United States
Environmental Protection
1=1 m m Agency
EPA/690/R-11/033F
Final
4-01-2011
Provisional Peer-Reviewed Toxicity Values for
2-Methoxyethanol
(CASRN 109-86-4)
and
2-Methoxyethanol Acetate
(CASRN 110-49-6)
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
Alan J. Weinrich, CIH, CAE
National Center for Environmental Assessment, Cincinnati, OH
DRAFT DOCUMENT PREPARED BY
SRC, Inc.
7502 Round Pond Road
North Syracuse, New York 13212
PRIMARY INTERNAL REVIEWERS
Dan D. Petersen, Ph.D., DABT
National Center for Environmental Assessment, Cincinnati, OH
Audrey Galizia, Dr. PH.
National Center for Environmental Assessment, Washington, DC
JeffS. Gift, PhD
National Center for Environmental Assessment, Research Triangle Park, NC
Jason C. Lambert, PhD, 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)

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TABLE OF CONTENTS
COMMONLY USED ABBREVIATIONS	ii
BACKGROUND	3
HISTORY	3
DISCLAIMERS	3
QUESTIONS REGARDING PPRTVS	4
INTRODUCTION	4
REVIEW 01 PERTINENT DATA	6
HUMAN STUDIES	6
Oral Exposure	6
Inhalation Exposure	6
ANIMAL STUDIES	9
Oral Exposure	9
Subchronic Studies	9
Reproductive Studies	22
Developmental Studies	37
Inhalation Exposure	44
Subchronic Studies	44
Reproductive Studies	45
Developmental Studies	46
OTHER STUDIES	50
Acute and Short-term Toxicity	51
Toxicokinetics	54
2-Methoxyethanol	54
2-Methoxyethanol Acetate	56
PBPK Models	57
Genotoxicity	57
DERIVATION OF SUBCHRONIC AND CHRONIC p-RfDS FOR
2-METHOXYETHANOL	58
DERIVATION OF SUBCHRONIC AND CHRONIC p-RfDs FOR
2-METHOXYETHANOL ACETATE	69
DERIVATION OF A SUBCHRONIC p-RfC FOR 2-METHOXYETHANOL	69
DERIVATION OF SUBCHRONIC AND CHRONIC p-RfCs FOR
2-METHOXYETHANOL ACETATE	76
PROVISIONAL CARCINOGENICITY ASSESSMENT FOR 2-METHOXYETHANOL
AND 2-METHOXYETHANOL ACETATE	78
WEIGHT-OF-EVIDENCE CLASSIFICATION	78
QUANTITATIVE ESTIMATES OF CARCINOGENIC RISK	78
REFERENCES	79
APPENDIX A. DETAILS OF BENCHMARK DOSE MODELING FOR
SUBCHRONIC AND CHRONIC p-RfDs	88
APPENDIX B. DETAILS OF BENCHMARK DOSE MODELING FOR
SUBCHRONIC p-RfC	94
l

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COMMONLY USED ABBREVIATIONS
BMC
benchmark concentration
BMD
benchmark dose
BMCL
benchmark concentration lower bound 95% confidence interval
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
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
p-OSF
provisional oral slope factor
p-RfC
provisional reference concentration (inhalation)
p-RfD
provisional reference dose (oral)
POD
point of departure
RfC
reference concentration (inhalation)
RfD
reference dose (oral)
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
11

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PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR
2-ME THOX YE THAN OL (CASRN 109-86-4) AND
2-METHOXYETHANOL ACETATE (CASRN 110-49-6)
BACKGROUND
HISTORY
On December 5, 2003, the U.S. Environmental Protection Agency's (EPA) Office of
Superfund Remediation and Technology Innovation (OSRTI) revised its hierarchy of human
health toxicity values for Superfund risk assessments, establishing the following three tiers as the
new hierarchy:
1)	EPA's Integrated Risk Information System (IRIS)
2)	Provisional Peer-Reviewed Toxicity Values (PPRTVs) used in EPA's Superfund
Program
3)	Other (peer-reviewed) toxicity values, including
~	Minimal Risk Levels produced by the Agency for Toxic Substances and Disease
Registry (ATSDR);
~	California Environmental Protection Agency (CalEPA) values; and
~	EPA Health Effects Assessment Summary Table (HEAST) values.
A PPRTV is defined as a toxicity value derived for use in the Superfund Program when
such a value is not available in EPA's IRIS. PPRTVs are developed according to a Standard
Operating Procedure (SOP) and are derived after a review of the relevant scientific literature
using the same methods, sources of data, and Agency guidance for value derivation generally
used by the EPA IRIS Program. All provisional toxicity values receive internal review by a
panel of six EPA scientists and external peer review by three independently selected scientific
experts. PPRTVs differ from IRIS values in that PPRTVs do not receive the multiprogram
consensus review provided for IRIS values. This is because IRIS values are generally intended
to be used in all EPA programs, while PPRTVs are developed specifically for the Superfund
Program.
Because new information becomes available and scientific methods improve over time,
PPRTVs are reviewed on a 5-year basis and updated into the active database. Once an IRIS
value for a specific chemical becomes available for Agency review, the analogous PPRTV for
that same chemical is retired. It should also be noted that some PPRTV documents conclude that
a PPRTV cannot be derived based on inadequate data.
DISCLAIMERS
Users of this document should first check to see if any IRIS values exist for the chemical
of concern before proceeding to use a PPRTV. If no IRIS value is available, staff in the regional
Superfund and Resource Conservation and Recovery Act (RCRA) program offices are advised to
carefully review the information provided in this document to ensure that the PPRTVs used are
appropriate for the types of exposures and circumstances at the Superfund site or RCRA facility
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in question. PPRTVs are periodically updated; therefore, users should ensure that the values
contained in the PPRTV are current at the time of use.
It is important to remember that a provisional value alone tells very little about the
adverse effects of a chemical or the quality of evidence on which the value is based. Therefore,
users are strongly encouraged to read the entire PPRTV document and understand the strengths
and limitations of the derived provisional values. PPRTVs are developed by the EPA Office of
Research and Development's National Center for Environmental Assessment, Superfund Health
Risk Technical Support Center for OSRTI. Other EPA programs or external parties who may
choose of their own initiative to use these PPRTVs are advised that Superfund resources will not
generally be used to respond to challenges of PPRTVs used in a context outside of the Superfund
Program.
QUESTIONS REGARDING PPRTVS
Questions regarding the contents of the PPRTVs and their appropriate use (e.g., on
chemicals not covered, or whether chemicals have pending IRIS toxicity values) may 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), or OSRTI.
2-Methoxyethanol (2-ME; see Figure 1) is a clear, colorless liquid that is used mainly as
a solvent and is miscible with water and other solvents. It is used as a solvent for many different
purposes such as varnishes, dyes, and resins, and as an additive in airplane deicing solutions
(ACGIH, 2006a). Similarly, 2-ME acetate (see Figure 2) is a colorless liquid used primarily as a
solvent in painting, furniture finishing, printing, semiconductor and other electronics industries
(ACGIH, 2006b). Both have high vapor pressures, so inhalation of the vapor is a likely route of
exposure. However, data from occupational studies indicate that the skin is the major route of
absorption in the workplace (Sparer et al., 1988; Piacitelli et al., 1990; Chang et al., 2004;
Kezic et al., 1997).
INTRODUCTION
Figure 1. 2-ME Structure
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Figure 2. 2-ME Acetate Structure
No RfD for 2-ME or 2-ME acetate is available on the IRIS (U.S. EPA, 2010a) database
or in the Drinking Water Standards and Health Advisories list (U.S. EPA, 2009). HEAST
(U.S. EPA, 2010b) lists a subchronic RfD of 0.01 mg/kg-day and a chronic RfD of
0.001 mg/kg-day for 2-ME, based on route-to-route extrapolation from an inhalation NOAEL of
31 mg/m for testicular effects in rabbits (Miller et al., 1983a), as originally derived in a Health
and Environmental Effects Profile (HEEP) for 2-ME (U.S. EPA, 1986). HEAST (U.S. EPA,
2010b) also lists subchronic and chronic RfDs of 0.02 and 0.002 mg/kg-day, respectively, for
2-ME acetate derived in a HEEP for 2-ME acetate (U.S. EPA, 1987). These RfDs were derived
by analogy to 2-ME based on in vitro evidence for metabolic hydrolysis of 2-ME acetate to
2-ME in nasal epithelium, lungs, liver, kidney, and blood of several animal species (Stott and
McKenna, 1985). The adjustment across chemicals was made by multiplying the RfD derived
for 2-ME (0.001 mg/kg-day) by the ratio of molecular weights for 2-ME and 2-ME acetate. In
addition to the HEEPs for 2-ME (U.S. EPA, 1986) and 2-ME acetate (U.S. EPA, 1987), the
Chemical Assessments and Related Activities (CARA) list (U.S. EPA, 1991, 1994a) reports a
Health Effects Assessment (HEA) for glycol ethers (U.S. EPA, 1984) that included 2-ME. HEA
declined to derive oral toxicity values due to inadequate data.
IRIS (U.S. EPA, 2010a) reports a RfC for 2-ME of 0.02 mg/m3 based on a NOAELhec of
17 mg/m3 for testicular effects in rabbits (Miller et al., 1983a) (modified by a total UF of 1000,
including a factor of 10 for extrapolation of subchronic effects data to chronic exposure, 10 for
protection of sensitive human populations, and a combined factor of 10 for interspecies
extrapolation [a dosimetric adjustment was used] and database deficiencies). The RfC
assessment, verified in 1990, was not reported in any EPA source document. HEAST
(U.S. EPA, 2010b) lists a subchronic RfC of 0.2 mg/m3 for 2-ME based on the same endpoint.
This assessment also was not attributed to any source document. CalEPA (2000a,b) derived
chronic inhalation reference exposure levels of 0.06 mg/m3 for 2-ME and 0.09 mg/m3 for 2-ME
acetate, extrapolated from Miller et al. (1983a) using an UF of 300. Occupational exposure
limits for 2-ME include an American Conference of Governmental Industrial Hygienists
(ACGIH, 2006a, 2010) threshold limit value-time weighted average (TLV-TWA) of 0.1 ppm
(0.3 mg/m3), a National Institute for Occupational Safety and Health (NIOSH, 2005)
recommended exposure limit-TWA of 0.1 ppm (0.3 mg/m ), and an Occupational Safety and
Health Administration (OSHA, 2010) permissible exposure limit-TWA (PEL-TWA) of 25 ppm
(80 mg/m3). For 2-ME acetate, ACGIH (2010, 2006b) recommends a TLV-TWA of 0.1 ppm
(0.5 mg/m ) derived by analogy to 2-ME. The NIOSH (2005) recommended exposure limit is
also 0.1 ppm, and the OSHA (2010) PEL-TWA is 25 ppm (120 mg/m3).
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An Environmental Health Criteria Document (WHO, 1990) that includes both 2-ME and
2-ME acetate is available, but no toxicity values were derived for either chemical. The chronic
toxicity and carcinogenicity of 2-ME and 2-ME acetate have not been assessed by ATSDR
(2010), IARC (2010), orNTP (2010, 2005).
Literature searches were performed in December 2006 and May 2010 for 2-ME (CASRN
109-86-4) and in July 2009 and May 2010 for 2-ME acetate (CASRN 110-49-6). For both
chemicals, literature searches included publications from the 1960s to the present for studies
relevant to the derivation of toxicity values. Databases searched included MEDLINE,
TOXLINE (with NTIS), BIOSIS, TSCATS/TSCATS2, CCRIS, DART, GENETOX, HSDB,
RTECS, Chemical Abstracts, and Current Contents (6 months prior to search date for each
chemical).
REVIEW OF PERTINENT DATA
Unless otherwise indicated, the statistical significance of the findings discussed below
was determined by the study authors.
HUMAN STUDIES
Oral Exposure
Young and Woolner (1946) reported a case of a 44-year-old man who ingested an
unknown but fatal amount (reported by the researchers to have been as much as one-half pint or
235 mL, purity unspecified) of a liquid thought to be 2-ME. Using the specific gravity of
965 mg/mL reported by the researchers for the ingested liquid, which matches the specific
gravity for pure 2-ME and a body weight of 70 kg, the ingested dose may have been up to
3240 mg/kg. Findings at autopsy were mainly hemorrhagic gastritis and toxic changes to the
liver and kidney.
Nitter-Hauge (1970) reported on two cases of accidental ingestion of-100 mL of 2-ME
of undisclosed purity. Using the density of 965 mg/mL for pure 2-ME and a body weight of
70 kg, the ingested dose was -1379 mg/kg in each case. Within 8-18 hours after ingestion, both
patients became confused and complained of general weakness and nausea. Both patients
exhibited rapid, deep respiration, as well as metabolic acidosis. Both cases made an uneventful
recovery.
No studies examining humans orally exposed to 2-ME acetate were located.
Inhalation Exposure
Human occupational studies, discussed below, have identified hematological and
neurological effects in workers exposed to 2-ME, although it has not been conclusively
demonstrated that 2-ME was responsible for the observed effects in any of these cases, because
in most cases, workers were exposed to a variety of chemicals. However, hematological effects
have been reported for 2-ME and 2-ME acetate in animals and in humans (Welch and Cullen,
1988; NIOSH, 1991; Larese et al., 1992; Shih et al., 2000, 2003).
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Cohen (1984) reported a single case of a worker presenting with subjective, reversible
neurological symptoms (apathy, fatigue, decreased appetite) and macrocytic anemia (an
asymptomatic hematological effect). The report followed both skin and inhalation exposure to a
mean airborne concentration of 35-ppm (110-mg/m3) 2-ME for 1-1.5 years. The symptomatic
findings were confounded by concurrent exposure to 1-5-ppm methyl ethyl ketone (2-butanone),
a known neurotoxicant at higher exposures, and 4.2-12.8-ppm propylene glycol monomethyl
ether (1-methoxypropanol).
Greenburg et al. (1938), Parsons and Parsons (1938), and Donley (1936) reported
hematological and neurological effects in several workers exposed to 2-ME while making
"fused" collar shirts. Neurological effects included encephalopathy, dizziness, fainting,
headache, weakness, ataxia, psychopathic disturbances, and personality changes. Anemia,
granulopenia, and respiratory tract irritation were found as well. The 2-ME content of the
solvents to which these workers were exposed ranged from <3 (with <3% dimethyl phthalate and
74% isopropanol) to 33% (with 67% denatured ethanol). Limited air analyses (single 2-2.5-hour
samples with pressing machines in operation and windows open or partially closed) in one plant
"3
with affected workers found air concentrations of 25-76 ppm (78-240 mg/m ) for 2-ME and
70-215 ppm for ethanol. The researchers considered it likely that actual exposure concentrations
were much higher due to worker habits (standing near dipping tanks, bending low over tanks
when working) and frequent clogging of exhaust ducts. Affected workers had been exposed for
>6 months. The central nervous system and hematological effects observed in this study have
been observed as well following exposure to ethanol or isopropanol alone, thus confounding the
possible association between 2-ME and the observed effects.
Case reports of five workers using 2-ME as a cleaning solvent for printing operations
described central nervous system depression, memory loss, narcolepsy, language difficulties, and
anemia following at least 7 months of exposure (Zavon, 1963). Experimental recreations of
work activities provided exposure estimates of 61-3960 ppm (190-12,300 mg/m3). Upon
institution of strict industrial hygienic practices including improvement of work area ventilation,
airborne 2-ME concentrations were reduced to -20 ppm (62 mg/m3). After exposure was
reduced, neurological symptoms were resolved, and hematological parameters returned to
normal in the case subjects.
A cross-sectional occupational exposure study (Cook et al., 1982) examined effects on
two groups of exposed workers at a facility involved in the manufacture and packaging of 2-ME.
Air sample measurements in the production area and packaging area, reported as 8-hour TWAs,
-3
were <0.42 ppm (<1.3 mg/m , unspecified number of samples collected in January 1976,
sampling times not reported) and 5.4-8.5 ppm (17-27 mg/m3, 8-hour TWA calculated from an
unspecified number of 2-hour samples collected from warehouse operators packaging 2-ME in
July 1980), respectively. However, the workers were exposed to various other chemicals as well,
including other ethylene glycol ethers; ethylene, propylene, and butylene oxides;
chlorobenzenes; brake fluids; and various organic amines. Control workers came from
manufacturing sites for alkanolamine and salicylic acid and may have been exposed to a variety
of other chemicals. There were a total of 53 exposed and 44 control workers eligible for the
study. The study group included 40 exposed workers and 25 controls, for which hematological
evaluations were made. Subgroups of six exposed and nine control workers were used for
fertility evaluations. Neurological effects were not evaluated. Duration of exposure was
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estimated for each participant from work history. These data were used in the analysis but were
not separately reported. Adjustments to the analysis were made for age and smoking status.
There were no statistically significant differences between groups in hematological parameters,
blood hormone concentrations, testicle size, sperm counts, or sperm motility. Study limitations,
including incomplete exposure information, lack of neurological data, and confounding
exposures among both exposed and control workers to other chemicals, preclude the
identification of a NOAEL from this study.
One shipyard was the subject of several publications where there were attempts to relate
2-ME exposures with urinary methoxyacetic acid (MAA) and to health effects. Welch and
Cullen (1988), McManus et al. (1989), Sparer et al. (1988), and Welch et al. (1988) reported
studies of shipyard painters exposed to a wide variety of paint solvents including
"3
2-ethoxyethanol and 2-ME. Mean 2-ME concentrations were 0.8 ±1.0 ppm (2.5 ±3.1 mg/m );
MAA was not found in any urine samples. These studies of shipyard painters indicated a
nonstatistically significant reduction in sperm parameters. However, the studies suffered from
methodological difficulties and the presence of confounding exposures, precluding identification
ofaLOAELorNOAEL.
Shih et al. (2000) reported hematological and spermatotoxic effects in a study of
53 copper-clad laminators in two semiconductor facilities. Control subjects were 121 laminate
workers with indirect exposure to 2-ME. When the two plants were considered together, the
hematological parameters (hemoglobinp < 0.0001), packed cell volumes (PCVs) (p < 0.0001),
and erythrocyte count (p < 0.001) were statistically significantly lower in exposed males than in
controls. Of the sperm parameters investigated, only sperm pH was statistically significantly
depressed (p < 0.005). The authors noted a positive correlation of airborne 2-ME with
erythrocyte counts (p < 0.003) and a positive correlation of urinary MAA with hemoglobin
(p < 0.001), packed erythrocytes (p < 0.001), and erythrocyte count (p < 0.001). The authors
concluded that at the current legal occupational exposure limit of 5 ppm (15 mg/m3) in Taiwan,
demonstrable hematological effects were seen, and there was a direct relationship between these
effects and MAA in urine, as well as 2-ME in air.
In a follow-up study of hematological effects at one plant, Shih et al. (2003) monitored
changes in hematological parameters initially, and at intervals of 3 and 6 months after the
implementation of aggressive engineering controls. The weekly geometric mean (GM) 8-hour
TWA 2-ME concentration for the exposed group before intervention was 9.62 ppm (geometric
standard deviation [GSD] = 4.75; n = 29; range 0.75-320), or 29.8 ± 14.7 mg/m . Three months
after intervention, the exposed group had a GM exposure of 2.34 ppm (GSD = 1.76; n = 29;
range 0.2-10) or 7.3 ± 5.5 mg/m3. Six months after intervention, the exposed group had a GM
exposure of 0.34 ppm (GSD = 2.69; n = 29; range 0.1-3.5) or 1.1 ± 8.3 mg/m3. Urinary MAA in
specimens collected after the last work shift of the workweek showed a GM of 50.7-mg/g
creatinine (GSD = 1.67; n = 29; range 24.3-139) at baseline. After the 3-month intervention, the
GM for MAA in the exposed group fell to 19.7-mg/g creatinine (GSD = 2.09; n = 29; range
4.60-54.9). After the 6-month intervention, the GM for MAA in the exposed group continued to
fall to 6.8-mg/g creatinine (GSD = 4.2; n = 29; range 0.95-25.2). As in the previous study, there
were statistically significant changes in exposed male workers compared to controls in
hemoglobin, packed erythrocytes, and erythrocyte count in males but not females. Airborne
2-ME at baseline before intervention was correlated with hematological changes in controls
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(p < 0.001). The correlation between MAA in urine at baseline and hematological effects in
male controls was more pronounced with hemoglobin (p = 0.005), packed erythrocytes
(p < 0.001), and erythrocytes (p < 0.001). Hematological changes from baseline to the reduced
exposures at 6 months progressed with increases seen in hemoglobin (p < 0.001), packed
erythrocytes (p < 0.001), and erythrocyte count (p < 0.001). The study confirmed that a GM
2-ME exposure of 0.34 ppm and a urinary MAA GM of 6.77 did not result in hematological
changes. Shih et al. (2003) demonstrated an 8 hours per day, 5 days per week, NOAEL of
1.1 ± 8.3 mg/m3 and LOAEL of 7.3 ± 5.5 mg/m3 for reversible hematologic effects in humans.
No data were located for humans exposed to 2-ME acetate by inhalation exposure.
ANIMAL STUDIES
Oral Exposure
Subchronic Studies
Nagano et al. (1979) gave groups of five male JCL-ICR mice gavage doses of 62.5, 125,
250, 500, 1000, or 2000 mg/kg-day of 2-ME or 2-ME acetate (purity not reported), 5 days/week,
for 5 weeks. A control group of 20 mice was used. Blood was collected after the final dose and
analyzed for erythrocyte and leukocyte counts, PCV, and hemoglobin concentrations. Testes
were excised, weighed, and subjected to histopathological examination. All endpoints were
compared statistically.
For 2-ME, Table 1 shows that statistically significantly reduced leukocyte counts
(54-77% lower than controls) were observed at >500 mg/kg-day, while erythrocyte counts and
hemoglobin concentrations were statistically significantly reduced (19-32 and 9-39% lower
than controls, respectively) at 1000 mg/kg-day (Nagano et al., 1979). Absolute and relative
vesicular and coagulating gland weights were statistically significantly lower than controls (37
and 24%), respectively) at 1000 mg/kg-day. Absolute and relative testes weights were
statistically significantly lower than controls (44-81%) and 45-80%), respectively) at
>250 mg/kg-day. A clear reduction in spermatocytes was observed in the seminiferous tubules
at 250 mg/kg-day (incidence data not reported). Seminiferous tubule atrophy was seen at
500 mg/kg-day, while complete absence of germ cells occurred at 1000 mg/kg-day (data not
shown). This study identified a LOAEL of 250-mg/kg-day 2-ME, with an associated NOAEL of
125 mg/kg-day, for testicular effects in mice.
For 2-ME acetate, Table 1 shows the statistically significantly reduced leukocyte counts
(71-79%o lower than controls) and hemoglobin concentrations (10%> lower than controls) were
observed at >1000 and 2000 mg/kg-day, respectively (Nagano et al., 1979). Absolute and
relative vesicular and coagulating gland weights were statistically significantly lower than
controls (35 and 32%>, respectively) at 2000 mg/kg-day. Absolute and relative testes weights
were statistically significantly lower than controls (63-76 and 64-75%), respectively) at
>500 mg/kg-day. A clear reduction in spermatocytes was observed in the seminiferous tubules
at 500 mg/kg-day (incidence data not reported). Tubule atrophy was seen at 1000 mg/kg-day,
while complete absence of germ cells occurred at 2000 mg/kg-day. This study identified a
5 days/week, 5-week LOAEL of 500 mg/kg-day, with an associated NOAEL of 250 mg/kg-day,
for testicular effects for 2-ME acetate exposure in mice.
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Table 1. Selected Changes in Male Mice Treated with 2-Methoxyethanol or
2-Methoxyethanol Acetate by Gavage 5 Days/Week for 5 Weeks

