally peer reviewed under contract to
Eastern Research Group, Inc.
110 Hartwell Avenue
Lexington, MA 02421-3136
Questions regarding the contents of this document may be directed to the U.S. EPA Office of
Research and Development's National Center for Environmental Assessment, Superfund Health
Risk Technical Support Center (513-569-7300).
l
2-Ethoxyethanol

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TABLE OF CONTENTS
COMMONLY USED ABBREVIATIONS	iii
BACKGROUND	1
DISCLAIMERS	1
QUESTIONS REGARDING PPRTVS	1
INTRODUCTION	2
REVIEW OF POTENTIALLY RELEVANT DATA (CANCER AND NONCANCER)	4
HUMAN STUDIES	14
Oral Exposures	14
Inhalation Exposures	14
Reproductive Studies	14
Other Studies	18
ANIMAL STUDIES	20
Oral Exposures	20
Sub chronic-duration Studies	20
Chronic-duration Studies	24
Reproductive and Developmental Studies	26
Inhalation Exposures	34
Sub chronic-duration Studies	34
Chronic-duration Studies	36
Developmental Studies	36
OTHER DATA (SHORT-TERM TESTS, MECHANISTIC STUDIES, OTHER
EXAMINATIONS)	48
Tests Evaluating Mutagenicity, Cytogenicity, and Embryotoxicity	48
Other Toxicity Studies	49
Metabolism/Toxicokinetic Studies	50
Immunotoxicity Studies	52
DERIVATION 01 PROVISIONAL VALUES	53
DERIVATION OF ORAL REFERENCE DOSES	54
Derivation of Subchronic Provisional RfD (Subchronic p-RfD)	54
Derivation of Chronic Provisional RfD (Chronic p-RfD)	57
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS	59
Derivation of Subchronic Provisional RfC (Subchronic p-RfC)	59
Derivation of Chronic Provisional RfC (Chronic p-RfC)	62
CANCER WEIGHT-OF-EVIDENCE (WOE) DESCRIPTOR	62
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES	63
Derivation of Provisional Oral Slope Factor (p-OSF)	63
Derivation of Provisional Inhalation Unit Risk (p-IUR)	63
APPENDIX A. PROVISIONAL SCREENING VALUES	64
APPENDIX B. DATA TABLES	65
APPENDIX C. BMD MODELING OUTPUTS FOR 2-EE	89
APPENDIX D. REFERENCES	95
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COMMONLY USED ABBREVIATIONS
BMC
benchmark concentration
BMCL
benchmark concentration lower bound 95% confidence interval
BMD
benchmark dose
BMDL
benchmark dose lower bound 95% confidence interval
HEC
human equivalent concentration
HED
human equivalent dose
IUR
inhalation unit risk
LOAEL
lowest-observed-adverse-effect level
LOAELadj
LOAEL adjusted to continuous exposure duration
LOAELhec
LOAEL adjusted for dosimetric differences across species to a human
MW
molecular weight
NOAEL
no-ob served-adverse-effect level
NOAELadj
NOAEL adjusted to continuous exposure duration
NOAELhec
NOAEL adjusted for dosimetric differences across species to a human
NOEL
no-ob served-effect level
OSF
oral slope factor
p-IUR
provisional inhalation unit risk
POD
point of departure
p-OSF
provisional oral slope factor
p-RfC
provisional reference concentration (inhalation)
p-RfD
provisional reference dose (oral)
RfC
reference concentration (inhalation)
RfD
reference dose (oral)
SD
standard deviation
SE
standard error
UF
uncertainty factor
UFa
animal-to-human uncertainty factor
UFC
composite uncertainty factor
UFd
incomplete-to-complete database uncertainty factor
UFh
interhuman uncertainty factor
UFl
LOAEL-to-NOAEL uncertainty factor
UFS
subchronic-to-chronic uncertainty factor
WOE
weight of evidence
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PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR
2-E THOX YE THAN OL (CASRN 110-80-5)
BACKGROUND
A Provisional Peer-Reviewed Toxicity Value (PPRTV) is defined as a toxicity value
derived for use in the Superfund Program. PPRTVs are derived after a review of the relevant
scientific literature using established Agency guidance on human health toxicity value
derivations. All PPRTV assessments receive internal review by a standing panel of National
Center for Environment Assessment (NCEA) scientists and an independent external peer review
by three scientific experts.
The purpose of this document is to provide support for the hazard and dose-response
assessment pertaining to chronic and subchronic exposures to substances of concern, to present
the major conclusions reached in the hazard identification and derivation of the PPRTVs, and to
characterize the overall confidence in these conclusions and toxicity values. It is not intended to
be a comprehensive treatise on the chemical or toxicological nature of this substance.
The PPRTV review process provides needed toxicity values in a quick turnaround
timeframe while maintaining scientific quality. PPRTV assessments are updated approximately
on a 5-year cycle for new data or methodologies that might impact the toxicity values or
characterization of potential for adverse human health effects and are revised as appropriate. It is
important to utilize the PPRTV database flittp://hhpprtv.ornl.gov) to obtain the current
information available. When a final Integrated Risk Information System (IRIS) assessment is
made publicly available on the Internet (www.epa.eov/iris). the respective PPRTVs are removed
from the database.
DISCLAIMERS
The PPRTV document provides toxicity values and information about the adverse effects
of the chemical and the evidence on which the value is based, including the strengths and
limitations of the data. All users are advised to review the information provided in this
document to ensure that the PPRTV used is appropriate for the types of exposures and
circumstances at the site in question and the risk management decision that would be supported
by the risk assessment.
Other U.S. Environmental Protection Agency (EPA) programs or external parties who
may choose to use PPRTVs are advised that Superfund resources will not generally be used to
respond to challenges, if any, of PPRTVs used in a context outside of the Superfund program.
QUESTIONS REGARDING PPRTVS
Questions regarding the contents and appropriate use of this PPRTV assessment should
be directed to the EPA Office of Research and Development's National Center for
Environmental Assessment, Superfund Health Risk Technical Support Center (513-569-7300).
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INTRODUCTION
2-Ethoxyethanol (2-EE), also commonly referred to as ethylene glycol monoethyl ether
or Cellosolve®, is a glycol ethyl ether that is used in many industrial processes as a solvent and
chemical intermediary in the production of ethylene glycol monoethyl ether acetate (NTP, 1993).
2-EE is a colorless liquid with a mild odor that is readily evaporated (NIOSH, 2003). The
empirical formula for 2-EE is C4H10O2 (see Figure 1), and Table 1 provides physicochemical
properties for 2-EE.
H H
H H
HO—C — C — O—C — C — H
H H
H H
Figure 1. 2-EE Structure (NTP, 1993)
Table 1. Physicochemical Properties Table (2-EE CASRN 110-80-5)a
Property (unit)
Value
Boiling point (°C)
135
Melting point (°C)
-70
Density (g/cm3)
Not available
Vapor pressure (kPa at 20°C)
0.5
pH (unitless)
Not available
Solubility in water (g/100 mL at 25°C)
Miscible
Relative vapor density (air =1)
3.1
Molecular weight (g/mol)
90.12
Flash point (°C)
44
Octanol/water partition coefficient (log Kow unitless)
-0.540
aNIOSH (2003).
No Reference Dose (RfD) or cancer assessment for 2-EE is included in the IRIS
database, but a Reference Concentration (RfC) of 0.2 mg/m3 (UFC = 300, modifying factor = 1)
is provided (U.S. EPA, 2011) based on decreased testis weight, degeneration of the seminiferous
tubules, and decreased hemoglobin in New Zealand White rabbits (Barbee et al., 1984b). The
Drinking Water Standards and Health Advisories List (U.S. EPA, 2006) does not list reference
values. A chronic RfD of 0.4 mg/kg-day (UFC = 1000), a subchronic RfD of 0.5 mg/kg-day
"3
(UFc = 100), and a subchronic reference concentration of 2 mg/m (UFc = 30) are reported in the
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HEAST (U.S. EPA, 2003). The Chemical Assessments and Related Activities (CARA) list
(U.S. EPA, 1994) includes a Health and Environmental Effects Profile (HEEP) for 2-EE esters,
which gives an acceptable daily intake of 21.1 mg/day for inhalation exposure (U.S. EPA, 1985).
The toxicity of 2-EE has not been reviewed by the ATSDR (2011) or the World Health
Organization (WHO, 2011). CalEPA (2008) has derived an acute reference exposure level
(REL) value of 0.1 ppm (0.370 mg/m3) for a 6-hour time-weighted average (TWA) exposure to
2-EE based on reproductive /developmental effects. CalEPA (2008) has also derived a chronic
REL of 0.02 ppm (0.070 mg/m3) based on effects in the reproductive and hematopoietic systems.
Additionally, 2-EE is listed under California Proposition 65 as a reproductive hazard. The
American Conference of Governmental Industrial Hygienists (ACGIH, 2010) has set an 8-hour
TWA occupational exposure limit of 5 ppm along with a skin notation; ACGIH has also set a
biological exposure index of 100 mg of 2-ethoxyacetic acid (2-EAA) per gram of creatinine in
urine. The National Institute for Occupational Safety and Health (NIOSH, 2005) has established
a 10-hour TWA of 0.5 ppm (1.8 mg/m ) along with a skin designation and an Immediately
Dangerous to Life or Health level of 500 ppm. The Occupational Safety and Health
Administration (OSHA, 2010) has established a permissible exposure limit 8-hour TWA of
"3
200 ppm (740 mg/m ) along with a skin designation (i.e., dermal contact should be avoided).
The HEAST (U.S. EPA, 2003) does not report a cancer weight-of-evidence classification.
The International Agency for Research on Cancer (IARC, 2011) has not reviewed the
carcinogenic potential of 2-EE. 2-EE is not included in the 12th Report on Carcinogens (NTP,
2011). CalEPA (2008) has not prepared a quantitative estimate of carcinogenic potential for
2-EE.
Literature searches were conducted on sources published from 1900 through March 2011
for studies relevant to the derivation of provisional toxicity values for 2-EE, CAS Number
110-80-5. Searches were conducted using EPA's Health and Environmental Research Online
(HERO) database of scientific literature. HERO searches the following databases: AGRICOLA;
American Chemical Society; BioOne; Cochrane Library; DOE: Energy Information
Administration, Information Bridge, and Energy Citations Database; EBSCO: Academic Search
Complete; GeoRef Preview; GPO: Government Printing Office; Informaworld; IngentaConnect;
J-STAGE: Japan Science & Technology; JSTOR: Mathematics & Statistics and Life Sciences;
NSCEP/NEPIS (EPA publications available through the National Service Center for
Environmental Publications (NSCEP) and National Environmental Publications Internet Site
(NEPIS) database); PubMed: MEDLINE and CANCERLIT databases; SAGE; Science Direct;
Scirus; Scitopia; SpringerLink; TOXNET (Toxicology Data Network): ANEUPL, CCRIS,
ChemlDplus, CIS, CRISP, DART, EMIC, EPIDEM, ETICBACK, FEDRIP, GENE-TOX,
HAPAB, HEEP, HMTC, HSDB, IRIS, ITER, LactMed, Multi-Database Search, NIOSH, NTIS,
PESTAB, PPBIB, RISKLINE, TRI, and TSCATS; Virtual Health Library; Web of Science
(searches Current Content database among others); World Health Organization; and Worldwide
Science. The following databases outside of HERO were searched for toxicity values: ACGIH,
ATSDR, CalEPA, EPA IRIS, EPA HEAST, EPA HEEP, EPA OW, EPA TSCATS/TSCATS2,
NIOSH, NTP, OSHA, and RTECS.
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REVIEW OF POTENTIALLY RELEVANT DATA
(CANCER AND NONCANCER)
Table 2 provides an overview of the relevant database for 2-EE and includes all
potentially relevant repeated short-term-, subchronic-, and chronic-duration studies. NOAELs,
LOAELs, and BMDL/BMCLs are provided in HED/HEC units for comparison except that oral
noncancer values are not converted to HEDs and are identified in parentheses as adjusted rather
than HED/HECs. Principal studies are identified. Entries for the principal studies are bolded.
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Table 2. Summary of Potentially Relevant Data for 2-EE (CASRN 110-80-5)
Category
Number of
Male/Female, Strain
Species, Study Type,
Study Duration
Dosimetry"
Critical Effects
NOAEL"
BMDL/
BMCLb
LOAEL'
Reference
(Comments)
Notes0
Human
1. Oral (mg/kg-d)a
None
2. Inhalation (mg/m3)a
Subchronic
None
Chronic
None
Developmental
None
Reproductive
Unreported number,
Chinese male factory
employees,
occupational exposure
(2-Ethoxyethanol
[2-EE] and
2-methoxyethanol
[2-ME]),
Unknown
Urinary metabolites higher with high
exposure; sperm counts and,
progressive motility lower; red blood
cell count, hemoglobin level, packed
cell volume, and white blood cell count
lower
Not
determinable
Not
determinable
Not
determinable
Wang et al.
(2003)

73/0 painters
(40 unexposed
controls),
occupational exposure
(2-EE and 2-ME),
cross-sectional study
Mean TWA
of
9.9 mg/m3
2-EE; mean
TWA of
2.6 mg/m3
2-ME
Increased semen pH and prevalance of
oligospermia; increased odds ratio for
lower sperm count per ejaculate
Not
determinable
Not
determinable
Not
determinable
Welch et al.
(1988)
PR
73/0 painters
(40 unexposed
controls),
occupational
exposure,
cross-sectional study
TWA of
9.9 mg/m3
2-EE; TWA
of
2.6 mg/m3
2-ME
No fertility effects
Not
determinable
Not
determinable
Not
determinable
Welch et al.
(1991)
PR
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Table 2. Summary of Potentially Relevant Data for 2-EE (CASRN 110-80-5)
Category
Number of
Male/Female, Strain
Species, Study Type,
Study Duration
Dosimetry3
Critical Effects
NOAEL"
BMDL/
BMCLb
LOAEL'
Reference
(Comments)
Notes0
Reproductive
37/0 manufacturing
plant employees
(39 controls),
occupational exposure
(roughly 50% ethanol
and 50% 2-EE),
cross-sectional
33 mg/m3
(average)
Sperm count per ejaculate decreased;
immature forms of sperm increased;
proportion of double-headed sperm
decreased
Not
determinable
Not
determinable
Not
determinable
Ratcliffe et al.
(1986)


1019/0 infertility or
subfertility cases
(475 fertile male
controls), case-control
study
Unknown
Testis volume, sperm motility, vitality,
concentration, morphology and
integrity of cell membrane decreased;
follicle stimulating hormone (FSH)
increased; association between
reproductive problems and ethoxyacetic
acid (EAA) in urine; association
between EAA-positive patients,
azoospermia, and oligospermia
Not
determinable
Not applicable
Not
determinable
Veulemans et
al. (1993)
PR

0/32 manufacturing
plant employees
(20 controls),
occupational
exposure, case-control
study
24
(average)
EAA in urine increased
Not
determinable
Not applicable
Not
determinable
Wang et al.
(2004)
PR

0/1712 semiconductor
manufacturing plant
employees and
0/1295 wives of
employees,
occupational
exposure,
retrospective cohort
Unknown
(mixtures of
glycol
ethers)
Spontaneous abortion and subfertility
increased in employees
Not
determinable
Not applicable
Not
determinable
Correa et al.
(1996)
PR
Carcinogenic
None
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Table 2. Summary of Potentially Relevant Data for 2-EE (CASRN 110-80-5)
Category
Number of
Male/Female, Strain
Species, Study Type,
Study Duration
Dosimetry3
Critical Effects
NOAEL"
BMDL/
BMCLb
LOAEL'
Reference
(Comments)
Notes0
Other studies
7/0 lithographers,
occupational
exposure, case study
Unknown
Stromal injury with deposition of a
granular periodic acid-Schiff positive
intracellular material and absolute
myeloid hypoplasia in the bone
marrow; marrow iron stores,
eosinophils, plasma cells, and mast
cells increased
Not
determinable
Not applicable
Not
determinable
Cullen et al.
(1983)
PR

94/0 ship painters
(55 controls);
cross-sectional study
(exposure to 2-EE and
2-ME)
TWA of
9.9 mg/m3
2-EE; TWA
of
2.6 mg/m3
2-ME (as
presented in
Sparer et al.
(1988)
Lowest quartile for hemoglobin values;
increased rate of anemia; 5 painters
abnormally low levels of
polymorphonuclear leukocytes
(significantly different than controls,
which showed no abnormal levels)
Not
determinable
Not applicable
Not
determinable
Welch and
Cullen (1988)
PR

94/0 ship painters
(55 controls);
cross-sectional study
(exposure to 2-EE and
2-ME)
TWA of
9.9 mg/m3
2-EE; TWA
of
2.6 mg/m3
2-ME (as
presented in
Sparer et al.
(1988)
Pyruvate kinase decreased
Not
determinable
Not applicable
Not
determinable
Cullen et al.
(1992)
PR
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Table 2. Summary of Potentially Relevant Data for 2-EE (CASRN 110-80-5)
Category
Number of
Male/Female, Strain
Species, Study Type,
Study Duration
Dosimetry"
Critical Effects
NOAEL"
BMDL/
BMCLb
LOAEL'
Reference
(Comments)
Notes0
Animal
1. Oral (mg/kg-day)a
Subchronic
10/10, F344/N rat,
diet, 7 d/wk, 13 wk
Males: 0,
109,205,
400, 792,
2240
(adjusted)
Females: 0,
122,247,
466,804,
2061
(adjusted)
Both sexes: mortality >2061 mg/kg-d
Males: decreased testis weights and
size at 792 mg/kg-d; testicular
degeneration >400 mg/kg-d;
decreased thymus weights
>205 mg/kg-d; prostate atrophy
>205 mg/kg-d
Females: decreased thymus weights
at 804 mg/kg-d, increased estrous
cycle length >247 mg/kg-d
(significant at 804 mg/kg-d)
NOAEL/LOAEL: thymus weights
and prostate atrophy
109
67 (prostate
atrophy)
205d
NTP (1993a)
PS,
PR
30/0, F344/N rat,
drinking water,
7 d/wk, 60 d
(followed by recovery
of 0, 30, or 56 d)
0, 407, 792,
2390
(adjusted)
Mortality at 2390 mg/kg-d; body
weights, body-weight gains decreased
in all groups; testicular weight
decreased, and testicular degeneration
with no recovery >792 mg/kg-d
NOAEL/LOAEL: testicular
degeneration
407d
398 (testicular
degeneration)
792d
NTP (1993b)
PR
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Table 2. Summary of Potentially Relevant Data for 2-EE (CASRN 110-80-5)
Category
Number of
Male/Female, Strain
Species, Study Type,
Study Duration
Dosimetry3
Critical Effects
NOAEL"
BMDL/
BMCLb
LOAEL3
Reference
(Comments)
Notes0
Subchronic
10/10, B6C3FJ
mouse, drinking
water, 13 wk
Males: 0,
587, 971,
2003,5123,
7284
(adjusted)
Females: 0,
722, 1304,
2725, 7255,
11,172
(adjusted)
Males: decreased absolute
(>5123 mg/kg-dl and relative (at
7284 mg/kg-d) testis weights with loss
of the germinal epithelium in the
seminiferous tubules >2003 mg/kg-d;
splenic hematopoiesis, lesions of the
small testes, and epididymides at
7284 mg/kg-d
Females: hypertrophy of the X-zone of
the adrenals6 >1304 mg/kg-d; estrous
cycle length increased >1304 mg/kg-d;
splenic hematopoiesis >2725 mg/kg-d
NOAEL/LOAEL: splenic
hematopoiesis in females
1304
1777 (splenic
hematopoiesis)
2725d
NTP (1993c)
PR
Chronic
50/50, F344 rat,
gavage, 103 wk
(followed by 1 wk
observation period)
0,357,714,
1429
(adjusted)
Both sexes: mortality >714 mg/kg-d;
body weights decreased >357 mg/kg-d;
stomach ulcerations
Males: testis size decreased at
1429 mg/kg-d; enlarged adrenal glands
>357 mg/kg-d
LOAEL: body weight decrease
None
Not run
357d
Melnick,
(1984a)
PR

50/50, B6C3Fi
mouse, gavage,
103 wk (followed by
1 wk observation
period)
0,357,714,
1429
(adjusted)
Mortality at 1429 mg/kg-d
Males: adrenal glands enlarged and
stomach ulceration at 1429 mg/kg-d
Not
determinable
Not run
Not
determinable
Melnick,
(1984b)
PR
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Table 2. Summary of Potentially Relevant Data for 2-EE (CASRN 110-80-5)
Category
Number of
Male/Female, Strain
Species, Study Type,
Study Duration
Dosimetry"
Critical Effects
NOAEL"
BMDL/
BMCLb
LOAEL'
Reference
(Comments)
Notes0
Reproductive
and
Developmental'
5/0, Sprague-Dawley
rat, gavage, 6 d/wk,
4 wk
0,86,171,
343,686
(adjusted)
Adult males: marked depletion of all
spermatid types at 686 mg/kg-d;
dose-related decrease in relative
epididymis weight at all doses;
relative testis weight decreased at
>343 mg/kg-d; body weight decreased
at >171 mg/kg-d; testicular pathology
with exfoliation of the germ cells into
the tubular lumen at_171 mg/kg-d;
NOAEL/LOAEL: testicular
pathology
86d
Not run for
testicular
pathology
171d
Yoon et al.
(2003)
PS,
PR

10/0 (5 pubertal and
5 adult),
Sprague-Dawley rat,
gavage, 6 d/wk, 4 wk
0, 43, 86,
171,343
(adjusted)
Adult males: abnormal
spermatogenesis; altered composition of
testicular germ cell populations,
decreased relative testis weight,
decreased relative epididymal weight,
and body weight at 343 mg/kg-d
Pubertal males: relative testes weight
and relative epididymal weight
increased in all dose groups; body
weights decreased at 343 mg/kg-d
NOAEL/LOAEL: testicular pathology
in males
17 ld
No fit
343d
Yoon et al.
(2001)
PR
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Table 2. Summary of Potentially Relevant Data for 2-EE (CASRN 110-80-5)
Category
Number of
Male/Female, Strain
Species, Study Type,
Study Duration
Dosimetry"
Critical Effects
NOAEL"
BMDL/
BMCLb
LOAEL'
Reference
(Comments)
Notes0
Reproductive and
Developmental
9/0, Sprague-Dawley
rat, gavage, 6 d/wk,
4 wk
0, 129
(adjusted)
Adult males: body weight, relative
adrenal gland, relative testis, and
relative epididymis weights decreased;
severe degeneration of seminiferous
tubules, germ cell necrosis, interstitial
Leydig cell hyperplasia and
hypertrophy; white blood cell, platelet
count, hematocrit, hemoglobin
concentration, mean corpuscular
hemoglobin concentration, plasma
protein content, plasma creatinine
concentration, and alkaline phosphatase
decreased at 129 mg/kg-d
NOAEL/LOAEL: testicular effects and
hematopoietic effects in males
None
Not run
129d
Yu et al.
(1999)
PR
Carcinogenic
None
2. Inhalation (mg/m3)a
Subchronic
15/15, Sprague-
Dawley rat,
inhalation, 6 h/d,
5 d/wk, 13 wk
0, 17, 68,
265
Males: no effects
Females: no effects
265d
Not run
Not
determinable
Barbee et al.
(1984a)
PR
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Table 2. Summary of Potentially Relevant Data for 2-EE (CASRN 110-80-5)
Category
Number of
Male/Female, Strain
Species, Study Type,
Study Duration
Dosimetry"
Critical Effects
NOAEL"
BMDL/
BMCLb
LOAEL3
Reference
(Comments)
Notes0
Subchronic
10/10, New Zealand
white rabbit,
inhalation, 6 h/d,
5 d/wk, 13 wk
0,17,68,
265
Males: body weights, and testes
weights decreased; hemoglobin,
hematocrit, and erythrocyte counts
decreased; focal degeneration of
seminiferous tubules with loss of
epithelium at 265 mg/kg-d
Females: body weights decreased (not
significant), hemoglobin, hematocrit,
and erythrocyte counts decreased at
265 mg/kg-d
NOAEL/LOAEL: testicular effects in
males and hematopoietic effects in
both sexes
68
Not run
265d
Barbee et al.
(1984b)
PS,
IRIS,
PR
Chronic
None
Developmental'
0/24, Wistar rat,
inhalation, 6 h/d,
GDs 6-15
0, 9, 47,
230
Maternal: hemoglobin, hematocrit, and
mean cell volume in red blood cells
decreased at 230 mg/m3
Maternal NOAEL/LOAEL:
hematopoietic effects
Fetal: reduced mean fetal body weight,
minor external, visceral, and skeletal
defects at 230 mg/m3
Fetal NOAEL/LOAEL: skeletal defects
Maternal: 47d
Fetal: 47d
Not run
Maternal:
230d
Fetal: 230d
Doe (1984a)
PR