Dose in mg/kg-day
Control
62.5
125
250
500
1000
2000
2-Methoxyethanol
Number of animals
examined
20
5
5
5
5
5
5
Hematology
WBC (/mm3)
3840 ± 1572a
3270±1714
4250 ±
2100
3470 ±2164
1770 ± 392b
1000 ± 221b
900
RBC (/mm3)
764 ± 79
772 ±31
694 ± 93
771 ± 93
760±110
617 ± 43b
517
Hgb (g/dL)
12.7 ±0.9
12.7 ±0.5
12.8 ±0.6
12.6 ±0.1
12.4 ±0.5
11.6 ± 1.0b
7.8
Organ weights
Testes, absolute (mg)
291 ±25
263 ± 32
300 ± 28
162 ± 37b
82 ± 13b
74 ± 8b
54
Testes, relative (%)
0.76 ±0.08
0.68 ±0.14
0.79 ±0.10
0.42 ± 0.10b
0.22 ± 0.03b
0.10 ± 0.02b
0.15
Vesicular and
coagulating glands,
absolute (mg)
376 ± 59
354 ±73
373 ±30
361 ±26
323 ± 66
237 ± 25b
258
Vesicular and
coagulating glands,
relative (%)
0.99 ±0.16
0.95 ±0.12
0.98 ±0.06
0.94 ±0.09
0.86 ±0.17
0.75 ± 0.03b
0.77
2-Methoxyethanol Acetate
Number of animals
examined
20
5
5
5
5
5
5
Hematology
WBC (/mm3)
3840 ± 1572
3430 ± 849
3980 ± 990
3890±1634
2940 ± 634
1030 ± 452b
750 ± 297b
Hgb (g/dL)
12.7 ±0.9
12.3 ±0.5
12.5 ±0.4
12.8 ± 1.2
12.7 ± 1.3
12.0 ±0.7
11.4 ± 0.4°
Organ weights
Testes, absolute (mg)
291 ±25
315 ±22
270 ± 23
289 ± 56
106 ± 10b
73 ± 7b
69 ± 5b
Testes, relative (%)
0.76 ±0.03
0.82 ±0.03
0.68 ±0.09
0.71 ±0.12
0.27 ± 0.01b
0.19 ± 0.02b
0.19 ± 0.02b
Vesicular and
coagulating glands,
absolute (mg)
376 ± 59
369 ± 70
353 ±53
331 ±40
356 ±86
328 ±48
214 ± 26b
Vesicular and
coagulating glands,
relative (%)
0.99 ±0.16
0.93 ±0.17
0.89 ±0.09
0.87 ±0.13
0.90 ±0.19
0.92 ±0.15
0.67 ± 0.04b
aMean± SD.
bSignificantly different from control atp< 0.01.
Significantly different from control atp< 0.05.
Source: Nagano et al. (1979).
The National Toxicology Program (NTP, 1993) conducted 13-week drinking water
toxicity studies in F344/N rats and B6C3F1 mice. In rats, 10 animals/sex/group were offered 0,
750-, 1500-, 3000-, 4500-, or 6000-ppm 2-ME (98% purity) in drinking water. Based on body
weight and water consumption data, average doses were calculated by the study authors to be 0,
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71, 165, 324, 715, or 806 mg/kg-day for males and 0, 70, 135, 297, 546, or 785 mg/kg-day for
females, respectively. Rats were observed twice daily for general health. Weekly evaluations
were made for clinical signs, water consumption, and body weights. Blood and urine samples
were collected at Week 13. Observed hematological and clinical chemistry parameters included
hematocrit, hemoglobin, and methemoglobin concentrations; total and nucleated erythrocyte,
reticulocyte, platelet, and total and differential leukocyte counts; mean corpuscular volume
(MCV); mean corpuscular hemoglobin (MCH); mean cell hemoglobin concentration (MCHC);
total bone marrow cellularity; and serum concentrations of urea nitrogen, creatinine, total
protein, albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine
kinase, and bile acids. Urinalysis included urine volume, specific gravity, and pH. At necropsy,
-38 tissues were collected from the control and high-dose groups and subjected to
histopathological evaluation. Lower-dose groups were examined for histopathological changes
of the bone marrow, ovary, preputial or clitoral gland, prostate gland, seminal vesicle, spleen,
stomach, thymus, and uterus. Males in the <3000-ppm groups were examined for epididymal
weights and measurement of spermatozoal parameters and sperm motility. Females in the
<4500-ppm groups were examined for body weights, estrous cycle length, and vaginal cytology.
All rats in the 6000-ppm groups died by Week 5 (males) or 7 (females), with 8/10 males
and 5/10 females in the 4500-ppm groups dying by the end of the study (NTP, 1993). No
mortality was seen at <3000 ppm in rats of either sex. Clinical signs from observations of all
rats, deemed by the study authors to be treatment-related, were tremors, diarrhea, emaciation,
abnormal posture, pallor, tachypnea, hypoactivity, and comatose state (relevant doses and
incidences not reported). Final mean body weights for males and females exposed to >1500 ppm
(6000 ppm not examined due to early mortality) were statistically significantly (p < 0.01)
decreased (10-56%) in a dose-related manner, relative to controls. Mean water consumption
was decreased in male rats (11-22%) exposed to >3000 ppm and in female rats (13—15%)
exposed to >1500 ppm; clinical signs of dehydration (altered posture and appearance) were seen
in these groups. In males exposed to >750 ppm, dose-related anemia was evident at Week 3 of
exposure, as determined by statistically significant decreases in hematocrit and hemoglobin (see
Table 2). However, by Week 13, hemoglobin in the 750-ppm group, hematocrit in the 750- and
3000-ppm groups, and erythrocyte counts in the 750-, 1500-, and 3000-ppm groups had
increased to levels that were not statistically significantly different from controls. Dose-related
decreases also were observed in reticulocytes, platelets, leukocytes, segmented neutrophils,
lymphocytes, and total bone marrow cellularity. MCV also exhibited dose-related decreases at all
but the highest dose.
In females, Table 3 shows statistically significant anemic effects, including decreased
hematocrit, hemoglobin, and erythrocytes generally were not seen in rats exposed to drinking
water concentrations below 1500 ppm (NTP, 1993). Statistically significant dose-related
decreases in leukocytes were seen at 750-1500 ppm. Statistically significant changes in clinical
chemistry in males were transient, with dose-related reductions in albumin and alkaline
phosphatase appearing at Weeks 1 or 3 in groups receiving >750 ppm (see Table 4). Bile acids
were statistically significantly higher than controls at Week 3, with dose-related increases being
observed at >750 ppm. However, concentrations of bile acids were similar to controls in all
groups after 13 weeks. In females, statistically significant dose-related decreases in multiple
clinical chemistry parameters were seen as early as Week 1 and through Week 13 in groups
given >750 ppm (see Table 5).
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Table 2. Selected Hematological Effects Observed in Male Rats Exposed to
2-Methoxyethanol in Drinking Water for 13 Weeks
Parameter"
Control
Concentration in ppm (Dose in mg/kg-day)
750 (71)
1500 (165)
3000 (324)
4500 (715)
6000 (806)
Hematocrit (%)
Week 1
46.1 ± 0.6a
45.8 ±0.5
46.2 ±0.6
44.3 ±0.4
45.4 ±0.3
43.6 ± 0.5b
Week 3
49.3 ±0.6
45.6 ± 0.6b
46.2 ± 0.6b
41.6 ± 0.5b
NT
NT
Week 13
48.1 ±0.4
46.9 ±0.6
45.4 ± 0.7b
46.0 ±0.8
31.6 ± 7.0b
NT
Hgb (g/dL)
Week 1
15.0 ±0.1
14.7 ±0.1
14.8 ±0.2
14.3 ± 0.1b
14.4 ± 0.2b
13.9 ± 0.2b
Week 3
16.0 ±0.2
14.9 ± 0.2b
14.9 ± 0.2b
13.8 ± 0.1b
NT
NT
Week 13
16.0 ±0.2
15.5 ±0.2
15.2 ± 0.2b
14.9 ± 0.2b
10.1 ± 1.9b
NT
Erythrocytes (106/jxL)
Week l
7.88 ±0.12
7.88 ±0.10
7.96 ±0.12
7.60 ± 0.07
7.70 ±0.11
7.44 ±0.11
Week 3
8.80 ±0.10
8.32 ±0.14
8.47 ±0.14
7.61 ± 0.10b
NT
NT
Week 13
9.44 ±0.11
9.40 ±0.13
9.20 ±0.13
9.08 ±0.16
5.94 ± 1.24
NT
Reticulocytes (106/jxL)
Week 1
0.22 ±0.03
0.27 ± 0.02
0.21 ±0.02
0.12 ±0.02
0.07 ± 0.01b
0.05 ± 0.01b
MCV (fL)
Week 3
56.1 ±0.3
54.9 ± 0.4b
54.6 ± 0.2°
54.8 ± 0.2°
NT
NT
Week 13
50.9 ±0.3
49.8 ±0.5
49.1 ± 0.2°
50.8 ±0.2
53.0 ± 1.0
NT
Platelets (103/|iL)
Week 1
937.5 ±31.3
864.8 ± 12. lb
791.8 ± 13.0°
492.5 ± 18.6°
338.1 ± 21.0°
276.2 ± 20.8°
Week 3
797.7 ± 13.3
730 ± 16.5°
568.7 ± 11.8°
267.7 ±7.9C
NT
NT
Week 13
582.4 ± 12.1
612.8 ± 18.0
490.9 ± 13.5°
401 ± 33.8°
265 ± 53.5°
NT
Leukocytes (103/jxL)
Week 1
7.87 ±0.51
7.45 ±0.45
7.05 ±0.37
4.94 ± 0.29°
3.37 ± 0.34°
2.92 ± 0.22°
Week 3
8.49 ±0.40
7.68 ±0.35
6.81 ± 0.46°
4.81 ± 0.19°
NT
NT
Week 13
7.49 ±0.63
8.51 ±0.73
6.47 ±0.61
6.18 ±0.54
1.80 ± 0.30b
NT
Segmented neutrophils (10 7|iL)
Week 1
0.96 ±0.12
0.75 ±0.08
1.04 ±0.13
0.77 ±0.12
0.51 ± 0.13b
0.39 ± 0.05°
Week 3
1.02 ±0.06
1.07 ±0.08
0.68 ± 0.08°
0.63 ± 0.11°
NT
NT
Week 13
1.26 ±0.20
1.19 ± 0.14
1.06 ±0.16
0.79 ± 0.07b
0.25 ± 0.06°
NT
Lymphocytes (103/jxL)
Week 1
6.97 ±0.56
6.80 ±0.38
5.71 ±0.32
4.09 ± 0.18°
2.78 ± 024°
2.50 ± 0.21°
Week 3
7.36 ±0.43
6.47 ±0.31
6.03 ± 0.41b
4.14 ± 0.20°
NT
NT
Week 13
6.09 ±0.45
7.17 ±0.61
5.32 ±0.51
5.19 ±0.45
1.51 ± 0.25b
NT
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Table 2. Selected Hematological Effects Observed in Male Rats Exposed to

2-Methoxyethanol in Drinking Water for 13 Weeks



Concentration in ppm (Dose in mg/kg-day)
Parameter"
Control
750 (71)
1500 (165)
3000 (324)
4500 (715)
6000 (806)
Total bone marrow cellularity (106/femur)
Week 1
70.6 ±2.7
NT
66.6 ±3.2
53.5 ± 2.9°
32.7 ± 2.0°
25.5 ± 1.2°
Week 3
66.1+2.9
82.2 ±3.6
75.1 ±3.9
53.2 ±2.4
NT
NT
Week 13
66.0 ±2.9
71.1 ±3.0
58.4 ±2.1
57.0 ± 2.2b
31.4 ± 12.2°
NT
aMean ± standard error.
bSignificantly different (p < 0.05) from controls by Dunn's or Shirley's test.
Significantly different (p < 0.01) from controls by Dunn's or Shirley's test.
NT = not tested.
Source: NTP (1993).
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Table 3. Selected Hematological Effects Observed in Female Rats Exposed
to 2-Methoxyethanol in Drinking Water for 13 Weeks
Parameter
Control
Concentration in ppm (Dose in mg/kg-day)
750 (70)
1500 (135)
3000 (297)
4500 (546)
6000 (785)
Hematocrit (%)
Week 1
46.8 ± 0.4a
45.6 ±0.7
44.8 ± 0.6b
43.2 ± 0.5°
43.7 ± 0.6°
43.4 ± 0.7°
Week 3
48.6 ±0.6
48.4 ±0.5
47.4 ±0.6
43.1 ± 1.2°
NT
NT
Week 13
44.5 ±0.4
43.8 ±0.4
42.2 ± 0.8b
41.5 ± 0.5°
40.7 ± 0.9°
NT
Hgb (g/dL)
Week 1
15.8 ±0.1
15.4 ±0.2
15.1 ± 0.2b
14.5 ±0.1°
14.8 ± 0.2°
14.9 ± 0.2°
Week 3
16.0 ±0.1
15.8 ±0.1
15.8 ±0.2
14.3 ± 0.2°
NT
NT
Week 13
15.2 ±0.1
14.8 ± 0.1°
14.5 ± 0.2°
13.7 ± 0.1°
13.6 ± 0.3°
NT
Erythrocytes (106/jxL)
Week l
8.14 ±0.09
7.94 ±0.12
7.86 ±0.12
7.43 ± 0.10°
7.66 ± 0.15°
7.57 ± 0.12°
Week 3
8.73 ±0.11
8.80 ±0.12
8.85 ±0.14
8.09 ±0.22
NT
NT
Week 13
8.34 ±0.09
8.30 ±0.09
8.24 ±0.13
8.13 ±0.12
7.91 ±0.18
NT
Reticulocytes (106/jxL)
Week 1
0.22 ± 0.02
0.15 ± 0.02b
0.09 ± 0.00°
0.05 ± 0.01°
0.03 ± 0.00°
0.03 ± 0.00c
MCV (fL)
Week 3
55.7 ±0.3
55.1 ±0.2
53.6 ± 0.3°
53.2 ± 0.2°
NT
NT
Week 13
53.3 ±0.3
52.8 ±0.2
51.3 ± 0.3°
50.9 ± 0.3°
51.6 ± 0.2°
NT
MCH (pg)
Week 13
18.3 ±0.2
17.9 ±0.1
17.6 ± 0.1°
16.9 ± 0.2°
17.3 ± 0.1°
NT
Platelets (103/|iL)
Week 1
852.8 ± 19.7
775.3 ± 14.6b
539.0 ± 12.9°
261.6 ± 10.6C
180.1 ±22.3C
159.9 ±21.7C
Week 3
861.4 ±20.1
658.0 ± 11.3°
531.1 ± 13.7°
349.6 ±20.7C
NT
NT
Week 13
658.9 ±24.3
650.6 ± 12.0
534.9 ±25.4C
400.7 ± 27.2°
376.0 ±32.0C
NT
Leukocytes (10 7|iL)
Week 1
9.24 ±0.36
7.35 ± 0.35°
5.80 ± 0.39°
4.49 ± 0.23°
3.51 ± 0.37°
3.45 ± 0.30°
Week 3
7.87 ±0.56
7.48 ±0.39
8.24 ±0.61
5.36 ± 0.52b
NT
NT
Week 13
7.14 ±0.23
6.76 ±0.18
5.74 ± 0.26°
4.16 ± 0.45°
4.62 ± 0.50°
NT
Segmented neutrophils (10 7|iL)
Week 1
1.07 ±0.19
0.69 ±0.07
0.75 ±0.08
0.54 ± 0.06°
0.42 ± 0.08°
0.43 ±0.10c
Week 13
0.94 ±0.14
0.97 ±0.12
0.75 ±0.06
0.48 ± 0.08°
0.53 ± 0.15b
NT
Lymphocytes (103/jxL)
Week 1
8.02 ±0.31
6.55 ± 0.34°
4.96 ± 0.36°
3.91 ± 0.19°
3.03 ± 0.33°
2.95 ± 0.24°
Week 13
6.08 ±0.34
5.63 ±0.12
4.80 ± 0.24°
3.56 ± 0.43°
4.00 ± 0.46°
NT
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Table 3. Selected Hematological Effects Observed in Female Rats Exposed

to 2-Methoxyethanol
in Drinking Water for 13 Weeks



Concentration in ppm (Dose in mg/kg-day)
Parameter
Control
750 (70)
1500 (135)
3000 (297)
4500 (546)
6000 (785)
Total bone marrow cellularity (106/femur)
Week 1
55.2 ±2.4
NT
43.6 ± 2.0°
25.9 ± l.lc
21.5 ± 1.4°
19.9 ± 1.3°
Week 3
46.2 ± 1.4
40.6 ± 1.8b
34.7 ± 1.5°
30.2 ± 2.7°
NT
NT
Week 13
38.9 ± 1.7
45.5 ± 1.3
42.6 ± 1.8
33.0 ±2.7
39.1 ±2.2
NT
aMean ± standard error.
bSignificantly different (p < 0.05) from controls by Dunn's or Shirley's test.
Significantly different (p < 0.01) from controls by Dunn's or Shirley's test.
NT = not tested.
Source: NTP (1993).
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Table 4. Selected Clinical Chemistry Effects Observed in Male Rats

Exposed to 2-Methoxyethanol in Drinking Water for 13 Weeks



Concentration in ppm (Dose in mg/kg-day)
Parameter
Control
750 (71)
1500 (165)
3000 (324)
4500 (715)
6000 (806)
Total protein (g/dL)
Week 13
6.6 ± 0.1a
6.4 ±0.1
6.2 ± 0.1b
6.0 ± 0.1b
5.0 ± 0.1b
NT
Albumin (g/dL)
Week 1
3.4 ±0.0
3.4 ±0.1
3.3 ±0.1
3.2 ± 0.1c
3.2 ± 0.1c
3.0 ± 0.1b
Week 3
3.7 ±0.0
3.5 ± 0.1c
3.6 ± 0.0C
3.3 ± 0.1b
NT
NT
Week 13
3.6 ±0.1
3.5 ±0.1
3.5 ±0.0
3.5 ±0.0
2.8 ± 0.3b
NT
Alkaline phosphatase (IU/L)
Week 1
442 ± 8
401± 10b
364± 10b
321± 14b
317± 18b
308 ± 8b
Week 3
271 ±5
281 ± 10
238 ± 8C
137 ±5b
NT
NT
Bile acids (|imol/L)
Week 3
9.30 ± 1.56
11.44 ± 1.36c
23.50 ±4.66b
33.78 ±7.85b
NT
NT
aMean ± standard error.
bSignificantly different (p < 0.01) from controls by Dunn's or Shirley's test.
Significantly different (p < 0.05) from controls by Dunn's or Shirley's test.
NT = not tested.
Source: NTP (1993).
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Table 5. Selected Clinical Chemistry Effects Observed in Female Rats