0/14-15,
Sprague-Dawley rat,
inhalation, 7 h/d,
GDs 7-13 or 14-20
0, 108
Fetal: neuromotor performance
decreased; elevations in brain chemistry
at 108 mg/m3
Fetal LOAEL: neurotoxicity
Fetal: none
Not run
Fetal: 108d
Nelson et al.
(1981)
PR
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Table 2. Summary of Potentially Relevant Data for 2-EE (CASRN 110-80-5)
Category
Number of
Male/Female, Strain
Species, Study Type,
Study Duration
Dosimetry"
Critical Effects
NOAEL3
BMDL/
BMCLb
LOAEL3
Reference
(Comments)
Notes0
Developmental'
0/24, Dutch rabbit,
inhalation, 6 h/d,
GDs 6-18
0,9,46,
161
Maternal: no effects
Fetal: minor visceral and skeletal
defects at 161 mg/m3
Fetal NOAEL/LOAEL: skeletal
defects
Maternal:
161d
Fetal: 46
4.23
Maternal:
not
determinable
Fetal: 161d
Doe (1984b)
PS,
PR
Carcinogenic
None
""Dosimetry: NOAEL, BMDL/BMCL, and LOAEL values are converted to human equivalent dose (HED in mg/kg-day) or human equivalent concentration (HEC in
mg/m3) units. All of the exposure values of long-term exposure (4 weeks and longer) are converted from a discontinuous to a continuous (weekly) exposure. Values for
inhalation (cancer and noncancer) and oral (cancer only) are further converted to an HEC/HED. Values from animal developmental inhalation studies are adjusted to a
24-hour continuous exposure, and then converted to human equivalent concentration (HEC in mg/m3) units. Following EPA guidance for Category 3 gases (U.S. EPA,
2009), concentrations were converted to adjust for continuous exposure by using the following equation:
ConcADj= concentrations in mg/Lx 1000 L/m3 x (hours exposed per 24 day) x (days dosed/ week) total days). Concentrations were calculated for an extrarespiratory
effect for a Category 3 gas. Because the blood:gas (air) partition coefficient lambda for humans is unknown, a default value of 1.0 is used for this ratio. ConcnECEXRESp =
ConcADix blood:gas (air) partition coefficient of 1.
bFor studies reporting a BMDL, the critical effect used as the POD is listed first.
°Notes: IRIS = utilized by IRIS, date of last update; PR = peer reviewed; PS = principal study.
dNOAEL or LOAEL values are determined from the data by the PPRTV authors.
"Hypertrophy can be considered an adaptive cell change and is not typically a viable toxicity endpoint.
Additional reproductive and developmental studies are summarized in Table 3 (oral studies) and Table 4 (inhalation studies).
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HUMAN STUDIES
Oral Exposures
The effects of oral exposure of humans to 2-EE have not been evaluated in any
subchronic-duration, chronic-duration, developmental, reproductive, or carcinogenic studies.
Inhalation Exposures
The effects of inhalation exposure of humans to 2-EE have been evaluated in six
primarily occupational studies evaluating various indicators of reproductive function
(Welch et al., 1988, 1991; Veulemans et al., 1993; Correa et al., 1996; Ratcliffe et al., 1986;
Wang et al., 2003, 2004). There are no clinical studies evaluating the effects of inhalation
exposure of humans to 2-EE exposure in short-term-, developmental, subchronic-, or
chronic-duration settings. Three other studies evaluated the hematological effects of inhalation
exposure of humans to 2-EE (Cullen et al., 1983; Welch and Cullen, 1988; Cullen et al., 1992).
Reproductive Studies
The metabolism of 2-EE, discussed more thoroughly in the following sections of this
document, is similar in both humans and animals. 2-EE metabolism is typical of the
biotransformation of ethers, with the main pathway being oxidation to the corresponding acid,
2-ethoxyacetic acid or EAA, as it shall be referred to in this document. Many sources attribute
the adverse effects of glycol ethers to these acid metabolites and measure those metabolites as an
indicator of exposure (Groesenken et al., 1988; Cheever et al., 1984; Medinsky et al., 1990;
Wang et al., 2003; Welch et al., 1988).
A number of cross-sectional, case-control, and retrospective cohort studies have
examined the association between occupational exposure to 2-EE and potential indicators of
male reproductive effects. In a poster session abstract, Wang et al. (2003) described their
investigation of the effects of exposure to combined glycol ethers (2-EE and 2-methoxyethanol
[2-ME]) among male workers (number not reported) in two Chinese factories manufacturing
photopolymer sensitization plates. Workers in areas of the plant that had low or no exposure to
glycol ethers served as the "unexposed" comparison group. No exposure metrics were reported.
Urine and semen samples were collected from participants. Urine testing measuring for the EAA
metabolite showed much higher levels in exposed workers. Analysis of semen samples revealed
that the exposed group had significantly lowered sperm count, progressive motility, and
percentage of sperm with normal morphology when compared to the unexposed group. Exposed
workers also had lowered red blood cell count, hemoglobin level, packed cell volume, and white
blood cell count compared to those of the unexposed workers. Blood hormone levels
(testosterone, luteinizing hormone, follicle stimulating hormone [FSH], prolactin, and estradiol)
were not different between the exposed and unexposed groups.
In a peer-reviewed cross-sectional study of male shipyard workers, Welch et al. (1988)
investigated the association between exposure to ethylene glycol ethers and reproductive effects.
Sparer et al. (1988) previously concluded that these workers were exposed principally to 2-EE
and 2-ME; Welch et al. (1988) also considered other potential exposures that may affect
reproduction, including lead and epichlorohydrin.
Welch et al. (1988) sampled for semen characteristics from a group that consisted of
73 male painters and 40 controls. Participants were given a questionnaire and physical
examination. Investigators evaluated participants for testicular size, presence of varicocele
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(abnormal dilation of the veins of the spermatic cord), and secondary sex characteristics. Urine
was collected at the time of interview and again at the sample-collection appointment and was
measured for the metabolites of 2-EE and 2-ME. Blood samples were obtained, and hormone
levels were measured. Semen samples (collected by participants at home) were analyzed for
sperm viability, velocity, motility, count, volume and pH, morphology, and morphometries.
An analysis of Welch et al. (1988) by Sparer et al. (1988) showed workers were exposed
to both inhaled 2-EE and 2-ME. For 2-EE, the TWA of inhalation exposure ranged from
3	3	3
0-80.5 mg/m with a mean of 9.9 mg/m and a median of 4.4 mg/m ; for 2-ME, the TWA ranged
3	3	3
from 0-17.7 mg/m with a mean of 2.6 mg/m and a median of 1.6 mg/m . Semen analysis
indicated that exposed men had significantly (p < 0.05) higher mean semen pH than unexposed
men (7.94 ± 0.15 compared to 7.88 ± 0.16). After smokers and nonsmokers were analyzed
separately, the exposed group had a significantly higher rate of oligospermia (p = 0.05), with an
odds ratio of 1.86 and confidence interval (CI) of 0.6-5.6 for this effect. Although the biological
significance of these findings is unknown, they indicate that unexposed controls may be more
likely than exposed painters to have reported a fertility problem. However, the study authors
posit that self-selection bias may have occurred in this study because both control and exposed
nonparticipating men were more likely to have experienced fertility problems compared to
participants. This bias may have underestimated the reproductive effects of exposure to shipyard
paints.
In a cross-sectional study, Ratcliffe et al. (1986) evaluated semen quality in 37 male
employees working with a binding slurry (roughly 50% ethanol and 50% 2-EE) used in the
preparation of ceramic shells for the manufacture of metal parts, with 2-EE being the only glycol
ether used. Workers were mixers of the binder slurry, hand-dippers and/or grabber operators
dipping the molds in the slurry, processors who handled the ceramic shells, supervisors, or
process engineers. All of the workers inhaled 2-EE circulated by fans and the air recirculation
system. The control group consisted of 39 men who worked in other areas of the plant; men who
had previously worked in the department with 2-EE were excluded. Participants were
interviewed and given a physical exam; semen samples were taken at the participants' homes.
Urine voids and spot samples were collected from subsamples of participants. Exposure was
estimated using bulk air and breathing zone samples collected in two different months. The
mean full-shift breathing zone exposure of 2-EE was 9 ppm ±5.6 ppm (33 ± 21 mg/m ) with a
range of 0-23.8 ppm (0-88 mg/m3). None of the blood samples contained detectable levels of
2-EE, whereas urine samples indicated some absorption of 2-EE. Although statistical testing
was not done on the urine data, EAA concentrations in workers showed higher levels in the
hand-dipper (most exposed) when compared to the supervisor (least exposed). No detectable
EAA was found in urine from controls. Sperm count per ejaculate was significantly (p = 0.047)
lower in exposed workers compared to unexposed. The exposed group had a significantly
(p = 0.001) higher proportion of immature forms of sperm and a significantly lowered proportion
of double-headed sperm. When workers in the higher-exposure jobs (e.g., hand-dipper) were
compared to the other workers, no significant differences in semen characteristics were found.
The study authors state, however, that the mean sperm concentrations of both groups were
significantly lower than that for other occupational populations. Also, the number of workers
was small, which may preclude the detection of an effect.
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In a peer-reviewed retrospective case-control study, Veulemans et al. (1993) evaluated
the relationship between exposure to ethylene glycol ethers and spermatogenic disorders in men.
The study authors recruited 1019 cases and 475 controls from the population of first-time
patients at an outpatient clinic for reproductive disorders between October 1985 and July 1990.
Cases had been diagnosed as infertile or subfertile; controls had been diagnosed as fertile.
Occupational history and information about potential exposure to spermatotoxic agents was
obtained using a questionnaire. Urine samples were collected and analyzed for methoxyacetic
acid (MAA) and EAA.
The reproductive disorder cases had significantly lower values for testis volume and
sperm motility, sperm vitality (% sperm living), sperm concentration, sperm morphology
(% normal), and integrity of the cell membrane (% normal), and a significantly (p < 0.0001)
higher concentration of FSH when compared to controls. EAA was detected in 45 patients
(39 cases and 6 controls) at levels ranging between 1.3 and 71.0 mg/L; the odds ratio was
statistically significant (3.11;/? = 0.004). When the study authors divided the study group
according to sperm concentration corrected for motility and morphology, there was a highly
statistically significant (chi-square value of 0.0087) association between EAA-positive patients
and subcategories of complete azoospermia and severe oligospermia. Though few patients'
urine contained MAA, the total number of positive EAA or MAA patients (40 cases versus
8 controls) retained a significant (p = 0.013) odds ratio of 2.39 for the detection of 2-EE
metabolites in the urine. After stratification for possible confounding by exposure to lead,
cadmium, insecticides, weed killers, asphalt and bitumen, carbon disulfide, and welding fumes,
the association between urinary EAA and case status remained significant (chi-square probability
of 0.011).
The study authors concluded that a highly significant association between impaired
fertility and EAA in urine as well as EAA and exposure to products containing solvents such as
paints was indicated. Additionally, although urinary EAA levels have been shown to be a
reflection of exposure, urinary metabolites are not always an accurate measure of past exposures,
and it is difficult in retrospective studies to determine whether the exposure preceded the effects.
In a peer-reviewed case-control study, Wang et al. (2004) investigated the effects of
exposure to 2-EE in female workers at factories that made photopolymer sensitization plates and
used 2-EE as paint thinner. The study included 32 female workers exposed to 2-EE and a control
group of 20 female workers at the same factories that were not exposed. Controls were matched
to exposed women based on age range, mean age, and alcohol and cigarette use. All of the
subjects underwent one-on-one interviews with physicians to gather demographic data, work and
childbirth histories, and any health complaints. The study authors collected blood samples for
analysis of blood cells, hormone levels, and plasma aminotransferases. Spot urine samples were
also collected at the end of the 8-hour work day and analyzed for EAA. Four workers in the
control group and 23 in the exposure group were given organic gas sampling badges to wear on
their chest pocket. These devices measured exposure to ambient 2-EE and other solvents over
6-8 hours of work.
The average period of employment for both the exposed and the control groups was just
over 2 years. Monitoring badges detected low concentrations of 2-EE (0.56 ppm or 2 mg/m3) in
"3
the control group and nearly 12 times higher concentrations (6.44 ppm or 24 mg/m ) in the
exposed workers. 2-EE was almost the only organic gas detected; two other chemicals were
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found at very low concentrations. Protective gloves or masks were not used during the survey
period; thus, exposure may have occurred through inhalation and also through the handling of
materials containing 2-EE. However, results of urinary analyses revealed that levels of EAA
were 40 times higher in the exposed group when compared to controls, although the range was
high, suggesting exposure to high 2-EE concentrations. None of the control urine samples
showed detectable levels of unmetabolized 2-EE, although 10 samples from the exposed group
contained 2-EE (geometric mean, 1.96-mg/g creatinine). There were no significant changes in
hematology or blood levels of prolactin. A number of patients in both groups reported irregular
menstruation, but no significant differences were noted in the exposed group versus the controls.
The study authors noted that the lack of findings regarding abnormal menstruation may
have been caused by the small study size. In addition, this study could not examine effects on
pregnancy or childbirth because most women in the study had already given birth prior to ever
being exposed to 2-EE. The frequency of the other effects, including swelling of the legs
(dropsy), was not different between the control and exposed groups.
Correa et al. (1996) conducted a peer-reviewed retrospective cohort study investigating
the association between exposure to ethylene glycol ethers at two semiconductor manufacturing
plants (designated I and II) and potential reproductive effects including spontaneous abortion and
subfertility in female workers and the wives of male workers. Ethylene glycol ethers are used in
these plants primarily as components of a photoresist film, which is used to coat silicon wafers.
Exposure occurred through inhalation and skin contact during cleaning and restocking of
photoresist containers and in the event of spills. Study participants had to have worked full time
for at least 6 months at the plant between 1980 and 1990. Interviews were completed by trained
interviewers using a standardized computer-assisted questionnaire. The specific ethylene glycol
ether compounds are identified in name only as diethylene glycol dimethyl ether or ethylene
glycol monoethyl ether acetate, the latter being metabolized to 2-EE. The study did not report
exposure levels. Rather, exposure was ranked as potentially low, intermediate, or high based on
the amount of work done with the photoresist mixture; the unexposed population included
workers in the clean room using no ethylene glycol ethers. Subfertility was defined as pregnancy
that took 1 year or more of unprotected intercourse to conceive.
A total of 1712 female employees and 1295 wives of male employees participated, with
1150 pregnancies among unexposed workers, 561 pregnancies among female semiconductor
manufacturers, and 589 pregnancies among wives of male semiconductor manufacturers.
Female employees experienced increased rates of spontaneous abortion with calendar year and
experienced slightly higher rates in Manufacturing Plant I that increased with the potential of
exposure to ethylene glycol ethers (chi-square value of 5,03, p = 0.02). The relative risk (RR) of
spontaneous abortion increased in female employees with age and potential exposure level. In
the high-exposure group, the adjusted RR was 2.9 (95% CI, 1.2-7.0) among female employees.
However, wives of male employees did not experience an increased risk.
Subfertility was significantly increased among female employees in an
exposure-dependent manner (chi-square value of 5.45,/? = 0.02). The adjusted odds ratios for
subfertility in female employees increased as exposure potential increased; the high-exposure
group had nearly a 5-fold higher risk (4.6, 95% CI: 1.6-13.3, with 22 pregnancies) than the
unexposed group (1.0 with 260 pregnancies). The study authors considered and ruled out
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potential selection bias and recall bias. However, this study is limited by a lack of any specific
exposure measurements. In addition, because the study was retrospective in nature, it is unclear
whether exposure preceded disease.
Other Studies
Cullen et al. (1983) performed a peer-reviewed study of lithographers occupationally
exposed to glycol ethers, including 2-EE, and other solvents used in multicolor offset printing.
The investigation began with a case report of a 39-year-old male worker who developed
profound pancytopenia (reduced number of red and white blood cells and platelets) and later
died; autopsy revealed hemorrhage of many organs and bone marrow that was highly depleted of
cellular elements. In the subsequent workplace evaluation, a staff industrial hygienist completed
a walk-through, worker interviews, and observation of work. Seven (male) out of 10 employees
agreed to participate in the study—5 pressmen and helpers, 1 foreman, and 1 ink mixer. A
physician administered a questionnaire and an examination to each participant. Blood was
collected, and bone marrow aspiration and biopsies were taken.
Workplace observations revealed consistent background inhalation exposure potential to
various substances, although exposure was only measured for one chemical (i.e., dipropylene
glycol monomethyl ether). Gloves were frequently not used, prolonged skin contact occurred at
times, and respirators were not used. Workers reported skin irritation, but no prolonged illness
or systemic disease was found. There were a variety of effects seen in the bone marrow aspirates
and biopsies. Three printers (which had the highest assumed exposures) had multifocal areas of
stromal injury with deposition of a granular periodic acid-Schiff (PAS) positive intracellular
material and absolute myeloid hypoplasia in the bone marrow; two also had ring sideroblasts
(possibly indicative of more specific myelodysplastic syndromes). Two cases had significant
increases in marrow iron stores, and other workers had significant increases in eosinophils,
plasma cells, and mast cells. Another case showed signs of increased intramedullary cell
turnover.
The study authors stated that the results strongly supported an association between
occupational exposures during multicolor offset printing and bone marrow injury. However, the
study was limited by a small population, the lack of preexisting data on marrow injury in the
general population with which to compare the data, and the lack of a matched control group with
which to compare marrow specimens.
In a peer-reviewed cross-sectional investigation of the shipyard workers discussed in
Sparer et al. (1988), investigators evaluated the potential for hematologic (Welch and Cullen,
1988) and male reproductive/fertility effects (Welch et al., 1988) from exposure to 2-EE and
2-ME. For the hematologic portion of the study, the study population included 94 male painters
and 55 unexposed controls in jobs with no work aboard the ships (clerks and draftsmen who had
not worked with ships in the past 10 years). The study authors gave participants a questionnaire
and physical examination and collected blood samples. The cohort for reproduction/fertility
portion of the study comprised 73 exposed and 40 unexposed workers. In addition to glycol
ethers, the study authors evaluated the potential of lead and benzene, which are known to affect
-3
hematological parameters. The maximum detected level of lead in the air was 11 mg/m ,
although the study authors maintained that blood lead levels were not at an amount normally
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associated with hematological effects among adults. In 1978, benzene levels in the workplace air
were low (ranging from 0.08-0.53 mg/m3). The current survey of paints and solvents indicated
that they did not contain benzene.
Results of a hematology analysis indicated that hemoglobin levels did not differ
significantly between the groups. However, when these data were rank-ordered and analyzed,
those in the lowest quartile were primarily painters (p = 0.02, two-tailed). Painters were also
more likely to be categorized as anemic based on hemoglobin level (p = 0.028), and the rate of
anemia was significantly (p = 0.04) different in painters compared to that in controls. Mean
values of polymorphonuclear leukocytes of painters were not significantly different than those of
controls. However, when the study authors further defined "normal" and "abnormal" levels
(with 1800 cells per microliter as the minimum considered "normal"), 0 controls and 5 painters
had low levels, which yielded a significant difference between groups. All five of these painters
had normal levels when hired.
The study authors stated that the significant differences between painters and controls in
distributions of hemoglobin and polymorphonuclear leukocytes may have been affected by race
and self-selection bias, but after considering these factors in depth, they concluded that the
hematological effects were likely attributable to ethylene glycol ethers.
The study authors also indicated that biologically important differences exist between
exposed and unexposed workers in sperm parameters (Welch et al., 1988). Semen samples of
73 exposed (painters) and 40 controls were analyzed for pH, volume, turbidity, liquidity,
viability, sperm density and count per ejaculate, motility, morphology, and morphometry. The
proportion of exposed men with a sperm count less than or equal to 20 million/cc was 13%, with
only 5% expected based on other population surveys. Also, the proportion of painters with
azoospermia was 5%, with only 1% expected based on other surveys. Nonsmoking painters
were more likely to have oligospermia (defined as a sperm count per ejaculate of <100 million),
with the odds ratio among the painters increased to 2.8 among the nonsmokers. In a later
peer-reviewed study, these authors reported on the reproduction function study of this cohort
using a questionnaire (Welch et al., 1991), which showed no effect of exposure on fertility
among the 74 married exposed painters when compared to that of 51 married controls even
though the groups differed as indicated in sperm count parameters.
A later peer-reviewed cross-sectional study (Cullen et al., 1992) of the same shipyard
painters involved a more in-depth investigation of the effects of exposure to 2-EE and 2-ME to
the bone marrow and circulating blood cells. This 18-month study took place 2 years after the
original investigations. Authors divided previous participants into three groups: Group I
consisted of painters with abnormal blood counts (10 painters with hemoglobin <14 g/dL or
absolute granulocyte count <1800 cells per microliter), Group II consisted of exposed painters
with normal counts (7 men from the prior survey), and Group III was the unexposed participants
(8 previously unsurveyed men).
The study authors conducted additional interviews and collected and analyzed blood
samples and bone marrow aspirates and biopsies. Liver, renal, and thyroid function tests were
performed to determine whether any alternate explanations for depressed blood counts existed;
all of the tests were normal in all of the subjects. Group I had a mean myeloid/erythroid ratio
significantly lower than that of Group II (2.25 ± 0.70 in Group I versus 2.96 ± 0.65 in Group II).
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However, based on an analysis of variance by demographic and exposure characteristics, the
only significant predictor of myeloid/erythroid ratio was race. In red cell enzyme and metabolite
analyses, there appeared to be a difference in pyruvate kinase levels between groups; men in
Group I had significantly (p = 0.05) lower levels (7.97 ± 2.57 and 6.97 ± 0.79 for Group I
[divided] versus 9.48 ± 1.05 for Group II and 8.89 ± 1.92 for Group III) compared to those of
controls. Analysis of sister chromatid exchange in peripheral blood lymphocytes yielded no
significant differences in groups. The only finding that indicated anything other than population
variation was the depression of red cell pyruvate kinase. This effect was noted by the study
authors as being the most consistent defect generally observed in acquired hematologic
disorders.
ANIMAL STUDIES
Oral Exposures
The effects of oral exposure of animals to 2-EE have been evaluated in
3 sub chronic-duration (NTP, 1993a,b,c), 2 chronic-duration (Melnick, 1984a,b), 4 developmental
(Wier et al., 1987a,b; Hardin et al., 1987; Lamb et al., 1984), and 10 reproductive (Yoon et al.,
2003, 2001; Yu et al., 1999; Oudiz and Zenick, 1986a; Horimoto et al., 1996, 2000; Hardin et al.,
1987; Lamb et al., 1984; Nagano et al., 1984; Weir et al., 1987) studies; no carcinogenic studies
are available. Table 3 presents the reproductive and developmental studies, and a general
summary and discussion of the three most sensitive key studies (Yoon et al., 2003, 2001;
Yu et al., 1999) are provided in the text below.
Subchronic-duration Studies
The NTP (1993a) study is selected as the principal study for deriving the subchronic
p-RfD. In 1993, the National Toxicology Program (NTP) published a report compiling several
different types of studies designed to investigate the toxicity of 2-EE. The report includes
subchronic-duration oral toxicity studies investigating the effects of 2-EE in F344/N rats and
B6C3Fi mice via drinking water for 13 weeks, which will be referred to in this review as NTP
(1993a) and NTP (1993c), respectively. Additionally, a stop-exposure study was conducted in
male F344/N rats, which will be referred to as NTP (1993b). The rat subchronic-duration
portion of the study (NTP, 1993a) is summarized first. The rat stop-exposure study (NTP,
1993b) and the mouse subchronic-duration study (NTP, 1993c) are summarized subsequently.
Furthermore, several mutagenicity, genotoxicity, cytogenicity, in vitro reproductive,
metabolism/toxicokinetic, mode-of-action/mechanistic, and immunotoxicity studies included in
the NTP report are presented in Table 5 (e.g., NTP, 1993d,e,f,g,h).
In a peer-reviewed good laboratory practice (GLP)-compliant subchronic-duration study,
2-EE (purity 99%) was administered in deionized drinking water to ten 5- to 6-week-old F344/N
rats (Taconic Farms) per sex per dose for 13 weeks (NTP, 1993a). Animals were treated with
doses of 0, 1250, 2500, 5000, 10,000, or 20,000 ppm in drinking water, which was available ad
libitum. Due to deaths (5/10 males and 7/10 females) in the 20,000-ppm dose group, exposure
was discontinued at 9 weeks. The study authors reported average daily doses of 0, 109, 205,
400, 792, or 2240 mg/kg-day for males and 0, 122, 247, 466, 804, or 2061 mg/kg-day for
females.
NTP (1993a) observed animals twice daily with weekly clinical observations; body
weights were taken for each animal prior to the study period and then weekly. Hematology
(hematocrit, hemoglobin, erythrocytes, mean cell volume, mean cell hemoglobin, mean cell
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hemoglobin concentration, platelets, reticulocytes, leukocyte count, differential nucleated
erythrocytes, methemoglobin, and total bone marrow cellularity) and clinical chemistry (urea
nitrogen, creatinine, total protein, albumin, alkaline phosphatase, alanine aminotransferase,
creatine kinase, and bile acids) analysis was conducted on all of the study animals at Weeks 1, 3,
and 13. Urinalysis (volume, specific gravity, and pH) was conducted at Week 13. All of the
animals were necropsied at the conclusion of the study. Organ weights were taken for heart,
right kidney, liver, lung, thymus, and right testis, which were also examined for gross lesions and
fixed in 10% formalin for microscopic examination. The study authors conducted
histopathological examinations (adrenal glands, femur and marrow, brain, esophagus, eyes, gross
lesions, heart, large and small intestines, lymph node, mammary gland, kidneys, larynx, liver,
lungs, nasal cavity, ovaries, pancreas, parathyroid glands, pituitary gland, pharynx, preputial or
clitoral glands, prostate gland, salivary gland, seminal vesicles, skin, spinal cord, sciatic nerve,
spleen, stomach, testes, thigh muscle, thymus, thyroid gland, tongue, trachea, urinary bladder,
uterus, and vagina) on all of the control animals, the high-dose groups, as well as animals in the
"higher dose groups" (including early deaths). Examinations in the lowest dose group were done
on the bone marrow, the epididymis, liver, ovaries, preputial or clitoral glands, prostate gland,
seminal vesicle, spleen, stomach, testes, thymus, uterus, and vagina. Males in the 0-, 205-, 400-,
and 792-mg/kg-day dose groups were examined for reproductive tissue weights and
spermatozoal effects. Females in the 0-, 247-, 466-, and 804-mg/kg-day dose groups were
examined for estrous cycle length and other cycle effects.
Statistical analysis was performed by NTP (1993a) using the parametric multiple
comparisons of Williams or Dunnett to compare organ and body weights of treated and control
animals. Clinical chemistry and hematology results were compared using the nonparametric
multiple comparisons of Shirley or Dunn. The results of the Jonckheere test of the data were
used to choose between the Dunn/Dunnett test (p > 0.10) and the Shirley/Williams test
(p < 0.10). Vaginal cytology results were transformed using the arcsine transformation and then
examined by multivariate analysis of variance.
NTP (1993a) reported decreased survivorship (50% in males and 30% in females) at
Week 9 in the animals exposed in the highest dose group (2240 and 2061 mg/kg-day for males
and females, respectively), at which time, further treatment was discontinued (see Table B. 1).
No other mortality was observed among treated or control animals. Clinical observations in
exposed rats included emaciation, diarrhea, abnormal posture, pallor, tachypnea, hypoactivity,
and comatose state, although the study authors did not specifically attribute these observations to
specific doses. Decreased final mean body weights as well as decreased body-weight gains were
also observed with increasing dose in males (e.g., 80% body weight and 66% body-weight gain
in the 792-mg/kg-day dose group relative to controls) and females (e.g., 87% body weight and
61%) body-weight gain in the 804-mg/kg-day dose group relative to controls) (see Table B.l).
Absolute and relative thymus weights were significantly decreased in males in the
205-mg/kg-day (i.e., 71 and 76%, respectively, relative to controls) and greater dose groups and
females in the 804-mg/kg-day dose group (i.e., 32 and 41%, respectively, relative to controls)
(see Table B.2). Absolute and relative testis weights were significantly decreased in males of the
792-mg/kg-day dose group (i.e., 44 and 59%, respectively, as compared to those of controls) (see
Table B.2). Testicular lesions were noted in the 792- and 2240-mg/kg-day dose groups. These
lesions were characterized by marked degeneration of the germinal epithelium in the
seminiferous tubules. Testicular degeneration in males in the 400-mg/kg-day dose group and
greater, as well as prostate atrophy in males in the 205-mg/kg-day dose group and greater, was
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observed (see Table B.3). Both testicular and prostate effects were noted to increase in severity
with dose, with the prostate atrophy appearing at a dose lower than the testicular effects,
indicating the prostate effects to be more sensitive. Other important chemical-related lesions in
males (see Table B.3) and females (see Table B.4) were noted in the spleen (hematopoiesis and
hemosiderin pigmentation), in the bone marrow, and in the liver (hemosiderin pigmentation in
Kupffer cells).
Hematological analysis conducted by NTP (1993a) at Week 1 showed mild anemia in the
male rats in the 792-mg/kg-day dose group (decreased red blood cell, hemoglobin, and
hematocrit concentrations), which progressed into moderate-to-marked anemia in the Weeks 3
and 13 analyses in both male rats (in the 400- and 792-mg/kg-day dose groups) and female rats
(in the 466- and 804-mg/kg-day dose groups), as characterized by decreased red blood cell and
hemoglobin concentrations. Mild and moderate leukopenia, which reached significance in the
males in the 792-mg/kg-day dose group and females in the 804-mg/kg-day dose group, was
observed in the males and females at Weeks 1 and 3, respectively, which transitioned to marked
leukocytosis at Week 13. Clinical chemistry results showed decreases in total protein, albumin,
and alkaline phosphatase levels. Bile acid concentrations as well as alkaline phosphatase activity
markedly increased during Week 3 in males in the 792- and 2240-mg/kg-day dose groups and
females in the 466-, 804-, and 2062-mg/kg-day dose groups, which also showed increased
alanine aminotransferase activity. Creatinine kinase activity increased in the females in the 247-,
466-, 804-, and 2062-mg/kg-day dose groups in Week 3. None of these effects persisted to
Week 13. Urinalysis showed significant decreases in the volume and pH of the males in the
792-mg/kg-day dose group at Week 13; however, no significant changes were observed in the
urine of females. The study authors also noted a decrease in average water consumption in
exposed animals, although this finding was not considered to be dose related.
NTP (1993a) performed sperm analysis (see Table B.5) showing sperm concentration
decreasing in male rats in the 205-, 400-, and 792-mg/kg-day dose groups (the 109-mg/kg-day
dose group was not examined) with spermatozoa measurements and motility decreasing to 1% of
controls in the 792-mg/kg-day dose group. Spermatozoa concentration was dramatically
decreased in the 792-mg/kg-day dose group to about 4% of the control value with the lower dose
groups also showing clear significant decreases relative to controls of 86% in the 205-mg/kg-day
dose group and 88% in the 400-mg/kg-day dose group. Females in the 804-mg/kg-day dose
group were found to have a significantly increased estrous cycle length. This change in estrous
cycle length was accompanied by an increased portion of the cycle in the diestrus stage.
On the basis of decreased thymus weights in male rats, NTP (1993a) identified a NOAEL
of 109 mg/kg-day. On the basis of histopathologic and hematopoietic effects in the female rats,
the study authors identified a NOAEL of 466 mg/kg-day. A LOAELadj of 205 mg/kg-day and a
corresponding NOAELadj of 109 mg/kg-day based on thymus weight changes and prostate
atrophy in males are identified. It is notable that more specific testicular effects, specifically the
spermatozoa concentration, were not examined at the 109-mg/kg-day dose.
NTP (1993b) conducted a peer-reviewed GLP-compliant, stop-exposure study; 2-EE
(purity 99%) was administered in deionized drinking water to thirty 6-week-old male F344/N
rats (Taconic Farms) per dose for 60 days. Animals were treated with doses of 0, 5000, 10,000,
or 20,000 ppm in drinking water, which was available ad libitum. The study authors reported
average daily doses of 0, 407, 792, or 2390 mg/kg-day. Ten animals were sacrificed
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immediately following the 60-day treatment period, following a 30-day recovery period, or
following a 56-day recovery period. Significant mortality was observed in the 2390-mg/kg-day
dose group, which led the study authors to add the 5 surviving animals in the 2240-mg/kg-day
dose group from the NTP (1993a) subchronic-duration rat study to the 10 surviving animals left
in the 2390-mg/kg-day dose group following the 60-day exposure period.
NTP (1993b) examined animals and collected data using the same methods as the NTP
(1993a) subchronic-duration rat study with the following exceptions: histologic analysis was
only performed on the testes, caput, and cauda of the left epididymis; no clinical pathologic
analysis (hematology, urinalysis, and clinical chemistry) was conducted; and no additional
reproductive endpoints were measured in any of the dose groups. The statistical analysis was
performed using the same methods as those used in the NTP (1993a) subchronic-duration rat
study.
NTP (1993b) reported dramatically decreased survivorship with 25 of the 30 animals that
were dead or sacrificed before the end of the 60-day exposure period in the animals exposed in
the 2390-mg/kg-day dose group (see Table B.6). Additionally, a single unscheduled death
following the treatment period occurred in each of the 792- and 2390-mg/kg-day dose groups.
Clinical observations in exposed rats included abnormal posture, diarrhea, emaciation, and
polyuria, although the study authors did not attribute these observations to specific doses. Mean
water consumption in the exposed groups was decreased as compared to that of the controls,
although the effect was not statistically significant. Decreased mean body weights as well as
decreased body-weight gains were also observed with increasing dose in all of the exposed
groups (e.g., 91% at body weight, and 84% body-weight gain, in the 792-mg/kg-day dose group
males at study termination, relative to controls). Animals in the 792- and 2390-mg/kg-day dose
groups showed increased weight gain as compared to those of the control during the recovery
period, although final body weights were still less than those of the control (e.g., 91% in the
792-mg/kg-day dose group relative to controls; data not shown). Chemical-related reduction in
testis weight (absolute and relative) was observed in the 792- and 2390-mg/kg-day dose groups
following the exposure period, 30 days recovery, and 56 days recovery (see Table B.7).
Additionally, absolute testis weight was significantly decreased (p < 0.01) in the 407-mg/kg-day
dose group following 56 days recovery. Moderate-to-marked testicular degeneration was
observed in rats exposed in the 792- and 2390-mg/kg-day dose groups following the exposure
period with no evidence of improvement after the 30- and 56-day recovery periods (see
Table B.8). Although no degeneration was noted in the 407-mg/kg-day dose group following the
exposure period, minimal degeneration was noted after the recovery periods. Sperm analysis
was not conducted in this study. No other treatment-related effects were noted by the study
authors. A LOAEL of 792 mg/kg-day based on evidence of testicular degeneration, which is
seen at increasing incidence and severity at the higher dose, is identified, with a corresponding
NOAEL of 407 mg/kg-day.
NTP (1993c) conducted a peer-reviewed GLP-compliant study, where 2-EE (purity 99%)
was administered in deionized drinking water to ten 5- to 6-week-old B6C3Fi mice (Taconic
Farms) per sex per dose for 13 weeks. Animals were treated with doses of 0, 2500, 5000,
10,000, 20,000, or 40,000 ppm in drinking water, which was available ad libitum. Unlike the
NTP (1993a) subchronic-duration rat study, no mortalities occurred in mice over the exposure
period. The study authors reported average daily doses of 0, 587, 971, 2003, 5123, or
7284 mg/kg-day for males and 0, 722, 1304, 2725, 7255, or 11,172 mg/kg-day for females.
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Animals were examined, and data were collected using the same methods as the NTP (1993a)
subchronic-duration rat study with the following exceptions: the gallbladder was subjected to
histopathologic analysis; no clinical pathologic analysis (hematology, urinalysis, and clinical
chemistry) was performed; and male and female reproductive endpoints (reproductive tissue
weights, spermatozoa, and estrous cycle effects) were measured in the 0-, 5000-, 10,000-, and
20,000-ppm dose groups. The statistical analysis was performed using the same methods as the
NTP (1993a) subchronic-duration rat study.
NTP (1993c) reported survivorship to be unaffected by treatment with 2-EE, although
body-weight gains in male and female mice in the 20,000- (59 and 57% of control, respectively)
and 40,000-ppm (52 and 46% of control, respectively) dose groups were decreased (see
Table B.9). The only clinical observation in exposed mice was emaciation in males and females
of the 20,000- and 40,000-ppm dose groups. Water consumption was variable and not attributed
to exposure. Absolute testis weights were significantly decreased in males in the
5123-mg/kg-day dose group (82% as compared to that of controls), and absolute and relative
testis weights were significantly decreased in the 7284-mg/kg-day dose group (16 and 19%,
respectively, as compared to those of controls) (see Table B.10). Chemical-related lesions of the
small testes and epididymides were found in the 7284-mg/kg-day dose group. Histopathologic
analysis identified changes in the spleen and testes of male mice and the spleen and adrenal
glands of female mice (see Tables B. 11 and B. 12). Degeneration of the testes in the
7284-mg/kg-day dose group was characterized by marked loss of the germinal epithelium in the
seminiferous tubules, similar to the effects observed in male rats (NTP, 1993a). Splenic
hematopoiesis was minimally to mildly increased in females (at doses >2725 mg/kg) and males
(7284-mg/kg-day dose group). Hypertrophy of the X-zone of the adrenals was noted at doses
>1304 mg/kg-day. No effects were seen in the bone marrow.
Sperm analysis showed that, in contrast to rat sperm, mouse sperm concentration was not
affected even in the highest group, although motility was somewhat decreased in the high-dose
(5123 mg/kg-day) group (see Table B.13). Females in all dose groups were found to have
increased estrous cycle length as compared to that of female controls (see Table B.13).
On the basis of testicular degeneration and increased hematopoiesis in the spleen in male
mice, the study authors identified a NOAEL of 5123 mg/kg-day. On the basis of on adrenal
gland hypertrophy and increased hematopoiesis in the spleen in the female mice, the study
authors identified a NOAEL of 1304 mg/kg-day. While hypertrophy in the adrenal gland was
observed at low-dose levels, hypertrophy can be considered an adaptive cell change and is not a
viable toxicity endpoint. A LOAELadj of 2725 mg/kg-day and a corresponding NOAELadj of
1304 mg/kg-day are identified based on effects on the increased hematopoiesis in the spleen in
the female mice.
Chronic-duration Studies
Melnick (1984a) published the results of a peer-reviewed 2-year oral toxicity study in
F344/N rats. 2-EE (>99% pure) was administered by gavage in deionized water at 0, 0.5, 1.0, or
2.0 g/kg of body weight in 5-mL/kg body-weight volume to 50 rats per sex per dose. Animals
were treated 5 times per week for 103 weeks, followed by a 1-week observation period. The
corresponding adjusted daily doses are 0, 357, 714, and 1429 mg/kg-day. Rats were acquired
from Charles River Laboratories at approximately 7 weeks of age. Animals were allowed food
and water ad libitum. Results demonstrated no measureable loss of the test compound when
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stored for 2 weeks at 25°C. Clinical observations of toxic effects were made twice daily. Body
weights were collected weekly for the first 13 weeks and then monthly until the end of the study
period. Necropsies were conducted on any moribund animals and on all of the surviving animals
at the end of the study period. Microscopic analysis was conducted on all of the animals in the
following tissues following necropsy: gross lesions, tissue masses, abnormal lymph nodes,
mandibular or mesenteric lymph nodes, salivary gland, thyroid, parathyroids, small intestine,
colon, liver, prostate/testes or ovaries/uterus, lungs, mainstream bronchi, heart, esophagus,
stomach, brain, thymus, trachea, pancreas, spleen, kidneys, adrenal glands, urinary bladder,
pituitary gland, mammary glands, sternebrae, and femur or vertebrae (with marrow). Despite
reporting statistical significance of the data, the study authors did not report the statistical
methods used to analyze the data. No information regarding compliance with GLP was
presented.
Melnick (1984a) reported decreased survival in the males and females in the
1429 mg/kg-day dose group, which caused this exposure level to be terminated at 17-18 weeks.
Stomach ulcers were found in many of the high-dose animals and thought to be a cause of death.
Additionally, males in this group showed reduced testicular size with no report of prostate effects
in those examined. Survival of the males in the 714-mg/kg-day dose group was significantly
reduced (p < 0.05) over the course of the 103-week exposure duration as compared to that of the
control, whereas no negative effect on survival was noted in the 357-mg/kg-day dose group. No
negative effects on survival in females of the 357- or 714-mg/kg-day dose groups were observed
(see Table B. 14). Dose-related decreases in body weight were also observed in the treated male
and female animals (see Table B.14). The study author reported that this trend became clear at
Week 15 and continued over the duration of the study. By Week 104 of the study, male rats in
the 357-mg/kg-day dose group weighed 81% of control, and those in the 714-mg/kg-day dose
group weighed 72% of control; female rats in the 357-mg/kg-day dose group weighed 76% of
control, and those in the 714-mg/kg-day dose group weighed 71% of control. Gross lesion
incidence in some organs was altered compared to that of controls; however, the quantitative
data were not provided, and the qualitative data combine the effects in the 357- and
714-mg/kg-day dose groups. The study author noted an increased incidence of enlarged adrenal
glands in treated males (357- and 714-mg/kg-day dose groups). This effect was not observed in
females. Treatment reportedly decreased the incidence of enlarged spleens and lesions of the
pituitary in treated males and females (357- and 714-mg/kg-day dose groups). Subcutaneous
tissue masses of the mammary glands were decreased in incidence in exposed females (357- and
714-mg/kg-day dose groups), and incidences of enlarged testes (as opposed to the reduced size
noted in the 2-g/kg dose group) were found to decrease in exposed males (357- and
714-mg/kg-day dose groups). The study author reported that the results of the histopathology
were not available at the time of publication. The mortality observed in males at 714 mg/kg-day
is a frank effect. The observed significant reduction in body weights in the exposed males and
females supports identification of a LOAELadj of 357 mg/kg-day. A NOAEL cannot be
identified because effects were seen at the lowest dose administered in the study.
Melnick (1984b) published the results of a peer-reviewed 2-year oral toxicity study in
B6C3Fi mice. 2-EE (>99% pure) was administered by gavage in deionized water at 0, 0.5, 1.0,
or 2.0 g/kg of body weight to 50 mice per sex per dose. Animals were treated 5 times per week,
for 103 weeks, followed by a 1-week observation period. The corresponding adjusted daily
doses are 0, 357, 714, and 1429 mg/kg-day. Mice were acquired from Charles River
Laboratories at approximately 8 weeks of age. Animals were allowed food and water ad libitum.
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Clinical observations of toxic effects were made twice daily. Body weights were collected
weekly for the first 13 weeks and then monthly until the end of the study period. Necropsies
were conducted on any moribund animals and on all of the animals surviving until the end of the
study period. Following necropsy, microscopic analysis was conducted in the following tissues
from all animals: gross lesions, tissue masses, abnormal lymph nodes, mandibular or mesenteric
lymph nodes, salivary gland, thyroid, parathyroids, small intestine, colon, liver, prostate/testes or
ovaries/uterus, lungs, mainstream bronchi, heart, esophagus, stomach, brain, thymus, trachea,
pancreas, spleen, kidneys, adrenal glands, urinary bladder, gallbladder, pituitary gland,
mammary glands, sternebrae, and femur or vertebrae (with marrow). Despite reporting statistical
significance, the methods used for the data analysis were not presented in the study. No
information regarding compliance with GLP was presented.
The investigator reported decreased survival in the males and females in the
1429-mg/kg-day dose group, which caused this exposure level to be terminated at 17-18 weeks.
Stomach ulcers were found in many of the high-dose male animals and thought to be a cause of
death, although no consistent incidence of gross lesions could be identified in the females.
Additionally, males in this group showed reduced testicular size. Survival of the mice in the
357- and 714-mg/kg-day dose groups was not negatively affected (see Table B. 15). No
dose-related changes in body weight were observed in the treated male and female animals.
Testis size was reduced in male mice compared to that of controls; however, the quantitative data
were not provided, and the qualitative data combine the effects in the 357- and 714-mg/kg-day
dose groups. The author reported that the results of the histopathology were not available at the
time of publication.
The mortality observed in the 1429-mg/kg-day dose group is a frank effect. Although the
reported changes in incidence of gross lesions were discussed, adequate quantitative and
dose-related data were not provided to support identification of a LOAEL or NOAEL. Also, it is
noted that subsequent studies (e.g., NTP, 1993a,b,c) clearly show that rats are more sensitive
than mice to the effects of 2-EE. This study is presented in this review as a supporting study and
will not be used to support derivation of a chronic p-RfD.
Reproductive and Developmental Studies
The study by Yoon et al. (2003) is selected as the principal study for deriving the
chronic p-RfD. In a peer-reviewed reproductive study, Yoon et al. (2003) administered doses of
0- (saline only), 100-, 200-, 400-, or 800-mg/kg 2-EE (purity unreported) by gavage to 5 groups
of 5 male Sprague-Dawley rats, 6 times per week, for 4 weeks. The adjusted daily doses are 0,
86, 171, 343, and 686 mg/kg-day. Rats were obtained from the Korea Food and Drug
Administration at 8 weeks of age. Animals were allowed to acclimate for 1 week before study
initiation. Throughout the study, rats were housed in plastic cages and given pellet food (brand
unspecified) and water ad libitum. The GLP compliance of this study was not provided.
Yoon et al. (2003) sacrificed rats after blood samples were obtained by heart puncture.
Testes and epididymides were weighed; testes were stored in citrate buffer at -80°C. Both
organs were examined under a light microscope after being fixed in 10%-sodium
phosphate-buffered formalin solution, embedded in paraffin, and stained with hematoxylin and
eosin. Thawed testes were minced and shaken for 30 minutes at room temperature. The
resulting cell suspension was filtered to discard tissue debris. Cells were counted using a
hemocytometer and light microscope. Testicular cells were then analyzed for DNA content by
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flow cytometry. Fluorescence was directly proportionate to the amount of stain absorbed, which
in turn, represented the DNA content of each cell. For this review, graphs are analyzed using
Coulter Multiparameter Data Acquisition and Display Software. Statistical analyses of data were
completed using one-way analysis of variance (ANOVA) with a significance level of p< 0.05.
Authors used the Scheffe test for significant differences between groups.
Yoon et al. (2003) reported that doses of 171 mg/kg-day and higher resulted in a
dose-dependent decrease in body weight, suggesting systemic toxicity (see Table B.16).
However, at 86 mg/kg-day, weight was increased compared to that of controls. At 343 and
686 mg/kg-day, the weights of the testes were reduced significantly (p < 0.01) to 64 and 49%
compared to controls, respectively (see Table B.17). Authors reported a dose-related decrease
(p < 0.05) in epididymis weight in all of the exposed groups. The numbers of testicular cells
were reduced (p < 0.01) to 44% at 343 mg/kg-day and to 20.0% at 686 mg/kg-day compared to
control (see Table B.18). Histopathological examination indicated that spermatogenesis was
affected in a dose-dependent manner. Considerable testicular pathology was noted at
>171 mg/kg-day. The group dosed at 171 mg/kg-day experienced exfoliation of the germ cells
into the tubular lumen, whereas the 343-mg/kg-day group showed some large cells with the
disappearance of spermatids and moderate testicular generation. A marked depletion of all of the
spermatid types was noted in the 686-mg/kg-day group, as well as a decrease in irregularly
shaped seminiferous tubules. This group also had increased spermatozoa aggregates in the
epididymal tubules and a marked reduction in the density of spermatozoa in the lumen. Flow
cytometry revealed that rats exposed to 2-EE experienced differences in proportions of testicular
cell types compared to those of controls. Mature haploid cells were reduced (p < 0.01) to 50% at
343 mg/kg-day and 4% at 686 mg/kg-day compared to control. Immature haploids were reduced
(p < 0.01) to 27% at 343 mg/kg-day and 11% at 686 mg/kg-day compared to control. The
relative proportions of diploid cells and tetraploid cells were significantly increased (p < 0.01) in
the 343-mg/kg-day group.
The altered ratios of sperm cell types, reported by Yoon et al. (2003), indicate that 2-EE
interferes with spermatogenesis in rats. The depletion in haploid cells was somewhat novel, and
authors noted this finding may be due to lethal effects to early cell types (spermatogonia). It is
not clear whether the effects on diploid cells and tetraploid cells are an artifact of the cell
counting method or an indicator of cytotoxicity. Overall, authors concluded that the study
supports the reproductive cytotoxicity of 2-EE in male rats. A LOAEL of 171 mg/kg-day is
identified based on testicular pathology (exfoliation of the germ cells in the testicular lumen),
with a corresponding NOAEL of 86 mg/kg-day.
In a peer-reviewed study, Yoon et al. (2001) investigated the effects of 2-EE on
spermatogenesis in male Sprague-Dawley rats. Twenty-five pubertal rats and 25 adult rats (5 per
dose group) were dosed with 0-, 50-, 100-, 200-, or 400-mg/kg 2-EE (purity unreported) by
gavage, 6 days per week, for 4 weeks. The adjusted daily doses are 0, 43, 86, 171, and
343 mg/kg-day. Rats were obtained from the Korea Food and Drug Administration. All of the
animals were acclimated for 1 week; at study initiation, pubertal rats were 5 weeks of age, and
adult rats were 9 weeks of age. Rats were provided with food and water ad libitum. Once a
week, investigators weighed and examined rats for behavioral effects. After blood samples were
drawn by heart puncture, rats were sacrificed, and the testes and epididymides were collected and
weighed. Testes were stored at -80°C in citrate buffer until analysis. The study report did not
indicate whether this study is GLP compliant.
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Yoon et al. (2001) thawed, minced, and mixed testes for 30 minutes using a magnetic
stirrer before analysis. The resulting cell suspension was filtered to remove debris, and cells
were stained with propidium iodide. Samples were kept in an ice bath until examined. Flow
cytometry was used to analyze the DNA content of the testicular cells. DNA content
corresponded to the amount of stain absorbed, as depicted by the degree of fluorescence. For
this review, histograms of fluorescence are analyzed using Coulter Multiparameter Data
Acquisition and Display Software for the relative proportions of haploid, diploid, and tetraploid
cells. Statistical analysis consisted of ANOVA at a significance level ofp < 0.05; the Scheffe
test was used for multiple comparisons of significant differences in groups.
Yoon et al. (2001) described significantly increased (p < 0.05) relative testis and
epididymis weights in pubertal rats treated with >43 mg/kg-day compared to those of controls
(see Table B. 19). Body weights were also reportedly decreased although the data were not
presented. Results of cellular analysis by flow cytometry indicated that spermatogenesis in
pubertal rats was not significantly affected by 2-EE. No other effects were noted in the exposed
pubertal groups.
Yoon et al. (2001) reported that adults administered 343-mg/kg-day 2-EE experienced
significantly decreased (p < 0.01) relative testis and epididymis weights compared to those of the
controls, unlike the trend observed in the pubertal rats (see Table B. 19). Body weights were also
reportedly decreased, although the data were not presented. Cellular analysis by flow cytometry
in adult rats treated with 343 mg/kg-day showed a significant decrease (p < 0.05) in the
proportions of mature (77%) and immature (52%) haploid cells as well as a significant increase
(p < 0.01) in the proportions of diploid and tetraploid cells (see Table B.20).
Yoon et al. (2001) concluded that adults appeared to be more sensitive to the effects of
2-EE administration than pubertal rats; however, both showed systemic toxicity at decreased
body weights. Although the results did not provide clear evidence of effects of 2-EE on
spermatogenesis in pubertal rats, 343-mg/kg-day 2-EE produced clear testicular toxicity in
adults. A LOAEL of 343 mg/kg-day is identified based on pathology of the testes of adults, with
a NOAEL of 171 mg/kg-day.
Yu et al. (1999) conducted a published peer-reviewed study in which groups of nine male
Sprague-Dawley rats were administered 2-EE (purity not reported) by gavage in olive oil. At
9 weeks of age, male, specific pathogen-free Sprague-Dawley rats weighing 315 ± 7 g were
separated into experimental groups that received either olive oil vehicle as a control or 2-EE. An
initial dose-finding experiment was conducted with 2-EE only administered via gavage at doses
of 0, 100, 150, 250, or 500 mg/kg, 6 times per week, for 4 weeks. The corresponding adjusted
daily doses are 0, 86, 129, 214, and 429 mg/kg-day. The dose-finding experiment sought to
cause measurable testicular atrophy with 2-EE while minimizing changes in weekly body-weight
gain, blood biochemistry, hematology, and organ pathology caused by 2-EE. The specific results
of this dose-finding experiment were not reported although the study authors chose to administer
150-mg/kg 2-EE in the main experiment also for 4 weeks, 6 days per week for which further
specifics and results are available (see below). The corresponding adjusted daily dose is
129 mg/kg-day.
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Arteriolar blood was collected and analyzed for aminotransferase, alanine
aminotransferase, alkaline phosphatase, glucose, urea nitrogen, total protein, total cholesterol,
creatinine, total bilirubin, red and white blood cell counts, and standard blood measures. Adrenal
glands, testes, epididymides, heart, lungs, kidneys, spleen, liver, and brain were removed,
weighed, fixed in 10% buffered formalin solution, embedded in paraffin, and stained with
hematoxylin and eosin for histological analysis. Statistical analyses of body weight, organ
weights, blood chemistry, and hematology data were conducted using multiple variance analysis
and Duncan multiple range tests. Compliance with GLP guidelines was not reported.
Among animals exposed to the 129-mg/kg-day dose regimen, Yu et al. (1999) reported
significantly decreased, right adrenal weight (80%, relative to control), left adrenal weight (72%,
relative to control), right and left testes weights (57%, relative to control), right epididymis
weight (77%), relative to control) and left epididymis weight (76%, relative to control) all at a
relative body weight decreased by only 5% in exposed animals (see Table B.21). Histology of
the testes showed "grossly damaged seminiferous tubules," with only 20% of the tubules
showing a normal appearance. Additionally, the germ cells in the tubules were necrotic, and the
Leydig cells showed signs of hyperplasia and hypertrophy. Hematology showed significant
reductions (p < 0.01) in platelets (74%, relative to control) and white blood cells (71%, relative
to control) (see Table B.22). Hematocrit (92%, relative to control), hemoglobin (93%, relative to
control), and mean corpuscular hemoglobin (92%, relative to control) were decreased, which are
indicators of bone marrow suppression. Blood biochemistry revealed a reduction (p < 0.01) in
plasma protein (89%, relative to control) and plasma creatinine concentration (82%, relative to
control), which can indicate decreased protein turnover as well as liver and kidney damage.
Alkaline phosphatase was decreased (59%, relative to control), but no change was noted in total
cholesterol.
A LOAEL based on testicular toxicity as well as changes in hematology of
129 mg/kg-day is identified. Because the effects were observed at the only dose administered, a
NOAEL cannot be identified.
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Table 3. Summary of Oral Reproductive and Developmental Studies for 2-EE (CASRN 110-80-5)
Study Type, Number of Male/Female,
Strain Species, Route of
Administration, Study Duration,
Methods
Dosimetry
(mg/kg-d)a,
Purity of
2-EE
Critical Effects
NOAEL
LOAEL
Reference
Rat
Reproductive, 5/0, Sprague-Dawley rat,
gavage, 6 d/wk, 4 wk; blood samples,
testes and epididymides collected and
weighed at study termination
0, 86, 171,
343, 686
(purity not
reported)
Body weight decreased >171 mg/kg-d; testis weight
decreased >343 mg/kg-d; testicular pathology at
>171 mg/kg-d; marked depletion of all of the
spermatid types at 686 mg/kg-d.
NOAEL/LOAEL: testicular pathology (exfoliation of
the germ cells in the testicular lumen)
86
171
Yoon etal.
(2003)
Reproductive, 10/0, Sprague-Dawley rat,
gavage, 6 d/wk, 4 wk; dose group:
5 pubertal rats and 5 adult rats; body
weights and clinical examination weekly;
testes and epididymides collected and
weighed at study termination
0, 43, 86, 171,
343 (purity not
reported)
Adult males: altered composition of testicular germ
cell populations; relative testes, relative epididymal,
and body weights decreased at 343 mg/kg-d
Pubertal males: no effect on testicular growth and
germ cell populations; increased relative testes,
relative epididymal, and decreased body weights
>43 mg/kg-d
NOAEL/LOAEL: testicular effects (adults)
171
343
Yoon etal.
(2001)
Reproductive, 9/0, Sprague-Dawley rat,
gavage, 6 d/wk, 4 wk; exposure dose from
a range-finding test (0, 86, 129, 214, and
429 mg/kg-d); organ weights, blood
chemistry, hematology, and clinical
pathology measured study termination
0, 129 (purity
not reported)
Adult males: body weight, relative adrenal gland,
relative testis, and relative epididymis weights
decreased; severe degeneration of seminiferous
tubules; germ cell necrosis, interstitial Leydig cell
hyperplasia, and decreased white blood cells, platelet
count, hematocrit, hemoglobin concentration, mean
corpuscular hemoglobin, plasma protein content,
plasma creatinine concentration, and alkaline
phosphatase at 129 mg/kg-d
LOAEL: hematopoietic and testicular effects
None
129
Yuetal. (1999)
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Table 3. Summary of Oral Reproductive and Developmental Studies for 2-EE (CASRN 110-80-5)
Study Type, Number of Male/Female,
Strain Species, Route of
Administration, Study Duration,
Methods
Dosimetry
(mg/kg-d)a,
Purity of
2-EE
Critical Effects
NOAEL
LOAEL
Reference
Reproductive, 9-10/0, Sprague-Dawley
rat, gavage, 2, 5, or 7 weeks; sacrificed at
(2, 5, or 7 wk; sperm motility and sperm
counts assessed; body weights, testis and
epididymis weights recorded; male rats in
7-wk group bred with untreated females
after Week 5; pregnant females sacrificed
on GD 14; numbers of implantation sites,
resorptions, and live fetuses recorded
0, 250, 500
(purity not
reported)
Adult males: decreased body-weight gains, decreased
sperm motility, decreased sperm counts, decreased
testis and epididymis weights >250 mg/kg-day; no
effects at 2 wk except slightly decreased testis weight
at 500 mg/kg-d; decreased pregnancy index, numbers
of implantations and live fetuses from males at
500 mg/kg-d
LOAEL: sperm motility
None
250
Horimoto et al.
(1996)
Reproductive, 19-20/0, Sprague-Dawley
rat, gavage, 35 d; males bred with
untreated females; males continued
treatment through breeding period
(49-52 d); subset sacrificed and
necropsied Day 36-39; subset sacrificed
and necropsied Day 50-53; pregnant
females sacrificed GD 14; body weights,
testes and epididymides weights recorded;
sperm motility and count analyzed
0, 250, 500
(purity not
reported)
Adult males: decreased sperm count, sperm motility,
body weights, body-weight gain and epididymis
weight (absolute and relative) >250 mg/kg-d;
decreased testis weight (absolute and relative)
>500 mg/kg-d; no motile sperm, almost no sperm,
decreases in the pregnancy index (30% of control),
number of implantation sites (24% of control), and
live births (27% of control) in those treated for 7 wk