Exposed to 2-Methoxyethanol in Drinking Water for 13 Weeks


Concentration in ppm (Dose in mg/kg-day)
Parameter
Control
750 (70)
1500 (135)
3000 (297)
4500 (546)
6000 (785)
Urea nitrogen (mg/dL)
Week 3
16.8 ± 0.4a
17.4 ±0.7
20.3 ± 0.7b
23.2 ± 0.7b
NT
NT
Week 13
22.3 ± 1.4
19.2 ± 0.6b
19.0 ±l.lb
18.8 ± 1.1°
18.4 ± 1.9°
NT
Creatinine (mg/dL)
Week 3
0.59 ±0.02
0.57 ±0.02
0.54 ±0.02
0.52 ± 0.02°
NT
NT
Week 13
0.55 ±0.02
0.51 ±0.02
0.47 ± 0.02b
0.48 ± 0.04b
0.52 ±0.04
NT
Total protein (g/dL)
Week 1
6.1 ±0.1
5.7 ± 0.1b
5.5 ± 0.1b
5.2 ± 0.1b
5.1 ± 0.1b
5.3 ± 0.1b
Week 3
6.0 ±0.1
5.7 ± 0.1°
5.6 ± 0.1°
5.4 ± 0.1b
NT
NT
Week 13
6.6 ±0.1
6.4 ±0.1
6.1 ±0.1b
5.9 ± 0.1b
5.8 ± 0.1b
NT
Albumin (g/dL)
Week 1
3.4 ±0.0
3.4 ±0.1
3.2 ± 0.0b
3.1 ± 0.1b
3.0 ± 0.1b
3.1 ± 0.1b
Week 3
3.6 ±0.1
3.5 ±0.1
3.5 ±0.1
3.2 ± 0.1b
NT
NT
Week 13
3.79 ±0.07
3.62 ±0.07
3.62 ± .003
3.57 ± 0.08°
3.46 ± 0.09b
NT
Alkaline phosphatase (IU/L)
Week 1
333 ±7
285 ± 7b
257 ± 7b
251 ±6b
227 ± 12b
242 ± 8b
Week 3
188 ±5
175 ±11
120 ± 5b
85 ± 4b
NT
NT
Week 13
192 ± 10
171 ± 10
157 ±12°
155 ± 13°
137 ±9b
NT
Bile acids (|imol/L)
Week 1
6.20 ±0.49
5.57 ±0.57
8.88 ± 1.95
22.70 ± 4.16b
13.33 ±2.46c
21.22 ± 3.84b
Week 3
11.75 ±2.46
23.00 ±5.21°
18.80 ±2.93
31.80 ± 7.12b
NT
NT
aMean ± standard error.
bSignificantly different (p < 0.01) from controls by Dunn's or Shirley's test.
Significantly different (p < 0.05) from controls by Dunn's or Shirley's test.
NT = not tested.
Source: NTP (1993).
Table 6 shows that statistically significant, dose-related decreases in relative thymus
weights among female rats and absolute thymus weights in male and females were observed in
all dose groups (NTP, 1993). The decreases from control were -25% at 750 ppm, ranging up to
-75% at 4500 ppm. In male rats, testis weights showed a similar pattern, with statistically
significant, dose-related decreases of 50-80% in absolute and relative weights at doses of 1500
to 4500 ppm. Weight changes in other organs (heart, lung, liver, and kidney) showed a pattern
of decreases in absolute weights and increases in relative weights, primarily at >3000 ppm; the
study authors attributed the findings in these organs to low body weights.
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Table 6. Selected Organ Weights Measured in Rats Exposed To 2-Methoxyethanol in
Drinking Water for 13 Weeks
Parameter
Sex
Concentration in ppm (Dose in mg/kg-day)
Control
750
(70-71)
1500
(135-165)
3000
(297-324)
4500
(546-715)
Absolute right testis weight
(g)
Male
1.398 ± 0.048s
1.411 ±0.019
0.603 ± 0.044b
0.442 ±0.032b
0.254 ±0.010b
Relative right testis weight
(g/g body weight)
Male
4.44 ± 0.15
4.81 ±0.09
2.31 ± 0.14b
2.07 ± 0.15b
1.89 ± 0.20c
Absolute thymus weight (g)
Male
0.268 + 0.026
0.198 ±0.017c
0.160 ± 0.016b
0.095 ±0.016b
0.072 ±0.005b
Relative thymus weight (g/g
body weight)
Male
0.85 ±0.08
0.67 ±0.05
0.61 ±0.06
0.45 ± 0.07b
0.53± 0.04
Absolute thymus weight (g)
Female
0.224 ±0.010
0.180 ± 0.012c
0.125 ±0.010b
0.084 ±0.008b
0.099 ± 0.01 lb
Relative thymus weight (g/g
body weight)
Female
1.19 ± 0.06
0.95 ± 0.06b
0.74 ± 0.06b
0.57 ± 0.06b
0.66 ± 0.07b
"Mean ± standard error
bSignificantly different from controls (p < 0.01) by Shirley's test.
Significantly different from controls (p < 0.05) by Shirley's test.
Source: NTP (1993).
Table 7 shows histopathological changes in the rat testes, which consisted of a
dose-related degeneration of the germinal epithelium in the seminiferous tubules that increased
in intensity from minimal at 750 ppm to marked at >3000 ppm (NTP, 1993). Chemical-related
fibrosis of the splenic capsule was seen in male and female rats and was most prominent in
animals in the 1500-4500-ppm groups. Only one (male) animal—dying by Week 5 in the
6000-ppm group—had time to develop splenic capsule fibrosis (see Table 7). The study authors
associated other microscopic changes with decreased body weight gain or stress-related
physiological illness. Spermatozoal measurements, including spermatid heads/g testis, sperm
motility, and sperm concentration, were statistically significantly and markedly decreased for
males exposed to 1500 or 3000 ppm (see Table 8). In females, mean estrous cycle length was
not affected by dose. However, the incidence of rats with estrous cycles of >12 days increased
with dose and was statistically significantly different from controls in the 3000-ppm group (see
Table 8). This study identified a 13-week LOAEL of 750 ppm (70-71 mg/kg-day) for testicular
lesions (degeneration of seminiferous tubules), reduced semen quality (reduced sperm
concentration), and decreased thymus weights in male rats and decreased thymus weights in
female rats; no NOAEL was identified in either gender.
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Table 7. Incidences of Selected Nonneoplastic Lesions Observed in Rats Exposed to
2-Methoxyethanol in Drinking Water for 13 Weeks
Lesion
Sex
Concentration in ppm (Dose in mg/kg-day)
Control
750
(70-71)
1500
(135-165)
3000
(297-324)
4500
(546-715)
6000
(785-806)
Degeneration of
seminiferous tubules
Male
0/10
7/10a
(1.0)b
10/103
(2.6)
10/10a
(4.0)
9/10a
(4.0)
10/10a
(4.0)
Splenic capsule fibrosis
Male
0/10
1/10
(1.0)
4/10a
(1.5)
10/103
(2.2)
5/9a
(1.2)
1/10
(1.0)
Splenic capsule fibrosis
Female
0/10
0/10
3/10
(1.0)
5/10a
(1.2)
0/10
0/10
'Significantly different from controls (p < 0.05) by Fisher's exact test, calculated for this assessment.
bParentheses indicate lesion severity averaged over all animals with lesion; 1 = minimal, 2 = mild, 3 = moderate, 4 = marked.
Source: NTP (1993).
Table 8. Reproductive Effects Observed in Rats Exposed to
2-Methoxyethanol in Drinking Water for 13 Weeks
Parameter
Sex
Concentration in ppm (Dose in mg/kg-day)
Control
750
(70-71)
1500
(135-165)
3000
(297-324)
4500
(546-715)
Spermatid heads (107/g testes)3
Male
9.14 ±0.32
8.63 ±0.33
1.79 ± 0.52b
0±0b
NT
Spermatid heads (107/testes)a
Male
13.69 ±0.63
12.84 ±0.48
1.41 ± 0.50b
o±ob
NT
Spermatid concentration
(mean/10 4 mL suspension)3
Male
68.43 ±3.17
64.20 ±2.42
7.03 ± 2.5 lb
o±ob
NT
Spermatozoal motility (%)a
Male
98.43 ±0.15
97.49 ±0.39
0±0b
o±ob
NT
Spermatozoal concentration
(106/g caudal epididymal
tissue)3
Male
755.4 ±25.6
655.8 ±
14.r
13.0 ± 3.4b
7.2 ± 2.2b
NT
Incidence of estrous cycle
longer
than 12 days
Female
1/10
NT
4/10
7/10d
3/5
aMean ± standard error.
bSignificantly different from controls (p < 0.01) by Shirley's test.
Significantly different from controls (p < 0.05) by Shirley's test.
dSignificantly different from controls (p < 0.05) by Fisher's exact test.
NT = not tested.
Source: NTP (1993).
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Mortality was not observed in either sex at any dose (NTP, 1993). Terminal body
weights of males exposed to 10,000 ppm and females exposed to 8000 or 10,000 ppm were
statistically significantly (p < 0.01) lower than controls (25% for males, 10-20% with increasing
dose for females). Water consumption was variable, but no treatment-related patterns were
evident. Hematologic and clinical chemistry evaluations were not performed in mice. Males
exposed to >8000 ppm showed statistically significantly (p < 0.05) increased relative weights of
the heart (9—30%), kidney (25-40%), and lung (13—26%). Treated females showed increased
relative weights of the hearts (21—29%) at >4000 ppm, kidneys (25-41%) at >8000 ppm, and
lungs (20-30%)) at >6000 ppm. The study authors attributed these increases in relative organ
weights to low body weights. Absolute and relative testis weights were decreased (16—81%) in
male mice exposed to >4000 ppm, while absolute and relative thymus weights were decreased in
males (16—50%) exposed to >8000 ppm and in females (23-46%) exposed to 10,000 ppm
(absolute only at 8000 ppm).
Upon histological examination, increased hematopoiesis was observed in the spleens of
all groups of treated mice except males in the lowest-dose group; the severity was minimal in all
groups (see Table 9) (NTP, 1993). Testicular degeneration was observed in males at 4000 ppm
(3/10). However, because of the small number of rats per dose group, the increased incidence
was not statistically significant until >6000 ppm, the concentration at which all rats exhibited this
effect (see Table 9). In the adrenal gland of 100% of the female mice in all dose-groups, there
was hypertrophy of the X-zone that increased in severity with dose from mild at 2000 ppm to
moderate-to-marked at 10,000 ppm. Spermatozoal concentrations were statistically significantly
lower than controls (reduced 26-79%) in males treated with >2000 ppm (see Table 10).
Although sperm motility was statistically significantly decreased in the 2000-ppm males relative
to controls, > 99% of sperm were judged to be motile. Thus, the statistically significant changes
in sperm motility at 2000 ppm were not considered toxicologically significant. Female mice in
the >6000-ppm groups differed significantly from controls in the relative frequency of time spent
in estrous stages (see Table 10). The LOAEL in mice was 2000 ppm for decreased spermatozoal
concentration and motility in male mice (295 mg/kg-day) and splenic hematopoiesis and adrenal
hypertrophy in females (492 mg/kg-day); no NOAEL was identified.
The Mellon Institute (1962) studied the effects of dietary exposure to 2-ME (purity not
reported) on DW albino rats. Groups of 10 rats/sex were exposed to 0, 0.01, 0.05, 0.25, or
1.25% of 2-ME in the diet for 3 months. Due to increased mortality, the 1,25%-groups were
terminated early (Day 18 of exposure). Based on food consumption and body-weight data, the
study authors calculated average daily doses of 7, 40, or 178 mg/kg-day for males and 8, 43, or
201 mg/kg-day for females in the 0.01-, 0.05-, and 0.25%-groups, respectively. Animals were
weighed weekly, and food consumption was measured monthly. At the conclusion of exposure,
animals were sacrificed, the liver and kidneys were weighed, and a gross examination of the
organs was conducted. The bladders were examined for concretions, and sections of
representative (unspecified) tissues were taken and fixed for examination.
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Table 9. Incidences of Selected Nonneoplastic Lesions Observed in Mice
Exposed to 2-Methoxyethanol in Drinking Water for 13 Weeks



Concentration in ppm (Dose in mg/kg-day)



2000
4000
6000
8000
10,000
Lesion
Sex
Control
(295-492)
(529-902)
(765-1194)
(992-1489)
(1367-1839)
Splenic hematopoiesis
Male
0/10
0/10
10/103
9/10a
9/10a
10/103




(1.0)b
(1.0)
(1.0)
(1.1)
Testicular degeneration
Male
0/10
0/10
3/10
10/103
10/103
10/103




(1.0)
(3.0)
(4.0)
(4.0)
Splenic hematopoiesis
Female
0/10
5/10a
10/103
8/10a
9/10a
10/103



(1.0)
(1.0)
(1.1)
(1.0)
(1.0)
Adrenal X-zone
Female
0/10
10/10a
9/9a
10/103
10/103
10/103
hypertrophy