NOAEL/LOAEL: severe male reproductive effects
250
500
Horimoto et al.
(2000)
Reproductive, 10/0, Long-Evans hooded
rat, gavage, 5 d/wk, 6 wk; males bred with
untreated females; semen collected
weekly; sperm motility and count
analyzed
0, 669 (purity
98%)
Adult males: sperm count decreased (30-40%) and
abnormal sperm morphology by Weeks 5 and 6;
sperm motility decreased by Week 6; three males
azoospermia no difference in swimming speeds of
motile sperm; decreases in testis, epididymis, and
caudae epididymis weights
LOAEL: sperm and testicular effects
None
669
Oudiz and
Zenick (1986a)
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Table 3. Summary of Oral Reproductive and Developmental Studies for 2-EE (CASRN 110-80-5)
Study Type, Number of Male/Female,
Strain Species, Route of
Administration, Study Duration,
Methods
Dosimetry
(mg/kg-d)a,
Purity of
2-EE
Critical Effects
NOAEL
LOAEL
Reference
Reproductive, 9/0, Long-Evans hooded
rat, tracheal intubation, 5 d/wk, 6 wk;
body weights, testes and epididymides
weights recorded; hematology evaluation;
semen collected weekly; sperm motility
and count analyzed
0, 669
(purity 99.9%)
Adult males: body-weight gain reduced, brain and
spleen weights increased; hemoglobin and hematocrit
decreased; reduced sperm parameters: sperm count
and percent normal morphology at Week 5; sperm
count, percentage normal morphology, and sperm
motility at Week 6; testicular lesions at Week 1
LOAEL: hematopoietic, sperm and testicular effects
None
669
Zenick et al.
(1984)
Mouse
Reproductive and developmental, 0/50,
CD-I mouse, gavage, GDs 6-13; body
weights taken GDs 6 and 17; nonbirthing
dams sacrificed at GD 22; litter size, birth
weight, neonatal growth and survival to
Postnatal Day 3
0, 3605
(purity not
reported)
Maternal: mortality: 10%
Fetal: mortality: 100%
3605 mg/kg-d: frank effect
Not applicable
Not applicable
Hardin et al.
(1987)
Reproductive and developmental, 20/20,
CD-I mouse, drinking water, 24 wk; body
weights recorded weekly; reproductive
endpoints during and at study termination;
crossover mating study: (1) mid- and
high-dose males bred control females,
(2) mid- and high-dose females bred
control males, and (3) control males bred
control females; sacrificed and necropsied
Week 24; litter size, birth weight, neonatal
growth, and survival
Males: 0,
1230, 2461,
4921
Females: 0,
1261, 2522,
5044
(purity not
reported)
Fetal: embryo mortality (100%) >4921 mg/kg-d;
number of litters, number of live pups per litter,
proportion of pups born alive, and live pup weight
decreased >2461 mg/kg-d
Fetal: crossover mating study: live pups per litter
decreased, and increased dead pups per litter in
2522 mg/kg-d females mated with control males; no
litters from 5044 mg/kg-d females mated with control
males; decreased fertile matings and numbers of live
pups per litter from 2461 mg/kg-d males paired with
control females
NOAEL/LOAEL: fertility and reproductive
performance
Fetal: 1230
Fetal: 2461
Lamb et al.
(1984)
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Table 3. Summary of Oral Reproductive and Developmental Studies for 2-EE (CASRN 110-80-5)
Study Type, Number of Male/Female,
Strain Species, Route of
Administration, Study Duration,
Methods
Dosimetry
(mg/kg-d)a,
Purity of
2-EE
Critical Effects
NOAEL
LOAEL
Reference
Reproductive, 8/0 (4 in control) JCL ICR
mouse, gavage, 5 d/wk, 5 wk; necropsied
at study termination; body weights, testes
and epididymides weights recorded;
sperm motility and count analyzed
0,357,714,
1429, 2857
(purity not
reported)
Adult males: testicular weights, atrophy of
seminiferous epithelium, spermatozoa, spermatids,
and spermatocytes decreased >714 mg/kg-d;
decreased testis weight, spermatozoa, and spermatids
>1429 mg/kg-d
NOAEL/LOAEL: sperm and testicular effects
357
714
Nagano et al.
(1984)
Developmental, 4-6/0, CD-I mouse,
gavage, GDs 8-14; sacrificed GD 18 and
necropsied; maternal body weights and
physical examinations on GDs 0, 8, 10,
12, 14, and 18; litter size, fetal weight,
fetal growth recorded
0, 1000, 1800,
2600, 3400,
4200 (purity
97%)
Maternal: lethargy, failure to right, uneven gait,
abnormal breathing, cold to the touch and/or red
vaginal discharge (4200 mg/kg-d only)
>3400 mg/kg-d; mortality 50% >3400 mg/kg-d;
decreased maternal body weight (GDs 8-14 and
14-18) >1800 mg/kg-d
Fetal: mortality 100% at 4200 mg/kg-d; decreased
fetal weights >1000 mg/kg-d; increased malformed
fetuses at 1800 and 2600 mg/kg-d
NOAEL/LOAELs maternal weights and increased
malformations in the fetus
Maternal: 1000
Fetal: none
Maternal: 1800
Fetal: 1000
Wier et al.
(1987a)
Developmental, 0/20-30 treated females
mated untreated males, CD-I mouse,
gavage, GDs 8-14; maternal body
weights and physical examinations on
GDs 0, 8, 10, 12, 14, and 18; litter size,
fetal weight, fetal growth recorded
0, 800, 1200
(purity 97%)
Fetal: decreased live-born pups at birth and survival
postbirth at 1200 mg/kg-d; malformations of forepaw
and kinked tail >800 mg/kg-d
LOAEL: malformation in pups
None
Fetal: 800
Wier et al.
(1987b)
aValues for oral reproductive and developmental studies are not converted to human equivalent doses (HEDs). Reproductive studies are presented as duration adjusted
doses (from 5-6 d per wk to continuous 7 d/wk). Doses for oral developmental studies are not adjusted beyond continuous daily as dosing is typically every day
throughout the developmental period.
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Inhalation Exposures
The effects of inhalation exposure of animals to 2-EE have been evaluated in two
subchronic-duration (Barbee et al., 1984a,b), no chronic-duration, six developmental (Doe,
1984a,b; Andrew and Hardin, 1984; Nelson et al., 1981, 1982), no reproductive, and no
carcinogenic studies. The developmental studies are presented in Table 4, with a general
summary and discussion of key studies in the text.
Subchronic-duration Studies
In a peer-reviewed subchronic-duration inhalation study, Barbee et al. (1984a) exposed
groups of 15 Sprague-Dawley rats per sex per concentration to 0 (air only), 25, 100, or 400 ppm
of 2-EE vapor (in air) (99.59% pure), for 6 hours per day, 5 times per week, for 13 weeks. The
analytical means of the 2-EE concentrations were 25, 103, and 403 ppm. The exposure
concentration adjustments for continuous exposure and unit conversion are 0, 17, 68, and
-3
265 mg/m . Rats were obtained from Charles River Breeding Laboratories and weighed between
149 and 275 g at study initiation. Animals were allowed to acclimate for 15 days before study
initiation. Animals had access to food and water ad libitum. Exposure was whole body and
conducted in a 10-m3 stainless steel and glass chamber that provided a complete air change every
3 minutes and a 99% equilibrium time of 15 minutes. Animals were observed twice per day for
clinical signs of toxicity. Investigators made physical examinations and weighed animals once
per week. The GLP compliance of this study was not provided.
Barbee et al. (1984a) conducted ophthalmic examination before the exposure period and
at termination of the overall exposure period. Hematology (red and white blood cell counts,
hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin,
platelet counts, total and differential leukocytes, reticulocyte count, and erythrocyte
morphology), clinical chemistry (albumin, globulin, albumin/globulin ratio, aspartate
transaminase, alanine aminotransferase, alkaline phosphatase, glucose, urea nitrogen, total
protein, total cholesterol, creatinine, total bilirubin, direct bilirubin, sodium, potassium, chloride,
calcium, and inorganic phosphorus), and urinalysis (specific gravity, pH, protein, glucose,
ketones, bilirubin, occult blood, urobilinogen, and microscopic examination for sediment)
examinations were performed at the termination of the exposure period. Rats were sacrificed
after blood samples were obtained, and full necropsies were performed. Body and organ weights
were measured for liver, kidneys, testes including epididymis, brain, spleen, thymus, adrenal
glands, and pituitary. The following tissues were collected, preserved, sectioned, and stained
from all of the necropsied animals: abdominal aorta, adrenal glands, bone marrow (sternum),
brain, eyes with Harderian gland and optic nerve, gonads, heart, intestine, colon, duodenum,
ileum, jejunum, kidneys, liver, lungs with trachea, lymph nodes, mammary glands, nasal
turbinates, pancreas, pituitary, prostate, salivary gland, sciatic nerve with muscle, seminal
vesicles, skin, spinal cord, and vagina. The sections from control and highly exposed animals
were examined microscopically. Sperm analysis was not performed. Statistical analyses of data
were completed using one-way ANOVA, the Bartlett test, the Dunnett test, the Kruskal-Wallis
test, the summed rank test, regression analysis, and the Jonckheere statistic test with a
significance level ofp< 0.05.
Barbee et al. (1984a) noted increased lacrimation and nasal discharges in all of the
exposed animals in Weeks 2-10, although a concentration-response was not observed.
Survivorship was not affected by exposure. No chemical-related effects were noted during the
ophthalmoscopic examination. The body weights of treated animals were not significantly
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affected by exposure (see Table B.23). Organ weights were largely unaffected by exposure with
the exception of decreased pituitary weight (p < 0.05) in the high-exposure males (86%, relative
to control) and females (92%, relative to control), and spleen weights (p < 0.01) in the
high-exposure females (85%, relative to control) (see Table B.24). The only changes noted in
the hematology were a decreased leukocyte count in high-exposure females, which the study
authors noted as being of unknown significance. Differences found in serum biochemistry were
not considered biologically significant. No differences were found during urinalysis. Gross
pathology and histopathology revealed no treatment-related effects.
Barbee et al. (1984a) concluded that no significant treatment-related effects were noted in
rats. The lack of effects noted in rats exposed to subchronic inhalation of 2-EE supports a
"3
NOAEL of 265 mg/m although no analysis was performed on the sperm, which is known to be a
target tissue by the oral route of 2-EE exposure.
In a peer-reviewed sub chronic-duration inhalation study, Barbee et al. (1984b) exposed
groups of 10 New Zealand White rabbits per sex per concentration to 0 (air only), 25, 100, or
400 ppm of 2-EE vapor (in air) (99.59% pure) 6 hours per day, 5 times per week, for 13 weeks.
The analytical means of the 2-EE concentrations were 25, 103, and 402 ppm. The exposure
concentration adjustments for continuous exposure and unit conversion are 0, 17, 68, and
265 mg/m3. Rabbits were obtained from Dutchland Laboratories (age unreported) and weighed
between 2.1 and 3.3 kg at study initiation. Animals were allowed to acclimate for 22 days before
study initiation. Animals had access to food and water ad libitum. Exposure was whole body
-3
and conducted in a 10-m stainless steel and glass chamber that provided a complete air change
every 3 minutes and a 99% equilibrium time of 15 minutes. Animals were observed twice per
day for clinical signs of toxicity. Investigators made physical examinations and weighed animals
once per week. The GLP compliance of this study was not provided. The study followed the
same methods as described for the Barbee et al. (1984a) study in rats with the addition of the
examination of the gallbladder from necropsied rabbits.
Barbee et al. (1984b) noted increased lacrimation and nasal discharges in all of the
exposed animals in Weeks 2-10 although a concentration-response was not observed. No
chemical-related effects were noted during the ophthalmoscopic examination. The body weights
of treated animals were slightly depressed, although effects were only significant (p < 0.05) in
high-exposure males (see Table B.25). Organ weights were largely unaffected by exposure with
the exception of decreased adrenal gland weight (p < 0.05) in the low-exposure males (72%,
relative to control) and decreased testis weights (p < 0.05) in the high-exposure males (78%,
relative to control) (see Table B.26). Hematology showed decreases in the hemoglobin,
hematocrit, and erythrocyte counts in high-exposure males and females, indicating anemia.
Although changes were noted in serum biochemistry, they were reportedly not biologically
significant. No differences were found during urinalysis. Gross pathology revealed no
treatment-related effects. Histopathology of the high-exposure males revealed minimal to slight
focal degeneration of the seminiferous tubules of the testes marked by a loss in epithelium
(3/10 animals). The study authors stated that spermatogenesis appeared normal as judged by
overall organ morphology although no specific sperm analysis was performed.
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Barbee et al. (1984b) concluded that subchronic-duration exposure to 2-EE resulted in
anemia that stemmed from the destruction of erythrocytes and testicular toxicity, which they
"3
used to identify a NOAEL of 68 mg/m . The effects seen in the high-exposure rabbits exposed
to subchronic-duration inhalation of 2-EE support a LOAEL of 265 mg/m3 and a corresponding
NOAEL of 68 mg/m3.
Chronic-duration Studies
No studies could be located regarding the effects of chronic-duration inhalation exposure
of animals to 2-EE.
Developmental Studies
Doe (1984) presented the results of a peer-reviewed inhalation developmental study of
2-EE in rats and rabbits. This study was sponsored by the Glycol Ethers Program Panel of the
Chemical Manufacturers Association and was intended to supplement a previous study
conducted for NIOSH. The study authors did not state whether the study followed GLP
guidelines. The portion of the study conducted in rats is referenced as Doe (1984a), and the
portion conducted in rabbits is referenced as Doe (1984b).
Nulliparous specific pathogen-free female Wistar-derived (Alpk/AP) rats (11-13 weeks
of age; initial number not specified) were paired with males (number and strain not specified)
until evidence of mating was found by a sperm-positive vaginal smear. The day that a
sperm-positive smear was detected was identified as Day 0 of pregnancy. 2-EE (purity >99%)
was administered by inhalation to females (24 per group) at concentrations of 0 (control), 10, 50,
or 250 ppm on GDs 6-15 for 6 hours per day. The overall atmospheric exposure concentrations
were measured as 9.9 ± 0.9, 50.8 ± 2.3, and 249.2 ± 10.4 ppm. The concentrations converted to
3	3
mg/m and adjusted for continuous exposure (6 hours per 24 hours) are 0, 9, 47, and 230 mg/m .
Doe (1984a) exposed whole animals using stainless steel exposure chambers of
"3
approximately 3.4 m . Each chamber consisted of six cage levels, and there were four cages per
level. Air (at a flow rate of 600 L/minute) entered at the front of the chamber and was extracted
in the back. Temperature was maintained at 22°C, and relative humidity was maintained at 50%.
Animal diets during the experiment were not described.
Females were observed daily for signs of clinical abnormalities. Body weight was
recorded on GDs 0, 5, 6-15, 16, and 21. Food consumption was also measured (frequency not
specified). On GD 21 of pregnancy, the rats were sacrificed and examined postmortem. Blood
samples were collected for hematological assessment, and the spleen, gravid uterus, and thymus
were weighed. The uterus was opened, and the number of corpora lutea in each ovary was
counted. Numbers of live fetuses, implantations, and early and late uterine deaths were also
recorded. Each live fetus was examined for gross abnormalities. Half of the fetuses in each litter
were examined for skeletal defects; the other half was examined for visceral defects. Visceral
assessments of fetuses included examination of the heart, abdomen, and thorax and
determination of sex.
Doe (1984a) reported no treatment-related effects on maternal body weight or food
consumption. In addition, no abnormalities related to treatment were observed during clinical or
"3
postmortem examinations. In dams exposed at the 230-mg/m level, the study authors noted
significant reductions in hemoglobin, hematocrit, and mean cell volume in red blood cells
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"3
compared to those of the control. Similar effects were not observed at the 9- and 47-mg/m
concentration levels. Preimplantation losses were higher in all of the treatment groups compared
to those of the control and were significantly higher in the 9- and 47-mg/m groups because
exposure began at GD 6; however, this effect may not be attributable to treatment (see
Table B.27). Postimplantation losses were also higher in all of the treatment groups compared to
those of the control; however, the differences were not statistically significant for any exposed
group. Mean numbers of live fetuses were lower in all of the exposed groups compared to those
of the control with the decreases being significantly lower in the 9- and 47-mg/m3 groups but not
in the 230-mg/m group. The study authors reported that mean live fetal weight in the
230-mg/m3 group was significantly reduced (92%, relative to control); however, mean fetal
weights for the other groups were comparable to that of the control (see Table B.27).
Occurrences of minor external and visceral defects, as well as skeletal defects, were significantly
-3
elevated (18.4 and 97.5%, respectively) in the 230-mg/m group compared to those in the control
group (11.7 and 46.3%, respectively) (see Table B.28). The elevated incidence of external and
visceral defects was associated with renal pelvic dilation, which occurred in 12.8% of offspring
in the 230-mg/m3 group compared to 6.8% in the control (see Table B.29). However, the study
authors noted that this endpoint is not indicative of teratogenicity. The elevated incidences of
skeletal defects were associated with increased partial nonossification of parts of the skull,
vertebrae, and sternebrae and other skeletal abnormalities (data not reported). The study authors
also noted that the number of limb malrotations was significantly increased in the 9-mg/m3 group
compared to that in the control group (see Table B.29), but the study authors believed that this
result had no toxicological significance because the effects were limited to the 9-mg/m3 group.
Cardiovascular abnormalities were not observed.
Doe (1984a) concluded that there was evidence of mild maternal toxicity associated with
2-EE due to significantly reduced hemoglobin, hematocrit, and mean cell volume in red blood
cells in the 230-mg/m3 group compared to those in the control group. There was a fetotoxic
"3
effect at the 230-mg/m level associated with retarded fetal growth and a significant increase
(p < 0.05) in minor skeletal defects and skeletal variants. A slight effect at the 47-mg/m3 level
was associated with increased (but not significant) incidences of minor skeletal defects (51%
compared to 46% in controls).
"3
The study authors identified 9 mg/m as a clear no-effect level in 2-EE-exposed rats;
however, the data do not support concentration-response effects in the 47-mg/m3 group. A
3	3
maternal LOAELadj of 230 mg/m with a corresponding NOAELadj of 47 mg/m is identified
based on hematopoietic effects. A fetotoxicity LOAELadj of 230 mg/m3 is identified based on
"3
increased skeletal malformations with a corresponding NOAELadj of 47 mg/m .
In a peer-reviewed and published developmental study, Nelson et al. (1981) evaluated the
possible functional effects in offspring of rats exposed to 2-EE during gestation. The study
exposed a total of 29 pregnant Sprague-Dawley rats to 100-ppm 2-EE (98-98.5%) purity) in an
inhalation chamber, for 7 hours per day, from GDs 7-13 and GDs 14-20. A pilot range-finding
study exposed pregnant Sprague-Dawley rats to 200-, 300-, 600-, 900-, or 1200-ppm 2-EE,
7 hours per day, from GDs 7-13 and GDs 14-20. Results of the dose-finding study were
complete resorptions of litters at 900 and 1200 ppm and increased mortality of pups as low as a
dose of 200 ppm. Thus, the study authors selected 100 ppm as the sole exposure of the main
study. Converted to mg/m3 and adjusted for the daily exposure (7 hours per 24 hours), the
"3
concentration is 108 mg/m . Fifty-nine pregnant animals were randomly assigned to one of four
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3	3
groups of exposure (7 hours per day) to 100-ppm 2-EE (369 mg/m ; 108 mg/m adjusted)
(15 animals on GDs 7-13, 14 animals on GDs 14-20, 15 animals each for control exposure on
GDs 7-13 and GDs 14-20).
Pups of the mother rats were weighed weekly and observed for abnormalities.
Neurochemical analysis was also performed. Behavioral tests were conducted to assess central
nervous system functions at several stages of development; rotorod tests, open field tests, activity
wheel, avoidance conditioning, and operant conditioning were conducted. Results of behavioral
testing of offspring exposed on GDs 7-13 included significantly impaired performance on the
rotorod test of neuromuscular ability, prolonged latency of leaving the start area of an open field,
and marginal superiority in avoidance conditioning starting on Day 34 of age. Results of animals
exposed on GDs 14-20 included significantly increased number of duration of shocks in
avoidance conditioning, starting on Day 60 of age, as well as, significantly decreased activity
compared to controls in a running wheel.
Results of neurochemical evaluation, of whole-brain samples from newborn pups, found
significant decreases in levels of norepinephrine in offspring from both exposure periods.
Significant elevations in acetylcholine, norepinephrine, and dopamine were found in the
cerebrums from 21-day-old offspring from GDs 7-13. The cerebellums were found to have a
more significant increase in acetylcholine, whereas the brainstem had an increase in
norepinephrine. The midbrain had excesses of acetylcholine, norepinephrine, and protein. The
21-day-old offspring from the GDs 14-20 exposure group revealed significant elevation in
acetylcholine, dopamine, and 5-hydroxytryptamine in the cerebrum relative to the concurrently
air-exposed offspring.
The study authors concluded that prenatal exposure to 108 mg/m results in behavioral
and neurochemical alternations in offspring. The study authors did not report a NOAEL or
LOAEL. A fetal LOAELadj of 108 mg/m is established based on developmental neurotoxicity.
Because effects were noted at the only concentration utilized in this study, a NOAEL cannot be
identified.
The study by Doe (1984b) is selected as the principal study for deriving the
subchronic p-RfC. Doe (1984b) conducted an inhalation developmental study in virgin female
Dutch rabbits (5-7 months of age; initial number not specified). Females were housed with
males of the same strain until evidence of mating was found by a vaginal smear containing
motile sperm. After mating, females received chorionic gonadotropin injections to encourage
ovulation. The day of mating was identified as GD 0. 2-EE (>99% pure) was administered by
inhalation to females (24 per group) at concentrations of 0 (control), 10, 50, or 175 ppm on
GDs 6-18 for 6 hours per day. The overall atmospheric exposure concentrations were measured
as 10.1 ± 0.03, 51 ± 4, and 175 ± 3 ppm. The concentrations converted to mg/m3 and adjusted
-3
for continuous exposure (6 hours per 24 hours) are 0, 9, 46, and 161 mg/m . Animals were
exposed and evaluated in the same manner as the reproductive and developmental study by Doe
(1984a), with the exception that animals were exposed from GDs 6-18. Body weights were
recorded on GDs 0, 5-19, 24, and 28, and rabbits were sacrificed on GD 29 and examined
postmortem.
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Doe (1984b) reported no treatment-related effects on maternal body weight or food
consumption. No abnormalities related to treatment were observed during clinical or
postmortem examinations. In addition, there appeared to be no treatment-related effects on litter
data for rabbits (see Table B.30). Occurrences of skeletal defects were significantly elevated in
-3
the 161-mg/m group compared to those in the control group (see Table B.31). The elevated
incidences of skeletal defects were associated with retarded ossification of the skeleton,
increased incidence of extra ribs, and other vertebrae abnormalities. Two major visceral effects
occurred in the 161-mg/m3 group—one fetus had a heart defect and another fetus had an
umbilical hernia (see Table B.32).
The study authors concluded that there was marginal evidence of fetotoxicity at the
"3
161-mg/m level due to the occurrence of major defects in two fetuses. There was no clear
evidence of maternal or fetal toxicity. The study authors identified 46-mg/m3 2-EE as the clear
-3
no-effect level for fetotoxicity in rabbits. A fetal LOAELadj of 161 mg/m with a corresponding
NOAELadj of 46 mg/m3 is identified based on increased incidence of skeletal defects in
offspring of exposed does. No effects were seen in the does following exposure, precluding
identification of a maternal LOAELadj, although a NOAELadj of 161 mg/m3 is identified.
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Table 4. Summary of Inhalation Developmental Studies for 2-EE (CASRN 110-80-5)
Study Type, Number of Male/Female,
Strain Species, Route of
Administration, Study Duration,
Methods
Dosimetry
(mg/m 3)a,
Purity of 2-EE
Critical Effects
NOAELb
LOAELb
Reference
Rat
Developmental, 0/24, Wistar rat,
inhalation, GDs 6-15(6 h/d), dams
weighed during study period; dams
sacrificed on GD 21; blood collected for
hematological assessment; uterus
examined; fetuses examined for skeletal
defects and visceral defects
0, 9, 47, 230,
(>99% purity)
Maternal: Decrease of hemoglobin, hematocrit, and
mean cell volume in red blood cells in the 230 mg/m3
exposure group
Maternal NOAEL/LOAEL: hematopoietic effects
Fetal: minor external, visceral, and skeletal defects
>230 mg/m3
Fetal NOAEL/LOAEL: skeletal defects
Maternal: 47
Fetal: 47
Maternal: 230
Fetal: 230
Doe (1984a)
Developmental, 0/30, Wistar rat,
inhalation, separate pregestational
exposure for 3 wk followed by exposure
GDs 1-19 (7 h/d); food consumption and
body weight of dams measured during
exposure; dams sacrificed GD 21; uterine
contents, maternal viscera, and fetal heads
were examined
0 (air-air),
61 (low-air),
103 (air-low),
164 (low-low),
262 (high-air),
392 (air-high),
653 (high-high)
(>99% purity)0
Maternal: relative spleen weights increased at 61,
392, and 653 mg/m3; gestational body weight
decreased, mean relative lung and kidney weights
increased, uterine involution (15/16), and corpora
lutea regression of ovaries (9/16) >392 mg/m3
Maternal NOAEL/LOAEL: gestational body weights
and histopathology in the uterus and ovaries
Fetal: intrauterine growth retardation, body weight,
decreased length, and reduced skeletal ossification
(with minor skeletal abnormalities) >103 mg/m3,
resorptions increased, 100% embryolethality
>392 mg/m3
Fetal NOAEL/LOAEL: growth retardation and
reduced skeletal ossification
Maternal: 262
Fetal: 61
Maternal: 392
Fetal: 103
Andrew and
Hardin (1984a)
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Table 4. Summary of Inhalation Developmental Studies for 2-EE (CASRN 110-80-5)
Study Type, Number of Male/Female,
Strain Species, Route of
Administration, Study Duration,
Methods
Dosimetry
(mg/m3)a,
Purity of 2-EE
Critical Effects
NOAELb
LOAELb
Reference
Developmental, 0/14-15,
Sprague-Dawley rat, inhalation,
GDs 7-13 or 14-20 (7 h/d); behavioral
testing to assess central nervous system
function (rotorod test, open field test,
activity wheel, avoidance conditioning,
and operant conditioning)
0, 108
(98.5% purity)
Fetal: Rotorod performance, open field activity, and
avoidance conditioning decreased; overall decreased
neuromotor performance; elevations in brain
chemistry in both exposures at 108 mg/m3
Fetal LOAEL: developmental neurotoxicity
Fetal: None
Fetal: 108
Nelson et al.
(1981)
Developmental, 0/16, Sprague-Dawley
rat, inhalation, GDs 7-13 (7 h/d); pups
weighed on Days 7, 14, 21, 28, and 35
postpartum; behavioral testing to assess
central nervous system function (rotorod
test, open field test, activity wheel,
avoidance conditioning, and operant
conditioning)
0,215
(98-98.5%
purity)
Maternal: maternal weight gain decreased; prolonged
pregnancy duration
Maternal LOAEL: body weight and gestational
duration
Fetal: rotorod performance, open field activity, and
avoidance conditioning decreased; overall decreased
neuromotor performance
Fetal LOAEL: behavioral effects
Maternal: None
Fetal: None
Maternal: 215
Fetal: 215
Nelson et al.
(1982)
Rabbit
Developmental, 0/24, Dutch rabbit,
inhalation, GDs 6-18 (6 h/d); dams
weighed during study period and
sacrificed GD 29; blood collected for
hematological assessment; uterus
examined; fetuses examined for skeletal
defects and visceral defects
0, 9, 46, 161
(>99% purity)
Maternal: no effects
Fetal: minor visceral, and skeletal defects at
161 mg/m3
Fetal NOAEL/LOAEL: skeletal defects
Maternal: 161
Fetal: 46
Maternal: None
Fetal: 161
Doe (1984b)
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Table 4. Summary of Inhalation Developmental Studies for 2-EE (CASRN 110-80-5)
Study Type, Number of Male/Female,
Strain Species, Route of
Administration, Study Duration,
Methods
Dosimetry
(mg/m3)a,
Purity of 2-EE
Critical Effects
NOAELb
LOAELb
Reference
Developmental, 0/29, New Zealand
rabbit, inhalation, GDs 1-18 (7 h/d); food
consumption and dams weighed during
study period and sacrificed GD 30;
uterine contents, maternal viscera, and
fetal heads examined
0, 172, 663
(>99% purity)
Maternal: relative liver weights increased
>172 mg/m3; mortality (17%), body weight
decreased, food consumption and relative kidney
weights increased, uterus, ovary and corpora lutea
effects at 663 mg/m3
Maternal NOAEL: 663 mg/m3 frank effect
Fetal: embryolethality, resorptions, major
malformations (ventral wall defects and fusion of
aorta with pulmonary artery), minor anomalies (renal
changes), and skeletal defects at >172 mg/mg3
Fetal LOAEL: fetal malformations and
embryolethality
Maternal: 172
Fetal: None
Maternal: None
Fetal: 172
Andrew and
Hardin (1984b)
aConversion Factors: MW = 90.12. Assuming 25°C and 1 atmosphere, Exposure mg/m3 = Exposure ppm x MW ^ 24.45 = 3.69. For developmental effects, this
concentration is adjusted for duration to a continuous exposure concentration; therefore, NO AE L/LO A EL A,,, = NOAEL/LOAEL and Exposureadj = Exposure hours
per day exposed 24. The NOAELhec was calculated for a gas: extrarespiratory effect, assuming periodicity was attained. Because blood:gas (air) lambda values are
unknown for the experimental animal species (a) and humans (h), a default value of 1.0 was used for this ratio. NOAELHec = NOAELadj x [blood:gas (air) lambda(a)
lambda(h)].
bNot reported by the study author(s) but determined from data for this review.
°Three exposure levels for pregestational exposure (air [0 mg/m3], low [115.18 mg/m3], and high [498.36 mg/m3]) and three exposure levels for gestational exposure (air
[0 mg/m3], low [217.16 mg/m3], and high [824.56 mg/m3]) were time-weighted averaged by duration to form the seven exposure groups shown in the table where
average body weight is the body weight provided by the study authors.
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Table 5. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Mutagenicity
Salmonella typhimurium TA97a, TA98,
TA100, and TA102 in buffer or S9 metabolic
fraction; 2-EE concentrations up to 20 |ig per
plate; incubation time unreported,
histidine-independent mutant colonies
counted
No significant increase in mutant
colonies was found with and without S9.
Negative for
mutagenic activity
Hoflack et al. (1995)
Mutagenicity
Salmonella typhimurium TA1535, TA1537,
TA98, and TA100 in buffer or S9 metabolic
fraction; 2-EE concentrations up to 104 |ig
per plate; incubation time unreported;
histidine-independent mutant colonies
counted
No significant increase in mutant
colonies was found with and without S9.
Negative for
mutagenic activity
Ong et al. (1980) as cited in
U.S. EPA (1981)
Mutagenicity
Streptomycin-dependent Escherichia coli
Sd-4-73; 2-EE concentrations of
0.01-0.024 mL per plate; incubation time
unreported; revertant mutant colonies
counted
No significant increase in mutant
colonies was found.
Negative for
mutagenic activity
Szybalski (1958) as cited in
U.S. EPA (1981)
Mutagenicity
Salmonella typhimurium (TA98, TA100,
TA1535, TA1537, and TA97) in buffer or S9
metabolic fraction; 2-EE concentrations up to
10,000 |ig per plate; incubated 2 d;
histidine-independent mutant colonies
counted
No significant increase in mutant
colonies was found with and without S9.
Negative for
mutagenic activity
NTP (1993d)
Mutagenicity
Mouse lymphoma L5178Y cells in media
alone or media with S9 metabolic fraction;
2-EE concentrations up to 5 |iL/mL:
incubated 4 h; trifluorothymidine-resistant
cells counted
No significant increase in mutant
colonies was found without S9;
however, slight increases were found
with S9.
Negative without
S9; weakly positive
with S9
NTP (1993e)