(2.1)
(2.9)
(3.1)
(3.7)
(3.6)
"Significantly different from controls (p < 0.05) by Fisher's exact test, performed for this assessment.
Parentheses indicate lesion severity averaged over all animals with lesions; 1 = minimal, 2 = mild, 3 = moderate, 4 =
marked.
Source: NTP (1993).
Table 10. Reproductive Effects Observed in Mice Exposed to
2-Methoxyethanol in Drinking Water for 13 Weeks
Parameter
Sex
Concentration in ppm (Dose in mg/kg-day)
Control
2000 (295)
4000 (529)
6000 (765)
Spermatid heads (107/g testes)
Male
19.44 ±0.63a
19.49 ±0.69
16.79 ±0.95b
1.49 ± 0.58c
Spermatid heads (107/testes)
Male
2.22 ±0.08
2.21 ±0.11
1.63 ±0.11°
0.04 ± 0.01c
Spermatid concentration
(mean/10 4 mL suspension)
Male
69.43 ±2.67
69.18 ±3.32
50.78 ±3.29c
1.20 ± 0.46c
Spermatozoal motility (%)
Male
99.29 ±0.07
99.06 ±0.08b
98.93 ±0.24
0±0C
Spermatozoal concentration
(106/g caudal epididymal tissue)
Male
1587.8 ±69.0
1181.0 ± 56.3C
1077.4 ±38.7C
335.9 ± 40.lc
Estrous cycle length (days)
Female
Control
6000 (1194)
8000 (1489)
10,000 (1839)
4.60 ±0.22
7.17 ± 0.83b
5.63 ± 0.47b
8.50 ± 1.50b
aMean ± standard error.
bSignificantly different from controls (p < 0.05) by Shirley's test.
Significantly different from controls (p < 0.01) by Shirley's test.
Source: NTP (1993).
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In the 1.25% (high-dose) group, three males and five females died during the first
3 weeks of dosing, body weights declined from preexposure levels (weights not reported), and
food consumption was less than half of control consumption rates (Mellon Institute, 1962).
Surviving animals in the high-dose group were sacrificed on Day 18 of dosing for tissue
examination. Mortality also occurred in the 0.01% (low-dose) group (one male and one female
dying on Days 19 and 84, respectively) and the 0.25%-group (two males and one female dying
on Days 31-76). There were no deaths in the 0.05%-group. Deaths observed at 0.01 and 0.25%
were not treatment related, as determined by the study authors, because gross examination of the
dead animals from these groups revealed peritonitis and/or lung infection in all cases, while
treatment-related mortality in the high-dose group was associated with starvation and did not
involve the lungs. Mean body-weight gain was statistically significantly (p < 0.05) decreased
relative to controls in the 0.05% (11% below controls) and 0.25% (34% below controls) groups
for male rats and in the 0.25% (37% below controls) group of female rats. A transient decrease
in weight gain was observed at 1 and 2 months in the 0.01% male rats, but this decrease was no
longer statistically significant at study termination. Food consumption by male and female rats
in the 0.25%-groups was statistically significantly decreased (p < 0.05) but not in rats at lower
doses. No changes in liver or kidney weights or histopathology relative to controls were seen at
exposure concentrations of <0.25% in either sex. In the 1.25% animals that were sacrificed
early, a marked testicular atrophy was evident, but this effect was not seen in the lower-exposure
groups. This study identified a 3-month NOAEL of 0.01% (7 mg/kg-day) and a LOAEL of
0.05%) (40 mg/kg-day) for decreased body-weight gain in male rats.
Reproductive Studies
Foster et al. (1984, 1983) reported on the testicular effects of 2-ME in groups of 6 male
Sprague-Dawley rats administered gavage doses of 0, 50, 100, 250, or 500 mg/kg-day for up to
11 days. To observe progression of effects on spermatocyte development, groups of six rats
were sacrificed at 6 and 24 hours after the initial dosing, and additional groups that received
repeated daily doses were sacrificed after 2, 4, 7, and 11 days. At sacrifice, the testes, seminal
vesicles, prostate, and liver were removed, weighed, and subjected to histopathological
examination. In a second study to determine the extent of recovery from testicular effects after
cessation of treatments, groups of 24 rats were gavaged with 0 or 500 mg/kg-day for 4 days.
Groups of six rats were sacrificed at 0, 2, 4, or 8 weeks after the last dosing and were examined
as described above. Intergroup differences in organ weights were compared statistically.
Incidence rates of histopathological lesions were not reported.
Body weights of treated groups reportedly did not differ statistically significantly from
controls at any time point (data not shown) (Foster et al., 1984, 1983). Relative testes weights
were statistically significantly decreased in the 500-mg/kg-day group starting on Day 2 and in
the 250-mg/kg-day group starting on Day 7 (see Table 11). On Day 11, the reduction from
controls was close to 50% in both dose groups. The relative weights of the prostate and seminal
vesicles were not consistently statistically different from controls, but both tissues showed
reductions of -30% versus controls in the 500-mg/kg-day group on Day 11. Relative liver
weights were decreased primarily in the 250- and 500-mg/kg-day groups for much of the study
(reductions of 10—20%) but did not differ from controls on Day 11. No histological effects were
observed at 50 mg/kg-day, but degeneration of pachytene spermatocytes was seen at
>100 mg/kg-day at 24 hours. Continued dosing resulted in progressive depletion of
spermatocytes, as well as maturation depletion of early spermatids. After 4 and 7 days at 500
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and 250 mg/kg-day, respectively, chromatin margination was seen in spermatid nuclei. By
Day 11, no spermatids or late spermatocytes were found at >250 mg/kg-day. Because early
spermatocyte degeneration was not consistently observed at 24 hours, an additional group of six
rats was gavaged with 500 mg/kg-day and sacrificed at 16 hours. In these animals,
mitochondrial swelling, vacuolation, and early nuclear chromatin condensation were observed in
the spermatocytes. In the recovery study (see Table 12), reduction in relative testicular weights
after 4 weeks of cessation from exposure was similar to that observed at the end of the
dose-response study (see Table 11). However, by Week 8, the relative tissue weights had
recovered to that of controls. This study identified a LOAEL of 100 mg/kg-day, with an
associated NOAEL of 50 mg/kg-day, for spermatocyte degeneration in rats.
NTP (1990) investigated the reproductive and developmental effects of 2-ME in the
drinking water of two generations of rats. In the first part of this study, 20 pairs (exposed
groups) or 40 pairs (control group) of Sprague-Dawley rats were provided with drinking water
containing 0, 0.01, 0.03, or 0.1% by volume (w/v) 2-ME (>99% pure) during a 6-week
cohabitation period. During this period, the F0 rats typically delivered and weaned one F1 litter
and remated for production of a second F1 litter. The investigators estimated doses of 0, 8.81,
23.56, or 75.77 mg/kg-day for males and 0, 12.65, 36.30, or 122.10 mg/kg-day for females.
In the second part of this study, control rats were cross-mated with rats provided with
drinking water containing 0.03% 2-ME. The investigators estimated doses of 0 or
16.71 mg/kg-day for the males and 0 or 31.29 mg/kg-day for the females.
In the final part of this study, reproductive performance was studied in the second
generation (Fl) breeding pairs from the first part of the study provided with drinking water
containing 0, 0.01, or 0.03% 2-ME. The investigators estimated doses of 0, 9.07, or
27.15 mg/kg-day for the males and 0, 14.96, or 40.78 mg/kg-day for the females. Experimental
observations for all parts of the study (F0 and Fl generations) included clinical signs, body
weights, organ weights (liver, kidney, ovary, seminal vesicles, testis, cauda, epididymis, prostate
gland), drinking water consumption, semen quality, testicular histology (in controls, 0.01%- and
0.03%-groups), fertility ratio (litters/breeding pairs), litters/pair, live pups/litter, and pup
viability, sex, and birth weights.
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Table 11. Relative Organ Weights Observed in Male Rats Administered
Gavage Doses of 2-Methoxyethanol for up to 11 Days
Tissue
Dose in mg/kg-day
0 (n = 6)
50 (n = 6)
100 (n = 6)
250 (n = 6)
500 (n = 6)
Testes3
6 Hours
100.0 ± 1.2b
101.6 ±4.5
96.5 ±2.4
102.8 ±4.9
93.4 ±3.3
24 Hours
100.0 ±7.0
108.7 ±4.3
122.3 ± 3.8C
99.7 ±2.2
94.1 ±2.6
Day 2
100.0 ±3.4
99.6 ±2.8
105.5 ±2.9
96.5 ±2.7
83.5 ± 6.5d
Day 4
100.0 ±2.8
95.3 ± 1.8
88.3 ±2.8
89.9 ±4.5
76.8 ± 5.3d
Day 7
100.0 ±3.9
105.3 ±2.5
102.5 ±3.2
81.9 ± 1.8d
62.4 ± 3.ld
Day 11
100.0 ±2.8
96.5 ± 1.7
88.0 ±3.4
54.7 ± 6.5d
52.1 ± 1.7d
Prostate
6 Hours
100.0 ± 11.4
124.3 ± 8.6
114.3 ± 10.0
85.6 ±4.4
93.3 ±8.9
24 Hours
100.0 ± 11.1
101.4 ±8.3
107.0 ±9.7
92.8 ± 11.6
101.4 ± 14.5
Day 2
100.0 ±5.5
88.0 ±4.0
93.3 ±9.3
92.3 ±6.4
74.4 ± 6.4°
Day 4
100.0 ±2.8
100.0 ±6.9
94.4 ±2.8
86.7 ± 14.7
94.7 ±6.7
Day 7
100.0 ±7.4
100.0 ±5.9
95.6 ± 10.2
84.7 ±6.9
83.3 ±4.2
Day 11
100.0 ±9.5
108.0 ±7.2
96.7 ±4.5
89.2 ±9.5
68.9 ± 4.1°
Seminal vesicles
6 Hours
100.0 ± 17.8
132.1 ± 14.2
139.3 ±21.4
100.9 ± 10.2
85.0 ±8.4
24 Hours
100.0 ± 12.8
105.1 ± 12.8
110.3 ±5.1
97.4 ± 10.5
105.3 ±5.2
Day 2
100.0 ±8.8
111.8 ± 5.9
114.7 ± 11.8
82.8 ±5.1
71.7 ± 16.2
Day 4
100.0 ±6.0
76.0 ±4.0
66.0 ± 6.0°
75.0 ±6.8
93.2 ± 11.4
Day 7
100.0 ± 10.0
115.0 ±7.5
102.5 ±7.5
118.8 ± 21.7
66.7 ±4.3
Day 11
100.0 ±8.1
127.5 ± 16.4
107.1 ± 12.7
80.3 ±4.0
71.1 ± 6.6
Liver
6 Hours
100.0 ± 1.8
96.1 ±2.0
94.5 ± 1.6
89.5 ± 3.8°
95.5 ± 1.3
24 Hours
100.0 ± 1.7
93.9 ±0.9
89.4 ± 3.3°
90.3 ±3.2C
88.1 ± 2.8°
Day 2
100.0 ±3.3
101.0 ± 1.8
95.7 ±2.5
93.8 ± 1.9
88.0 ± 3.7°
Day 4
100.0 ±2.4
100.0 ± 1.6
91.8 ± 3.8
81.0 ± 1.0d
79.0 ± 2.4d
Day 7
100.0 ±2.2
106.5 ±2.5
99.7 ± 1.7
85.1 ± 1.2d
91.2 ± 2.5°
Day 11
100.0 ±2.4
97.4 ±2.4
92.7 ± 1.8
94.8 ±4.7
101.4 ±3.8
ag/100 g body weight.
bMean ± standard error.
Significantly different (p < 0.05) from controls by Dunnett's test.
Significantly different (p < 0.01) from controls by Dunnett's test.
Sources: Foster etal. (1984, 1983).
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Table 12. Relative Organ Weights Observed in Male Rats Administered
Gavage Doses of 2-Methoxyethanol for 4 Days Followed by
Recovery of up to 8 Weeks
Tissue
Control (n = 6)
500 mg/kg-day (n = 6)
Testes3
0 Weeks
0.985 ±0.028b
0.752 ±0.031c
2 Weeks
0.473 ±0.017
0.221 ±0.01c
4 Weeks
0.448 ±0.010
0.255 ±0.019c
8 Weeks
0.690 ±0.038
0.643 ±0.037
Prostate
0 Weeks
0.072 ±0.002
0.073 ± 0.002
2 Weeks
0.077 ±0.007
0.081 ±0.006
4 Weeks
0.109 ±0.009
0.080 ±0.009
8 Weeks
0.114 ± 0.010
0.105 ±0.007
Seminal vesicles
0 Weeks
0.050 ±0.003
0.031 ±0.004d
2 Weeks
0.153 ±0.010
0.127 ±0.009
4 Weeks
0.205 ± 0.020
0.231 ±0.007
8 Weeks
0.179 ±0.036
0.289 ±0.026e
Liver
0 Weeks
4.821 ±0.115
3.850 ±0.061c
2 Weeks
4.800± 0.156
4.760 ±0.067
4 Weeks
4.368 ± 0.171
4.062 ±0.084
8 Weeks
3.675 ±0.095
3.651 ±0.069
Hg/100 gbody weight.
bMean ± standard error.
Significantly different (p < 0.001) from controls by Student's /-test.
Significantly different (p < 0.01) from controls by Student's t-test.
Significantly different (p < 0.05) from controls by Student's /-test.
Sources: Foster etal. (1984, 1983).
NTP (1990) observed profound effects on reproduction. Male rats exposed to 0.1% had
decreased sperm concentrations and motility, and increased percentage of abnormal sperm (see
Table 13). Only one litter was born to F0 rats treated with 0.1% 2-ME; F0 rats treated with
0.03% 2-ME showed statistically significant decreases in live pups/litter, pup viability, and pup
body weights (see Table 14), as well as an increase in cumulative days to litter (see Table 15).
There were no effects on reproduction in F0 rats provided drinking water containing 0.01%. In
the cross-mated pairs (controls and 0.03% treated animals), there were no effects on mating or
fertility. However, reproductive performance was affected in the mating of 0.03% males with
control females, as shown by a statistically significant decrease in proportion of pups born alive
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A similar, but nonsignificant, reduction in the same endpoint was seen in the mating of 0.03%
females with control males. At necropsy, body weights were statistically significantly reduced
15% in the 0.1% males, and the weights of the individual organs were all statistically
significantly reduced as well. In the 0.03% males, body weights were reduced only slightly from
controls (3%, nonsignificant), but organ weights (adjusted for body weights by analysis of
covariance) were statistically significantly reduced by 7—9% for liver, seminal vesicles, cauda,
and epididymis. Necropsy body and organ weights were not reduced in the 0.01% males or in
the F0 females at any dose. Testicular histology was comparable between control, 0.01-, and
0.03%-groups of F0 males.
Table 13. Sperm Parameters of F0 Male Rats Given 2-Methoxyethanol in
Drinking Water
Sperm Parameter
Control
(0)
0.01%
(9 mg/kg-day)
0.03%
(24 mg/kg-day)
0.1%
(76 mg/kg-day)
Sperm motility (%)
81.8± 1.8
(20 f
83.7 ± 2.3
(20)
84.5 ± 4.6b
(20)
40.8 ± 10.4b c
(16)
Sperm concentration (106/g caudal
tissue)
492.8 ±24.5
(20)
465.8 ± 18.5
(20)
425.0 ±28.9
(20)
186.3 ±50.3b
(20)
Abnormal Sperm (%)
1.01 ±0.13
(20)
0.82 ±0.12
(20)
1.04 ± 0.15d
(19)
6.22 ± 1.76e
(9)
aMean ± standard error («).
bSignificantly different (p < 0.05) from controls.
°A few dead sperm were observed in both the right and left cauda in 4/20 rats.
dFew intact sperm observed in 1/20 rats.
Tew or no intact sperm observed in 11/20 rats.
Source: NTP (1990).
Table 14. Reproductive Effects in F0 Female Rats Given 2-Methoxyethanol in
Drinking Water
Reproduction Parameter
Control
(0)
0.01%
(9-13 mg/kg-day)
0.03%
(24-36 mg/kg-day)
0.1%
(76-122 mg/kg-day)
Fertility (number fertile/number cohabited)
38/38
20/20
16/18
l/20a
Litters/breeding pair
3.79 ± 0.19b
4.25 ±0.30
3.88 ±0.34
1.00 ±0
Proportion of live pup births
0.95± 0.01
0.97 ±0.01
0.74 ± 0.07a
0.89 ±0
Proportion of pups surviving to PND 4 (males)
0.99 ±0.01
0.99 ±0.01
0.53 ± 0.1 la
NT
Proportion of pups surviving to PND 4 (females)
0.97 ±0.01
0.98 ±0.01
0.69 ± 0.10a
NT
Pup body weight at PND 21 (g) (males)
51.93 ± 1.28
51.83 ±0.88
42.83 ± 1.89a
NT
Pup body weight at PND 21 (g) (females)
49.79 ± 1.16
49.92 ± 1.07
40.59 ± 1.39a
NT
Significantly different (p < 0.05) from controls.
bMean ± standard error.
NT = not tested.
Source: NTP (1990).
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Table 15. Cumulative Days to Litter for F0 Rats Given 2-Methoxyethanol in
Drinking Water
Treatment
Group
Control
(0)
0.01%
(9-13 mg/kg-day)
0.03%
(24-36 mg/kg-day)
0.1%
(76-122 mg/kg-day)
Litter 1
27.3 ± 1.2a
27.2 ± 1.6
35.4 ± 3.4b
151.0 ± 0
Litter 2
53.5 ±2.5
52.6 ± 3.5
69.8 ± 8. lb
NT
Litter 3
113.0± 1.9
112.5 ±0.3
121.3 ±4.0
NT
Litter 4
138.6 ±2.4
138.9 ±2.1
149.1 ±4.2b
NT
Litter 5
157.7 ± 1.7
159.6 ± 1.2
160.0 ± 1.2
NT
aMean ± standard error.
bSignificantly different (p < 0.05) from controls.
NT = not tested.
Source: NTP (1990).
In the F1 matings, the proportion of live pups sired by the 0.03%-group males (95%) was
statistically significantly less than controls (99%) (NTP, 1990). There were no effects on litter
parameters in F1 rats provided the 0.01% concentration. Sperm concentration and motility were
statistically significantly decreased among F1 male rats in the 0.01% group, and the percent of
abnormal sperm was increased at 0.03% (see Table 16). Necropsy body and organ weights were
statistically significantly reduced in the 0.03% F1 males and females but not the 0.01% F1 males
or females. The incidence of testicular degeneration in the F1 males was 2/10 (minimal
severity), 7/10 (minimal-mild severity), and 5/10 (minimal-moderate) in the control, 0.01-, and
0.03%-groups, respectively. The incidence in the 0.01%-group was statistically significantly
higher than controls; though incidence also increased at 0.03%, the increase at this concentration
did not quite achieve statistical significance. The observation of effects at lower doses in the F1
than in the F0 generation suggests that immature rats are more susceptible than mature rats to the
reproductive effects of 2-ME. The 0.01% concentration, 9.07 mg/kg-day in the F1 males, is a
LOAEL for testicular degeneration and reduced sperm density in male rats. A NOAEL in male
rats was not identified.
NTP (1989, 1988a,b) performed two-generation reproductive assessment studies in three
strains of mice (C57BL/6, CD-I, and C3H) using a continuous breeding protocol similar to that
used for rats (NTP, 1990). Drinking water concentrations of 2-ME (purity >99%) used in the
mouse studies were 0, 0.03, 0.1, or 0.3% by weight (w/v). The period of cohabitation was
generally sufficient to allow delivery of five consecutive litters. Usually offspring from the fifth
litter were used for evaluation of reproductive performance in the F1 generation. Male and
female F1 mice were exposed to 0, 0.03, or 0.1% 2-ME in drinking water. Exposure began
7 days prior to being paired for 14 weeks of cohabitation. F1 animals were mated at
74 ± 10 days of age. They were weighed at weaning, first day of cohabitation, and weekly
thereafter. Reproductive measurements included fertility index, number of litters/mating pair,
proportion of pups born alive, and pup survival and body-weight gain to Day 21. At necropsy,
liver, kidney, and right ovaries (females) or liver, kidney, seminal vesicle, right testis, right
cauda, epididymis, and prostate gland (males) were weighed. F0 and F1 males were examined
for semen quality and testicular histopathology.
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Table 16. Reproductive Effects in F1 Male Rats Given 2-Methoxyethanol in
Drinking Water
Sperm Parameter
Control
(0)
0.01%
(9 mg/kg-day)
0.03%
(27 mg/kg-day)
Sperm motility (%)
84.30 ± 1.34 (20)a
87.25 ± 1.48 (19)bd
77.87 ±4.38 (20)cd
Sperm density
(106/g caudal tissue)
640.56 ±31.72 (20)
532.03 ± 39.06 (20)d
496.29 ±52.62 (20)d
Abnormal sperm3 (%)
0.53 ± 0.06 (20)
0.68 ± 0.09 (19)b
0.94± 0.10 (17)cd
Degeneration of seminiferous tubules
2/10
7/10e
5/10
aMean ± standard error («).
bNo sperm observed in either the right or left cauda for 1/20 rats.
°No sperm observed in right cauda of 3/20 rats.
dSignificantly different (p < 0.05) from controls.
"Significantly different (p < 0.05) from controls by Fisher's exact test (one-tailed), performed for this review.
Source: NTP (1990).
NTP (1989) estimated doses of 0, 64, 219, or 636 mg/kg-day for the male C3H mice and
0, 63, 235, or 645 mg/kg-day for the females, corresponding to drinking water concentrations of
0, 0.03, 0.1, or 0.3% 2-ME, respectively. A reduction in the fertility index was exhibited, with
F0 mice receiving 0.3% 2-ME producing no litters (see Table 17). Mean necropsy body weights
were statistically significantly reduced (by 7—8%), relative to controls, for the 0.3% males and
females. These same males had statistically significantly lower testis (-50%), cauda (-13%),
and epididymis (—25%) weights, compared to controls (with or without adjustment for body
weights by analysis of covariance). Sperm density and motility were statistically significantly
lowered, while the percentage of morphologically abnormal sperm was increased in the
0.3%) males. Sperm motility was also statistically significantly decreased in the 0.1% males.
Further, the number of pups born alive and the body weights of male pups on Postnatal Day
(PND) 4 were statistically significantly reduced for the 0.1% F0 mice (see Table 17). F0 males
in the 0.3%-group had statistically significantly higher incidences of seminiferous tubule
degeneration than controls (see Table 17), with lesion severity ranging from moderate to severe
(compared to minimal lesions in the controls). Treated males also exhibited an accumulation of
sloughed cells and degeneration of ductal epithelium in the epididymis.
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Table 17. Reproductive Effects Observed in Two Generations of Mice
Exposed to 2-Methoxyethanol in Drinking Water
Parameter
Sex
Control
(0)
0.03%
(63-64
mg/kg-day)
0.1%
(219-235
mg/kg-day)
0.3%
(636-645
mg/kg-day)
F0 mice
Fertility index
(No. fertile/No. cohabitated x 100)
Both
93%
89%
84%
0%a
Pups born alive (%)
Both
81%
78%
52%a
NT
Male pup body weight at Day 4 (g)b
Male
3.45 + 0.13
3.57 + 0.12
2.79 + 0.02a
NT
Sperm motility (%)
Male
92%
93%
85%a
26%a
Sperm density (106 cells)b
Male
1298 ± 54a
1323 + 63
1216 + 86
518+ 156a
Morphologically abnormal sperm (%)
Male
6%
7%
8%
50%
Seminiferous tubule degeneration
Male
19/30
NE
NE
30/303
F1 mice
Fertility index
(No. fertile/No. cohabitated x 100)
Both
73%
87%
0%a
NT
Sperm density (106 cells)b
Male
1596+ 128a
1520+ 122
761 + 322a
NT
Morphologically abnormal sperm (%)
Male
4%
4%
25 %a
NT
Seminiferous tubule degeneration0
Male
6/11
(minimal)
10/15
(moderate)
4/4
(severe)
NT
aSignificantly different from controls (p < 0.05).
bMean ± standard error.
°Relative severity described in parentheses
NE = not examined; NT = not tested: no litters at this dose
Source: NTP (1989).
F1 C3H mice exposed to 0.1% 2-ME produced no litters (see Table 17); no effects on
0.03% F1 litters or pups were observed (NTP, 1989). Mean necropsy body weights in F1 males
from the 0.1 %-group were 15% lower than in controls. This was not reported to be statistically
significant by the researchers, although a two-tailed /-test conducted for this review showed a
statistically significant difference from controls (p = 0.016). The absolute weights of
reproductive organs were also decreased in the 0.1 % males, including testis, cauda, seminal
vesicles, and epididymis. After adjustment for body weights by analysis of covariance, only
weights of the cauda and epididymis were statistically significantly reduced. Weights of the
liver and kidney were increased after adjustment, but this was secondary to the decreases in body
weights; absolute weights of these organs were slightly lower than in controls. Mean necropsy
body weights in the 0.03% F1 males did not differ from controls, and there were no changes in
absolute organ weights in this group. There was a statistically significant increase in adjusted
kidney weights in the 0.03% F1 males, but the change from controls was small (+4.8%,
<1 standard deviation [SD]) and was not accompanied by any corresponding effect on absolute
kidney weights, suggesting that this was not a biologically relevant change. In F1 females at
necropsy, body weights were statistically significantly reduced by 23% in the 0,1 %-group, but
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there was no difference from controls in the 0.03%-group. Organ weights in F1 females were
reduced in the 0.1%-group but did not differ from controls after adjustment for body weights.
F1 males in the 0.1%-group had statistically significantly decreased sperm counts and increased
occurrences of morphologically abnormal sperm (see Table 17). Seminiferous tubule
degeneration was observed in 55, 67, and 100% of the males examined in the control, 0.03-, and
0,1%-groups, respectively; although none of these changes achieved statistical significance
because of the high incidence among controls, they appeared to show a dose-response trend (see
Table 17). Interstitial cell hyperplasia was seen in treated animals but not in controls. The
0.03%) dose (64 mg/kg-day) is a NOAEL, and the 0.1% dose (219 mg/kg-day) is a LOAEL for
C3H mice based on testicular lesions, reduced sperm quality, fetal toxicity, and impaired
fertility.
In the study of C57BL/6 mice, NTP (1988a) estimated doses of 0, 53, 170, or
505 mg/kg-day for the males and 0, 54, 174, or 543 mg/kg-day for the females, corresponding to
drinking water concentrations of 0, 0.03, 0.1, and 0.3% 2-ME, respectively. Table 18 reports
selected reproductive effects. Treatment of F0 mice with 0.3% 2-ME statistically significantly
decreased the fertility index, resulting in no live pups. Treatment with 0.3% also extended
gestation, with cumulative days to first and second litters of 57 and 71 days, respectively,
compared to 26 and 54 days in controls (data not shown). In the 0.1%-group, the percentages of
pups born alive and pup viability were statistically significantly decreased. Pup survival also
was decreased at the low dose of 0.03%. Sperm motility and density were statistically
significantly reduced in the 0.3%-group; the appearance of abnormal sperm was statistically
significantly increased in both the 0.1- and 0.3%-groups (see Table 18). Table 19 shows that
F0 males in the 0.3%-group had statistically significant decreases in body and organ weights at
necropsy; weights of the testis (29%) and epididymis (11%) remained statistically significantly
decreased after adjustment for body weights, by analysis of covariance. F0 females had no
statistically significant changes in body or organ weights associated with treatment (see
Table 19). Mild degeneration of the seminiferous tubules was seen in 57% of control F0 males,
while the 0.3% males exhibited 100% moderate-to-severe occurrence of this lesion (see
Table 18). Sloughed cell accumulation was also observed in treated males.
In F1 C57BL/6 mice, the fertility index was reduced in a dose-related manner, with no
litters produced by the 0.1%-group (NTP, 1988a). Dose-related increases in morphologically
abnormal sperm and decreases in sperm density were observed, including a statistically
significant decrease in sperm density at 0.03% (see Table 18). Table 20 shows that, at necropsy,
there were no differences from controls in F1 female or male body weights. In females, ovary
weights (see Table 20) were statistically significantly reduced in the 0.1%-group (—50%), with or
without adjustment for body weights, by analysis of covariance. In the males, the most notable
organ-weight changes were decreases in prostate weights (—35%, with or without adjustment for
body weights) in the 0.1%-group (see Table 19). Small decreases in seminal vesicle weights
were found in both the 0.1% (15%) and 0.03% (6%) groups after adjustment for body weights.
There was also an increase in adjusted kidney weights at 0.1% (see Table 20). Seminiferous
tubule degeneration of minimal severity was observed in control and treated groups of F1 males.
Seminiferous tubule degeneration occurred in 60% of controls, 70% of the 0.03%-group, and
100%) of the 0.1%-group (not statistically significant [p > 0.05] by Fisher's exact test, calculated
for this assessment) (see Table 18). The severity of the lesions was not different between groups.
The low dose of 0.03% (53 mg/kg-day) is a LOAEL for mice based on reductions in sperm
quality and pup viability. A NOAEL was not identified.
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Table 18. Reproductive Effects Observed in Two Generations of Mice
Exposed to 2-Methoxyethanol in Drinking Water
Parameter
Sex
Control
(0)
0.03%
(53-54
mg/kg-day)
0.1%
(170-174
mg/kg-day)
0.3%
(505-543
mg/kg-day)
F0 mice
Fertility index (number
fertile/number cohabitated x
100)
Both
85%
93%
86%
25 %a
Pups born alive (%)
Both
92%
91%
82%a
0%a
Male pup survival to Day 4
(%)
Male
85%
53%a
39%a
NT
Female pup survival to Day
4 (%)
Female
87%
61%
31%a
NT
Sperm motility (%)
Male
91%
92%
91%
61%a
Sperm density (106)b
Male
1847 ±110a
1540 ±91
1633 ±56
1221±101a
Morphologically abnormal
sperm (%)
Male
30%
32%
38%a
96%a
Seminiferous tubule
degeneration
Male
16/30
NE
NE
30/303
F1 mice
Fertility index (number
fertile/number cohabitated x
100)
Both
70%
50%
0%a
NT
Pups born alive (%)
Both
94%
77%
NT
NT
Sperm density (106)b
Male
1776 ±70
1590 ±76a
1379 ±372
NT
Morphologically abnormal
sperm (%)
Male
26%
28%
44%a
NT
Seminiferous tubule
degeneration
Male
12/20
14/20
6/6
NT
aSignificantly different from controls (p < 0.05).
bMean ± standard error.
NE = not examined; NT = not tested: no litters at this dose.
Source: NTP (1988a).
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Table 19. Body and Organ Weights3 in F0 Mice Following Treatment with
2-Methoxyethanol in Drinking Water
Dose (mg/kg-day)
Parameter
0 (Control)
0.03%
(53-54 mg/kg-day)
0.1%
(170-174 mg/kg-day)
0.3%
(505-543 mg/kg-day)
Females
Body (g)
28.916 ±0.359(27)b
30.091 ±0.597(30)
28.912 ±0.349(29)
28.102 ±0.471(30)
Rt. Ovary (mg)
5.774 ±0.215(27)
5.853 ± 0.295(30)
6.072 ± 0.357(29)
6.030 ±0.236(30)
Liver (g)
1.591 ±0.046(27)
1.607 ±0.049(30)
1.634 ±0.029(29)
1.543 ±0.035(30)
Kidneys (g)°
0.410 ±0.007(27)
0.418 ±0.007(30)
0.420 ± 0.006(29)
0.410 ±0.005(30)
Males
Body (g)
33.178 ±0.657(29)b
31.681 ±0.647(29)
31.254 ±0.645(30)
30.094 ± 0.693(28)d
Liver (g)
1.667 ±0.036(29)
1.535 ±0.035(29)d
1.689 ±0.046(30)
1.590 ±0.036(28)
Kidneys (g)°
0.517 ±0.008(29)
0.490 ± 0.008(29)d
0.491 ±0.009(30)
0.473 ±0.010(28)d
Seminal Vesicles (g)
0.493 ±0.013(29)
0.495 ±0.013(29)
0.469 ±0.015(30)
0.453 ±0.012(28)d
R. Testis (g)
0.111 ±0.002(29)
0.107 ±0.002(29)
0.106 ±0.002(30)
0.075 ± 0.004(28)d
R. Cauda (mg)
13.979 ±0.343(29)
14.307 ±0.992(29)
13.360 ±0.358(30)
12.832 ±0.410(28)d
R. Epididymis (mg)
41.617 ±0.665(29)
40.428 ± 0.635(29)
39.220 ± 0.752(30)d
35.939 ±0.764(28)d
Prostate Gland (mg)
14.903 ± 0.700(29)
15.324 ±0.858(29)
13.003 ±0.719(30)
15.789 ±0.751(28)
"Mean weights ± standard error.
bNumber of animals providing the data indicated in parenthesis.
°Kidneys were weighed with the adrenal glands attached.
dSignificantly different (p < 0.05) from the control group.
Source: NTP (1988a)
Table 20. Body and Organ Weights" in F1 Mice Following Treatment with
2-Methoxyethanol in Drinking Water
Treatment
Group
Parameter
0 (Control)
0.03%
(53-54 mg/kg-day)
0.1%
(170-174 mg/kg-day)
Females
Body (g)
24.451 ±0.240(20)b
24.803 ±0.359(18)
23.990 ±0.826(06)
Rt. Ovary (mg)
6.320 ±0.383(20)
5.856 ±0.329(18)
3.017 ±0.347(06)c
Liver (g)
1.428 ±0.033(20)
1.510 ±0.053(18)
1.303 ±0.087(06)
Kidneys (g)d
0.352 ± 0.005(20)
0.362 ±0.007(18)
0.434 ± 0.068(06)
Males
Body (g)
25.432 ± 0.320(20)b
26.111 ±0.370(20)
25.632 ± 0.795(06)
Liver (g)
1. 326 ±0.031(20)
1.408 ±0.044(20)
1.395 ±0.060(06)
Kidneys (g)d
0.393 ± 0.007(20)
0.423 ± 0.009(20)°
0.501 ±0.046(06)c
Seminal Vesicles (g)
0.285 ± 0.006(20)
0.276 ± 0.006(20)
0.244 ±0.014(06)c
R. Testis (g)
0.104 ±0.002(20)
0.101 ±0.003(20)
0.101 ±0.004(06)
R. Cauda (mg)
9.575 ± 0.264(20)
9.830 ±0.338(20)
9.333 ± 1.146(06)
R. Epididymis (mg)
31.530 ±0.476(20)
31.230 ±0.693(20)
35.650 ±5.147(06)
Prostate Gland (mg)
11.905 ±0.400(20)
11.570 ±0.800(20)
7.900 ± 1.843(06)
aMean weights ± standard error.
bNumber of animals providing the data indicated in parenthesis.
Significantly different (p < 0.05) from the control group.
dKidneys were weighed with the adrenal glands attached.
Source: NTP (1988a)
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Doses estimated for the CD-I mice were 0, 60, 198, or 540 mg/kg-day for the males and
0, 58, 194, or 584 mg/kg-day for the females, corresponding to drinking water concentrations of
0, 0.03, 0.1, and 0.3% 2-ME, respectively (NTP, 1988b). Treatment with 0.3% in F0 mice
resulted in a statistically significantly reduced fertility index, increased percentage of
morphologically abnormal sperm, low percentage of pups born alive, and no survival of
offspring to PND 4 (see Table 21). The cumulative days to litter were extended in the
0.3%-group, with cumulative littering of the first through fourth litters occurring at Gestation
Days (GDs) 36, 75, 88, and 106 for the treated pairs, compared to GDs 23, 43, 65, and 86 for the
controls. F0 mice treated with 0.1% exhibited statistically significantly decreased proportion of
pups born alive and decreased pup survival to PND 4 (see Table 21). At necropsy, body weights
did not differ statistically significantly from controls for males or females. Weights of the testis
(16%) lower), epididymis (13%> lower), and cauda (16%> lower) were statistically significantly
reduced in the 0.3%> males compared to controls (with or without adjustment for body weights by
analysis of covariance). F0 males in the 0.3%>-group had degeneration of the seminiferous
tubules in 77% of animals, compared to 63%> in controls (not statistically significant \p > 0.05]
by Fisher's exact test, calculated for this assessment), including observations of the accumulation
of sloughed cells and degeneration of ductal epithelium. Effects observed in the F1 generation of
CD-I mice treated with 0.1%> include a statistically significant decrease in the fertility index and
increase in incidence of seminiferous tubule degeneration (see Table 21). The proportion of
pups born alive to F1 parents was statistically significantly decreased in both the 0.1- and
0.03%-groups. However, it should be noted that the 95%> live birth rate to F1 mice in the
0.03%-group is slightly higher than the rate among F0 controls. Body weights of treated F1 mice
at necropsy did not differ from controls for either sex. In males at 0.1%>, weights of the cauda
(20%) and epididymis (10%>) were reduced, with or without adjustment for body weights. On
the basis of a slight but statistically significant decrease in pups born alive, the low dose of
0.03% (60 mg/kg-day) is a LOAEL for F1 CD-I mice. A NOAEL is not identified.
In all three strains of mice, the effects were more severe in the F1 generation compared
with the F0 generation, suggesting that immature mice are more sensitive than older mice to the
reproductive effects of 2-ME. Overall, these NTP (1989, 1988a,b) data suggest the lowest dose
of 53 mg/kg-day in male C57BL/6 mice, corresponding with a 0.03% concentration in drinking
water, is a LOAEL for statistically insignificant reproductive effects, including reduced survival
of male offspring in F0 and reduced sperm quality in F1 males; a NOAEL was not identified.
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Table 21. Reproductive Effects Observed in Two Generations of Mice
Exposed to 2-Methoxyethanol in Drinking Water
Parameter
Sex
Control
0.03%
(58-60
mg/kg-day)
0.1%
(194-198
mg/kg-day)
0.3%
(540-548
mg/kg-day)
F0 mice
Fertility index (number
fertile/number cohabitated x 100)
Both
100%
100%
100%
30%a
Pups born alive (%)
Both
93%
94%
87%a
14%a
Male pup survival to Day 4 (%)
Male
100%
92%
69%a
0%a
Female pup survival to Day 4
(%)
Female
94%
99%
72%a
0%a
Morphologically abnormal sperm
(%)
Male
4%
5%
4%
18%a
Seminiferous tubule degeneration
Male
19/30
NE
NE
23/30
F1 mice
Fertility index (number
fertile/number cohabitated x 100)
Both
85%
80%
35%a
NT
Pups born alive (%)
Both
100%
95 %a
90%a
NT
Seminiferous tubule degeneration
Male
6/20
11/20
13/20a
NT
aSignificantly different from controls (p < 0.05).
NE = not examined; NT = not tested: no litters at this dose.
Source: NTP (1988b).
A 12-week study in male Dutch rabbits examined effects of 2-ME in drinking water.
Foote et al. (1995) exposed groups of six male Dutch rabbits to 2-ME (purity not reported) in
drinking water, resulting in daily doses of 0, 12.5, 25, 37.5, or 50 mg/kg-day, for 5 days/week,
over a 12-week period. Semen was collected twice weekly for 12 weeks and analyzed by
computer-assisted sperm analysis for both sperm morphology and motility. At the end of the
12-week exposure, animals still producing sufficient sperm were tested for fertility. Profound
reductions in sperm production, including azospermia (absence of spermatozoa in the semen),
were observed in five of the six animals in the 37.5- and 50-mg/kg-day groups (measured after 9
and 6 weeks of exposure, respectively). Table 22 shows decreases in other parameters of semen
quality relative to control rabbits that were seen at Weeks 10 or 12 at these two highest doses.
Semen quality parameters that decreased included average path velocity, track speed, straight
line velocity, percent sperm motility, and progressive sperm motility at 50 mg/kg-day, and total
number of sperm per ejaculate at 37.5 and 50 mg/kg-day.1 Exposure to 25 mg/kg-day caused no
microscopic changes in sperm morphology but statistically significantly altered several indices
of semen quality, including the percentage of progressively motile sperm and the ratios of
' Foote et al. (1995) appears to have compared the Week-10 data for the 37.5-mg/kg-day dose with Week-12 data for
controls. Because the control data at Week 10 were notably lower for all parameters except total sperm per
ejaculate, only this parameter of sperm quality is significantly lower than the Week-10 controls at 37.5 mg/kg-day.
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straight line velocity to average path velocity and track speed. No effects were seen in any
parameters in rabbits exposed to 12.5 mg/kg-day. Sperm produced from rabbits after 12 weeks
of exposure showed no reduction in the ability to fertilize ova in vitro. This study identified a
NOAEL of 12.5 mg/kg-day and a LOAEL of 25 mg/kg-day for decreased semen quality of
rabbits exposed for 12 weeks.
Table 22. Semen Quality in Male Dutch Rabbits Given 2-Methoxyethanol in
Drinking Water, 5 Days/Week, for 12 Weeks
Semen Quality Parameter
Dose
(mg/kg-day)
Control
12.5
25
37.5
50
Week
1
Week
10b
Week
12
Week
1
Week
12
Week
1
Week
12
Week
1
Week
10
Week
1
Week
12
Average path velocity (|im/s)
112
97
114
114
109
114
99
127
101
119
112a
Track speed (|im/s)
138
121
144
140
135
142
131
150
125
146
133a
Straight line velocity (|im/s)
96
83
96
99
91
97
79
113
89
99
90
Ratio of straight line
velocity/average path velocity
0.85
0.86
0.82
0.86
0.82
0.84
0.78a
0.88
0.87
0.82
0.80
Ratio of straight line
velocity/average track speed
0.71
0.68
0.67
0.71
0.68
0.70
0.6 la
0.76
0.71
0.69
0.69
Sperm motility (%)
83
74
79
82
71
81
76
87
67
81
40a
Progressive sperm motility (%)
51
40
49
52
41
49
36a
62
38
51
28a
Total sperm/ejaculate (106)
181
280
261
366
172
225
183
198
152a
254
18a
aSignificantly different from control rabbits at Week 12, or at Week 10 for 37.5 mg/kg-day (p < .05). SDs were not
reported.
bControl values at Week 10 are interpolated from Foote et al. (1995), Figure 2.
Source: Foote et al. (1995).
An identical exposure of male Dutch rabbits to 2-ME in the drinking water for 12 weeks
was followed by testicular histological evaluation (Berndtson and Foote, 1997). Groups of six to
seven sexually active, mature male Dutch rabbits were exposed to 0, 12.5, 25, 37.5, or
50 mg/kg-day of 2-ME in the drinking water, 5 days/week, for 12 weeks in two sequential
replicates. Animals were weighed weekly. At the end of the treatment period, animals were
sacrificed. Testes were removed and weighed; one testis was prepared for histological
examination, and the other used for determination of spermatid number.
In the 50-mg/kg-day groups, five of seven animals showed a severe disruption of
spermatogenesis (Berndtson and Foote, 1997). A dose-related increase in disrupted seminiferous
tubules was seen at doses >37.5 mg/kg-day. Statistically significant (p < 0.05) reductions in the
yield of round spermatids per old primary spermatocyte, per young primary spermatocytes, or
per spermatogonium, as well as the mean numbers of elongated spermatids, were seen at doses
of 37.5 and 50 mg/kg-day. At lower doses, reductions were seen but did not attain statistical
significance. This study identified a NOAEL of 25 mg/kg-day and a LOAEL of 37.5 mg/kg-day
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for effects on spermatogenesis in male rabbits. The higher NOAEL identified in this study (in
which semen quality was not evaluated) relative to Foote et al. (1995) suggests that semen
quality may be a more sensitive indicator of adverse effects in rabbits than histological
evaluation.
Nagano et al. (1984) evaluated the reproductive tissues and hematology in male JCL-ICR
mice, Syrian golden hamsters, and guinea pigs given gavage doses of 2-ME (purity not reported)
5 days/week for 5 weeks. At sacrifice, blood was collected for hematological evaluation
(leukocyte counts). Testes, seminal vesicles, and the coagulating gland were removed, weighed,
and subjected to histopathological examination.
The mice (five/group) were treated with 0, 62.5, 125, 250, 500, 1000, or 2000 mg/kg-day
of 2-ME (Nagano et al., 1984). Data were presented in graphic, rather than tabular, form.
Inspection of the graphical data figures revealed a dose-related decrease in relative testicular
weights, which was markedly different from controls, in mice treated with >250 mg/kg-day. The
investigators reported a dose-related atrophy of the seminiferous epithelium; but, dose-response
data were not presented, and the threshold for this effect cannot be identified. Evaluation of the
graphical data indicated that leukocyte counts in mice receiving >250 mg/kg-day decreased with
increasing dose; however, no control data were shown for comparison. The limited data
precluded identification of a NOAEL or a LOAEL.
Nagano et al. (1984) treated hamsters (four/group) with 0, 62.5, 125, 250, or
500 mg/kg-day of 2-ME. Dose-related reductions in relative testicular (7-80%) and combined
seminal vesicle and coagulating gland (2-24%) weights, and leukocyte counts (9—19%) were
observed. No data were reported for SDs from the means, precluding statistical comparisons
between treatment groups. The limited data precluded identification of a NOAEL or LOAEL.
Guinea pigs (three/group) were treated with 0, 250, or 500 mg/kg-day (Nagano et al.,
1984). Marked reductions in relative testicular weights {15-11%) and leukocyte counts
(55-56%)) were observed in treated groups. No data were reported for deviations from the
means, precluding statistical comparisons between treatment groups. The limited data precluded
identification of a NOAEL or LOAEL.
Chapin et al. (1985a) gave male F344 rats (20/group) daily gavage doses of 0-, 50-, 100-,
or 200-mg/kg-day 2-ME (purity not reported) for 5 days. The males were then individually
cohabitated with two females per week, for 8 weeks, after which they were housed singly for
8 more weeks. After this second 8-week period, they were again cohabitated with two females
each for 5 days. Statistically significantly (p < 0.05) reduced fertility (approximately a 2-fold
reduction in live fetuses/pregnant female, compared to controls) was seen in the 200-mg/kg-day
group beginning at Week 4 and in the 100-mg/kg-day animals at Week 5 only. Separate groups
of males (96/group/time point) were exposed similarly but were not allowed to mate. At weekly
intervals, nine animals per group were subjected to bilateral efferent duct ligation. The following
morning, animals were sacrificed, and the testis, epididymis, prostate, and seminal vesicle
weights were recorded. A sample of sperm was taken from the distal cauda of the epididymis for
analysis. No changes in weights of the seminal vesicles or prostate were seen at any dose or
time. Evaluation of spermatogenesis revealed statistically significantly (p < 0.05) reduced
numbers of total and motile sperm and increased numbers of morphologically abnormal sperm in
the > 100-mg/kg-day groups beginning at 3 weeks of exposure and in the 50-mg/kg-day group
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during Week 5 only. Data were presented in graphical form; actual values for spermatogenesis
were not reported. A LOAEL of 50 mg/kg-day was identified for transient alterations in sperm
number and morphology in rats; no NOAEL was identified.
In a follow-up study (Chapin et al., 1985b), male F344 rats (nine/group) were given daily
gavage doses of 0-, 50-, 100-, or 200-mg/kg-day 2-ME (99.5% pure) for 5 days and then
observed for 8 weeks. Weekly testicular histology was performed. Biochemical activities of
P-glucuronidase, acid phosphatase, sorbitol dehydrogenase, and lactate dehydrogenase in the
testes were measured. A dose-related increase in histological alterations of the testes was seen,
with the 200-mg/kg-day animals showing pronounced damage and cell death, the 100-mg/kg-day
animals showing a high degree of alteration (appearance of numerous Giant cells and spermatid
heads near the basement membrane, absence of round spermatids) but little cell death, and the
50-mg/kg-day animals showing a transient effect, lasting from Weeks 5-7, in spermatogenesis
(testicular lesions were not compared statistically). 2-ME treatment also resulted in statistically
significantly (p > 0.05) increased activities of P-glucuronidase and acid phosphatase and reduced
activities of sorbitol dehydrogenase and lactate dehydrogenase in testicular homogenates. Data
were presented in graphical form; no actual incidences of testicular histopathology or
biochemical activities were reported. A LOAEL of 50 mg/kg-day was identified for testicular
histopathology in this 5-day study; no NOAEL was identified.
Dodo et al. (2009) treated 10 female Sprague-Dawley rats per group with 0, 30, 100, or
300 mg/kg-day of 2-ME in drinking water for 2 or 4 weeks, to assess toxicity. Other groups of
10 female rats were treated similarly 2 weeks prior to mating through GD 6, to assess fertility. In
the toxicity study, continuous diestrous and hypertrophy of the corpora lutea and other alterations
in ovarian morphology were observed in rats treated with >100 mg/kg-day. "Minimal"
irregularities in the estrous cycle were observed at the low dose of 30 mg/kg-day; estrous
changes appeared to correlate well with histopathological changes in the ovaries. Adrenal
weights were statistically significantly decreased at 300 mg/kg-day after 2 weeks, and at
>30 mg/kg-day following 4 weeks treatment. In the female fertility study, continuous diestrous
was observed at all doses. At 300 mg/kg-day, mating was postponed, and no rats became
pregnant although 7/10 copulated. These data indicate a 4-week LOAEL of 30 mg/kg-day for
estrous cycle alterations in female rats, with no NOAEL.
Developmental Studies
Scott et al. (1989) examined the maternal and developmental effects of repeated gavage
doses of 2-ME in macaques. Groups of 6-14 pregnant macaques (Macaca fascicularis) were
given gavage doses of 0-, 12-, 24-, or 36-mg/kg-day 2-ME (99.9% pure) in 15 ml water during
organogenesis on GDs 20-45. A second control group of 3 animals was given ethanol,
equivalent to the highest dose of 2-ME. On GD 100 or upon abortion if prior to GD 100,
maternal body weights were measured, and the fetus from each mother (there were no multiple
pregnancies) was collected by Caesarean section. Table 23 summarizes selected data. Anorexia
and maternal body-weight loss occurred in a dose-related manner in all treated groups. Loss of
appetite was so severe in the two high-dose groups that the researchers sometimes gave the
animals nutrition by gavage. The macaques regained their appetite after the end of treatment,
and body weights were generally similar to controls by the time of Caesarian section. The data
for hematological parameters (red blood cell [RBC], hemoglobin, and hematocrit) measured on
GD 45 showed no statistically significant effects of treatment. Chemical-related embryo
lethality claimed 0/6, 0/3 (ethanol controls), 3/13 (23%), 3/10 (30%), and 8/8 (100%) embryos in
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macaques treated with 0-, 12-, 24-, or 36-mg/kg-day 2-ME, respectively. One additional
embryonic death in each of the low- and mid-dose groups was not considered treatment related
by the researchers (one was attributed to a severe fight between the mother and a cage mate and
the other to a spontaneous abortion). Although no spontaneous abortions were observed in
controls in this study, the authors estimated that spontaneous embryonic death occurs in about
10-20% of pregnancies inM fascicularis. In the study authors' experience, spontaneous
abortion of M. fascicularis results in excessive vaginal bleeding, rapid reduction in uterine size,
and expulsion of uterine contents. However, the dead embryos observed in this study were held
within the uterus, with minimal autolysis, until hysterotomy at GD 100. This led the study
authors to conclude that the observed embryo lethality was chemical related (except for the two
cases noted above, which were not counted in the incidence data). From the 36-mg/kg-day dose,
one of the dead fetuses had bilateral forelimb malformations (missing digit). Among the fetuses
that were not aborted, fetal body and organ weights were not statistically different from controls
in the 12- and 24-mg/kg-day groups (no data for the 36-mg/kg-day group, as all fetuses in that
group were aborted). The lowest dose tested, 12 mg/kg-day, is a 25-day LOAEL for both
maternal effects (anorexia and reduced body weights) and embryo lethality in macaques in this
study.
Table 23. Selected Maternal and Developmental Effects of Gavage Doses of
2-Methoxyethanol During Organogenesis on GDs 20-45 in Female Macaques
Parameter
Dose (mg/kg-day