Mutagenicity
Adult male flies (Drosophila melanogaster)\
2-EE by diet for 2 days or single injection;
live numbers of offspring counted after
mating for two generations
No effects were noted in the germ cells
of exposed adult male flies in any
exposure group.
Negative for
induction of
sex-linked recessive
mutations
NTP (1993g)
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Table 5. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Cytogenicity
Chinese hamster ovary cells in media alone
or media with S9 metabolic fraction; 2-EE
concentrations up to 9510 |iL/mL: incubated
10.5 or 25.5 h; sister chromatid exchanges
per cell and chromosomal aberrations
counted
Sister chromatid exchanges were found
at 3170 and 9510 ng/mL with and
without S9. Chromosomal aberrations
were found in cells treated without S9.
No cell cycle delay was noted.
Positive for sister
chromatid
exchanges at high
doses; positive for
chromosomal
aberrations without
S9
NTP (1993f)
Cytogenicity
Chinese hamster ovary cells in media alone
or media with S9 metabolic fraction; 2-EE
concentrations up to 30 mg/mL; incubated
10.5 or 25.5 h; sister chromatid exchanges
per cell and chromosomal aberrations
counted
Cytotoxicity was found at 30 mg/mL
(30% survival). Metabolic cooperation
was impaired at 10 mg/mL. A 50%
recovery in cellular communication was
noted after dosing.
Blocked cellular
communication
before becoming
cytotoxic
Loch-Caruso et al. (1984)
Genotoxicity and
Cytogenicity
Chinese hamster lung V79 cells evaluated for
chromosomal aberrations, micronuclei in
polychromatic erythrocytes, alteration of
mitotic division apparatus and aneuploidy,
and inhibition of intracellular communication
between cells; other in vitro tests:
chromosomal aberrations in human
lymphocytes; micronuclei in polychromatic
erythrocytes in mouse bone marrow; and
morphological transformation of Syrian
hamster embryo cells.
Results were negative for chromosomal
aberrations; positive for enhancing
chromosomal aberrations in combination
with a known clastogen, induction of
morphological transformation of Syrian
hamster embryo cells, aneuploidy and
spindle malformations, equivocal for
inducing sister chromatid exchanges,
micronuclei induction in vitro and
negative in vivo.
Positive for inhibiting intercellular
communication.
Weakly positive for
genotoxicity and
epigenetic effects
Elias et al. (1996)
In vitro reproductive
Postimplantation rat embryos (GD 10.5) in
serum; 2-EE concentrations of
1.0-15.0 (iL/mL; incubated 40 h; toxicity
observed
Exposed embryos showed retarded dose-
dependent growth and development
beginning at 7.3 |iL/mL.
Positive for
embryotoxicity;
NOAEL of
14.2 pL/mL
Brown-Woodman et al.
(1994, 1995)
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Table 5. Other Studies
Test
Materials and Methods
Results
Conclusions
References
In vitro reproductive
Postimplantation rat embryos (GD 9.5) in
serum; 2-EE concentrations of
6.25-100 mM; incubated 2 d; toxicity
observed
Exposed embryos showed significant
reduction in protein explanted/embryo
ratio (12.5 mM), a high frequency of
unrotated embryos (50 mM) and
completely inhibited embryo
development (100 mM).
Positive for
embryotoxicity;
NOAEL of
6.25 mM
Giavani et al. (1993)
In vitro reproductive
Primary pachytene spermatocytes in buffer;
concentrations of 10-mM 2-EE or 1 and
10-mM EAA; incubation unreported; 02
consumption and ATP production monitored
2-EE exposed embryos showed no
effects. EAA interfered with the
energetic metabolism of pachytene
spermatocytes.
2-EE: negative
EAA: altered
spermatocyte
metabolism
Oudiz and Zenick (1986b)
In vivo short-term
reproductive
10 male Wistar rat, gavage; 0-, 100-, 200-,
and 400-mg/kg-d 2-EE for 14 consecutive
days
Sperm and testicular effects
>100 mg/kg-d; hematological effects
>200 mg/kg-d.
Positive for
gonadotoxicity and
hematotoxicity
Adedara and Farombi (2010)
Metabolism/
toxicokinetic
6 male Sprague-Dawley rat, gavage;
230 mg/kg of ethanol or ethoxy 14C-labeled
2-EE; urinary excretion of metabolites, and
composition of testes measured
Elimination of 14C was primarily
through urinary excretion within 96 h of
dosing (76-80%). Main pathway of
metabolism was oxidation, with
following conjugation with glycine. The
major metabolite EAA and Y-cthoxy-
acetyl glycine composed 73-76% of the
2-EE dose. Half-time was 9.9 h and
12.5 h, depending on the label. EAA
found in testes.
Major metabolites:
EAA and .Y-ethoxy-
acetyl glycine
EAA found in testes
Cheever et al. (1984)
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Table 5. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Metabolism/
toxicokinetic
5 male humans; inhalation; 200 min; male
Wistar rat (unreported number) gavage; 2-EE
concentrations of 10 mg/m3, 20 mg/m3, or
40 mg/m3; urinary excretion of EAA and
inhaled and exhaled 2-EE measured
The half-life of EAA elimination was
42.0 ± 4.7 h in humans compared to
7.20 ± 1.54 h in rats. Total recovery
was dose-dependent in rats and
amounted to between 13.4% and 36.8%,
while in humans, the recovery was
estimated between 30 and 35%
regardless of concentration. Based on
higher recovery in humans than rats at
low doses, the metabolic conversion to
EAA appears more important in humans
than in the rat.
Blood
concentrations
could be three times
higher in man than
rat.
Groeseneken et al. (1988)
Metabolism/
toxicokinetic
Human blood; head space vial of
physiological saline or olive oil; 2 |il of
2-EE; gas chromatographic analysis
Study authors reported the partition
coefficients of 2-EE to be:
water/air—23069; blood/air—22093;
water/blood—1.044; oil/air—962;
oil/water—0.042; and oil/blood—0.044.
Evidence of high
levels of respiratory
uptake, uniform
tissue distribution
Johanson and Dynesius
(1988)
Metabolism/
toxicokinetic
physiologically based pharmacokinetic
model for pregnant rats effects to a pregnant
woman; vapor exposures of 50 and 100 ppm
(6 h per d); 5 d (GDs 11-15) and controlled
exposure in a pregnant woman published by
Groeseneken et al. (1988); exposure of 2-EE
(8 h per d); 5 d per week; 270 d; calculated
for a 58-kg pregnant woman to match
internal venous blood concentration
The human inhaled concentration
equivalent to rat NOAEL for
EGEEA/2-EE (50 ppm) was predicted to
be 25 ppm by using the maternal blood
average daily area under the curve
(AUC) and 40 ppm by using the
maximum concentration achieved in
maternal blood (Cmax). Similarly, the rat
LOEL for EGEEA (100 ppm) was
determined to be 55 ppm using the
maternal blood average daily AUC and
80 ppm using the maternal blood Cmax.
AUC metric:
NOAEL of 8.3 ppm
(designated for 8 h
only) and 18.3 ppm
(adjusted for
continuous
exposure)
Gargas et al. (2000)
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Table 5. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Metabolism/
toxicokinetic
8- to 10-wk-old adult male Sprague-Dawley
rat; unreported administration method;
113-mM 2-EE or up to 9.85 mM EAA;
metabolites of mitochondria from livers and
testes identified
2-EE did not affect mitochondrial
respiration. EAA caused decreased
state-3 succinate oxidation and a
respiratory control ratio above 3.85 mM.
2-EE: negative
EAA: positive for
mitochondrial
toxicity
Beattie and Brabec (1986)
Metabolism/
toxicokinetic
Primary mixed Sertoli and germ testis cells
in media; concentrations 50 mM 2-EE, 2 mM
MAA or 10 mM EAA; incubated 72 h;
toxicity observed and metabolites identified
2-EE produced no sign of cellular
toxicity. MAA and EAA caused
degeneration of the pachytene and
dividing spermatocytes.
2-EE: negative
MAA and EAA:
positive for
testicular toxicity
Gray et al. (1985)
Mode of action/
mechanistic
36 male/0 female rats (strain unreported);
gavage; 2-EE at 250, 500 or 1000 mg/kg-d or
2-ME at 50, 100, 250 or 500 mg/kg-d; 11 d;
testicular histology examined
2-EE and 2-ME primarily damaged
spermatocytes undergoing maturation
and division. 2-ME was estimated to be
5-fold more potent than 2-EE.
2-EE and 2-ME:
positive for
spermatocyte
toxicity
Foster etal. (1984)
Immunotoxicity
0 male/6-8 female Hybrid B6C3Fi (C57B1/6
female x C3H) and CD2F1 (BALB/c female
x DBA/2 inbred) mice; gavage; 2-EE at 600,
1200, or 2400-mg/kg; Days -12, -8, -5, -1;
mouse lymphoid leukemia L1210 tumors
(3 x 106, 1 x io5, 3 x 103, or 1 x 102)
implanted Day 0
Allogenic mice treated with 2-EE and
tumor cells showed increased
survivorship as compared to control.
Positive for
antileukemic
activity
Houchens et al. (1984)
Immunotoxicity
20 male/0 female Fisher rats; drinking water;
2-EE at 2500 or 5000-ppm (231-255 or
438-459 mg/kg-d); 9 wk; F344 rat leukemia
cells (2.5 x io7) implanted Day 0
The 5000-ppm dose reduced effects of
leukemia by approximately 50%, with
100% survival.
Positive for
antileukemic
activity
NTP (1993h)
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OTHER DATA (SHORT-TERM TESTS, MECHANISTIC STUDIES, OTHER
EXAMINATIONS)
Data evaluating the genotoxicity, cytogenicity, and embryotoxicity are included in
Table 5 (Hoflack et al., 1995; Ong et al., 1980, as cited in U.S. EPA, 1981; Szybalski, 1958, as
cited in U.S. EPA, 1981; NTP, 1993d,e,f,g; Loch-Caruso et al., 1984; Elias et al., 1996;
Brown-Woodman et al., 1994, 1995; Giavani et al., 1993; Oudiz and Zenick, 1986b). Table 5
also summarizes studies investigating the kinetics and metabolism of 2-EE in human, animal,
and in vitro systems (Cheever et al., 1984; Gargas et al., 2000; Groeseneken et al., 1988;
Johanson and Dynesius, 1988; Beattie and Brabec, 1986; Gray et al., 1985) and the immunotoxic
effects of 2-EE (Houchens et al., 1984; NTP, 1993h). A general discussion of the data is
presented in this section; the key studies pertaining to the physiologically based pharmacokinetic
(PBPK) model (Gargas et al., 2000) are presented in greater detail in the text.
Tests Evaluating Mutagenicity, Cytogenicity, and Embryotoxicity
Mutagenicity, cytogenicity, and embryotoxicity tests have been conducted for 2-EE in
several strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102, TA1535, and
TA1537), Escherichia coli, mouse lymphoma L5178Y cells, Drosophila melanogaster, and in
vitro reproductive studies (Hoflack et al., 1995; Ong et al., 1980, as cited in U.S. EPA, 1981;
Szybalski, 1958, as cited in U.S. EPA, 1981; NTP, 1993d,e,f,g; Loch-Caruso et al., 1984;
Elias et al., 1996; Brown-Woodman et al., 1994, 1995; Giavani et al., 1993; Oudiz and Zenick,
1986b). The results from tests evaluating genotoxicity and/or mutagenicity have been largely
negative in prokaryotic systems exposed to 2-EE (Hoflack et al., 1995; Ong et al., 1980, as cited
in U.S. EPA, 1981; Szybalski, 1958, as cited in U.S. EPA, 1981; NTP, 1993d). However, there
have been some positive genotoxic and cytotoxic results in some eukaryotic mammalian cell
cultures (NTP, 1993e,f; Loch-Caruso et al., 1984; Elias et al., 1996). In vitro reproductive
studies showed embryotoxicity and developmental impairment following 2-EE exposure
(Brown-Woodman et al., 1994; Giavani et al., 1993); effects on spermatocytes were not observed
following 2-EE exposure (Oudiz and Zenick, 1986b).
Genotoxicity and cytotoxicity tests have been conducted for 2-EE in Chinese hamster
ovary (CHO) cells and Salmonella typhimurium with and without S9 activation, Escherichia coli,
Chinese hamster lung V79 cells, human lymphocytes, human and mouse erythrocytes, mouse
bone marrow, Syrian hamster embryo cells, rat embryos, and primary pachytene rat
spermatocytes (Hoflack et al., 1995; Ong et al., 1980, as cited in U.S. EPA, 1981; Szybalski,
1958, as cited in U.S. EPA, 1981; NTP, 1993f; Loch-Caruso et al., 1984; Elias et al., 1996;
Brown-Woodman et al., 1994, 1995; Giavani et al., 1993; Oudiz and Zenick, 1986b). Sister
chromatid exchanges occurred in CHO cells at 2-EE concentrations of 3170 and 9510 |ig/mL
both with and without S9 activation (NTP, 1993f). Chromosomal aberrations also occurred in
CHO cells, but this effect was only significant without S9 activation (NTP, 1993f). Although
positive results were seen in Chinese hamster ovary cells (NTP, 1993f; Loch-Caruso et al.,
1984), only weakly positive results were obtained from studies of Chinese hamster lung
V79 cells (Elias et al., 1996).
Cytotoxic effects ranging from inhibition of cellular communication (measured in vitro
by an assay that depends on the transfer of metabolites via gap junctions, i.e., metabolic
cooperation) at concentrations of 10 mg/mL to approximately 70% cytotoxicity at 30 mg/mL
were also observed in CHO cells (Loch-Caruso et al., 1984). Cells were able to recover from
effects on cellular communication in vitro at 2-EE concentrations of up to 20 mg/mL
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(Loch-Caruso et al., 1984). Specifically, Loch-Caruso et al. (1984) outlined the potentially
harmful effects of decreased gap junction communication. Briefly, gap junction communication,
although not yet clearly defined, is thought to have an important function in morphogenesis. The
study authors hypothesized that these effects may be related to significantly lengthened gestation
observed in rats and mice exposed to 2-EE. Increased length of parturition or labor and other
effects to organs such as the heart and the intestines and the male reproductive system that are
dependent on gap junction communication may also arise through this putative mechanism of
action.
Cultured rat embryos have also been examined for embryotoxicity in vitro.
Concentrations, as low as 12.5 mM, caused effects on embryo protein content (Giavani et al.,
1993). Effects on growth and development occurred in a dose-dependent manner
(Brown-Woodman et al., 1994, 1995; Giavani et al., 1993). There were no cytotoxic effects of
2-EE exposure on primary pachytene rat spermatocytes in vitro, but there were significant effects
from exposure to EAA, a metabolite of 2-EE, which the study authors noted could contribute to
some of the toxic effects on spermatocytes observed in vivo (Oudiz and Zenick, 1986a,b).
Other Toxicity Studies
The metabolism of administered 2-EE (Cheever et al., 1984), a PBPK model
(Gargas et al., 2000), and the effects of oral 2-EE exposure in leukemia inhibition studies in mice
and rats have been evaluated (Houchens et al., 1984; NTP, 1993h).
In a peer-reviewed short-term-duration reproductive study, Adedara and Farombi (2010)
administered doses of 0- (saline only), 100-, 200-, and 400-mg/kg-day 2-EE (purity unreported)
by gavage to four groups of 10 adult male Wistar rats for 14 consecutive days. Rats were
obtained from the University of Ibadan, Ibadan, Nigeria. Throughout the study, rats were housed
in plastic suspended cages and given pellet food (brand unspecified) and water ad libitum. The
GLP compliance of this study was not reported.
Twenty-four hours after the last treatment, rats were sacrificed, body weights were
recorded, and blood was collected for hematological analysis. Testes, epididymides, seminal
vesicles, and prostate glands were removed and weighed. Testes and epididymis samples were
fixed, sectioned, and stained with hematoxylin and eosin for microscopy. For biochemical
assays, glutathione (GSH), vitamin C, malondialdehyde (MDA), and lactate dehydrogenase
(LDH) levels as well as superoxide dismutase (SOD), catalase (CAT), and glutathione-
S-transferase activities were measured in the testes and epididymal spermatozoa. Epididymal
spermatozoa number and motility were assessed, and daily spermatozoa production and testicular
spermatozoa number were quantified. Spermatozoa were also assayed for morphological
abnormalities and percentage viability. Statistical analyses were performed using one-way
ANOVA followed by Student's t-test.
Adedara and Farombi (2010) reported no significant changes in the weights of the testes,
epididymides, seminal vesicles, and prostate glands of rats exposed to 2-EE. Body-weight gain
was significantly (p < 0.05) decreased in the 200- and 400-mg/kg-day groups compared to
controls. At doses >100 mg/kg-day, significant (p < 0.05) decreases were observed in
epididymal and testicular spermatozoa number, daily spermatozoa production, and spermatozoa
motility, and total spermatozoa abnormalities were significantly (p < 0.05) increased. No effect
was observed on the spermatozoa live-dead ratio at all doses tested. There was a significant
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(p < 0.05) decrease in testicular GSH levels and SOD and CAT activities, as well as a significant
(p < 0.05) increase in MDA levels and GST and LDH activities in the 200- and 400-mg/kg-day
groups compared to controls. Vitamin C content in the testes was unchanged. In the
spermatozoa, there was a significant (p < 0.05) decrease in SOD and LDH activities as well as
GSH and vitamin C levels at doses >100 mg/kg-day, and CAT and GST activities in the 200- and
400-mg/kg-day groups. MDA was significantly (p < 0.05) increased in spermatozoa at doses
>100 mg/kg-day. Histopathological analysis of the testes revealed treatment-related lesions
(e.g., congestion and hemorrhage at the interstitium of the seminiferous tubules, erosion of the
germinal epithelium, and necrosis of germinal cells with reduced number of Sertoli cells) at all
doses tested, but the epididymides were only mildly affected at 400 mg/kg-day. Hematological
analysis showed that white blood cells, platelets, neutrophils, and mean corpuscular hemoglobin
concentration were significantly (p < 0.05) lower, whereas lymphocytes were increased in the
200- and 400-mg/kg-day groups compared to controls.
Adedara and Farombi (2010) reported a LOAEL of 100 mg/kg-day based on sperm and
testicular effects. A NOAEL cannot be identified.
Metabolism/Toxicokinetic Studies
Studies have investigated the kinetics and metabolism of 2-EE in human, animal, and in
vitro systems (Cheever et al., 1984; Gargas et al., 2000; Groeseneken et al., 1988; Johanson and
Dynesius, 1988; Beattie and Brabec, 1986; Gray et al., 1985).
The metabolism of 2-EE is understood to involve initial oxidation by alcohol
dehydrogenase followed by aldehyde dehydrogenase-forming acid metabolites, principally
ethoxy acetic acid or EAA, which are measurable in the urine (Groeseneken et al., 1988;
Cheever et al., 1984). Studies by Beattie and Brabec (1986) and Gray et al. (1985) have
implicated major metabolites of 2-EE in mitochondrial and cellular toxicity, respectively; the
particular toxic mechanism of action remains unclear. Johanson and Dynesius (1988), to better
understand the absorption and distribution potential of 2-EE, experimentally calculated several
partition coefficients, which led the study authors to the conclusion that 2-EE could be highly
absorbed through inhalation and that it would distribute in a relatively uniform manner in the
tissues once absorbed. Importantly, Groeseneken et al. (1988) compared the metabolism and
kinetics of exposed humans with those of rats. This study provided important quantitative data,
utilized by Gargas et al. (2000) to develop a PBPK model of inhaled 2-EE for purposes of
cross-species extrapolation of fetal effects from pregnant rats to the pregnant woman. Further
description of the Gargas et al. (2000) study is presented below.
Gargas et al. (2000) developed a PBPK model for inhaled 2-EE for purposes of
cross-species extrapolation of fetal effects from pregnant rats to the pregnant woman. The basis
underlying this extrapolation is to estimate external human exposures equivalent with the rat
based on internal measures of dose. PBPK models utilize the anatomical and physiological
structure and functions of the organism (animal or human) by representing blood flows,
pulmonary ventilation rate, and organ volumes as compartments. The model also utilizes
metabolic values to estimate the kinetics of a toxic species, including concentrations of a given
chemical species at specific organs or sites. General and chemical-specific toxicokinetic values
used in the construction of the models were generated from measurements made by other
researchers as documented in Gargas et al. (2000). These parameters included estimates of in
vivo metabolic transformation rates of 2-EE to EAA and EAA to ethylene glycol for both
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humans and rats from comparative in vitro studies with species-specific hepatocytes
(Green et al., 1996). Fetal and placental tissues were grouped with the richly perfused
compartment, as well as rat fetal and maternal blood concentrations for 2-EE and 2-EAA. They
were assumed to be valid correlates of one another, based on observations with related glycol
ethers (2-ME and its principal metabolite, 2-MAA; Welsch et al., 1995), showing that rat
maternal blood and rat fetal tissue concentrations are nearly identical or proportional.
The rat model constructed by Gargas et al. (2000) was validated by comparing model
outputs with measurements made by Gargas et al. (2000) in whole-body vapor inhalation
exposures of pregnant Sprague-Dawley rats at 50 and 100 ppm, 6 hours per day, to the acetate of
2-EE1 for 5 consecutive days (GDs 11-15). These comparisons included EAA concentrations in
maternal venous blood and fetal tissue during and following GD 15 for pregnant rats exposed to
100- or 5-ppm 2-EE/EGEEA on GDs 11-15. The human model thus constructed was validated
with the human experiments of Groeseneken et al. (1988) by predicting values for (1) the rate of
urinary excretion of EAA from human volunteers exposed to 20-mg/m3 2-EE, (2) as in (1) with
"3
28-mg/m 2-EE, (3) exhaled breath concentrations of 2-EE from these human volunteers exposed
to 20-mg/m3 2-EE, and (4) as in (3) with 28-mg/m3 2-EE. In all of these experiments with both
rats and humans volunteers, the modeled outputs were shown to be very near and comparable to
the observed values.
On the precept that a common internal dose would produce similar effects in similar
tissues, the validated rat model was used to estimate the internal venous blood concentration in
the pregnant rats at a steady state under the exposure conditions of 50- and 100-ppm 2-EE for
6 hours per day. The validated human model was then used to find the external exposure
concentrations of 2-EE of 8 hours in duration, 5 days per week, for 270 days to a 58-kg pregnant
woman that would match this internal venous blood concentration. These concentrations, which
may be termed human equivalent concentrations, were obtained for two different measures of
tissue dose, i.e., maternal venous blood: the area under the curve (AUC) and the maximum
concentration achieved in maternal blood (Cmax)- The human inhaled concentration equivalent to
the rat exposure of 50-ppm 2-EE from the study of Doe (1984) was estimated to be somewhat
lower at 25 ppm using the maternal blood average daily AUC and 40 ppm using the Cmax- The
human inhaled concentration equivalent to the rat exposure of 100-ppm 2-EE was likewise
estimated at 55 ppm using the maternal blood average daily AUC and 80 ppm using the maternal
blood Cmax-
It is these concentrations, preferably the lower AUC values, which could be utilized in
deriving the HEC values for the rat study of Doe (1984a). There are, however, a number of
limitations present, both with the data used and with the model itself. The PBPK model
simulation was applied to the human data of Groesneken et al. (1988). These data are considered
marginal and could be better judged if more human data (which are available from
Groesneken et al., 1987a,b) were used in the development of the model. Also, these human data
were obtained from male workers with the weight adjustment to the pregnant female made only
on body-weight allometry. As mentioned above, a number of sensitive
physiochemical/physiological parameters in the human model were calculated based on related
'The acetate is rapidly and nearly completely hydrolyzed to the subject ether, 2-EE, and thence to the putative active
metabolite 2-EAA in both rats (Gargas et al., 2000; Stott and McKenna, 1985) and in humans (Groeseneken et al.,
1987a,b).
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compounds, e.g., the acetate of 2-EE and 2-ME, or based on in vitro measurements. Also, the
data sets used in developing the model are rather small (the human sample size was five).
Lastly, the model simulation was for a work shift (8 hours) not for a continuous exposure as is
required for derivation of p-RfC values, thus necessitating further manipulation of the modeled
estimates. In consideration of the multiple aforementioned limitations, these estimated values
cannot be used in developing the HEC values from the rat study of Doe (1984a).
Foster et al. (1984) undertook an investigation into the mechanism of toxicity of glycol
ethers, including both 2-ME and 2-EE, to the testicular tissues of rats. Groups of male rats
(n = 36) were treated orally for 11 days with either 2-ME at 50, 100, 250, or 500 mg/kg-day or
with 2-EE at 250, 500, or 1000 mg/kg-day. Groups of controls were given an equivalent volume
of water. At sequential times in the treatment regime (6 and 24 hours after a single dose and 1,
2, 4, 7, and 11 days after repeated daily doses), groups of animals were sacrificed, and testicular
histology was examined. The major site of damage following treatment with either of these
glycol ethers was shown to be the primary spermatocytes undergoing maturation and division.
Further, 2-ME was estimated to be about 5-fold more potent than 2-EE. Equimolar doses of
either MAA (4 days at 500 mg/kg) or EAA (11 days at 500 mg/kg)—the primary metabolites of
these glycol ethers—also induced injury similar to the corresponding parent. Administration of
MAA but not 2-ME to in vitro germ cell cultures resulted in adverse effects analogous to those
seen in vivo. These investigators also showed that inhibitors of aldehyde dehydrogenase
administered before a toxic dose of 2-ME (500 mg/kg) afforded complete protection against
testicular toxicity. These results indicate that it is likely the acetic acid metabolite (or possibly
the corresponding aldehyde) and not the parent glycol ether that is responsible for the testicular
damage observed. Aldehydes are also known to have profound effects on cells undergoing
division. However, the precise biochemical lesion affecting the spermatocytes undergoing
division was not elucidated.
Immunotoxicity Studies
Houchens et al. (1984) published the results of a cell-mediated immunity assay in a
peer-reviewed study in which syngeneic mice and mice allogenic for the leukemia cell tumor
were pretreated with 2-EE and then injected with leukemia cells. All syngeneic mice died from
the effects of the leukemia treatment 8-9 days following the injection. Similarly, allogenic mice
pretreated with water instead of 2-EE died an average of 8 days following leukemia cell
treatment. However, the allogenic mice pretreated with 2-EE survived for more than 43 days, on
average, with survival between 80-100% per treatment group. The study authors concluded that
the increased survivorship of the 2-EE pretreated mice may indicate a preventative effect of 2-EE
or a stimulation of the immune system.
A published peer-reviewed study by NTP (1993h) investigated the activity of 2-EE in a
cellular leukemia transplant model in rats to examine the effect of treatment on cancer
progression. 2-EE given orally (2.5 mg/mL in drinking water) to transplant recipients showed a
roughly 50% reduction in clinical, morphological, and histopathological signs of leukemia.
While the mechanism of action was not explored in this study, the effects of 2-EE occurred at
levels below which toxicity was observed.
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DERIVATION OF PROVISIONAL VALUES
Table 6 presents a summary of noncancer reference values. Table 7 presents a summary of cancer values.
Table 6. Summary of Noncancer Reference Values for 2-EE (CASRN 110-80-5)
Toxicity Type
(units)
Species/Sex
Critical Effect
Reference
Value
POD Method
POD
UFC
Principal Study
Subchronic p-RfD
(mg/kg-d)
Rat/M
Prostate atrophy
1 x 10"1
BMDLioo/0
33.7
300
NTP (1993a)
Chronic p-RfD
(mg/kg-d)
Rat/M
Pathological effects in the testes
9 x 10~2
NOAEL
86
1000
Yoonetal. (2003)
Subchronic p-RfC
(mg/m3)
Rabbit pups
(both sexes)
Increased percentage of rabbit
offspring showing major skeletal
defects
4 x 10~2
bmcl5o/oHEC
4.23
100
Doe (1984b)
RfC
(IRIS)3 (mg/m3)
Rabbit/M
Decreased testis weight, degeneration
of the seminiferous tubules, and
decreased hemoglobin
2 x 10"1
NOAEL
68
300
Barbee et al. (1984b)
"All the reference values obtained from IRIS are indicated with the latest revision date (05/01/1991).
Table 7. Summary of Cancer Reference Values for 2-EE (CASRN 110-80-5)
Toxicity Type
Species/Sex
Tumor Type
Cancer Value
Principal Study
p-OSF
None
p-IUR
None
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DERIVATION OF ORAL REFERENCE DOSES
Derivation of Subchronic Provisional RfD (Subchronic p-RfD)
The study by NTP (1993a) is selected as the principal study for deriving the
subchronic p-RfD. This 90-day study is peer reviewed and performed according to GLP
principles, and the results were reviewed by the NTP Pathology Working Group (PWG). The
study meets the standard of study design and performance, with numbers of animals (10 per sex
per dose), examination of potential toxicity endpoints, and presentation of information. Details
are provided in the "Review of Potentially Relevant Data" section. The observed atrophy of the
prostate in the NTP (1993a) study represents the most appropriate and most sensitive effect for
developing a subchronic p-RfD among the acceptable studies. The study authors do not discuss
the methods for evaluating the severity of prostate atrophy, nor did they use this endpoint in the
derivation of the NOAEL. However, this endpoint is considered toxicologically relevant and is
the most sensitive endpoint of the available acceptable subchronic-duration studies for 2-EE.
Oral administration of 2-EE is shown within the literature database to elicit varied effects.
There was a significant decrease in thymus weight observed in a subchronic-duration study in
male rats by NTP (1993a), and hematopoietic effects were also observed in male rats (Yu et al.,
1999). However, reproductive effects, particularly in the testes, were noted in a number of
studies. A significant decrease in testis weight coupled with testicular degeneration was
observed in a stop-exposure study in male rats by NTP (1993b). Testicular pathology was
observed in a reproductive study in adult male rats by Yoon et al. (2001), and testicular effects
were observed in a reproductive study in male rats by Yu et al. (1999). Prostate atrophy is
similar in that it is also a toxicologically relevant reproductive effect, and compared to testicular
effects, it is a more sensitive endpoint. The dose at which prostate atrophy was identified is
within 2-fold of the doses at which testicular effects occurred. This adds further confidence in
the selection of prostate atrophy as the critical effect.
A duration adjustment is not required because the study authors adjusted for continuous
exposure. A POD of 67 mg/kg-day is identified using BMD analysis. This POD is protective
against other effects from subchronic-duration exposure, including those seen in the testes and
epididymides of rats and mice at slightly higher dose levels (Yoon et al., 2001; Yu et al., 1999;
Horimoto et al., 1996, 2000; Zenick et al., 1984; NTP, 1993a,b; Nagano, 1984). It is also noted
that prostate effects were not reported in the available chronic-duration rat study performed by
Melnick (1984a).
All EPA Benchmark Dose Software (BMDS version 2.1.2) dichotomous models were fit
to the data for incidence of prostate atrophy. The high-dose data (2240 mg/kg-day) were
excluded due to the observed 50% (5/10) chemical-related mortality in that dose group. A
benchmark response (BMR) of 10% extra risk was used to estimate the BMD, as recommended
by EPA (2000). Table 8 presents BMD input data for the incidence of prostate atrophy in male
rats after 13 weeks of oral exposure via drinking water. Appendix C presents the BMD model
output.
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Table 8. Prostate Atrophy in the Male F344/N Rat Following a 13-Week Oral Exposure to
2-EE Used for BMD Analysis"
Doseb
(mg/kg-d)
Number of Rats
Incidence
0
10
0
109
10
0
205
10
6
400
10
7
792
10
10
aNTP (1993a).
bThe highest dose group data (2240 mg/kg-day) were excluded due to the observed frank effect of 50% mortality.
Table 9 summarizes the BMD modeling results for the male prostate atrophy. The
Log-Probit model fit the data best compared to other models. The Logistic and Probit models
are eliminated because they failed thep-walue criteria (i.e., />-score less than 0.1). The
Log-Probit model, as well as the Multistage, Gamma, Log-Logistic, and Weibull models, have a
goodness-of-fit p-w alue greater than 0.1. Among the models that pass the /;-value criteria, the
Multistage model provides the lowest AIC value of 33.368. Therefore, the BMDLi0% of
33.7 mg/kg-day from the Multistage model is used as a POD for the derivation of the subchronic
p-RfD (see Table 9).
Table 9. Model Predictions for Prostate Atrophy in the Male F344/N Rat Following a
13-Week Oral Exposure to 2-EEa
Model
Goodness of Fit
77-value
AICb for Fitted
Model
BMDio%
(mg/kg-d)
BMDLio%
(mg/kg-d)
Conclusions
Gamma
0.23
34.868
108.3
45.1

Logistic
0.09
37.578
113.1
72.7
/?-score 4 < 0.1
Log-Logistic
0.24
34.783
113.3
61.2

Log-Probit
0.27
34.303
118.3
66.9

Multistage
0.31
33.368
102.2
33.7
Lowest AIC
Probit
0.097
37.037
110.7
70.8
/?-score 4 < 0.1
Weibull
0.22
35.301
93.6
38.4

aNTP (1993a).
bValues <0.10 fail to meet conventional goodness-of-fit criteria.
AIC = Akaike's Information Criteria; BMD = benchmark dose; BMDL = lower confidence limit (95%) on the BMD
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The subchronic p-RfD is based on the BMDLio%of 33.7 mg/kg-day for prostate atrophy
calculated from male rats exposed to 2-EE for 13 weeks (NTP, 1993a) and is derived as follows:
Subchronic p-RfD = BMDLio% + UFc
= 33.7 mg/kg-day -^300
= 1 x 10-1 mg/kg-day
The composite uncertainty factor (UFC) for the subchronic p-RfD for 2-EE is 300 as
explained in Table 10.
Table 10. Uncertainty Factors for Subchronic p-RfD of 2-EE
UF
Value
Justification
ufa
10
A UFa of 10 is applied for interspecies extrapolation to account for potential toxicokinetic
and toxicodynamic differences between rats and humans. There are no data to determine
whether humans are more or less sensitive than rats to the reproductive effects of 2-EE.
ufd
3
A UFd of 3 is selected because the database includes four acceptable developmental studies in
mice (Wier et al., 1987a,b; Hardin et al., 1987; Lamb et al., 1984), but there are no acceptable
two-generation reproductive studies. Although thymus effects are observed in rats in a
subchronic-duration study, these effects are not observed following chronic-duration
exposure, and there is no other indication of immunotoxicity. Thus, there is likely no
potential for immunotoxicity, and the absence of a comprehensive immunotoxicity study is
not a concern.
UFh
10
A UFh of 10 is applied for intraspecies differences to account for potentially susceptible
individuals in the absence of information on the variability of response to humans.
ufl
1
A UFl of 1 is applied because the POD was developed using a NOAEL.
UFS
1
A UFS of 1 is applied because a subchronic-duration study was utilized.
UFC
<3000
300

The confidence of the subchronic p-RfD for 2-EE is medium as explained in Table 11.
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Table 11. Confidence Descriptors for Subchronic p-RfD for 2-EE
Confidence Categories
Designation"
Discussion
Confidence in study
H
Confidence in the key study is high. NTP (1993a) examined
appropriate reproductive toxicity endpoints and used 10 males per
dose group. The study was peer reviewed and is GLP compliant.
The study examined multiple effects, and a thorough description of
general methods and data was provided. However, the
methodology for characterization of the critical effect
used—prostate atrophy—is not entirely clear.
Confidence in database
M
The database includes subchronic-duration toxicity studies in two
species (rat and mouse), chronic-duration toxicity studies in two
species (rat and mouse), developmental toxicity studies in one
species (mouse), and no two-generation reproductive studies.
Confidence in subchronic
p-RfDb
M
The overall confidence in the subchronic p-RfD is medium.
"L = low, M = medium, H = high.
bThe overall confidence cannot be greater than the lowest entry in the table.
Derivation of Chronic Provisional RfD (Chronic p-RfD)
The study by Yoon et al. (2003) is selected as the principal study for deriving the
chronic p-RfD. This 28-day study is a published peer-reviewed study and meets the standard of
study design and performance, with numbers of animals (five per dose), examination of potential
toxicity endpoints, and presentation of information. Details are provided in the "Review of
Potentially Relevant Data" section. The critical effect is the pathology findings in the testes,
which consisted of exfoliation of the germ cells in the testicular lumen in male Sprague-Dawley
rats administered 2-EE by gavage for 4 weeks. While the subchronic duration of this study is
only 28 days, the study is supported by other observations that the testes appear to be a target of
2-EE. This study also notes that testicular toxicity due to this compound was dose dependent
with indices of testicular pathology (i.e., decreased testis weight, exfoliation of germ cells in the
tubular lumen, etc.). These study results are supported by an earlier study by Yoon et al. (2001)
that also reported similar testicular pathology in treated adult male rats. Available data from
other oral studies support the testes (and related prostate tissues) as a target organ (with
particular effects noted in sperm) for toxicity in rats and mice (Yu et al., 1999; Horimoto et al.,
1996, 2000; Zenick et al., 1984; NTP, 1993a,b; Nagano, 1984) as well as humans (Wang et al.,
2003; Ratcliffe et al., 1986; Veulemans et al., 1993, Welch et al., 1988).
Although prostate atrophy was more sensitive compared to testicular effects following
subchronic-duration exposure to 2-EE (NTP, 1993a), prostate effects were examined but not
observed following chronic-duration exposure at higher doses and in multiple animal species,
thus precluding its choice as a critical effect for deriving a chronic p-RfD. Therefore, the next
most sensitive endpoint that was also observed following chronic-duration exposure was
pathological effects in the testes observed in the 28-day Yoon et al. (2003) study. Although the
principal study for the chronic p-RfD was shorter in duration than that for the subchronic p-RfD
(13 weeks vs. 28 days), the duration of exposure is not a concern with respect to the testicular
effects observed in the study by Yoon et al. (2003) because testicular toxicity is the most
appropriate sensitive endpoint that occurs in the potentially most sensitive rat strain (i.e.,
Sprague-Dawley). This is evidenced by the higher LOAEL (400 mg/kg-day) for testicular
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effects observed after a 13-week exposure in F344/N rats (NTP, 1993a) versus a LOAEL of
171 mg/kg-day following a 28-day exposure in Sprague-Dawley rats (Yoon et al., 2003).
Additionally, induction of testicular damage by chronic-duration exposure to 2-EE is also
evident in studies where chronic-duration exposure (at comparatively higher doses than the
subchronic-duration studies) induced testicular atrophy in both F344/N rats and B6C3Fi mice
(Melnick, 1984a,b). Therefore, the critical effect is pathology of the testes observed in rats by
Yoon et al. (2003).
The testicular pathology data from Yoon et al. (2003) are not amenable to BMD
modeling. Thus, the NOAELadj of 86 mg/kg-day based on pathological effects in the testes of
male Sprague-Dawley rats exposed to 2-EE for 4 weeks (Yoon et al., 2003) is chosen as the POD
to derive the chronic p-RfD. The chronic p-RfD for 2-EE, based on the NOAELadj, is derived as
follows:
Chronic p-RfD = NOAELadj ^ UFc
= 86 mg/kg-day 1000
= 9 x 10~2 mg/kg-day
The UFc for the chronic p-RfD for 2-EE is 1000, as explained in Table 12.
Table 12. Uncertainty Factors for Chronic p-RfD of 2-EE
UF
Value
Justification
ufa
10
A UFa of 10 is applied for interspecies extrapolation to account for potential toxicokinetic
and toxicodynamic differences between rats and humans. There are no data to determine
whether humans are more or less sensitive than rats to the reproductive effects of 2-EE.
ufd
3
A UFd of 3 is applied because the database includes four acceptable developmental studies
in mice (Wier et al., 1987a,b; Hardin et al., 1987; Lamb et al., 1984), but there are no
acceptable two-generation reproduction studies.
UFh
10
A UFh of 10 is applied for intraspecies differences to account for potentially susceptible
individuals in the absence of information on the variability of response to humans.
ufl
1
A UFl of 1 is applied because the POD was developed using a NOAEL.
UFS
3
A partial UFS of 3 is applied for using data from a subchronic-duration study to assess
potential effects from chronic-duration exposure. Although there are data on testicular
effects from chronic-duration studies (Melnick, 1984a,b) in F344/N rats at comparatively
higher doses, these studies were not performed in the potentially most sensitive rat strain
(i.e., Sprague-Dawley), therefore a partial UFS is warranted.
UFC <3000
1000

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The confidence of the chronic p-RfD for 2-EE is medium as explained in Table 13.
Table 13. Confidence Descriptors for Chronic p-RfD for 2-EE
Confidence Categories
Designation"
Discussion
Confidence in study
M
Confidence in the key study is medium. Yoon et al. (2003)
examined appropriate reproductive toxicity endpoints, although
only five male rats per dose group were used. The study was peer
reviewed. GLP compliance is unknown. The study examined
multiple effects, and a thorough description of methods and data
was provided. The data used as the critical effect are well
supported within the database. The key endpoint of pathology in
the testes is seen in multiple independent studies and in two species
(rat and mouse).
Confidence in database
M
The database includes subchronic-duration toxicity studies in two
species (rat and mouse), chronic-duration toxicity studies in two
species (rat and mouse), developmental toxicity studies in one
species (mouse), and no two-generation reproductive studies.
Confidence in subchronic
p-RfDb
M
The overall confidence in chronic p-RfD is medium.
aL = low, M = medium, H = high.
bThe overall confidence cannot be greater than lowest entry in the table.
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS
Derivation of Subchronic Provisional RfC (Subchronic p-RfC)
IRIS (U.S. EPA, 2011) has provided an RfC based on the critical effect of decreased
testis weight, seminiferous tubule degeneration, and decreased hemoglobin following
subchronic-duration exposure of New Zealand White rabbits to 2-EE reported by Barbee et al.
(1984b). However, when compared on the basis of duration-adjusted concentrations, major and
minor fetal skeletal defects in the offspring of Dutch rabbits described by Doe (1984b) in a
developmental inhalation study are a more sensitive endpoint than testicular effects in adult New
Zealand White rabbits reported by Barbee et al. (1984b) following subchronic-duration exposure
3	3
(LOAELs of 161 vs. 265 mg/m , respectively, with associated NOAELs of 46 and 68 mg/m ,
respectively). The occurrence of fetal skeletal defects is also supported in a developmental
inhalation study in Wistar rats where a dose-dependent increase in minor skeletal defects was
observed with a LOAEL of 230 mg/m3 and an associated NOAEL of 47 mg/m3 (Doe, 1984a).
"3
Maternal effects from the Doe (1984a) studies also support a LOAEL of 230 mg/m with an
associated NOAEL of 47 mg/m3, based on reported hematology effects in the exposed does;
however, the quantitative data were not presented in the study report.
The characteristics of 2-EE indicate that it is a Category 3 gas, and thus exhibits systemic
toxicity (U.S. EPA, 2009). Because Category 3 gases cause extrarespiratory effects, the
concentrations in the study were converted to adjusted doses (to account for continuous
exposure) and then to HEC concentrations utilizing a default blood:gas (air) partition coefficient
of 1 because the actual value is unknown.
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The following dosimetric adjustments are made for unit conversion and inhalation
exposure in adjusting for continuous exposure for extrarespiratory effects. The example
calculation given below is for the inhalation exposure on GDs 6-18 in the Doe (1984a) study:
NOAELadj = NOAEL x (molecular weight + 24.45) x (hours per day + 24) x
(days exposed + total days)
= 50 ppm x (90.12 g/mol 24.45) x (6 hours/day 24) x
(13 days exposed -M3 total days)
= 50 ppm x 3.68 x 0.25 x 1
= 46 m«/m3
NOAELhec = NOAELadj x blood:gas partition coefficient
= 46 mg/m3 x 1
= 46 mg/m3
where blood:gas partition coefficient = 1
BMD modeling could not be applied to the less sensitive testis weight or seminiferous
tubule generation endpoints from Barbee et al. (1984b); an abnormally large standard deviation
was reported for one of the testis weight values, and no quantitative data on seminiferous tubule
generation were provided in the study.
Although the Guidelines for Developmental Toxicity Risk Assessment (U.S. EPA, 1991)
suggest that analysis of endpoints of developmental toxicity preferably be performed on a
per-litter basis, the major fetal skeletal defect data from Doe (1984b) are presented on a per-pup
basis. However, this does not necessarily preclude these data from BMD modeling. Using the
number of pups showing any major skeletal defects and the percentage of pups showing major
skeletal defects, the sample size of each exposure group was calculated for use in BMD
modeling. The EPA Benchmark Dose Software (BMDS version 2.1.2) dichotomous variable
models were fit to the major fetal skeletal defect data, and a BMR of a 5% extra risk was used
based on current EPA practice for developmental toxicity endpoints. Table 14 presents BMD
input data for the incidence of major fetal skeletal defects in offspring of Dutch rabbits exposed
to 2-EE via inhalation on GDs 6-18. The BMD model output is presented in Appendix C.
Table 14. Major Skeletal Defects in Offspring of Dutch Rabbits Exposed to 2-EE via
Inhalation on GDs 6-18 Used for BMD Analysis"
Dose
(mg/m3, HEC)
Number of Offspring
Responseb
0
136
51.5
9
140
60.0
46
96
64.6
161
134
79.1
aDoe (1984b).
Percentage of offspring showing any major defects.
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Table 15 summarizes the BMD modeling results for major fetal skeletal defects. The
Log-Logistic model fit the data best compared to other models. All models have a goodness of
fitp-walue greater than 0.1, and the Log-Logistic model provides the lowest AIC value of
643.971.
Table 15. Model Predictions for Major Skeletal Defects in Offspring of Dutch Rabbits
Exposed to 2-EE via Inhalation on GDs 6-18a
Model
Goodness of
Fit /7-value
AIC for Fitted
Model
BMCs%Hec
(mg/m3)
BMCL5%Hec
(mg/m3)
Conclusions
Gamma
0.54
644.252
10.43
7.42

Logistic
0.48
644.471
12.75
9.66

Log-Logistic
0.62
643.971
6.99
4.23
Lowest AIC and lowest
BMCL in a range of
4.23-20.27
Log-Probit
0.30
645.407
28.24
20.27

Multistage
0.53
646.942
10.91
7.69

Probit
0.47
644.516
13.20
10.14

Weibull
0.54
644.252
10.43
7.42

Quantal-Linear
0.54
644.252
10.43
7.42

aDoe (1984b).
AIC = Akaike's Information Criteria; BMC = benchmark concentration; BMCL = lower confidence limit (95%) on
the BMC
The BMC5o/oHec and BMCL5°/oHec associated with the best-fitting model (Log-Logistic)
were 6.99 and 4.23 mg/m3, respectively (see Table 15). Therefore, the BMCL5o/oHEC of
4.23 mg/m calculated from the major fetal skeletal defect data observed in rabbits in Doe
(1984b) was chosen as the POD, and a subchronic p-RfC is derived as follows:
Subchronic p-RfC = BMCL5o/oHec ^ UFc
= 4.23 mg/m3 - 100
= 4 x 1 () 2 mg/m3
The UFc for the subchronic p-RfC for 2-EE is 100 as explained in Table 16.
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Table 16. Uncertainty Factors for Subchronic p-RfC of 2-EE
UF
Value
Justification
UFa
3
A UFa of 3 is applied for animal-to-human extrapolation to account for the toxicodynamic
portion of the UFAbecause the toxicokinetic portion (10°5) has been addressed in the
dosimetric conversions.
ufd
3
A UFd of 3 is applied, because the database includes six acceptable developmental studies
in rats and rabbits (Doe, 1984a,b; Andrew and Hardin, 1984a,b; Nelson et al., 1981, 1982),
but there are no acceptable two-generation reproduction studies.
UFh
10
A UFh of 10 is applied for intraspecies differences to account for potentially susceptible
individuals in the absence of information on the variability of response to humans.
ufl
1
A UFl of 1 is applied because the POD was developed using a BMCL5%hec.
UFS
1
A UFS of 1 is applied because a subchronic-duration study was utilized.
UFC <3000
100

The confidence of the subchronic p-RfC 2-EE is medium as explained in Table 17.
Table 17. Confidence Descriptors for Subchronic p-RfC for 2-EE
Confidence Categories
Designation"
Discussion
Confidence in study
M
Confidence in the key study is medium. Doe (1984b) examined
appropriate developmental toxicity endpoints. The study was peer
reviewed, although GLP compliance is unknown. The key
endpoint of fetal skeletal defects is seen in two species (rabbit and
rat).
Confidence in database
M
The database includes subchronic-duration toxicity studies in two
species (rat and rabbit), no chronic-duration toxicity studies,
developmental toxicity studies in two species (rat and rabbit), and
no two-generation reproductive studies.
Confidence in subchronic
p-RfCb
M
The overall confidence in the p-RfC is medium.
aL = low, M = medium, H = high.
bThe overall confidence cannot be greater than the lowest entry in the table.
Derivation of Chronic Provisional RfC (Chronic p-RfC)
IRIS (U.S. EPA, 2011) has provided an RfC. No additional studies that may be relevant
have been discovered. A chronic p-RfC is not developed.
CANCER WEIGHT-OF-EVIDENCE (WOE) DESCRIPTOR
Table 18 identifies the cancer weight-of-evidence (WOE) descriptor for 2-EE. Under
EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005), 2-EE is classified as
"Inadequate Information to Assess Carcinogenic Potential. "
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Table 18. Cancer WOE Descriptor for 2-EE
Possible WOE
Descriptor
Designation
Route of Entry
Comments
"Carcinogenic to
Humans "
N/A
N/A
No human cancer studies are available.
"Likely to Be
Carcinogenic to
Humans "
N/A
N/A
No strong animal cancer data are available.
"Suggestive Evidence
of Carcinogenic
Potential"
N/A
N/A
There is not enough evidence from human and
animal studies to be suggestive of
carcinogenicity.
"Inadequate
Information to Assess
Carcinogenic
Potential"
Selected
Inhalation and
oral
Adequate information is not available to assess
carcinogenic potential.
"Not Likely to Be
Carcinogenic to
Humans "
N/A
N/A
No strong evidence of noncarcinogenicity in
humans is available.
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES
Derivation of Provisional Oral Slope Factor (p-OSF)
No human or animal studies examining the carcinogenicity of 2-EE following oral
exposure have been identified. Therefore, derivation of a provisional oral slope factor is
precluded.
Derivation of Provisional Inhalation Unit Risk (p-IUR)
No human or animal studies examining the carcinogenicity of 2-EE following inhalation
exposure have been identified. Therefore, derivation of a provisional inhalation unit risk is
precluded.
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APPENDIX A. PROVISIONAL SCREENING VALUES
No screening values are presented.
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APPENDIX B. DATA TABLES
Table B.l. Survival, Weight Loss, and Water Consumption in F344/N Rats Following
Oral Administration of 2-EE via Drinking Water for 13 Weeks3
Parameter
Exposure Group (Average Daily Dose, mg/kg-d)b
0 ppm
1250 ppm
(109)
2500 ppm
(205)
5000 ppm
(400)
10,000 ppm
(792)
20,000 ppm
(2240)e
Male rats
Sample size
10
10
10
10
10
10
Survival
10
10
10
10
10
5
Initial body weight (g)°
142
142 (100)
146 (103)
144(101)
142 (100)
143 (101)
Final body weight (g)d
333
331 (99)
325 (98)
315 (95)
268 (80)
204 (61)
Body weight change (g)°
191
189 (99)
179 (94)
171 (90)
127 (66)
61 (32)
Water consumption (g/d)°
21.2
20.7 (98)
19.4 (92)
18.3 (86)
16.6 (78)
18.4 (87)
Parameter
Exposure Group (Average Daily Dose, mg/kg-d)b
0 ppm
1250 ppm
(122)
2500 ppm
(247)
5000 ppm
(466)
10,000 ppm
(804)
20,000 ppm
(2061)e
Female rats
Sample size
10
10
10
10
10
10
Survival
10
10
10
10
10
3
Initial body weight (g)°
123
123(100)
124(101)
127 (103)
126 (102)
126 (102)
Final body weight (g)d
197
194 (98)
190(96)
186 (94)
171 (87)
185 (94)
Body weight change (g)°
74
71 (96)
66 (89)
59 (80)
45 (61)
59 (80)
Water consumption (g/d)°
17.9
16.3 (91)
16.2(91)
14.8(83)
12.4(69)
14.6 (82)
aNTP (1993a).
bDoses are converted from ppm intake using the following equation: DoseADi = dose x consumption per day x (R
body weight) x (days dosed total days).
°Mean, (% change relative to controls calculated for this review).
dMean, (% change relative to controls calculated by the study authors).
"Exposure terminated at Week 9 due to decreased survivorship.
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Table B.2. Selected Organ Weights and Organ-weight Ratios in F344/N Rats
Following Oral Administration of 2-EE via Drinking Water for 13 Weeks"
Parameter
Exposure Group (Average Daily Dose, mg/kg-d)b
0 ppm
1250 ppm
(109)
2500 ppm
(205)
5000 ppm
(400)
10,000 ppm
(792)
20,000 ppm
(2240)f
Male rats
Sample size
10
10
10
10
10
5
Necropsy body weight (g)°
315
309 (98)
296 (94)e
295 (94)d
236 (75)°
-
Absolute right testis weight
(g)c
1.394
1.431(103)
1.443 (104)
1.342 (96)
0.618 (44)e
-
Relative right testis weight
(mg/g body weight)0
4.43
4.64 (105)
4.89(110)
4.56 (103)
2.62 (59)d
-
Absolute thymus weight (g)°
0.299
0.270 (90)
0.213 (71)°
0.258 (86)°
0.154 (52)e
-
Relative thymus weight (mg/g
body weight)0
0.95
0.87 (92)
0.72 (76)e
0.87 (92)d
0.65 (68)e
-
Parameter
Exposure Group (Average Daily Dose, mg/kg-d)b
0 ppm
1250 ppm
(122)
2500 ppm
(247)
5000 ppm
(466)
10,000 ppm
(804)
20,000 ppm
(2061)f
Female rats
Sample size
10
10
10
10
10
3
Necropsy body weight (g)°
185
183 (99)
177 (96)
173 (94)e
149 (81)e
-
Absolute thymus weight (g)°
0.214
0.210 (98)
0.221 (103)
0.186 (87)
0.069 (32)e
-
Relative thymus weight (mg/g
body weight)0
1.16
1.15 (99)
1.25 (108)
1.07 (92)
0.47 (41)e
-
aNTP (1993a).
bDoses are converted from ppm intake using the following equation
(1 body weight) x (days dosed total days).
°Mean, (% change relative to controls calculated for this review).
Significantly different (p < 0.05) from the control group by Dunn's
"Significantly different (p < 0.01) from the control group by Dunn's
fExposure terminated at week 9 due to decreased survivorship.
: DoseADi = dose x consumption per day x
or Shirley's test,
or Shirley's test.
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Table B.3. Incidence and Severity of Selected Histopathologic Lesions in Male 344/N Rats
Following Oral Administration of 2-EE via Drinking Water for 13 Weeks"