0 (Control)0
0 (Ethanol
Control)
12
24
36
Mean maternal body weight (kg)
GD 20
3.45 ±0.17
3.53 ± 0.16
3.79 ±0.15
3.48 ±0.12
4.25 ±0.33
GD 45
3.47 ± 0.18
3.63 ±0.11
3.72 ±0.11
3.28 ±0.15
3.88 ±0.35
GD 100a
4.02 ±0.28
4.23 ±0.22
4.40 ±0.14
4.27 ±0.26
NAd
Maternal Anorexiab
0/6
0/3
3/13
6/10
8/8
Chemical-related Embryo Lethality
0/6
0/3
3/13e
3/10e
8/8
Bilateral forelimb malformations
(missing digit)
0/6
0/3
0/13
0/10
1/8
"Final maternal body weight measured at GD 100 or at spontaneous abortion of fetus.
bAuthors reported anorexia to increase in severity with dose: "slight" at 12 mg/kg-day and "severe" at 36 mg/kg-day.
Includes data from three macaques from a previous study by these authors in the same laboratory.
dAll pregnancies aborted; no maternal-weight data reported.
eOne additional embryonic death in each of the low- and mid-dose groups was attributed by the researchers to a
severe fight between the mother and a cage mate or to a spontaneous abortion.
Source: Scott etal. (1989).
Nelson et al. (1989) performed two developmental experiments in which pregnant
Sprague-Dawley rats were fed liquid diets containing 2-ME (purity not reported) on GDs 7-18.
In the first experiment, groups of 10 rats were fed diets containing 0, 0.006, 0.012, 0.025, 0.05,
0.1, 0.25, or 0.5% 2-ME. The investigators estimated corresponding doses of 0, 16, 31, 73, 140,
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198, 290, or 620 mg/kg-day. Animals were observed daily for clinical signs. The dams were
weighed and sacrificed for examination of the uterine contents on GD 20. Maternal effects
included statistically significantly reduced mean body-weight gain (p < 0.05), ranging from
20% less than controls in the 140-mg/kg-day group to frank weight loss from preexposure levels
in the 620-mg/kg-day group. Overt signs of toxicity (diarrhea, respiratory difficulties, alopecia,
and malaise) were seen in the 620-mg/kg-day group. Statistically significantly increased embryo
lethality occurred in rats fed 73 mg/kg-day. No data were reported for higher dose groups
because all fetuses were resorbed. In the 73-mg/kg-day group, 44% of litters were resorbed,
while 100%) of litters were resorbed in > 140-mg/kg-day group. No litter resorption was seen in
rats fed <73 mg/kg-day. In groups that littered successfully (see Table 24), fetal body weights
were statistically significantly reduced in a dose-related manner. A LOAEL of 16 mg/kg-day
was identified for reduced fetal body weights, with no developmental NOAEL. The maternal
NOAEL and LOAEL were 73 and 140 mg/kg-day, respectively, for decreased weight gain.
Table 24. Developmental Effects in Rats Fed Liquid Diets Containing
2-Methoxyethanol on GDs 7-18
Effect
Dose (mg/kg-day)
Control
16
31
73
>140a
Male fetal body weights (kg)
3.3
2.9b
2.8b
2.3b
NT
Female fetal body weights (kg)
3.3
2.8b
2.7b
2.5b
NT
Embryo lethality (%>)°
11
7
14
92
NT
"Rats in the >140-mg/kg-day groups did not litter.
bSignificantly different from controls (p < 0.05).
°No statistical analysis of this endpoint was performed by the researchers.
NT = not tested.
Source: Nelson et al. (1989).
In the second experiment, groups of 12 rats were fed diets containing 0, 0.006, 0.012, or
0.014%) 2-ME on GDs 7-18 for evaluation of behavioral parameters in the 42- to 63-day-old
offspring (Nelson et al., 1989). The investigators estimated corresponding doses of 0, 17, 33, or
40 mg/kg-day. Dams were observed for mortality, clinical signs, and weight gain. The
behavioral parameters observed in the offspring were figure-8 activity (test of general activity),
the Cincinnati maze (a problem-solving test), the startle response (reflex test), and conditioned
lick suppression (operant conditioning). There were no effects on maternal body-weight gain or
lactation. A statistically significant and dose-related increase in gestation length was noted in all
treated groups (see Table 25). Statistically significantly increased mortality of offspring during
the pre- and postweaning periods was observed in rats fed the 33- and 40-mg/kg-day diets (see
Table 25). Too few offspring from rats fed the 40-mg/kg-day diet survived for behavioral
testing. The only behavioral effect attributed to treatment was statistically significantly
increased error (p < 0.05) in the Cincinnati maze test in the offspring of rats fed 33 mg/kg-day.
In this experiment, 17 mg/kg-day, the lowest dose tested, was a LOAEL for increased gestation
length; no NOAEL was identified.
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Table 25. Gestational and Postnatal Effects in Rats Fed Liquid Diets
Containing 2-Methoxyethanol on GDs 7-18
Effect
Dose (mg/kg-day)
Control
17
33
40
Length of gestation (days)
21.9
22.4a
22.8a
22.9a
% Pups dead at
Birth
2
1
7
14
PNDs 1-25
9
8
51a
83a
PNDs 26-63
0
0
3
NT
Birth weight (g)
5.6
6.2
6.1
5.8
"Significantly different from controls (p < 0.05).
NT = not tested.
Source: Nelson etal. (1989).
Toraason and coworkers (Toraason and Breitenstein, 1988; Toraason et al., 1986, 1985)
investigated the effects on the fetal heart after gavage treatment of pregnant Sprague-Dawley rats
with 2-ME (purity not reported) on GDs 7-19. In the first study, groups of 8-11 rats were
treated with 0-, 25-, 50-, or 100-mg/kg-day 2-ME on GDs 7-13 (Toraason et al., 1985).
Maternal body weights were recorded daily. Fetal electrocardiograms (EKGs) were recorded on
GD 20, after which, the fetuses were examined for external and visceral abnormalities.
Treatment with 100 mg/kg-day resulted in early resorption of all fetuses; therefore, fetal effects
could not be studied in this group. Apparent dose-related reductions in live litter size and
maternal body weights were observed in rats treated with 25 and 50 mg/kg-day, as well as an
increase in fetal resorptions at 50 mg/kg-day, but the changes were not statistically significant.
There were also slight, apparently dose-related reductions in mean fetal body weights in both
groups of treated rats, but these changes, too, were not statistically significant. A statistically
significant and dose-related increase in the percent of fetuses with prolonged QRS intervals
(probably reflecting intraventricular conduction delay) was seen in both treated groups
(p < 0.05). Several fetuses from the 50-mg/kg-day group exhibited one or more cardiovascular
malformations. Double aortic arch was exhibited in 1/74 fetuses in the 25-mg/kg-day group.
The occurrence of double aortic arch in the 25-mg/kg-day group cannot be unequivocally
attributed to 2-ME exposure, as this particular anomaly was not seen in any of the 38 fetuses
(from six different litters) treated with 50 mg/kg-day. The investigators noted no correlation
between altered EKG and the presence of cardiovascular anomalies in individual animals. In this
study, 25 mg/kg-day is considered a developmental LOAEL for slight changes in the fetal EKG
and a maternal LOAEL for decreased body weights. No developmental or maternal NOAEL
was identified.
In the second study, Toraason et al. (1986) explored the developmental effect of 2-ME on
the heart in rats. Doses of 0- or 25-mg/kg-day 2-ME (purity not reported) were given on
GDs 7-13 or 13-19 to groups of 13 pregnant Sprague-Dawley rats. Ornithine decarboxylase
(ODC) activity in the heart was used as an indicator of cardiac development. ODC activity
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following isoproterenol administration was used as an indicator of cardiac autonomic nervous
system response in the offspring. ODC measurements, with or without previous treatment with
isoproterenol, were made on PNDs 3, 9, 16, and 22. Treatment had no effect on maternal body
weights, litter weights, number of viable offspring, or the heart or body weights of the offspring
through PND 22. Prolonged gestation was observed in both treated groups but was statistically
significant (p < 0.05) only in those treated on GDs 7-13. Statistically significantly reduced ODC
activity on PND 3 occurred in rats treated on GDs 7-13. Isoproterenol pretreatment had no
effect on ODC activity. The 25-mg/kg-day dose is a developmental LOAEL, associated with
biochemical evidence of retarded cardiac development; no developmental NOAEL was
identified. The maternal NOAEL is 25 mg/kg-day but is based on limited observations.
In a study designed to determine whether the effects on EKG observed in the 20-day
fetuses persisted into the postnatal period, Toraason and Breitenstein (1988) treated groups of
25-30 rats with 0, 50, or 75 mg/kg-day on GDs 7-13. EKG recordings were made when the
pups were 3 and 6 weeks old. A statistically significant, dose-related decrease in maternal body
weight gain was observed in both treated groups, which was attributed to statistically
significantly reduced litter size (increased resorptions). Statistically significantly prolonged
gestation and a statistically significant (p < 0.05) and dose-related decrease in postnatal survival
were also observed in both treated groups. No offspring from dams treated with 75 mg/kg-day
survived more than 3 days. Statistically significantly increased QRS or T-wave intervals were
observed in male and female 50-mg/kg-day offspring at both periods of measurement.
Statistically significantly decreased body weights and increased relative heart weights were
observed when the 50-mg/kg-day offspring were sacrificed at 8 weeks of age. The low dose of
50 mg/kg-day was a maternal and developmental LOAEL associated with prolonged gestation,
increased fetal resorptions, high pup lethality, decreased postnatal weights, increased relative
heart weights, and EKG changes in pups; no NOAEL was identified.
Sleet and Ross (1997) exposed groups of 4-10 pregnant CRL:CD rats on GD 13 to a
single gavage dose of 0, 50, 100, or 250 mg/kg-day of 2-ME (purity not reported). Dams and
their litters were sacrificed on either Day 15 or 20, and body weights were recorded for dams and
fetuses. Gross observations were made for embryonic/fetal abnormalities. No maternal lethality
occurred during the study; maternal body weights, corrected for gravid uterine weights, were not
different between control and treated groups. On Day 15, embryonic body weights in all
exposed groups were statistically significantly decreased relative to controls, and the incidence
of limb-bud dysmorphogenesis was statistically significantly (p < 0.05) elevated in a dose-related
manner, occurring in 36, 65, and 98% of embryos (75-100% of litters from treated dams) in the
50-, 100-, and 250-mg/kg-day groups, respectively. Embryonic limb-bud paddle area was
decreased following exposure to >100 mg/kg-day, while interdigital distance (distance between
developing paw digits) was statistically significantly increased at both 50 and 100 mg/kg-day
(measurements could not be made for the 250-mg/kg-day group due to the distorted state of the
condensing and noncondensing regions of the limb bud). Intralitter differences were not reported
for these endpoints. Exposure to 100 mg/kg-day resulted in decreased fetal body weights, while
exposure to 250 mg/kg-day resulted in decreased fetal body weights and an increase in the
incidence of malformations of the digits. This study did not identify a developmental NOAEL; a
LOAEL of 50 mg/kg-day was identified for decreased embryonic weights and limb-bud
malformations. The high dose of 250 mg/kg-day was a NOAEL for maternal effects.
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Nagano et al. (1984, 1981) examined maternal and fetal effects of gavage doses of 2-ME
given to JCL-ICR mice from GDs 7-14. Groups of 21-24 mice were given 0, 31.25, 62.5, 125,
250, 500, or 1000 mg/kg-day of 2-ME (purity not reported) in distilled water. Dams were
observed daily for clinical signs, and body weights were measured every 2-4 days. Dams were
sacrificed on GD 18. Blood was taken from dams for leukocyte counting, while observations
were made for live, dead, and resorbed fetuses. Gross and microscopic observations were made
of skeletal abnormalities. Maternal effects included statistically significantly (p < 0.05) reduced
body-weight gain in mice receiving >250 mg/kg-day (-38-45% lower terminal body weights
than controls) and leukocytopenia (46% fewer leukocytes than controls) in the 1000-mg/kg-day
group. There were no live pups born in the 1000-mg/kg-day group, 0.3% of pups were born
alive in the 500-mg/kg-day group, and 47% of pups were born alive in the 250-mg/kg-day group.
Groups receiving <125 mg/kg-day had >89% of pups born alive. Exencephaly was statistically
significantly (p < 0.05) increased (19% of fetuses, compared to 0.03% in controls) in the
250-mg/kg-day group. Other fetal effects included statistically significant dose-related increases
in incidences of skeletal anomalies in all treated groups (see Table 26). The ribs, vertebrae, and
fingers were affected. Skeletal anomalies appeared to be the most sensitive indicator of
developmental toxicity in the mice. The lowest dose tested—31.25 mg/kg-day—was a
developmental LOAEL for skeletal anomalies in mice; the maternal NOAEL and LOAEL were
125 and 250 mg/kg-day, respectively, for reduced maternal weight gain.
Table 26. Skeletal Anomalies in Pups Born to Mice Given Gavage Doses of
2-Methoxyethanol on GDs 7-14



Dose (mg/kg-day)
Anomaly
Control
31.25
62.5
125
250
Number of fetuses examined
173
174
229
178
77
Cervical ribs
1
7
23
55a
44a
Lumbar ribs
63
76
131
170a
64a
Bifurcated/split cervical vertebrae
46
74b
108a
101a
68a
Supernumerary lumbar vertebrae
0
1
11
63a
39a
Asymmetrical sternebrae
14
27
61a
50a
29
Fused ribs
0
0
1
48b
71a
Fused cervical vertebrae
0
0
1
8
32a
Agenesis of cervical vertebrae
0
0
0
2
13a
Fused thoracic vertebrae
0
0
0
28a
63a
Fused lumbar vertebrae
0
0
0
41a
60a
Fused postlumbar vertebrae
0
0
0
5
55a
Spina bifida occulta
1
2
19b
25a
17a
Abnormal fingers (oligodactyly)
0
0
0
0
17a
Significantly different from controls (p < 0.01).
bSignificantly different from controls (p < 0.05).
Sources: Nagano etal. (1984, 1981).
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Greene et al. (1987) treated groups of three to four pregnant CD-I mice by gavage with 0,
100, 175, 250, 350, 400, 450, or 500 mg/kg-day of 2-ME (analytical grade, purity not reported)
in distilled water on GD 11. The purpose of the study was to identify the threshold of
cytotoxicity in the limb buds and anomalies of the digits in the offspring. Maternal effects were
not reported. Histological evidence of cell death in the limb buds was apparent in fetuses treated
with >100 mg/kg-day. A statistically significant and dose-related increase in malformations of
the forepaw occurred at doses >175 mg/kg-day. The lowest dose tested—100 mg/kg-day—was
identified as a fetal LOAEL for limb-bud cell death. Maternal effects cannot be determined from
this study.
Horton et al. (1985) examined maternal and fetal toxicity in mice given 2-ME on various
days of gestation. Groups of 10 pregnant CD-I mice were given gavage doses of 0-, 100-, 175-,
250-, 300-, 350-, 400-, 450-, or 500-mg/kg-day 2-ME (purity not reported) in distilled water.
Some mice were given a single dose on GDs 9, 10, 11, 12, or 13; others were treated on 2 or 3
consecutive days at various times during GDs 7-11. Dams were observed for clinical signs and
changes in body-weight gain. Offspring were observed for embryo lethality and gross
abnormalities. Treatment caused no apparent maternal toxicity, although some (unspecified)
groups had reduced body-weight gain, which the authors attributed to reduced litter size due to
embryo lethality (Horton et al., 1985). The incidence of skeletal anomalies, particularly of the
digits, increased statistically significantly and in a dose-related manner in mice treated with
>175 mg/kg-day on GD 11. Embryo lethality was statistically significantly increased in mice
receiving 250 mg/kg-day on at least two GDs. Fetal body weights were statistically significantly
(p < 0.05) reduced in mice given a single dose of >250 mg/kg-day on GD 11. The
developmental NOAEL and LOAEL for skeletal abnormalities were 100 and 175 mg/kg-day,
respectively; the data were not sufficiently reported to identify the NOAEL and LOAEL for
maternal toxicity.
Hardin and Eisenmann (1987) treated groups of 14 or 16 pregnant CD-I mice on GD 11
with 0- or 304-mg/kg-day gavage doses of 2-ME (>99% pure) in distilled water. Dams were
sacrificed on GD 18 and examined for maternal body weights and gravid uterus weights. Live
and dead fetuses were counted. Live fetuses were weighed and observed for paw malformations.
Maternal and fetal body weights and number of live fetuses were not affected by treatment.
Statistically significant (p < 0.001) increases in the incidence of paw defects occurred in treated
mice (14/16 litters and 88.5% of the fetuses, compared to 2/14 litters and 13% of fetuses in the
control group). The hind limbs were affected more frequently than the forelimbs. Syndactyly
(fused digits) was the predominant anomaly. In this study, 304 mg/kg-day was a developmental
LOAEL for paw defects; no NOAEL or maternal LOAEL was identified.
One study was located that investigated the developmental toxicity of 2-ME acetate. As
part of a large preliminary assessment of chemicals for developmental toxicity, groups of
50 pregnant CD-I mice were given gavage doses of 0- or 1225-mg/kg-day 2-ME acetate (purity
and dosing vehicle not specified) for 8 days on GDs 6-13 (Hardin et al., 1987). The treatment
concentration was determined during prior dose-finding studies to be the LDio (details not
reported). Dams were observed for mortality, and body weights were measured prior to
treatment and again on GD 18. Live and dead pups were counted, and litters were weighed on
PNDs 1 and 3. Uteri were examined for the presence of implantation sites in dams that failed to
litter. Maternal body weights were also measured on PND 3. There was no maternal mortality,
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and no changes in body weights were reported. No pups were born alive. No results were
reported for presence or absence of implantation sites. A developmental LOAEL of
1225 mg/kg-day in mice was identified for 2-ME acetate in this study.
Inhalation Exposure
Unless otherwise indicated, all inhalation exposures discussed below were whole-body
exposures conducted using 2-ME in exposure chambers rather than nose-only exposures. No
inhalation studies were available for animals exposed to 2-ME acetate.
Subchronic Studies
Miller et al. (1983a) exposed rats and rabbits to airborne 2-ME. Groups of
Sprague-Dawley rats (10/sex/group) and New Zealand white rabbits (5/sex/group) were exposed
to concentrations of 0-, 30-, 100-, or 300-ppm (0-, 93-, 310-, or 930-mg/m3) 2-ME, 6 hours/day,
5 days/week, for 13 weeks. Observations for clinical signs were made daily, while body weights
were recorded weekly. Blood samples were collected at 4 and 12 weeks. Hematological tests
included measurements of hemoglobin, PCV, MCV, MCH, MCHC, and RBC, total and
differential leukocyte (white blood cell [WBC], and platelet counts. Evaluated clinical chemistry
parameters included blood urea nitrogen (BUN), AST, alkaline phosphatase, glucose, total
protein, albumin, globulins, and total bilirubin concentrations. Urinalysis was performed on rats
(only) at 12 weeks and included measurements of bilirubin, ketones, glucose, protein, pH,
urobilinogen, and specific gravity. At necropsy, weights of livers, kidneys, brains, spleens,
thymuses, and testes were measured. Including the nasal turbinates, 39 tissue types were
collected and subjected to histopathological evaluation.
No mortality occurred in the rats. Miller et al. (1983a) reported statistically significant
body weight decreases among 300-ppm males (13% below controls) and 100- and 300-ppm
females (9-18% below controls). Both sexes of 300-ppm groups exhibited pancytopenia, thymic
atrophy (incidence not reported), and decreases in thymus (42-66% lower than controls) and
liver (15-17%) lower than controls) weights and serum concentrations of total protein, albumin,
and globulin. In the 300-ppm males, decreased testicular weights (59%> lower than controls),
small flaccid testes, and severe degeneration of the seminiferous tubules (incidence not reported)
were observed. No gross or histological changes were seen in the 30- or 100-ppm males or
females. A NOAEL of 100 ppm (310 mg/m3) and a LOAEL of 300 ppm (930 mg/m3) were
identified for rats on the IRIS database (U.S. EPA, 2010a) based on testicular degeneration.
In rabbits, two of five males exposed to 300 ppm and two of five females in each of the
100- and 300-ppm groups died or were sacrificed moribund (Miller et al., 1983a). The
researchers considered the deaths to be of uncertain relationship to 2-ME exposure (one was due
to inner ear infection, one to pneumonia, one to acute hemorrhagic enteritis, one to inanition, and
two to undetermined causes). Males and females in the 300-ppm group exhibited statistically
significant (p < 0.05) body-weight reduction (9— 13%> below controls), pancytopenia, and relative
thymus-weight reduction (35—59%>) and atrophy (incidence not reported). All males in the
300-ppm group had small flaccid testes by gross observation (see Table 27), and mean testicular
weights were statistically significantly and markedly reduced in this group (73%> lower than
controls). Some males in the 100- and 30-ppm groups also showed slight-to-moderate decreases
in gross testes size, although mean testicular weights in these groups did not differ from controls.
Histopathological examination of males revealed testicular degeneration that increased in
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incidence and severity with exposure concentration. Severe diffuse seminiferous tubule
degeneration occurred in all males at 300 ppm, while more moderate degeneration was observed
in the three affected males at 100 ppm, and relatively minor changes (thinner than normal
germinal epithelium with few spermatozoa) were observed in the one male affected at 30 ppm
(see Table 27). No gross or histological effects on reproductive organs were observed in treated
females. IRIS (U.S. EPA, 2010a) identified a NOAEL of 30 ppm (93 mg/m3) and a LOAEL of
100 ppm (310 mg/m ) for rabbits in this study, based on testicular effects in males.
Table 27. Gross and Histological Testicular Changes of Male New Zealand
White Rabbits Exposed to Airborne 2-Methoxyethanol, 6 Hours/Day,
5 Days/Week, for 13 Weeks
Testicular Parameter
Controla
30 ppm
(93 mg/m3)
100 ppm
(310 mg/m3)
300 ppm
(930 mg/m3)
Gross reduction in testes size
0/5
2/5
4/5b
5/5b
Seminiferous tubule degeneration
0/5
1/5
3/5
3/3b
"Control incidence implied, but not explicitly reported by researchers.
bSignificantly different (p < 0.05) from controls by Fisher's exact test, performed for this review.
Source: Miller etal. (1983a).
In a follow-up study, Miller et al. (1982, as summarized on the IRIS database [U.S. EPA,
2010a]) further investigated the toxicity of 2-ME exposure in male rabbits. Groups of 10 male
New Zealand white rabbits were exposed to airborne concentrations of 0-, 3-, 10-, or 30-ppm (0-,
9-, 31-, or 93-mg/m3) 2-ME, 6 hours/day, 5 days/week, for 13 weeks. Observations were made
for clinical signs, and body weights were recorded. At necropsy, testicular weights were
recorded, and major organs (including the testes) were examined for gross and histological
abnormalities. No statistically significant differences were observed between the control and any
treated groups for any of the endpoints examined. The high exposure concentration of 30 ppm
(93 mg/m3) was identified as a NOAEL on IRIS (U.S. EPA, 2010a). The results of this study
support identification of 30 ppm as a NOAEL in the previous study by Miller et al. (1983a).
Reproductive Studies
Rao et al. (1983) examined fertility effects in rabbits exposed to 2-ME for 13 weeks.
Groups of 20-30 male and female Sprague-Dawley rats were exposed to airborne concentrations
of 0-, 30-, 100-, or 300-ppm (0-, 93-, 310-, or 930-mg/m3) 2-ME, 6 hours/day, 5 days/week, for
13 weeks. The exposed rats were then bred with nonexposed partners. Clinical signs were
observed daily and body weights collected weekly from all rats during the exposure period, after
which, male weights were collected weekly and female weights collected at littering. Observed
litter parameters included gestation duration; litter size; pup viability on PNDs 1, 4, 7, 14, and
21; pup weights; and gross abnormalities. Mated males and females were sacrificed at 23 and
15 weeks, respectively, following cessation of exposure. Organ weights were collected for liver,
kidney, brain, spleen, thymus, and testes. Histological examinations were performed on spleen,
thymus, bone marrow, lymph nodes, testes, epididymides, ovaries, and uteri. A subset of
unexposed females were mated with exposed males and sacrificed 12 days after last cohabitation.
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Dominant lethality was assessed by observing the male fertility index, rate of preimplantation
loss, and resorption rate. A second subset of exposed males (0 or 300 ppm) and unexposed
females were bred at 13 and 19 weeks after cessation of exposure to assess recovery of fertility.
Terminal body weights were statistically significantly reduced (8-13% decrease from
controls) in both sexes of the 300-ppm groups. None of the treated female groups exhibited
reproductive effects. Fertility was reduced in the 300-ppm males, as only 4/20 unexposed
females were impregnated by the exposed males from this group, compared to 29/30 pregnancies
from matings with control males. In the four impregnated dams, complete fetal resorption was
observed for all conceptuses. After the 13-week postexposure recovery period, 55% of 300-ppm
males were fertile. High-dose males also exhibited decreased testicular size and atrophy of the
seminiferous tubules. No effects on fertility or testicular weights were observed in the 30- or
100-ppm males. A NOAEL of 100 ppm (310 mg/m3) and an associated LOAEL of 300 ppm
(930 mg/m3) were identified on the IRIS database (U.S. EPA, 2010a) for reduced fertility and
testicular atrophy in male rats, which is consistent with the results of the Miller et al. (1983a) rat
study.
Developmental Studies
Nelson et al. (1984a) examined the teratogenic effects of 2-ME in pregnant rats. Groups
of 8-34 pregnant Sprague-Dawley rats were exposed to airborne concentrations of 0-, 50-, 100-,
or 200-ppm (0-, 155-, 310-, or 620-mg/m3) 2-ME, for 7 hours/day, on GDs 7-15. On GD 20,
dams were weighed and sacrificed. There were no overt signs of toxicity in the dams. Observed
developmental endpoints included number of resorption sites, live fetuses, fetal body weights,
and gross and microscopic skeletal and soft tissue abnormalities. All of the litters in the
200-ppm group were resorbed. In the 100-ppm group, 50% of all fetuses were resorbed, with
resorptions occurring in all litters. A statistically significant, 3-fold increase in fetal resorptions
was observed in the 50-ppm group. This group also exhibited fetal body weights 20% lower
than controls. Statistically significant increases in fetuses with cardiac malformations were
observed in the 100-ppm group, while increased skeletal malformations were observed in the 50-
and 100-ppm groups (see Table 28). This study identified a LOAEL of 50 ppm (155 mg/m3) for
fetal resorptions and skeletal malformations in rats. A NOAEL was not identified.
Table 28. Fetal Body Weights and Incidences of Malformations in Pups
Born to Rats Exposed to Airborne 2-Methoxyethanol, for 7 Hours/Day on

GDs 7-15

Malformation
Control
50 ppm (155 mg/m3)
100 ppm (310 mg/m3)
Fetal body weight—males (g)
3.46
2.84a
2.29a
Fetal body weight—females (g)
3.64
2.91a
2.49a
Cardiac IV septal defect
0/270
4/103
20/65a
Wavy ribs
0/137
28/53a
16/3 la
aSignificantly different from controls (p < 0.05).
Source: Nelson etal. (1984a).
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Hanley et al. (1984) conducted a study of developmental toxicity from 2-ME in rats,
mice, and rabbits. Groups of 24-32 pregnant rats, mice, and rabbits were exposed to
99.96% pure 2-ME for 6 hours/day on GDs 6-15 (mice and rats) or 6-18 (rabbits). Fischer 344
rats and New Zealand white rabbits were exposed to airborne concentrations of 0, 3, 10, or
50 ppm (0, 9.3, 31, or 155 mg/m ), while CF-1 mice were exposed to airborne concentrations of
0, 10, or 50 ppm (0, 31, or 155 mg/m3). Daily observations were made for clinical signs, while
body weights and food and water consumption were measured at 3-day intervals. On GDs 18,
21, or 29 (for mice, rats, and rabbits, respectively), dams were sacrificed, and maternal body,
liver, spleen, and thymus weights were recorded. Blood samples were collected for analysis of
RBC and WBC counts, hemoglobin, PCV, MCV, MCH, and MCHC. Counts were made of
corpora lutea (rats and rabbits); live, dead, and resorbed fetuses; and implantation sites. Fetuses
were weighed, measured, and examined for gross and microscopic abnormalities.
In rats, no statistically significant effects on maternal body or organ weights were
observed (Hanley et al., 1984). Statistically significant decreases in hemoglobin and PCV (3-6
and 4-6%, respectively) were observed in all treated adult groups, and decreased RBC counts
(5%>) occurred in the 50-ppm group. Statistically significantly increased incidences of fetuses
with lumbar spurs and delayed ossification of the vertebral centra were also observed in the
50-ppm group (see Table 29). Thus, a developmental NOAEL and LOAEL of 10 and 50 ppm
(31 and 155 mg/m3), respectively, were identified for minor skeletal abnormalities in rats. The
"3
maternal NOAEL was 50 ppm (155 mg/m ).
Table 29. Incidences of Select Skeletal Variations in Fetuses of Rats
Exposed to Airborne 2-Methoxyethanol for 6 Hours/Day on GDs 6-15
Malformation
Control
3 ppm
(9 mg/m3)
10 ppm
(31 mg/m3)
50 ppm
(155 mg/m3)
Lumbar spurs
18/287
(21/29 )a'b
13/283
(10/28)
20/293
(13/28)
5i mr
(26/30)
Delayed ossification
of vertebral centra
4/287
(4/29)
3/283
(3/28)
6/293
(5/28)
19/307°
(13/30)
aParentheses indicate litter incidences.
bThe number of affected litters cannot exceed the number of affected fetuses, so the litter incidence reported by the
researchers (and duplicated here) for lumbar spurs in controls is likely in error.
Significantly different from control (p < 0.05).
Source: Hanley et al. (1984).
In mice, Hanley et al. (1984) reported a statistically significant decrease in maternal
body-weight gain (18%) from GDs 12 to 15 in the 50-ppm group. Statistically significantly
increased fetal incidences of extra lumbar ribs and unilateral testicular hypoplasia also were
observed in the 50-ppm group (see Table 30). Thus, a maternal and developmental NOAEL and
"3
LOAEL of 10 and 50 ppm (31 and 155 mg/m ), respectively, were identified for transient
decreases in maternal body weight gain and fetal skeletal and soft tissue abnormalities in mice.
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Table 30. Incidences of Select Alterations in Fetuses of Mice Exposed to
Airborne 2-Methoxyethanol for 6 Hours/Day on GDs 6-15
Malform ation/lesion
Control
10 ppm
(31 mg/m3)
50 ppm
(155 mg/m3)
Extra lumbar ribs
48/317
(14/26)a
49/260
(14/23)
82/25 lb
(21/24)
Unilateral testicular hypoplasia
2/165
(2/26)
3/136
(3/23)
8/132b
(6/24)
aParentheses indicate litter incidences.
bSignificantly different from controls (p < 0.05).
Source: Hanley et al. (1984).
In rabbits, a decrease in body-weight gain (42% compared with controls) occurred in the
50-ppm group during exposure (Hanley et al., 1984). A statistically significantly higher fetal
resorption rate (500% increase) and lower fetal mean body weights (9%) were both observed in
the 50-ppm group when compared with controls. Of all 50-ppm fetuses, 63% exhibited at least
one gross malformation, with abnormalities in the extremities (shortened or missing digits)
appearing most often (see Table 31). Table 31 notes other observed malformations occurring in
the 50-ppm group. A maternal and developmental NOAEL and LOAEL of 10 and 50 ppm (31
"3
and 155 mg/m ), respectively, were identified for decreases in maternal body-weight gain and
numerous skeletal and soft tissue abnormalities in fetal rabbits.
Doe et al. (1983) studied the developmental effects of 2-ME (99% pure) in rats that were
allowed to give birth. Groups of 20 pregnant Wistar rats were exposed to airborne
concentrations of 0-, 100-, or 300-ppm (0-, 310-, or 930-mg/m3) 2-ME, for 6 hours/day, on
GDs 6-17. Dams were allowed to birth, and pups were observed for 3 days. Maternal body
weights were measured throughout the study. Numbers of live and dead pups and pup body
weights were recorded on Postpartum Days 1 and 3. Dams not birthing by GD 24 were
sacrificed and evaluated for pregnancy status. Body-weight gain in the 300-ppm group was
statistically significantly less (weights not reported) than controls throughout the study
(Doe et al., 1983). None of the 300-ppm dams produced litters. Only 9/20 100-ppm dams
littered, with gestation extending 1.6 days longer than controls. The mean number of live
pups/litter (eight fewer/litter), total live pup ratio (22% lower), and pups surviving to Postpartum
Day 3 (29% lower) were statistically significantly reduced in the 100-ppm group. This study
identified a LOAEL of 100 ppm (310 mg/m3) for decreased live-pup birth and pup survival in
rats. No NOAEL was identified.
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Table 31. Incidences of Select Malformations in Fetuses of New Zealand
White Rabbits Exposed to Airborne 2-Methoxyethanol for
6 Hours/Day on GDs 6-18