Exposure Group (Average Daily Dose, mg/kg-d)b
Parameter

0 ppm
1250 ppm
(109)
2500 ppm
(205)
5000 ppm
(400)
10,000 ppm
(792)
20,000 ppm
(2240)d
Sample size
10
10
10
10
10
5
Liver
Degeneration0
0
0
0
0
0
5 (2.4)

Pigmentation0
0
0
0
0
10 (1.0)
5 (1.0)

Hematopoiesis0
0
0
0
0
9(1.7)
0
Bone marrow
Cellular
depletion0
0
0
0
0
0
5 (3.6)

Hyperplasia0
0
0
0
0
10 (2.7)
0
Spleen
Hematopoiesis0
0
0
0
10 (2.0)
10 (3.2)
0

Pigmentation0
0
0
0
0
0
5 (2.6)

Atrophy0
0
0
0
0
0
4 (2.3)
Thymus
Atrophy0
0
of
oe
0
4 (2.0)
2 (4.0)°
Testes
Degeneration0
0
0
0
10(1.1)
10(3.5)
5 (4.0)
Prostate
Atrophy0
0
0
6(1.3)
7 (1.4)
10 (2.0)
5 (3.4)
aNTP (1993a).
bDoses are converted from ppm intake using the following equation: Dosc adj = dose x consumption per day x
(1 body weight) x (days dosed total days).
Incidence, (average severity of the number of animals with lesions: 1 = minimal, 2 = mild, 3 = moderate, 4 =
marked).
dExposure terminated at Week 9 due to decreased survivorship.
"Sample size was 3 for this measurement.
fSample size was 2 for this measurement.
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Table B.4. Incidence and Severity of Selected Histopathologic Lesions in Female F344/N
Rats Following Oral Administration of 2-EE via Drinking Water for 13 Weeks"


Exposure Group (Average Daily Dose, mg/kg-d)b
Parameter

0 ppm
1250 ppm
(122)
2500 ppm
(247)
5000 ppm
(466)
10,000 ppm
(804)
20,000 ppm
(2061)d
Sample size
10
10
10
10
10
7
Liver
Degeneration0
0
0
0
0
0
6(1.8)

Pigmentation0
0
0
0
0
10(1.0)
7(1.0)

Hematopoiesis0
0
0
0
0
9.0 (2.0)
0
Bone marrow
Cellular
depletion0
0
0
0
0
0
7(3.3)

Hyperplasia0
0
0
0
0
10 (3.0)
0
Spleen
Hematopoiesis0
0
0
0
0
10 (2.5)
0

Pigmentation0
0
0
0
0
0
7 (2.7)

Atrophy0
0
0
0
0
0
6 (2.2)
Thymus
Atrophy0
0
e
e
0
10 (1.3)
6 (4.0)°
Uterus
Atrophy0
0
0
0
0
9 (2.7)
7 (3.7)
aNTP (1993a).
bDoses are converted from ppm intake using the following equation: DoseADi = dose x consumption per day x
(1 body weight) x (days dosed total days).
Incidence, (average severity of the number of animals with lesions: 1 = minimal, 2 = mild, 3 = moderate,
4 = marked).
dSample size was 7 for this measurement.
eNot applicable; tissue not examined for animals in this dose group.
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Table B.5. Summary of Reproductive Tissue and Estrous Cycle Analysis in F344/N Rats
Following Oral Administration of 2-EE via Drinking Water for 13 Weeks"
Parameter
Exposure Group (Average Daily Dose, mg/kg-d)b
0 ppm
1250 ppm
(109)
2500 ppm
(205)
5000 ppm
(400)
10,000 ppm
(792)
20,000 ppm
(2240)
Male rats
Spermatid heads (107/g
testis)0
8.980 ±
0.352
-
9.630 ±
0.273 (107)
9.410 ±
0.376 (105)
1.610 ±
0.399° (18)
-
Spermatid heads (107/testis)°
13.12 ±0.58
-
14.63 ±0.50
(112)
13.27 ±0.60
(101)
1.17 ± 0.3 le
(9)
-
Spermatid count
(mean/10-4 mL suspension)0
65.58 ±2.90
-
73.15 ±2.49
(112)
66.35 ±3.00
(101)
5.83 ± 1.57°
(9)
-
Spermatozoal motility (%)°
96.55 ± 1.02
-
97.88 ±0.67
(101)
97.07 ±0.93
(101)
0.56 ±0.44°
(1)
-
Spermatozoal concentration0
(106/g caudal epididymal
tissue)0
763.9 ±23.1

658.3 ± 14.8°
(86)
669.0 ±25.2°
(88)
27.2 ±5.2°
(4)

Parameter
Exposure Group (Average Daily Dose, mg/kg-d)
0 ppm
1250 ppm
(122)
2500 ppm
(247)
5000 ppm
(466)
10,000 ppm
(804)
20,000 ppm
(2061)d
Female rats
Estrous cycle length (days)0
5.40 ±0.15
-
5.83 ±0.40
(108)
5.83 ± 0.26s
(108)
6.50 ±0.43^
(120)
-
Estrous stages: Diestrus (% of
cycle)
36.7
-
37.3
42.5
55.0
-
Estrous stages: Proestrus
(% of cycle)
15.0
-
11.0
15.8
10.0
-
Estrous stages: Estrus (% of
cycle)
39.2
-
44.1
30.0
25.8
-
Estrous stages: Metestrus
(% of cycle)
9.2
-
7.6
11.7
9.2
-
aNTP (1993a).
bDoses are converted from ppm intake using the following equation: Dosc AI , [ = dose x consumption per day x
(1 body weight) x (days dosed total days).
°Mean ± standard error (% change relative to controls calculated for this review).
Significantly different (p < 0.05) from the control group by Shirley's test.
"Significantly different (p < 0.01) from the control group by Shirley's test.
fEstrous cycle longer than 12 days or unclear in 4/10 animals.
8Estrous cycle longer than 12 days or unclear in 1/10 animals.
hNot applicable; tissue not examined for animals in this dose group.
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Table B.6. Survival, Weight Loss, and Water Consumption in Male F344/N Rats
Following Oral Administration of 2-EE via Drinking Water for 60 Days3
Parameter
Exposure Group (Average Daily Dose, mg/kg-d)b
0 ppm
5000 ppm
(407)
10,000 ppm
(792)
20,000 ppm
(2390)c
Male rats
Sample size
30
30
30
35
Survival
10
10
9
5
Initial body weight (g)e
164
164 (100)
165 (101)
161 (98)
Day 60 body weight (g)f
302
284 (94)
255 (84)
157 (52)
Final body weight (g)f
388
361 (93)
353 (91)
277 (71)
Body weight change (g)e
224
197 (88)
188 (84)
116(52)
Water consumption (g/d)e
21.2
19.3 (91)
17.5 (83)
19.9 (94)
aNTP (1993b).
bDoses are converted from ppm intake using the following equation: Dosc adj = dose x consumption per day x
(1 body weight) x (days dosed total days).
Twenty rats in this group died at or before Day 60; one rat died after Day 60. Because of the excessive mortality of
rats administered 20,000-ppm 2-ethoxyethanol in the stop-exposure and rat subchronic-duration NTP (1993a)
study, the five surviving rats from the subchronic-duration study were moved to the 20,000-ppm stop-exposure
group at Day 60.
dNumber surviving at the end of the recovery period; number surviving does not include animals sacrificed after
60 days of treatment or 30 days of recovery.
eMean, (% change relative to controls calculated for this review).
fMean, (% change relative to controls calculated by the study authors).
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Table B.7. Selected Organ Weights and Organ-weight Ratios in Male F344/N Rats Following
Oral Administration of 2-EE via Drinking Water for 60 Days3
Parameter
Exposure Group (Average Daily Dose, mg/kg-d)b
0 ppm
5000 ppm
(407)
10,000 ppm
(792)
20,000 ppm
(2390)c
60-d treatment period
Sample size
10
10
10
4
Necropsy body weight (g)d
306 ±7
285 ± 6e (93)
259 ± 5f (85)
138 ± 21f (45)
Absolute right testis weight (g)d
1.368 ±0.019
1.400 ±0.016 (102)
0.609 ± 0.044f (45)
0.361 ±0.096f (26)
Relative right testis weight
(mg/g body weight)d
4.48 ±0.09
4.93 ±0.10 (110)
2.37 ± 0.19f (53)
2.51 ± 0.27e (56)
Absolute epididymis weight (g)d
0.441 ±0.012
0.420 ±0.014 (95)
0.228 ±0.012f (52)
0.114 ±0.018f (26)
Relative epididymis weight
(mg/g body weight)d
1.44 ±0.03
1.48 ±0.06 (103)
0.88 ± 0.04f (61)
0.83 ± 0.06f (58)
30-d recovery period
Sample size
10
10
10
5
Necropsy body weight (g)d
339 ±8
339 ±6 (100)
303 ±3f(89)
237 ± 37f (70)
Absolute right testis weight (g)d
1.460 ±0.030
1.415 ±0.021 (97)
0.652 ±0.029f (45)
0.395 ±0.038f (27)
Relative right testis weight
(mg/g body weight)d
4.32 ±0.05
4.19 ±0.10 (97)
2.15 ±0.10f (50)
1.72 ± 0.10f (40)
Absolute epididymis weight (g)d
0.507 ±0.018
0.497 ±0.017 (98)
0.311 ±0.015f (61)
0.204 ±0.014f (40)
Relative epididymis weight
(mg/g body weight)d
1.49 ±0.04
1.47 ±0.05 (99)
1.03 ± 0.05f (69)
0.91 ± 0.1 lf (61)
56-d recovery period
Sample size
10
10
9
5
Necropsy body weight (g)d
384 ±6
362 ± 8e (94)
352 ±6f (92)
272 ± 29f (71)
Absolute right testis weight (g)d
1.486 ±0.022
1.362 ±0.026f (92)
0.678 ± 0.044f (46)
0.444 ±0.023f (30)
Relative right testis weight
(mg/g body weight)d
3.88 ±0.07
3.77 ±0.06 (97)
1.92 ± 0.12f (49)
1.72 ± 0.23f (44)
Absolute epididymis weight (g)d
0.533 ±0.015
0.544 ±0.021 (102)
0.319 ±0.019fg (60)
0.255 ±0.024f (48)
Relative epididymis weight
(mg/g body weight)d
1.39 ±0.04
1.51 ±0.06 (109)
0.91 ± 0.05f'8 (65)
0.95 ± 0.04f (68)
aNTP (1993b).
bDoses are converted from ppm intake using the following equation: Dosc AI , [ = dose x consumption per day x
(1 body weight) x (days dosed total days).
Twenty rats in this group died at or before Day 60; one rat died after Day 60. Because of the excessive mortality of
rats administered 20,000-ppm 2-EE in the stop-exposure and rat subchronic-duration NTP (1993a) study, the five
surviving rats from the subchronic-duration study were moved to the 20,000-ppm stop-exposure group at Day 60.
dMean± S.E. (% change relative to controls conducted for this review).
"Significantly different (p < 0.05) from the control group by the Dunn's or Shirley's test.
Significantly different (p < 0.01) from the control group by the Dunn's or Shirley's test.
8Sample size was 10 fortius measurement.
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Table B.8. Incidence and Severity of Testicular Degeneration in Male F344/N Rats
Following Oral Administration of 2-EE via Drinking Water for 60 Days3
Time
Exposure Group (Average Daily Dose, mg/kg-d)b
0 ppm
5000 ppm
(407)
10,000 ppm
(792)
20,000 ppm
(2390)c
60-d treatment periodd
0/10
0/10
10/10 (2.9)
24/24 (4.0)
30-d recovery periodd
0/10
6/10 (1.0)
11/11 (2.7)
5/5 (4.0)
56-d recovery periodd
0/10
7/10 (1.0)
9/9 (2.7)
5/5 (4.0)
aNTP (1993b).
bDoses are converted from ppm intake using the following equation: DoscAI , [ = dose x consumption per day x
(1 body weight) x (days dosed total days).
Twenty rats in this group died at or before Day 60; one rat died after Day 60. Because of the excessive mortality of
rats administered 20,000-ppm 2-ethoxyethanol in both the stop-exposure and rat subchronic-duration NTP (1993a)
study, the five surviving rats from the subchronic-duration study were moved to the 20,000-ppm stop-exposure
group at Day 60.
incidence, (average severity: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked).
Table B.9. Survival, Weight Loss, and Water Consumption in B6C3Fi Mice
Following Oral Administration of 2-EE via Drinking Water for 13 Weeks"
Parameter
Exposure Group (Average Daily Dose, mg/kg-d)b
0 ppm
2500 ppm
(587)
5000 ppm
(971)
10,000 ppm
(2003)
20,000 ppm
(5123)
40,000 ppm
(7284)
Male mice
Sample size
10
10
10
10
10
10
Survival
10
10
10
10
10
10
Initial body weight (g)°
22.7
23.7 (104)
23.5 (104)
22.8 (100)
23.4 (103)
23.9 (105)
Final body weight (g)d
39.2
41.7(106)
43.1 (110)
41.0(105)
33.2 (85)
32.5 (83)
Body weight change (g)°
16.5
18.0(109)
19.6(119)
18.2(110)
9.8 (59)
8.6 (52)
Water consumption (g/d)°
6.7
7.6 (113)
6.5 (97)
6.3 (94)
7.8(116)
5.2 (78)
Parameter
Exposure Group (Average Daily Dose, mg/kg-d)b
0 ppm
2500 ppm
(722)
5000 ppm
(1304)
10,000 ppm
(2725)
20,000 ppm
(7255)
40,000 ppm
(11,172)
Female mice
Sample size
10
10
10
10
10
10
Survival
10
10
10
10
10
10
Initial body weight (g)°
19.3
19.0(98)
18.9 (98)
19.1 (99)
19.1 (99)
19.0 (98)
Final body weight (g)d
32.0
34.0 (106)
34.1(107)
30.2 (94)
26.4 (83)
24.9 (78)
Body weight change (g)°
12.7
15.0(118)
15.2(120)
11.1 (87)
7.3 (57)
5.9 (46)
Water consumption (g/d)°
8.7
7.5 (86)
6.9 (79)
6.9 (79)
8.7(100)
6.1 (70)
aNTP (1993c).
bDoses are converted from ppm intake using the following equation: Doscadj = dose x consumption per day x
(1 body weight) x (days dosed total days).
°Mean, (% change relative to controls conducted for this review).
dMean, ( % change relative to controls as calculated by the study authors).
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Table B.10. Selected Organ Weights and Organ-weight Ratios in Male B6C3Fi Mice
Following Oral Administration of 2-EE via Drinking Water for 13 Weeks"
Parameter
Exposure Group (Average Daily Dose, mg/kg-d)b
0 ppm
2500 ppm
(587)
5000 ppm
(971)
10,000 ppm
(2003)
20,000 ppm
(5123)
40,000 ppm
(7284)
Male mice
Sample size
10
10
10
10
10
10
Necropsy body weight (g)°
38.9
40.9 (105)
43.0(111)
40.5 (104)
33.6 (86)d
31.9 (82)e
Absolute right testis weight (g)°
0.119
0.124(104)
0.123 (103)
0.119(100)
0.097 (82)e
0.019 (16)e
Relative right testis weight
(mg/g body weight)0
3.08
3.05 (99)
2.86 (93)
2.95 (96)
2.88 (94)
0.59 (19)e
aNTP (1993c).
bDoses are converted from ppm intake using the following equation: Doscadj = dose x consumption per day x
(1 body weight) x (days dosed total days).
°Mean, (% change relative to controls calculated for this review).
Significantly different (p <0.05) from the control group by Dunn's or Shirley's test.
"Significantly different (p <0.01) from the control group by Dunn's or Shirley's test.
Table B.ll. Incidence and Severity of Selected Histopathologic Lesions in Male B6C3Fi
Mice Following Oral Administration of 2-EE via Drinking Water for 13 Weeks"
Parameter
Exposure Group (Average Daily Dose, mg/kg-d)b
0 ppm
2500 ppm
(587)
5000 ppm
(971)
10,000 ppm
(2003)
20,000 ppm
(5123)
40,000 ppm
(7284)
Sample size
10
10
10
10
10
10
Spleen
Hematopoiesis0
0
d
0
0
0
10 (1.6)
Testes
Degeneration0
0
d
0
0
0
10 (4.0)
aNTP (1993c).
bDoses are converted from ppm intake using the following equation: Doscadj = dose x consumption per day x
(1 body weight) x (days dosed total days).
"Incidence, (Average severity of the number of animals with lesions: 1 = minimal, 2 = mild, 3 = moderate,
4 = marked).
dNot applicable; tissue not examined for animals in this dose group.
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Table B.12. Incidence and Severity of Selected Histopathologic Lesions in Female B6C3Fi
Mice Following Oral Administration of 2-EE via Drinking Water for 13 Weeks3
Parameter
Exposure Group (Average Daily Dose, mg/kg-d)b
0 ppm
2500 ppm
(722)
5000 ppm
(1304)
10,000 ppm
(2725)
20,000 ppm
(7255)
40,000 ppm
(11,172)
Sample size
10
10
10
10
10
10
Spleen
Hematopoiesis0
0
d
0
1 (1.0)
9(1.3)
10 (1.8)
Adrenal gland
X-zone,
hypertrophy0
0
d
1 (2.0)
8(1.8)
10 (2.8)
9 (2.4)
aNTP (1993c).
bDoses are converted from ppm intake using the following equation: DoscAI , [ = dose x consumption per day x
(1 body weight) x (days dosed total days).
Incidence, (Average severity of the number of animals with lesions: 1 = minimal, 2 = mild, 3 = moderate,
4 = marked).
dNot applicable; tissue not examined for animals in this dose group.
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Table B.13. Summary of Reproductive Tissue and Estrous Cycle Analysis in B6C3Fi Mice
Following Oral Administration of 2-EE via Drinking Water for 13 Weeks"
Parameter
Exposure Group (Average Daily Dose, mg/kg-d)b
0 ppm
2500 ppm
(587)
5000 ppm
(971)
10,000 ppm
(2003)
20,000 ppm
(5123)
40,000 ppm
(7284)
Male mice
Spermatid heads (107/g
testis)0
19.160 ±
0.745
_g
19.340 ±
0.767(101)
19.970 ±
0.961 (104)
18.710 ±
1.018(98)
_g
Spermatid heads (107/testis)0
2.26 ±0.10
_g
2.27 ±0.15
(100)
2.39 ±0.10
(106)
1.72 ± 0.12d
(76)
_g
Spermatid count
(mean/10-4 mL suspension)0
70.68 ±3.16
_g
70.85 ± 4.74
(100)
74.68 ±3.18
(106)
53.68 ±3.88d
(76)
_g
Spermatozoal motility (%)°
98.65 ±0.24
_g
98.40 ±0.30
(100)
97.92 ±0.25
(99)
97.35 ±0.45d
(99)
_g
Spermatozoal concentration
(106/g caudal epididymal
tissue)0
1126.7 ±
55.7
_g
1036.2 ±
94.5 (92)
1133.2 ±
63.4(101)
1139.7 ±
91.0(101)
_g
Parameter
Exposure Group (Average Daily Dose, mg/kg-d)b
0 ppm
2500 ppm
(722)
5000 ppm
(1304)
10,000 ppm
(2725)
20,000 ppm
(7255)
40,000 ppm
(11,172)
Female mice
Estrous cycle length (days)0
4.30 ± 0.11
_g
4.85 ± 0.15d
(113)
5.25 ±0.23°
(122)
5.50 ± 0.47e'f
(128)
_g
Estrous stages: Diestrus (% of
cycle)
31.7
_g
27.5
32.5
40.8
_g
Estrous stages: Proestrus
(% of cycle)
23.3
_g
20.8
18.3
19.2
_g
Estrous stages: Estrus (% of
cycle)
29.2
_g
41.7
37.5
33.3
_g
Estrous stages: Metestrus
(% of cycle)
15.8
_g
10.0
11.7
6.7
_g
aNTP (1993c).
bDoses are converted from ppm intake using the following equation: DoscAI , [ = dose x food consumption per day x
(1 body weight) x (days dosed total days).
°Mean ± standard error (% relative to controls).
Significantly different (p < 0.05) from the control group by Shirley's test.
"Significantly different (p < 0.01) from the control group by Shirley's test.
fEstrous cycle longer than 12 days or unclear in 1/10 animals.
8Not applicable; tissue not examined for animals in this dose group.
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Table B.14. Survivorship and Mean Body Weights in F344/N Rats Exposed to 2-EE by
Gavage for 103 Weeks"
Parameter
Exposure Group (Adjusted Daily Dose, mg/kg-d)b
0 g/kg (0)
0.5 g/kg (357)
1*0 g/kg (714)
2.0 g/kg (1429)
Male rats
Sample size
50
50
50
50
Survivorship
30
38
18d
f
Mean body weights (g)°
421
339 (81)
304 (72)
f
Parameter
Exposure Group (Adjusted Daily Dose, mg/kg-d)b
0 g/kg (0)
0.5 g/kg (357)
1*0 g/kg (714)
2.0 g/kg (1429)
Female rats
Sample size
50
50
50
50
Survivorship
26
46e
25
f
Mean body weights (g)°
315
239 (76)
225 (71)
f
aMelnick (1984a).
bDoses are converted from % of food to ppm by multiplying by 10,000 (1% = 10,000 ppm), and then ppm intake in
food is adjusted using the following equation: Doscadj = Dose x food consumption per day x (1 body weight) x
(days dosed total days).
°Meanbody weight at 104 weeks, (% change relative to controls as calculated by the study author).
Significantly decreased relative to control (p < 0.05) as reported by the study author.
"Significantly increased relative to control (p < 0.01) as reported by the study author.
fNot applicable; tissue not examined for animals in this dose group.
Table B.15. Survivorship and Mean Body Weights in B6C3Fi Mice Exposed to 2-EE by
Gavage for 103 Weeks3
Parameter
Exposure Group (Average Daily Dose, mg/kg-d)b
0 g/kg (0)
0.5 g/kg (357)
1*0 g/kg (714)
2.0 g/kg (1429)
Male mice
Sample size
50
50
50
50
Survivorship
36
28
33
-
Mean body weights (g)°
44
40 (91)
41 (93)
-
Parameter
Exposure Group (Adjusted Daily Dose, mg/kg-d)b
0 g/kg (0)
0.5 g/kg (357)
1*0 g/kg (714)
2.0 g/kg (1429)
Female mice
Sample size
50
50
50
50
Survivorship
36
38
32
d
Mean body weights (g)°
40
38 (95)
39 (98)
d
"Melnick (1984b).
bDoses are converted from g/kg body weight to mg/kg-day by converting grams to milligrams and multiplying by
days dosed total days using the following equation: DoseADj = dose x (1000 mg lg) x (days dosed total
days).
°Meanbody weight at 104 weeks, (% change relative to controls as calculated by the study authors).
dNot applicable; tissue not examined for animals in this dose group.
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Table B.16. Body-Weight Gain in Male Sprague-Dawley Rats Exposed to 2-EE for
4 Weeks a'b
Body-weight Gain
(g)
Exposure Group (Average Daily Dose, mg/kg-day)c
0 mg/kg (0)
100 mg/kg (86)
200 mg/kg (171)
400 mg/kg (343)
800 mg/kg (686)
Sample size
5
5
5
5
5
Week 1
15.7
22.5
11.8
3.91
0.00
Week 2
33.9
41.4
20.9
24.8
0.094
Week 3
61.4
84.4
50.2
33.4
20.8
Week 4
72.08
80.8
66.5
47.2
25.7
"Yoonetal. (2003).
bData extracted from study graph(s) using Coulter Multiparameter Data Acquisition and Display Software.
Doses are converted from g/kg body weight to mg/kg-day by converting grams to milligrams and multiplying by
days dosed total days using the following equation: Doscadj = dose x (1000 mg lg) x (days dosed total days).
Table B.17. Mean Testis and Epididymis Weights in Male Sprague-Dawley Rats Exposed
to 2-EE for 4 Weeksa'b
Parameter
Exposure Group (Average Daily Dose, mg/kg-d)c
0 mg/kg (0)
100 mg/kg (86)
200 mg/kg (171)
400 mg/kg (343)
800 mg/kg (686)
Sample size
5
5
5
5
5
Testis (mg/100 g
bw)d
337.5 ± 14.52
320.21 ±22.18
(95)
318.3 ± 13.76
(94)
217.6 ±32.89f (64)
165.19 ±26.77f
(49)
Epididymis
(mg/100 gbw)d
123.6 ±8.072
113.9 ± 9.463e (92)
107.03 ± 10.58f
(87)
95.41 ±9.463f (77)
82.94 ±6.401f
(67)
aYoon et al. (2003).
bData extracted from study graph(s) using Coulter Multiparameter Data Acquisition and Display Software.
Doses are converted from g/kg body weight to mg/kg-day by converting grams to milligrams and multiplying by
days dosed total days using the following equation: DoseADj = dose x (1000 mg 1/g) x (days dosed total
days).
dMean ± SD (% relative to controls).
"Statistically significantly different from control (p < 0.05) as reported by the study authors.
Statistically significantly different from control (p < 0.01) as reported by the study authors.
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Table B.18. Testicular Cell Numbers in Male Sprague-Dawley Rats Exposed to 2-EE for
4 Weeksa'b
Parameter
Exposure Group (Average Daily Dose, mg/kg-d)c
0 mg/kg (0)
100 mg/kg (86)
200 mg/kg (171)
400 mg/kg (343)
800 mg/kg (686)
Totald
20.54 ±4.057
21.72 ±3.934
(106)
20.81 ±3.872
(101)
9.084 ± 2.828e (44)
4.057 ± 1.600e
(20)
Mature haploidd
43.75 ±4.321
43.62 ±3.086
(100)
39.78 ±3.086
(91)
21.74 ±6.800e (50)
1.854 ± 1.852e
(4)
Immature haploidd
25.27 ±3.717
26.511 ±3.725
(105)
25.90 ± 1.856
(102)
6.700 ± 3.260e (27)
2.791 ±0.939e
(11)
Diploidd
17.77 ±3.615
18.45 ± 1.205
(104)
19.12 ±3.615
(108)
42.70 ± 6.024e
(240)
71.08 ±9.639e
(400)
S-phased
1.460 ± 1.171
0.9757 ±0.329
(67)
0.9675 ±0.1463
(66)
1.728 ±0.4390
(118)
1.208 ±0.5852
(83)
Tetraploidd
4.157 ±2.174
5.073 ± 1.957
(122)
5.557 ± 1.522
(134)
14.30 ±2.610d
(344)
9.346 ±6.087
(225)
aYoon et al. (2003).
bData extracted from study graph(s) using Coulter Multiparameter Data Acquisition and Display Software.
Doses are converted from g/kg body weight to mg/kg-day by converting grams to milligrams and multiplying by
days dosed total days using the following equation: DoseADj= Dose x (1000 mg lg) x (days dosed total
days).
dNumber (1 x 106) ± SD.
Statistically significantly different from control (p < 0.01) as reported by the study authors.
Table B.19. Relative Testis and Epididymis Weight in Male Sprague-Dawley Rats Dosed
by Gavage with 2-EE for 4 Weeks"'
Parameter
Exposure Group (Average Daily Dose, mg/kg-d)c
0 mg/kg (0)
50 mg/kg (43)
100 mg/kg (86)
200 mg/kg (171)
400 mg/kg (343)
Pubertal
Epididymis11
101 ± 10
116 ±4e
111 ± 5e
124 ±6f
113 ±6e
Testis'1
406 ±21
452 ± 8e
426 ± 19e
445 ± 27e
440 ± 12e
Adult
Epididymis11
125 ±7
129 ± 19
111 ±21
113 ± 11
111 ± 13f
Testis'1
352 ±35
380 ±56
354 ±47
349 ± 19
233 ± 57f
"Yoonetal. (2001).
bData extracted from study graph(s) using Coulter Multiparameter Data Acquisition and Display Software.
Doses are converted from g/kg body weight to mg/kg-day by converting grams to milligrams and multiplying by
days dosed per total days using the following equation: DoseADj= dose x (1000 mg 1 g) x (days dosed total
days).
dWeight (mg/lOOgbw) ± SD.
"Statistically significantly different from control (p < 0.05) as reported by the study authors.
Statistically significantly different from control (p < 0.01) as reported by the study authors.
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Table B.20. Relative Alteration of Testicular Cell Populations in Male Sprague-Dawley Rats