3 ppm
10 ppm
50 ppm
Malformation
Control
(9 mg/m3)
(31 mg/m3)
(155 mg/m3)
Limb defects
0/173
1/172
1/187
55/145b

(0/23 )a
(1/23)
(1/24)
(16/22)
Digit defects
0/173
0/172
0/187
17/145b

(0/23)
(0/23)
(0/24)
(8/22)
Ventral wall defects
0/173
0/172
0/187
1 l/145b

(0/23)
(0/23)
(0/24)
(4/22)
Cardiovascular defects
0/95
0/93
0/101
34/80b

(0/23)
(0/23)
(0/24)
(15/22)
Coarctation of the aortic arch
0/95
0/93
0/101
13/80b

(0/23)
(0/23)
(0/24)
(6/22)
Aplastic/hypoplastic spleen
0/95
0/93
0/101
26/80b

(0/23)
(0/23)
(0/24)
(13/22)
Renal defects
0/95
2/93
1/101
29/80b

(0/23)
(2/23)
(1/24)
(14/22)
Missing bones
0/173
0/172
0/187
1 l/145b

(0/23)
(0/23)
(0/24)
(5/22)
aParentheses indicate incidence/litter.
bSignificantly different from controls (p < 0.05).
Source: Hanley et al. (1984).
Nelson et al. (1984b) examined the reproductive and neurodevelopmental effects of
2-ME in a cross-breeding study in rats. There were 15 male Sprague-Dawley rats exposed to
airborne concentrations of 25-ppm (78-mg/m3) 2-ME, (>98% pure) 7 hours/day, 7 days/week,
for 6 weeks. No male controls were used. These rats were then mated with unexposed females.
In a separate experiment, groups of 15 pregnant Sprague-Dawley rats were exposed to airborne
concentrations of 0- or 25-ppm (0- or 78-mg/m3) 2-ME, for 7 hours/day ,on GDs 7-13 or 14-20.
Parental body weights were recorded. Food and water consumption were recorded in the
pregnant female-only experiment. At birth, litters were evaluated for litter number and allowed
to nurse with the biological mothers. From each litter, four male and four female pups were
randomly selected for further observation. Pup body weights were taken on Postpartum Days 7,
14, 21, 28, and 35. On Postpartum Days 10-90, one male and female from each litter were
selected for behavioral evaluation via six tests for neuromuscular function, activity, and learning.
At birth and Postpartum Day 21, representative pups (n > 10) were sacrificed, and the brains
were removed and analyzed for concentrations of protein, acetylcholine, dopamine,
norepinephrine, and 5-hydroxytryptamine.
No treatment-related effects were observed for parental body weights or water or food
consumption (Nelson et al., 1984b). Litter sizes and pup body-weight gain through Postpartum
Day 90 were not affected by treatment. Of the behavioral parameters measured, only avoidance
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behavior in pups from dams exposed on GD 7-13 was statistically significantly affected.
Avoidance was tested by placing pups in shuttle boxes in which an electrical shock could be
delivered via the metal grid floor on one side of the box. A warning tone was sounded, followed
by a 5-second delay and administration of a shock. Pups from the treated dams experienced
statistically significantly (p < 0.01 using Wilcoxson test) fewer number and shorter duration of
electrical shocks compared to controls. No treatment-related behavioral changes were observed
in pups sired by treated males. In contrast, brain tissue analysis revealed statistically significant
differences of all monitored neurochemicals in various segments of the brain in both paternally
and maternally exposed 21-day pups. Increases or decreases in neurochemicals were not
consistent for various brain segments. The toxicological significance of these changes or their
association (if any) with treatment-related changes in avoidance-conditioning is unclear. This
study identified a LOAEL of 25 ppm (78 mg/m ) for neurobehavioral changes in rats exposed in
utero.
OTHER STUDIES
Data from occupational studies indicate that the dermal route is the major route of
absorption in the workplace (Sparer et al., 1988; Piacitelli et al., 1990; Chang et al., 2004;
Kezic et al., 1997). In addition, physicochemical properties of 2-ME and 2-ME acetate,
especially their solubility in both water and lipids, indicate the likelihood of important dermal
absorption of the liquid. Dugard (1984) found the dermal penetration rate of 2-ME in vitro in
human abdominal skin to be 2.82 mg/cm /hour. Dermal contact with either the liquid or its
concentrated aqueous solutions raised the biological concentrations of MAA statistically
significantly above the concentrations reached during inhalation-only exposure at 5 ppm
(Johanson, 1988). Dermal absorption of dilute aqueous solutions of glycol ethers and their
acetates, including 2-ME and 2-ME acetate, were reported to be higher than for neat compounds
(Johanson, 1988). Studies of volunteers exposed to vapors and liquid 2-ME showed extensive
dermal absorption of both the vapor and the liquid, with uptake of the vapor estimated to be 55%
of the total dose. Dermal uptake of undiluted 2-ME placed in a 27-cm2 glass chamber on the
volar forearm for 60 minutes exceeded the uptake by an 8-hour inhalation exposure to 5 ppm
(Kezic et al., 1997) by 100 times. Thus, dermal absorption appears to be a principal route of
exposure to the liquid, as well as the vapor.
Starek et al (2008) injected groups of 5, 12-week old Wistar rats, initially weighing about
300 g each, with 2-ME in saline solution at daily doses of 0, 1.25, 2.5, or 5 millimoles (mM)/kg
for 29 days. These correspond to doses of 0, 95, 190, and 380 mg/kg-day. While control rats
gained weight, all treated rats lost weight; mean body weights correlated negatively with dose
(p < 0.0001). All 2-ME doses resulted in decreased RBC, PCV, and HGB, and increased
reticulocyte counts. These data indicate a 29-day LOAEL of 95 mg/kg-day for hematological
effects in rats injected with 2-ME.
Bagchi and Waxman (2008) reviewed the impact of 2-ME and its active biological
oxidation product, MAA, on testicular gene expression. MAA primarily affects tissues with
rapidly dividing cells and high rates of energy metabolism, including testes, thymus and the
fetus. Testicular toxicity is one of the most prominent consequences of 2-ME and MAA
exposure, which results from apoptosis of primary spermatocytes and is associated with changes
in the expression of various genes and signaling pathways. Of particular importance are the
genes that code for oxidative stress response factors, protein kinases, and nuclear hormone
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receptors. Nuclear receptors and protein kinases regulate multiple cellular processes and are
critical for signaling events required for spermatogenesis. De-regulation of their activity by
2-ME or MAA leads to inappropriate signaling in testicular cells. Oxidative stress in
spermatocytes exposed to MAA triggers mitochondrial release of cytochrome C, activation of
caspases, and ultimately, apoptosis.
Tonkin et al. (2009) gavaged groups of five ~10-week old male Wistar rats with 0-, 50-,
or 150-mg/kg/day 2-ME (99.9% pure) in sterilized water for 3 days and collected testes for
histopathological (left testicle) and gene-expression analysis (right testicle). Histopathological
changes in the testes, consisting of degeneration and necrosis of spermatocytes and reductions in
spermatocyte numbers, were observed only in high-dose (150 mg/kg-day) animals. Microarray
analysis of testicular samples from these animals revealed a large number of differentially
expressed genes from animals exposed to 50- or 150-mg/kg 2-ME (>900 each at >1.5-fold
changed). Expression Analysis Systematic Explorer (EASE) analysis of these genes
demonstrated enrichments in gene protein transport, endocytosis, protein kinase activity, cell
cycle, and meiosis. Quantitative polymerase chain reaction confirmed increased expression of
the actin-binding protein cortactin and the transcription factor Wilm's tumor 1 (Wtl) following
2-ME exposure. Increased localization of cortactin in abnormal spermatocytes was also
observed by immunohistochemistry, consistent with a possible role for this protein in the
mechanism of toxicity.
Acute and Short-term Toxicity
Doe et al. (1983) studied the effects of 2-ME in male rats. Groups of 10 male Wistar rats
were exposed to airborne concentrations of 0-, 100-, or 300-ppm (0-, 310-, or 930-mg/m3) 2-ME
(-99% pure), 6 hours/day, for 10 consecutive days. Daily clinical observations were made. On
Day 10, rats were sacrificed and subjected to postmortem evaluation; blood was collected for
hematological analysis (hemoglobin, hematocrit, MCH, and total RBC and WBC counts), and
testes and thymus were removed for histological evaluation. Body-weight gain in the 300-ppm
males was statistically significantly reduced (body weights not reported). Statistically significant
decreases were observed for all hematological parameters, testicular size, and thymus weights,
while spermatocytic degeneration was statistically significantly increased (no quantitative data
reported) in the 300-ppm group. No hematological, gross, or histological differences were
observed in controls or 100-ppm rats. This study identified a NOAEL and LOAEL of 100 and
300 ppm (310 and 930 mg/m ), respectively, for hematological, testicular, and spermatocytic
effects.
Hong et al. (1988) gave male and female B6C3F1 mice (group size not reported) daily
gavage doses of 0-, 50-, 100-, or 250-mg/kg-day 2-ME (99.6% purity) for 4 consecutive days.
The mice were evaluated for hematology (hemoglobin, hematocrit, MCV, and erythrocyte and
lymphocyte counts) and bone marrow cellularity at 1, 5, and 14 days after the last treatment.
Seven mice/sex/group were sacrificed the day after the last treatment for body- and organ-weight
measurements and histopathological examination of lung, heart, liver, kidneys, adrenal glands,
spleen, thymus, stomach, bone marrow, urinary bladder, small and large intestines, and uterus or
testes. No treatment-related effects were seen for body weights or relative liver, spleen, kidney,
or thymus weights. Relative testes weights were statistically significantly reduced in the
250-mg/kg-day group (24% less than controls). Mild-to-moderate degeneration of seminiferous
tubules was observed in the 250-mg/kg-day males. Erythrocyte counts and hematocrit were
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statistically significantly reduced in >100-mg/kg-day females, while lymphocyte counts were
statistically significantly reduced in >50-mg/kg-day males (see Table 32). Bone marrow
cellularity was statistically significantly reduced in >100-mg/kg-day males on Days 1 and 14 and
in females treated with >50 mg/kg-day on Day 1, >100 mg/kg-day on Day 5, and 250 mg/kg-day
on Day 14 (comparative values not reported). A LOAEL of 50 mg/kg-day was identified for
hematological effects in mice; no NOAEL was identified.
Table 32. Selected Hematological Effects in Mice Given Gavage Doses of