Dosed by Gavage with 2-EE for 4 Weeks"'


Exposure Group (Average Daily Dose, mg/kg-d)c
Parameter
0 mg/kg (0)
50 mg/kg (43)
100 mg/kg (86)
200 mg/kg (171)
400 mg/kg (343)
Mature haploid
Pubertal
35 ±6
40 ±5
39 ±2
40 ± 1
40 ±6
Adultd
39 ±2
40 ±4
39 ±2
35 ±5
30 ± 6e
Immature haploid
Pubertal"1
28 ±4
27 ±4
28 ±3
29 ±5
27 ±4
Adultd
29 ±3
27 ±4
26 ± 1
27 ±6
15 ± 8e
Diploid
Pubertald
25 ±6
21 ±4
22 ±3
21 ±4
22 ±4
Adultd
23 ±3
24 ±2
24 ±2
26 ±5
38 ± 5f
S-phase
Pubertald
1.4 ±0.33
1.4 ±0.40
1.2 ±0.35
1.3 ±0.42
1.3 ±0.18
Adultd
1.4 ±0.33
1.3 ±0.19
1.3 ± 0.12
1.4 ±0.20
2 ± 0.70
Tetraploid
Pubertald
6.3 ±2.5
6.6 ±2.2
5.8 ± 1.4
7.3 ± 1.7
7 ± 3.1
Adultd
3.7 ±0.6
3.9 ±1.1
4.3 ±0.84
5.9 ±2.2
9.4 ± 2.0f
aYoon et al. (2001).
bData extracted from study graph(s) using Coulter Multiparameter Data Acquisition and Display Software.
Doses are converted from g/kg body weight to mg/kg-day by converting grams to milligrams and multiplying by days
dosed per total days using the following equation: DoseADj = dose x (1000 mg 1 g) x (days dosed total days).
Percentage of population (%) ± SD.
"Statistically significantly different from control (p < 0.05) as reported by the study authors.
Statistically significantly different from control (p < 0.01) as reported by the study authors.
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Table B.21. Body and Organ Weights in Male Sprague-Dawley Rats Dosed by Gavage with
2-EE for 4 Weeks"
Parameter
Exposure Group (Average Daily Dose, mg/kg-d)b
0 mg/kg (0)
150 mg/kg (129)
Body weight (g)°
385 ± 17
364 ± 17d (95)
Adrenal gland (R) (mg/100 gbody weight)0
9.8 ±2.4
7.8 ±2.8 (80)
Adrenal gland (L) (mg/100 gbody weight)0
12.1 ±4.9
8.7 ±2.1° (72)
Testis (R) (mg/100 gbody weight)0
425 ± 28
244 ± 32e (57)
Testis (L) (mg/100 gbody weight)0
433 ±25
247 ±38° (57)
Epididymis (R) (mg/100 gbody weight)0
149 ±22
114 ± 15e (77)
Epididymis (L) (mg/100 gbody weight)0
148 ±23
112 ± 15° (76)
"Yuetal. (1999).
bDoses are converted from mg/kg body weight to mg/kg-day by multiplying by days dosed ^ total days using the
following equation: DoseADj = dose x (days dosed total days).
°Mean ± SD (% change relative to controls calculated for this review)
Statistically significantly different from control (p < 0.05) as reported by the study authors.
"Statistically significantly different from control (p < 0.01) as reported by the study authors.
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Table B.22. Hematology and Blood Chemistry in Male Sprague-Dawley Rats Dosed
by Gavage with 2-EE for 4 Weeks3
Grade
Exposure Group (Average Daily Dose, mg/kg-d)b
0 mg/kg (0)
150 mg/kg (129)
White blood cells (103/mm3)°
5.8 ±0.1
4.1 ± 0.8d (71)
Red blood cells (106/mm3)0
8.1 ±0.4
8.3 ±0.6 (102)
Hematocrit (%)°
49.8 ±2.8
45.6 ± 3.6d (92)
Hemoglobin (g/dL)°
15.2 ±0.4
14.2 ± 0.8d (93)
Mean corpuscular volume (|i3)c
54.2 ±2.0
55.2 ±3.5 (102)
Mean corpuscular hemoglobin (pg)°
18.9 ±0.9
17.3 ± 0.5d (92)
Mean corpuscular hemoglobin
concentration (%)°
30.7 ± 1.1
31.3 ±1.7 (102)
Platelet counts (103/jx3)°
1010 ±72
750 ± 144d (74)
Total protein (mg/dL)°
6.6 ±0.3
5.9 ± 0.2d (89)
Blood urea nitrogen (mg/dL)°
17.7 ± 1.5
18.9 ±2.8 (107)
Creatinine (mg/dL)°
0.61 ±0.0
0.50 ± 0.0d (82)
Total bilirubin (mg/dL)°
0.20 ±0.0
0.19 ±0.1 (95)
Glucose (mg/dL)°
127 ±25
125 ± 12 (98)
Total cholesterol (mmol/L)°
59 ±8
54 ± 7 (92)
Aspartate aminotransferase (units/L)c
137 ±25
125 ±23 (91)
Alanine aminotransferase (units/L)°
39 ±3
34 ± 6 (87)
Alkaline phosphatase (units/L)°
377 ± 82
223 ± 76d (59)
aYu et al. (1999).
bDoses are converted from mg/kg body weight to mg/kg-day by multiplying by days dosed ^ total days using
the following equation: Doscadj = dose x (days dosed total days).
°Mean ± SD (% change relative to controls calculated for this review).
Statistically significantly different from control (p < 0.01) as reported by the study authors.
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Table B.23. Body-Weight Gain in Sprague-Dawley Rats Exposed to 2-EE Vapor for
13 Weeks"
Body-weight Gain
(g)
Exposure Group (Human Equivalent Concentration, mg/m3)b
0 ppm (0)
25 ppm (17)
100 ppm (68)
400 ppm (265)
Male rats
Sample size
15
15
15
15
Week 0C
249 ± 11
254 ± 9 (102)
250 ± 10(100)
251 ±14 (101)
Week 3°
358 ±21
356 ± 19(99)
367 ±22 (103)
365 ± 24 (102)
Week 6°
404 ± 36
403 ± 27 (100)
423 ±36 (105)
415 ±27 (103)
Week 9°
460 ± 46
453 ± 30 (98)
468 ±44 (102)
448 ±41 (97)
Week 13°
475 ± 53d
472 ± 38 (99)
481 ±38 (101)
471 ±56 (99)
Terminal0
442 ± 49d
446 ±34 (101)
454 ± 39 (103)
439 ± 48 (99)
Female rats
Sample size
15
15
15
15
Week 0C
171 ±8
169 ± 6 (99)
169 ± 7 (99)
170 ± 8 (99)
Week 3°
218± 11
208 ± 22 (95)
210 ± 13 (96)
215 ±8 (99)
Week 6°
247 ± 15
253 ±30 (102)
246 ± 20 (100)
249 ± 15 (101)
Week 9°
264 ± 18
262 ± 18 (99)
259 ±21 (98)
270 ± 28 (102)
Week 13°
278 ± 23
275 ± 23d (99)
267 ± 26 (96)
282 ± 38d (101)
Terminal0
252 ± 23
253 ±21d(100)
244 ± 23 (97)
250 ± 20d (99)
aBarbee et al. (1984a).
bDoses are converted using conversion factors: MW = 90.12 and assuming 25°C and 1 atmosphere. HECexresp =
(ppm x MW ^ 24.45) x (hours per day exposed ^ 24) x (days per week exposed ^ 7) x blood:gas (air) partition
coefficient.
°Mean ± SD (% change relative to controls calculated for this review).
dSample size is 14 rats.
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Table B.24. Mean Pituitary and Spleen Weights in Sprague-Dawley Rats Exposed to 2-EE


Vapor for 13 Weeks"


Exposure Group (Human Equivalent Concentration, mg/m3)b
Parameter
0 ppm (0)
25 ppm (17)
100 ppm (68)
400 ppm (265)
Male rats
Sample size
15
15
15
15
Pituitary (g)°
10.9 ± 1.4d
10.7 ± 1.3 (98)
10.2 ± 1.0 (94)
9.4 ± l.O'1'6 (86)
Spleen (g)°
0.67 ± 0.13d
0.68 ±0.09 (101)
0.71 ±0.11 (106)
0.62 ±0.08 (93)
Female rats
Sample size
15
15
15
15
Pituitary (g)°
15.6 ±2.6
14.5 ± 1.9(93)
13.7 ± 1.7 (88)
14.4 ± 2.8e (92)
Spleen (g)°
0.52 ±0.07
0.46 ± 0.05 (88)
0.46 ± 0.07e (88)
0.44 ±0.06^ (85)
aBarbee et al. (1984a).
bDoses are converted using conversion factors: MW = 90.12 and assuming 25°C and 1 atmosphere. HECEXresp =
(ppm x MW ^ 24.45) x (hours per day exposed ^ 24) x (days per week exposed ^ 7) x blood gas partition
coefficient.
°Mean ± SD (% relative to controls).
dSample size is 14 rats.
Statistically significantly different from control (p < 0.05) as reported by the study authors.
Statistically significantly different from control (p < 0.01) as reported by the study authors.
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Table B.25. Body-weight Gain in New Zealand White Rabbits Exposed to 2-EE Vapor for
13 Weeks"
Body-weight Gain
(g)
Exposure Group (Human Equivalent Concentration, mg/m3)b
0 ppm (0)
25 ppm (17)
100 ppm (68)
400 ppm (265)
Male rabbits
Sample size
10
10
10
10
Week 0C
2900 ± 100
2900 ± 100 (100)
2900 ± 100 (100)
2900 ± 200 (100)
Week 3°
3200 ± 200
3100 ±200 (97)
3100 ±200 (97)
3000 ± 100e (94)
Week 6°
3500 ± 200
3400 ± 100 (97)
3300 ± 100 (94)
3200 ± 200f (91)
Week 9°
3600 ± 200
3500 ± 100 (97)
3500 ± 100 (97)
3300 ± 200f (92)
Week 13°
3900 ± 300
3700 ± 200e (95)
3800 ± 200 (97)
3500 ± 200f (90)
Terminal0
3700 ± 300
3500 ± 200® (95)
3600 ± 200 (97)
3400 ± 200f (92)
Female rabbits
Sample size
10
10
10
10
Week 0C
2400 ± 200
2400 ± 100 (100)
2400 ± 200 (100)
2400 ± 200 (100)
Week 3°
2800 ± 200
2700 ± 200 (96)
2900 ± 200 (104)
2700 ± 200 (96)
Week 6°
3300 ± 200
3000 ±300 (91)
3200 ± 200 (97)
2900 ± 400d'e (88)
Week 9°
3400 ± 300
3200 ± 300d (94)
3400 ± 300 (100)
3100 ±300 (91)
Week 13°
3800 ± 300
3400 ± 400d'e (89)
3700 ± 400 (97)
3400 ± 300d (89)
Terminal0
3700 ± 300d
3300 ± 400d'e (89)
3500 ± 300 (95)
3300 ± 300d (89)
aBarbee et al. (1984b).
bDoses are converted using conversion factors: MW = 90.12 and assuming 25°C and 1 atmosphere. HECEXresp =
(ppm x MW ^ 24.45) x (hours per day exposed ^ 24) x (days per week exposed ^ 7) x blood:gas (air) partition
coefficient.
°Mean ± SD (% relative to controls).
dSample size is 9 rabbits.
"Statistically significantly different from control (p < 0.05) as reported by the study authors.
Statistically significantly different from control (p < 0.01) as reported by the study authors.
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Table B.26. Mean Adrenal Gland, Testis, and Brain Weights in New Zealand White

Rabbits Exposed to 2-EE Vapor for 13 Weeks"


Exposure Group (Human Equivalent Concentration, mg/m3)b
Parameter
0 ppm (0)
25 ppm (17)
100 ppm (68)
400 ppm (265)
Male rabbits
Sample size
10
10
10
10
Adrenal (g)°
424 ± 86d
304 ±78e (72)
364 ± 86 (86)
351 ±89 (115)
Testis (g)°
8.19 ±0.82
8.73 ± 119s (107)
8.73 ±0.69 (107)
6.36 ± 0.99f (78)
Brain (g)°
9.50 ±0.57
9.50 ±0.42 (100)
9.71 ±0.35 (102)
9.29 ±0.39 (98)
Female rabbits
Sample size
10
10
10
10
Adrenal (g)°
324 ± 79
318 ± 30d (98)
289 ± 54 (89)
282 ± 60 (89)
Brain (g)°
9.20 ±0.55
9.42 ±0.37 (102)
9.20 ±0.48 (100)
9.33 ±0.39 (99)
aBarbee et al. (1984b).
bDoses are converted using conversion factors: MW = 90.12 and assuming 25°C and 1 atmosphere. HECEXresp =
(ppm x MW ^ 24.45) x (hours per day exposed ^ 24) x (days per week exposed ^ 7) x blood:gas (air) partition
coefficient.
°Mean ± SD (% relative to controls).
dSample size is 9 rabbits.
Statistically significantly different from control (p < 0.05) as reported by the study authors.
Statistically significantly different from control (p < 0.01) as reported by the study authors.
8The SD value reported here reflects the number reported in the study. There is no way to confirm this value.
Table B.27. Litter Data for Female Wistar Rats Exposed to 2-EE via Inhalation on
GDs 6-15a
Parameter
(Human Equivalent Concentration, mg/m3)b
0 ppm (0)
10 ppm (9)
50 ppm (47)
250 ppm (230)
No. pregnant
23/24
24/24
23/24
21/24
Preimplantation loss (%)
2.4
9.7d
14.3°
6.2
Postimplantation loss (%)
5.5
7.6
8.9
12.6
Mean no. of live fetuses
12.2
10.6°
10.8°
11.1
Mean live fetus weight (g)
5.1
5.2
5.1
4.7d
aDoe (1984a).
developmental studies are adjusted for hourly but not for weekly exposure. Conversion Factors: MW = 90.12.
Assuming 25°C and 1 atmosphere; HECexresp = (ppm x MW ^ 24.45) x (hours per day exposed ^ 24) x blood:gas
(air) partition coefficient.
Significantly different from control (p < 0.05); statistical test methods were not specified.
'The significance of this result cannot be confirmed. Study text indicated that this result was statistically significant;
however, Table 4 in the study did not. This represents a discrepancy in the article.
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Table B.28. Fetal, Visceral, External, and Skeletal Defects in Offspring of Wistar Rats
Exposed to 2-EE via Inhalation on GDs 6-15a


(Human Equivalent Concentration, mg/m3)b
Parameter
0 ppm (0)
10 ppm (9)
50 ppm (47)
250 ppm (230)
External and Visceral Defects
No. (%) showing any minor defects
33 (11.7)
41 (16.1)
29 (11.6)
43 (18.4)°
No. (%) showing any major defects
0
0
0
1 (0.4)
Skeletal Defects
No. (%) showing any minor defects
68 (46.3)
52 (39.7)
66 (51.2)
119 (97.5)°
No. (%) showing any major defects
0
0
0
0
aDoe (1984a).
bDevelopmental studies are adjusted for hourly but not for weekly exposure. Conversion Factors: MW = 90.12.
Assuming 25°C and 1 atmosphere; HECexresp = (ppm x MW ^ 24.45) x (hours per day exposed ^ 24) x blood:gas
(air) partition coefficient.
Significantly different from control (p < 0.05); statistical test methods were not specified.
Table B.29. Incidence of Specific Visceral and External Defects in Offspring of Wistar Rats
Exposed to 2-EE via Inhalation on GDs 6-15a
Parameter
Exposure Group (Human Equivalent Concentration, mg/m3)b
0 ppm (0)
10 ppm (9)
50 ppm (47)
250 ppm (230)
Minor Defects
No. (%) renal pelvic dilation
19 (6.8)
25 (9.8)
22 (9.8)
30 (12.8)°
No. (%) hydroureter
13 (4.6)
26 (2.4)
4 (1.6)
10 (4.3)
No. (%) bladder distended
0
1 (0.4)
0
0
No. (%) dermal hemorrhage
2 (0.7)
2 (0.8)
0
0
No. (%) limb malrotation
0
9 (3.5)°
2 (0.8)
3(1.3)
No. (%) lateral ventricles of
brain dilated
0
0
1 (0.8)
0
Major Defects
Right ureter/kidney fused to left
kidney, right kidney vestigial
0
0
0
1 (0.4)
aDoe (1984a).
'Developmental studies are adjusted for hourly but not for weekly exposure. Conversion Factors: MW = 90.12.
Assuming 25°C and 1 atmosphere; HECexresp = (ppm x MW ^ 24.45) x (hours per day exposed ^ 24) x blood:gas
(air) partition coefficient.
Significantly different from control (p < 0.05); statistical test methods were not specified.
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Table B.30. Litter Data for Female Dutch Rabbits Exposed to 2-EE via Inhalation on
GDs 6-18a
Parameter
Exposure Group (Human Equivalent Concentration, mg/m3)b
0 ppm (0)
10 ppm (9)
50 ppm (46)
175 ppm (161)
No. pregnant
21/24
21/24
16/24
22/24
Preimplantation loss (%)
19.5
17.6
22.1
25.7
Postimplantation loss (%)
5.7
8.4
8.8
5.7
Mean no. of live fetuses
6.5
6.6
6.0
6.1
Mean live fetus weight (g)
34.1
34.2
35.7
36.1
aDoe (1984b).
bDevelopmental studies are adjusted for hourly but not for weekly exposure. Conversion Factors: MW = 90.12.
Assuming 25°C and 1 atmosphere; HECEXresp = (ppm x MW ^ 24.45) x (hours per day exposed ^ 24) x blood:gas
(air) partition coefficient.
Table B.31. Fetal, Visceral, External, and Skeletal Defects in Offspring of Dutch Rabbits
Exposed to 2-EE via Inhalation on GDs 6-18a


Exposure Group (Human Equivalent Concentration, mg/m3)b
Parameter
0 ppm (0)
10 ppm (9)
50 ppm (46)
175 ppm (161)
External and Visceral Defects
No. (%) showing any minor defects
6 (4.4)
8 (5.8)
4 (4.2)
2(1.5)
No. (%) showing any major defects
1 (0.7)
1 (0.7)
0
2(1.5)
Skeletal Defects
No. (%) showing any minor defects
44 (32.4)
72 (52.2)
35 (36.5)
87 (64.5)°
No. (%) showing any major defects
70 (51.5)
84 (60.0)
62 (64.6)
106 (79.1)°
aDoe (1984b).
'Developmental studies are adjusted for hourly but not for weekly exposure. Conversion Factors: MW = 90.12.
Assuming 25°C and 1 atmosphere; HECexresp = (ppm x MW ^ 24.45) x (hours per day exposed ^ 24) x blood:gas
(air) partition coefficient.
Significantly different from control (p < 0.05); statistical test methods were not specified.
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Table B.32. Incidence of Specific Visceral in Offspring of Dutch Rabbits Exposed to 2-EE via
Inhalation on GDs 6-18a
Parameter
Exposure Group (Human Equivalent Concentration, mg/m3)b
0 ppm (0)
10 ppm (9)
50 ppm (46)
175 ppm (161)
Right subclavian artery, absent,
aorta and heart reduced in size
(%)
0
0
0
1 (0.7)
Extreme pelvic dilatation of
both kidneys
1 (0.7)
0
0
0
Umbilical hernia
0
0
0
1 (0.7)
aDoe (1984b).
bDevelopmental studies are Adjusted for hourly but not for weekly exposure. Conversion Factors: MW = 90.12.
Assuming 25°C and 1 atmosphere; HECexresp = (ppm x MW ^ 24.45) x (hours per day exposed ^ 24) x blood:gas
(air) partition coefficient.
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T3
0)
+->
O
it
<
c
o
¦-4-'
o
ro
0.8
0.6
0.4
0.2
APPENDIX C. BMD MODELING OUTPUTS FOR 2-EE
Multistage Model with 0.95 Confidence Level
Multistage
BMDL
100 200 300 400 500 600 700 800
dose
16:51 05/17 2011
Figure C.l. Multistage BMD Model for Male Prostate Atrophy Data (NTP [1993a])
Text Output for Multistage BMD Model for Male Prostate Atrophy Data (NTP [1993a])
Multistage Model. (Version: 3.2; Date: 05/26/2010)
Input Data File: C:/USEPA/BMDS2l/Data/mst_2EE_prostate_atrophy_Mst-BMR10-
Restrict.(d)
Gnuplot Plotting File: C:/USEPA/BMDS21/Data/mst_2EE_prostate_atrophy_Mst-
BMR10-Restrict.pit
Tue May 17 16:51:03 2011
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose/sl-beta2*dose/s2) ]
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The parameter betas are restricted to be positive
Dependent variable = Incidence
Independent variable = Dose
Total number of observations = 5
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background =	0
Beta(l) =	0
Beta (2) = 1.6426e+014
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background -Beta(l)
have been estimated at a boundary point, or have been specified by
the user,
and do not appear in the correlation matrix )
Beta(2)
Beta (2)	1
Parameter Estimates
95.0% Wald Confidence
Interval
Variable	Estimate	Std. Err. Lower Conf. Limit Upper Conf.
Limit
Background	0	* * *
Beta(1)	0	* * *
Beta(2)	1.00902e-005	* * *
* - Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-12.8388
-15.6842
-34.4972
# Param's
5
1
1
Deviance Test d.f.
5.6909
43.3169
P-value
0.2235
<.0001
AIC:
33.3684
Goodness of Fit
Scaled
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Dose	Est._Prob. Expected Observed	Size	Residual
0.0000
0.0000
0.000
0.000
10
0. 000
109.0000
0.1130
1.130
0.000
10
-1.129
205.0000
0.3456
3.456
6.000
10
1. 692
400.0000
0.8010
8.010
7.000
10
-0.800
792.0000
0.9982
9.982
10.000
10
0.134
Chi^2 = 4.79	d.f. = 4	P-value = 0.3092
Benchmark Dose Computation
Specified effect =	0.1
Risk Type	=	Extra risk
Confidence level =	0.95
BMD =	102.185
BMDL =	33.6961
BMDU =	130.329
Taken together, (33.6961,	130.329) is a 90	% two-sided confidence
interval for the BMD
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Log-Logistic Model with 0.95 Confidence Level
Log-Logistic
0.8
0.7
0.6
0.5
0.4 BMDL IBMD
0	20 40 60 80 100 120 140 160
dose
15:08 12/21 2010
Figure C.2. Log-Logistic BMD Model for Major Fetal Skeletal Defect Data (Doe [1984b])
Text Output for Log-Logistic BMD Model for Major Fetal Skeletal Defect Data (Doe
[1984b])
Logistic Model. (Version: 2.13; Date: 10/28/2009)
Input Data File: C:/USEPA/BMDS2l/Data/lnl_2EE_skeletal_Lnl-BMR05-Restrict.(d)
Gnuplot Plotting File: C:/USEPA/BMDS21/Data/lnl_2EE_skeletal_Lnl-BMR05-
Restrict.pit
Tue Dec 21 15:08:43 2010
BMDS Model Run
The form of the probability function is:
P[response] = background+(1-background)/[1+EXP(-intercept-siope*Log(dose))]
Dependent variable = Percent_Positive
Independent variable = Dose
Slope parameter is restricted as slope >= 1
Total number of observations = 4
Total number of records with missing values = 0
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Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
User has chosen the log transformed model
Default Initial Parameter Values
background =	0.515
intercept =	-4.71869
slope =	1
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -slope
have been estimated at a boundary point, or have been specified by
the user,
and do not appear in the correlation matrix )
background intercept
background	1	-0.5 9
intercept	-0.59	1
Interval
Variable
Limit
background
intercept
slope
Parameter Estimates
Estimate
0.538916
-4.88826
1
Std. Err.
Indicates that this value is not calculated.
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-319.51
-319.985
-331.646
# Param's	Deviance	Test d.f.	P-value
4
2	0.950696	2	0.6217
1	24.2718	3	<.0001
AIC:
643.971
Dose
Goodness of Fit
Est._Prob. Expected Observed	Size
Scaled
Residual
0.0000
9.0000
46.0000
161.0000
Chi^2 =0.95
0.5389
0.5682
0.6576
0.7917
73.293
79.548
63.129
106.081
d.f. = 2
70.040	136
84.000	140
62.016	96
105.994	134
P-value = 0.6226
-0.559
0.760
-0.239
-0.019
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Benchmark Dos*
Specified effect
Risk Type
Confidence level
BMD
BMDL
Computation
0. 05
Extra risk
0. 95
6.98538
4.23203
FINAL
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Cullen, MR; Solomon, LR; Pace, PE; et al. (1992) Morphologic, biochemical, and cytogenetic
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Horimoto, M; Tassinari, MS; Isobe, Y; et al. (1996) Effects of ethylene glycol monoethyl ether
(EGME) on epididymal sperm parameters and male fertility in Sprague-Dawley (SD) rats.
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