2-Methoxyethanol for 4 Days

Hematological Parameter
Control
50 mg/kg-day
100 mg/kg-day
250 mg/kg-day
Males
Erythrocytes
7.9 ± 0.1a
7.9 ±0.1
8.0 ±0.1
7.9 ±0.1
Hematocrit
37.5 ±0.5
37.2 ±0.4
36.8 ±0.4
36.8 ±0.7
Lymphocytes
4.9 ±0.3
3.1 ±0.1b
2.6 ± 0.2b
1.9 ± 0.2b
Females
Erythrocytes
8.3 ±0.1
8.1 ± 0.1
7.7 ± 0.2b
7.7 ± 0.1b
Hematocrit
38.3 ±0.8
37.4 ±0.5
35.3 ± 0.8b
35.2 ± 0.2b
Lymphocytes
4.0 ±0.2
3.8 ±0.2
4.8 ±0.7
3.9 ±0.3
aMean ± standard error for five mice/group.
bSignificantly different from controls (p < 0.01).
Source: Hong et al. (1988).
Immunotoxicity was identified in several acute studies. Williams et al. (1995) gave male
Fischer 344 rats (six/group) four daily gavage doses of 0, 25, 50, 100, or 200 mg/kg-day of 2-ME
(purity not reported). Animals were immunized with sheep erythrocytes (SRBCs) or
trinitrophenyl-lipopolysaccharide (TNP-LPS) on Day 1 or 2 and assessed for a primary antibody
response to SRBCs or TNP-LPS using a direct plaque-forming cell (PFC) assay. The
lymphoproliferative response was assessed by in vitro mitogenic response of splenic
lymphocytes to a variety of T- and B-cell mitogens. Rats were sacrificed 24 hours after the final
dose. Body, spleen, and thymus weights were measured. Body weights were not affected by
treatment. Rats in the 200-mg/kg-day group showed statistically significantly (p < 0.05)
decreased spleen weights, and rats given >50 mg/kg-day exhibited statistically significantly
(p < 0.05) decreased thymic weights. A statistically significantly (p < 0.05) increased
lymphoproliferative response to in vitro mitogen stimulation occurred in the 200-mg/kg-day
group. Doses of >50 mg/kg-day resulted in a statistically significantly (p < 0.05) decreased PFC
response of spleen cells in rats immunized with SRBCs on Day 1 and TNP-LPS on Day 2 of
dosing. No body or organ weights, or immune responses were reported. This study identified a
NOAEL of 25 mg/kg-day and a LOAEL of 50 mg/kg-day for decreased thymus weights and
decreased primary antibody responses in rats.
Smialowicz et al. (1991) gave male Fischer 344 rats (six to eight/sex/group) daily gavage
doses of 0-, 50-, 100-, or 200-mg/kg-day 2-ME (purity not reported) for 10 days. Rats were
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immunized with SRBC or TNP-LPS on Day 1 or 2 and assessed for a primary antibody response
to SRBC or TNP-LPS using a direct PFC assay. Rats were sacrificed 48 hours after the last
treatment. Body, spleen, testes, and thymus weights were recorded. Multiple T- and B-cell
mitogens were used to assess the in vitro lymphoproliferative response of splenic lymphocytes.
Splenocytes were collected and assessed for in vitro interleukin-2 (IL-2) and natural killer cell
activity. Lymph node cells were collected and assessed for in vitro cytotoxic T-lymphocyte
production. Treatment did not affect body or spleen weights or natural killer cell activity.
Statistically significantly (p < 0.05) decreased testes weights were observed in the
200-mg/kg-day group. A statistically significant (p < 0.05) dose-related decrease in thymus
weights was seen in the >50-mg/kg-day groups. Histological examination of the testes revealed
degeneration of the seminiferous tubules in the >100-mg/kg-day groups. Rats in the
>50-mg/kg-day groups showed a statistically significantly (p < 0.05) decreased splenic
lymphoproliferative response to mitogen stimulation, as well as a decreased PFC response to
TNP-LPS. IL-2 production was statistically significantly (p < 0.05) reduced in all treated
groups. In a separate test in which groups of six male and female rats were given 10 daily
gavage doses of 0-, 25-, 50-, 100-, or 200-mg/kg-day 2-ME, males were more sensitive to the
decreased PFC response to TNP-LPS than females, with statistically significant (p < 0.05)
reduction in PFC response observed in the >50-mg/kg-day males and >100-mg/kg-day females.
Males given 200 mg/kg-day exhibited a decreased ability to expel worms following infection
with Trichinella spiralis larvae. No quantitative values were reported. This study identified a
LOAEL of 50 mg/kg-day for decreases in thymus and testes weights and ex vivo immunologic
responses; no NOAEL was identified.
Smialowicz et al. (1992a) also gave male Fischer 344 rats (six/group) daily gavage doses
of 0-, 50-, 100-, 200-, or 400-mg/kg-day 2-ME (purity not reported) in distilled water for
2 consecutive days following a single immunization with TNP-LPS; 3 days later, the primary
antibody response to TNP-LPS was determined with a PFC assay. In all treated groups, 2-ME
caused a statistically significant (p < 0.05) decrease in PFC response to TNP-LPS (32% lower
than controls). This effect was blocked by coadministration of 4-methylpyrazole, an alcohol
dehydrogenase inhibitor. The study authors suggested that 4-methylpyrazole protection against
reduced PFC response indicates that oxidative metabolism of 2-ME is required for
immunotoxicity. This study identified a LOAEL of 50 mg/kg-day for decreased primary
antibody response in male rats; no NOAEL was identified.
Smialowicz et al. (1992b) gave female Fischer 344 rats and C57BL/6J mice (six/group)
daily gavage doses of 0-, 50-, 100-, 200-, or 400-mg/kg-day 2-ME (purity not reported) in
distilled water for 10 consecutive days. The animals were weighed and sacrificed 48 hours after
the last dose, and the spleen and thymus were removed and weighed. The lymphoproliferative
response of splenic lymphocytes was determined following in vitro stimulation with multiple B-
and T-cell mitogens. In rats, 2-ME treatment did not affect body or spleen weights. Thymus
weights were decreased in a dose-related manner, with statistically significant differences from
controls occurring at >100 mg/kg-day. A decreased lymphoproliferative response in rat splenic
lymphocytes was seen in all dose groups. In rats injected with TNP-LPS on Day 9 of 2-ME
exposure, statistically significantly (p < 0.05) decreased PFC responses were seen in the
>100-mg/kg-day groups. In contrast, exposure of mice to 2-ME did not affect body, spleen, or
thymus weights or lymphoproliferative responses. Likewise, no effect on PFC response was
seen in mice exposed to TNP-LPS on Day 9 of exposure to 2-ME. No quantitative values were
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reported. These studies identified a LOAEL of 50 mg/kg-day for decreased lymphoproliferative
response in female rats; a NOAEL for rats was not identified. A NOAEL of 400 mg/kg-day, the
highest dose tested, was identified for changes in body weights, organ weights, and
lymphoproliferative response in female mice.
Toxicokinetics
2-Methoxyethanol
At air concentrations <5 ppm, metabolism is a first-order process, and the relationship
between uptake and biological concentrations of metabolites is linear (Groeseneken et al., 1989).
2-ME is rapidly and extensively absorbed in the lungs, with 76% of an inhaled dose translocated
across pulmonary tissues in humans, resulting in a measured uptake rate of 97 |ig/min
(Groeseneken et al., 1989). No data were given regarding gastrointestinal uptake of 2-ME.
Partition coefficients for 2-ME at body temperature are as follows (Johanson and Dynesius,
1988):
blood-gas 32,800
water-gas 35,900
oil-gas 529
Initially, it is widely distributed across body tissues with the exception of adipose tissues,
in which a very low tissue:blood partition coefficient of 0.02 has been measured. Radiolabel
studies have shown 2-ME to initially distribute rapidly throughout the body's water and soft
tissues and subsequently localize in the liver, kidney, bone marrow, and epididymis. In pregnant
mice, 2-ME rapidly crosses the placenta into the conceptus. 2-ME is predominantly oxidized to
MAA via methoxyacetaldehyde. In human volunteers, 85% of a 4-hour inhalation exposure to
16-mg/m3 2-ME was metabolized to MAA. MAA appeared in the urine shortly after the start of
exposure to 2-ME or 2-ME acetate; the concentration rose during the exposure. The
concentration usually peaked 4-8 hours after the end of exposure and remained measurable for at
least 5 days after a single exposure (Groeseneken et al., 1989). The decline of MAA
concentration was reported to be monophasic with an apparent elimination half-life of
77.1 hours, measured in human volunteers at rest and "calculated from the slope of the linear part
of the log-linear excretion rate-time curve" (Groeseneken et al., 1989). Volunteers were still
excreting MAA at one-third of the peak rate 5 days after a 4-hour experimental exposure. In a
workplace study, Shih et al., 2001 reported that MAA rose daily in urine specimens taken at the
end of the shift throughout the week. Seven days after no exposure, MAA in the urine was still
elevated, indicating accumulation of MAA over the workweek. In workplace settings, the
half-life has been close to that established in laboratory studies, about 77 hours.
Alcohol dehydrogenase (in mice and rats) and aldehyde dehydrogenase (in mice, rats,
hamsters, rabbits, guinea pigs, and humans) in the testes are capable of 2-ME oxidation to
2-MAA. This metabolite produces testicular toxicity in male rats (Foster et al., 1987) and
embryo toxicity in female rats at doses equivalent to those of 2-ME and 2-ME acetate that
produce the same effects (Rawlings et al., 1985). Welsch (2005) also concluded that 2-MAA is
the putative toxicant for reproductive and developmental effects in animals. Ethanol reduces the
metabolism of ethylene glycol monoethyl ether (EGEE) and ethylene glycol monoethyl ether
acetate (EGEEA) in rats and probably affects the metabolism of 2-ME and 2-ME acetate in
humans, due to competitive inhibition of alcohol dehydrogenase (Johanson, 1988). 2-ME also
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has been shown to be demethylated in humans to ethylene glycol. It may also be conjugated to a
glucuronide or sulfate. Elimination occurs via urinary excretion of the metabolites. There are
10 urinary metabolites that have been identified, including ethylene glycol, glycolic acid,
glycine, methoxyethanol-P-D-glucuronide, methoxyethylsulfate, MAA, methoxyacetyl-
P-D-glucuronide, methoxy-A-acetylglycine, methoxycitrate, and methoxybutenoic acid.
Shih et al. (1999) monitored 27 workers exposed to 2-ME in a semiconductor copper
laminate circuit board plant and 30 nonexposed controls for 2-ME exposure for the entire
workweek, using personal sampling. Eighteen of the 27 exposed workers, whose exposures were
confined to airborne exposure as well as vapor only dermal exposures to 2-ME, showed a good
correlation between both end of the workweek MAA and the mean weekly exposure to 2-ME.
The authors found a weekly mean concentration of 4.46 ppm (SD = 2.56) and a mean Friday
afternoon MAA of 46.5-mg/g creatinine (SD = 33.5). Even after 7 days away from work, the
mean before-shift urinary MAA concentration was elevated, demonstrating that the half-life of
MAA is close to that established in laboratory studies, about 77 hours. Of the 30 "control"
workers monitored, exposure measurements for three workers near the heat press operation
ranged from 0.2-0.3 ppm. These three workers were the only ones who had detectable
concentrations of MAA in their urine in the range of 0.4-0.5 mg/L.
Shih et al. (2000, 2003) conducted two studies on the effects of 2-ME exposure on
hematological and spermatotoxicity. In the first study, Shih et al. (2000) monitored 53 workers
from two semiconductor copper laminate circuit board assembly plants for 2-ME exposure on
Friday using personal samplers. These workers also submitted an end-of-shift urine specimen
for MAA analysis. A group of 121 lamination workers with indirect exposure to 2-ME were
selected as controls. The GM air concentration of 2-ME in the first plant was 3.98 ppm
(GSD = 2.88, n = 55 personal samples), with a range of 0.65 to 30.1 ppm, while the GM 2-ME
concentration in the second plant was 4.27 ppm (GSD = 2.19,n = 11 personal samples) with a
range of 1.7 to 20.0 ppm. The GM urinary MAA concentrations were 19.95-mg/g creatinine
(GSD = 2.19, n = 30) in the first plant and 20.89-mg/g creatinine (GSD = 2.19, n = 15) in the
second plant. The air concentrations reported for the control group were between nondetectable
and 0.28 ppm, n = 9. The GM urinary concentration of MAA in the control facility was
1.26-mg/g creatinine (GSD = 1.62, n = 32) with a range from nondetectable to 4.22-mg/g
creatinine.
In the second study, Shih et al. (2003) studied the same group of 29 impregnation
workers in a copper laminated circuit board plant. An initial survey was conducted in February
1997 with follow-up studies conducted in April and in August of the same year. The aim of the
study was to examine the air concentrations of 2-ME, urinary MAA concentrations, and
hematological effects following an aggressive workplace improvement program. There were
three groups: an exposed group (n = 29), a low-exposure group of heat-press workers (n = 32),
and a nonexposed control group (n = 58) of administrative workers. Full-shift 8-hour TWA
personal air samples were collected on Fridays for exposed groups and randomly among the
administrative workers. End-of-shift urine specimens were collected from exposed workers and
randomly among the administrative workers. Nine of the 32 heat-press operators were randomly
selected for personal air sampling. Ten randomly selected area air samples were collected from
the 58 administrative workers. After improved engineering controls were instituted, the GM
concentrations of 2-ME declined in the 29 workers from the initial survey of 9.62 ppm
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(GSD = 4.75; range 0.75-320) to 2.34 ppm (GSD = 1.76, range 0.2-10) in April to 0.34 ppm
(GSD = 2.69; range, 0.1-3.5) in August, confirming the impact of engineering controls to reduce
exposure. During the same corresponding intervals, urinary end-of-shift MAA in the same
29 workers dropped from an initial GM baseline of 50.7-mg/g creatinine (GSD = 1.67; range,
24.3-139) to a GM of 19.7-mg/g creatinine (GSD = 2.09; range, 4.6-54.9) in April and a mean
of 6.77-mg/g creatinine (GSD = 4.19; range, 0.95-25.2) in August. The comparison group of
nine randomly selected heat-press workers had an initial baseline GM exposure of 0.08 ppm
(GSD = 5.09; range not detected (ND) to 0.80, n = 9 randomly selected workers). No 2-ME was
detected in the air to which administrative controls were exposed. The heat-press workers' mean
MAA was 0.53-mg/g creatinine (GSD = 3.40; range not detected to 4.22, n = 32). These results
from the heat press operators show a GM of 0.53-mg/g creatinine MAA at a GM 2-ME air
concentration of 0.08 ppm.
In another study by this group, Chang et al. (2004) examined the protective effectiveness
of gloves in 74 exposed workers in the same semiconductor copper laminated circuit board plant
as before, along with 80 nonexposed controls. As in other plants, there were two groups of
workers, "regular operations" with lower exposures and "special operations" with higher
exposures, as well as more contact with liquid 2-ME. One 8-hour TWA 2-ME sample was
collected on the last day of the workweek. The exposures were quite constant throughout the
workweek. The GM exposure in the regular operations group was 2.14 ppm (GSD = 2.01; range
0.57-9.28, n = 49) and 8.13 ppm (GSD = 1.62; range 3.18-15.64, n = 25) in special operations,
with the lowest detected exposure of 0.57 ppm. Urine specimens collected at the end of the last
shift on Friday showed GMs of 5.44-mg/g creatinine (GSD = 3.59; range 0.52-40.7, n = 49) for
the regular operations group and 72.6-mg/g creatinine (GSD = 2.04; range 16.39-178.0, n = 25)
for the special operations group. The authors did not show correlations between 2-ME and MAA
in urine. The amount of MAA in the urine of "regular operations" workers was much less than
reported in previous papers by the same group, perhaps an indication of less exposure through
the dermal route.
2-Methoxyethanol Acetate
2-ME acetate is more soluble in oil and less soluble in water than 2-ME. 2-ME acetate in
blood is immediately hydrolyzed to 2-ME (Johanson and Dynesius, 1988). No data are available
for describing the absorption, distribution, metabolism, and elimination of 2-ME acetate in vivo
in humans or animals. However, in vitro studies in animal tissues suggest hydrolysis of the
acetate to 2-ME. In mice, nasal mucosal epithelium homogenates exhibited equimolar
hydrolysis of 2-ME acetate to 2-ME and acetic acid via carboxylesterase, with apparent Vmax
and Km values of 0.9 mM/min and 13.7 mM, respectively (Stott and McKenna, 1985). No
differences were observed between sexes, but carboxylesterase activity in mice was 13% lower
than dogs, 19% higher than rats, and 147%) higher than rabbits. Further, the specific activity of
carboxylesterase for 2-ME acetate hydrolysis in homogenized mouse nasal mucosa
(667 nmoles/mg protein/min) was similar to that of liver (677 nmoles/mg protein/min) and
higher than kidney, lung, or blood (266, 177, and 84 nmoles/mg protein/min, respectively).
Bogdanffy et al. (1987) showed that carboxylesterase activity was 3-6 times higher in olfactory
epithelium compared to respiratory epithelium. In rat plasma, 2-ME acetate is quickly
hydrolyzed by plasma esterases to 2-ME, with an in vitro half-life of-12 minutes (Hoffman,
1984, as cited in ECETOC, 1984).
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Groeseneken et al. (1989) determined that inhaled 2-ME acetate is exhaled as 2-ME after
hydrolysis. ACGIH (2006b) concluded that the systemic effects of inhalation exposure to 2-ME
acetate occurred at lower concentrations than were likely to cause respiratory tract effects.
In vivo animal studies of glycol ethers support the notion that hydrolysis of 2-ME acetate
to 2-ME is rapid and extensive. Oral exposure of mice to propylene glycol monomethyl ether
(Miller et al., 1983b) or its acetate (Miller et al., 1984) resulted in almost identical profiles of
metabolite production and urinary elimination, suggesting that the acetate is rapidly and
extensively hydrolyzed. For 2-ME acetate, the occurrence of reduced testes weights and
increased testicular atrophy and tubule degeneration at >500 ppm in mice was also seen at
approximately equimolar exposures of >250-ppm 2-ME (Nagano et al., 1979). This suggests
equivalent internal dosimetry of 2-ME and its metabolites in animals exposed to the acetate, and
that the generation of acetic acid from the oral exposures tested does not contribute substantially
to 2-ME acetate toxicity.
PBPK Models
Several studies described the development and application of PBPK models for
estimation of internal 2-ME dosimetry across species. Welsch et al. (1995) developed single and
multiple-compartment physiological models in the mouse (for oral or i.v.-administered 2-ME)
capable of predicting maternal plasma, embryo, and extraembryonic fluid concentrations of
2-ME and its putative reproductive toxicant, 2-MAA. The multicompartment model was scaled
to rats (using rat-specific values for physiological and metabolic characteristics) and humans (via
linkage to a previously published human model for pregnancy [O'Flaherty et al., 1992]). The
human model was not exercised against actual 2-ME or 2-MAA data from humans. Hays et al.
(2000) developed a PBPK model to simulate oral and i.v. exposures of 2-ME in the rat. This
model is capable of predicting blood and tissue concentrations of both 2-ME and 2-MAA.
Gargas et al. (2000) further developed the Hays et al. (2000) rat model to include inhalation
exposures and dynamic physiological changes associated with pregnancy. Predictions of
maternal blood 2-ME and fetal 2-ME and 2-MAA concentrations were similar to observations.
This model was allometrically scaled to humans and exercised against human urinary 2-MAA
data but not in pregnant humans. The available PBPK models were designed to predict internal
dosimetry as it related to maternal 2-ME exposures and organogenesis but do not have the
capability for predicting male reproductive endpoints, such as testicular concentrations of 2-ME
or 2-MAA.
Genotoxicity
Available data on the genotoxicity of 2-ME have been mixed. Tests of mutagenic
activity in the Ames test using Salmonella typhimurium strains TA98, TA100, TA102, TA1535,
TA1537, and TA1538 with or without an S-9 activation system have been negative
(Hoflack et al., 1995; Guzzie et al., 1986; McGregor et al., 1983; Ong, 1980). 2-ME was
negative in the cell culture test for unscheduled DNA synthesis (McGregor et al., 1983;
Guzzie et al., 1986) and point mutations in mouse lymphoma L5178Y cells or Chinese hamster
ovary cells (Ma et al., 1993; McGregor, 1984). Weakly positive results (Chapin et al., 1985b;
McGregor et al., 1983) and negative results (Rao et al., 1983) in the dominant lethal test in rats,
strong positive results in mouse sperm abnormalities (Chapin et al., 1985a,b; McGregor et al.,
1983), and inconsistent results in the sex-linked recessive lethal test in Drosophila
(McGregor et al., 1983) were reported for 2-ME. Sister chromatid exchange was observed in
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mouse bone marrow cells following i.p. injection (Arashidani et al., 1998) but not following
gavage (Au et al., 1993) or in vitro exposure of human peripheral blood (Arashidani et al., 1998)
or Chinese hamster ovary cells (Chiewchanwit and Au, 1994).
DERIVATION OF SUBCHRONIC AND CHRONIC
p-RfDS FOR 2-METHOXYETHANOL
Available human oral studies consist of case reports of acute accidental ingestion. As
such, they are not suitable for use as key studies in the derivation of a p-RfD.
Available studies in animals have reported adverse effects of 2-ME on a number of
endpoints following oral exposure (see Table 33). Studies in adult animals have identified
changes in body-weight gain, decreased organ weights (thymus, spleen, and testes), anemia, and
diminished immune function as sensitive endpoints (NTP, 1993; Smialowicz et al., 1992a,b,
1991; Hong et al., 1988; Nagano et al., 1984; Mellon Institute, 1962). Oral exposure to 2-ME
has also been shown to have adverse effects on reproductive function and reproductive organs,
including testicular and seminiferous tubule degeneration, morphologically altered sperm and
decreased sperm number, and ovarian histopathology and estrous cycle alterations (Berndtson
and Foote, 1997; Foote et al., 1995; NTP, 1993; Smialowicz et al., 1991; NTP, 1990, 1989,
1988a,b; Hong et al., 1988; Chapin et al., 1985a,b; Nagano et al., 1984; Dodo et al., 2009).
2-ME also induced adverse effects on the developing organism including fetal death, decreased
fetal/pup body weights, increased gestation length, and developmental malformations, including
cardiovascular malformations and alterations of the digits (Sleet and Ross, 1997; Nelson et al.,
1989; Scott et al., 1989; Toraason and Breitenstein, 1988; Greene et al., 1987; Hardin and
Eisenmann, 1987; Toraason et al., 1986, 1985; Horton et al., 1985; Nagano et al., 1981).
Reproductive or developmental effects have been reported in rats, mice, hamsters, guinea pigs,
rabbits, and macaques. Tonkin et al. (2009) provided some elucidation of the MOA for testicular
toxicity using gene-expression analysis.
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Table 33. Summary of Oral Toxicity Studies of 2-Methoxyethanol and 2-Methoxyethanol Acetate in Animals
Reference
Description
NOAEL
(mg/kg-day)
LOAEL
(mg/kg-day)
Observed Responses
2-Methoxyethanol acetate
Nagano et al. (1979)
A gavage study in male JCL-ICR mice
exposed for 5 weeks, 5 days/week
Daily doses: 0, 62.5, 125, 250, 500, 1000, or
2000 mg/kg-day
250
(179, adjusted
for continuous
exposure)
500
(357, adjusted for
continuous
exposure)
Reduced testes weights and spermatocyte
counts
(Data shown in Table 1)
2-Methoxyethanol
Short-term systemic studies
Nagano et al. (1979)
A gavage study in male JCL-ICR mice
exposed for 5 weeks, 5 days/week
Daily doses: 0, 62.5, 125, 250, 500, 1000, or
2000 mg/kg-day
125
(89, adjusted for
continuous
exposure)
250
(179, adjusted for
continuous
exposure)
Reduced testes weights and spermatocyte
counts
(Data shown in Table 1)
Hong et al. (1988)
A gavage study in male and female B6C3F1
mice exposed for 4 days
Daily doses: 0, 50, 100, or 250 mg/kg-day
ND
50
Reduced lymphocytes in males
(Data shown in Table 32)
Immunotoxicity studies
Williams et al. (1995)
A gavage study in male F344 rats exposed
for 4 days
Daily doses: 0, 25, 50, 100, or
200 mg/kg-day
25
50
Decreased thymus weight and primary antibody
response
Smialowicz et al.
(1991)
A gavage study in male F344 rats exposed
for 10 days
Daily doses: 0, 50, 100, or 200 mg/kg-day
ND
50
Decreased thymus and testes weights and
primary antibody response
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Table 33. Summary of Oral Toxicity Studies of 2-Methoxyethanol and 2-Methoxyethanol Acetate in Animals
Reference
Description
NOAEL
(mg/kg-day)
LOAEL
(mg/kg-day)
Observed Responses
Smialowicz et al.
(1992a)
A gavage study in male F344 rats exposed
for 2 days
Daily doses: 0, 50, 100, 200, or
400 mg/kg-day
ND
50
Decreased primary antibody response
Smialowicz et al.
(1992b)
A gavage study in female F344 rats and
female C57BL/6J mice exposed for 10 days
Daily doses: 0, 50, 100, 200, or
400 mg/kg-day
Rats: ND
Mice: 400
Rats: 50
Mice: ND
Rats: Decreased lymphoproliferative response
Mice: No treatment-related changes in body or
organ weights or lymphoproliferative response
Systemic studies
NTP (1993)
A drinking water study in F344/N rats
exposed for 13 weeks
Drinking water concentrations: 0, 750,
1500, 3000, 4500, or 6000 ppm
Estimated doses (mg/kg-day)a:
Males: 0, 71, 165, 324, 715, or 806
Females: 0, 70, 135, 297, 546, or 785
ND
Male: 71
(750 ppm in
drinking water)
Female: 70
(750 ppm in
drinking water)
Male: Testicular lesions and reduced semen
quality; decreased thymus weight
Female: Decreased thymus weight
(Data shown in Tables 6-8)
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Table 33. Summary of Oral Toxicity Studies of 2-Methoxyethanol and 2-Methoxyethanol Acetate in Animals
Reference
Description
NOAEL
(mg/kg-day)
LOAEL
(mg/kg-day)
Observed Responses
NTP (1993)
A drinking water study in B6C3F1 mice
exposed for 13 weeks
Drinking water concentrations: 0, 2000,
4000, 6000, 8000, or 10,000 ppm
Estimated doses (mg/kg-day)a:
Males: 0, 295, 529, 765, 992, or 1367
Females: 0, 492, 902, 1194, 1489, or 1839
ND
Male: 295
(2000 ppm in
drinking water)
Female: 492
(2000 ppm in
drinking water)
Male: Decreased spermatozoal concentration
Female: Adrenal hypertrophy and splenic
hematopoiesis
(Data shown in Tables 9 and 10)
Mellon Institute
(1962)
A dietary exposure study in DW albino rats
exposed for 3 months
Dietary concentrations: 0, 0.01, 0.05, 0.25%
Estimated doses (mg/kg-day)a:
Males: 0, 7, 40, or 178
Females: 0, 8, 43, or 201
7 (0.01% diet)
40 (0.05% diet)
Decreased body weight gain
Reproductive studies
Foster et al. (1984,
1983)
A gavage study in male Sprague-Dawley
rats exposed for 11 days
Daily doses: 0, 50, 100, or 200 mg/kg-day
50
100
Spermatocyte degeneration
Chapin et al. (1985a)
A gavage study in male F344 rats exposed
for 5 days
Daily doses: 0, 50, 100, or 200 mg/kg-day
ND
50
Reduced sperm counts and increased incidences
of abnormal sperm morphology
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Table 33. Summary of Oral Toxicity Studies of 2-Methoxyethanol and 2-Methoxyethanol Acetate in Animals
Reference
Description
NOAEL
(mg/kg-day)
LOAEL
(mg/kg-day)
Observed Responses
Chapin et al. (1985b)
A gavage study in male F344 rats exposed
for 5 days
Daily doses: 0, 50, 100, or 200 mg/kg-day
ND
50
Increased severity of testicular histopathology
NTP (1990)
A two-generation drinking water study in
Sprague-Dawley rats cohabitating for 6
weeks
Drinking water concentrations: 0, 0.01,
0.03, or 0.1%
Estimated doses (mg/kg-day)a:
F0 Males: 0, 9, 24, or 76
F1 Males: 0,9, or 27
F0 Females: 0, 13, 36, or 132
F1 Females: 0, 15, or 41
ND
9 (F1 males)
(0.01% dw)
Increased testicular degeneration, decreased
sperm density
(Data shown in Table 16)
NTP (1988a,b; 1989)
A two-generation drinking water study in
C57BL/6, CD-I, and C3Hmice cohabitating
for 6 weeks
Drinking water concentrations: 0, 0.03, 0.1,
or 0.3%
Estimated doses (mg/kg-day)a:
F0 Males: 0, 53-64, 170-219, or 505-636
F0 Females: 0, 54-63, 174-235, or
543-645
CD-I: ND
C57BL/6: ND
C3H: 64
(0.03% dw)
CD-I: 60
(0.03% dw)
C57BL/6: 53
(0.03% dw)
C3H: 219
(0.1% dw)
CD-I: Reduced number of live pups/litter in
F1 males
C57BL/6: Reduced survival of F0 and F1 male
offspring, reduced sperm quality
C3H: Testicular lesions, reduced sperm
quality, fetal toxicity, impaired fertility
(Data shown in Tables 17-19)
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Table 33. Summary of Oral Toxicity Studies of 2-Methoxyethanol and 2-Methoxyethanol Acetate in Animals
Reference
Description
NOAEL
(mg/kg-day)
LOAEL
(mg/kg-day)
Observed Responses
Berndtson and Foote
etal. (1997)
A drinking water study in male Dutch
rabbits, 5 days/week, for 12 weeks
Doses: 0, 12.5, 25, 37.5, or 50 mg/kg-day
25
(18, adjusted for
continuous
exposure)
37.5
(27, adjusted for
continuous
exposure)
Reduced spermatids
Foote et al. (1995)
A drinking water study in male Dutch
rabbits, 5 days/week, for 12 weeks
Doses: 0, 12.5, 25, 37.5, or 50 mg/kg-day
12.5
(9, adjusted for
continuous
exposure)
25
(18, adjusted for
continuous
exposure)
Reduced sperm motility and semen quality
(Data shown in Table 22)
Developmental studies
Nelson et al. (1989)
A liquid diet study in pregnant
Sprague-Dawley rats exposed on GDs 7-18
Maternal: 73
(0.025% diet)
Maternal: 140
(0.05% diet)
Maternal: Reduced body weight gain

Liquid dietary concentrations: 0, 0.006,
0.012, 0.025, 0.05, 0.1, 0.25, or 0.5%
Fetal: ND
Fetal: 16
(0.006% diet)
Fetal: Reduced body weight

Estimated doses (mg/kg-day)a:
0, 16,31,73, 140, 198, 290, or
620 mg/kg-day


(Data shown in Table 24)
Nelson et al. (1989)
A liquid diet study in pregnant
Sprague-Dawley rats exposed on GDs 7-18
Maternal: ND
Maternal: 17
(0.006% diet)
Maternal: Increased length of gestation

Liquid dietary concentrations: 0, 0.006,
0.012, or 0.14%
Fetal: ND
Fetal: 17
(0.006% diet)
Fetal: Increased length of gestation; no effect
on neurobehavioral battery

Estimated doses (mg/kg-day)a:
0, 17, 33, or 40 mg/kg-day


(Data shown in Table 25)
Sleet and Ross (1997)
A gavage study in pregnant CRL:CD rats
exposed on GD 13
Daily doses: 0, 50, 100, or 250 mg/kg-day
Maternal: 250
Fetal: ND
Maternal: ND
Fetal: 50
Maternal: No effects identified
Fetal: Limb-bud malformations; decreased
embryonic weights
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Table 33. Summary of Oral Toxicity Studies of 2-Methoxyethanol and 2-Methoxyethanol Acetate in Animals
Reference
Description
NOAEL
(mg/kg-day)
LOAEL
(mg/kg-day)
Observed Responses
Toraason et al. (1985)
A gavage study in pregnant Sprague-Dawley
rats exposed on GDs 7-13
Daily doses: 0, 25, 50, or 100 mg/kg-day
Maternal: ND
Fetal: ND
Maternal: 25
Fetal: 25
Maternal: Reduced body weight
Fetal: Slight changes in EKG
Toraason et al. (1986)
A gavage study in pregnant Sprague-Dawley
rats exposed on GDs 7-13 or 13-19
Daily doses: 0 or 25 mg/kg-day
Maternal: 25
Fetal: ND
Maternal: ND
Fetal: 25
Maternal: No effects identified
Fetal: Retarded cardiac development
Toraason and
Breitenstein (1988)
A gavage study in pregnant Sprague-Dawley
rats exposed on GDs 7-13
Daily doses: 0, 50, or 75 mg/kg-day
Maternal: ND
Developmental:
ND
Maternal: 50
Developmental: 50
Maternal: Reduced body weight and prolonged
gestation
Developmental: Fetal resorptions, pup
lethality, decreased postnatal weights, increased
relative heart weights, and EKG changes
Nagano et al. (1981)
A gavage study in pregnant JCL-ICR mice
exposed on GDs 7-14
Daily doses: 0, 31.25, 62.5, 125, 250, 500,
or 1000 mg/kg-day
Maternal: 125
Fetal: ND
Maternal: 250
Fetal: 31.25
Maternal: Reduced body weight
Fetal: Dose-related increase in skeletal
anomalies
(Data shown in Table 26)
Greene et al. (1987)
A gavage study in pregnant CD-I mice
exposed on GD 11
Daily doses: 0, 100, 175, 250, 350, 400,
450, or 500 mg/kg-day
Maternal: ND
Fetal: ND
Maternal: ND
Fetal: 100
Maternal: Not reported
Fetal: Histological evidence of limb-bud cell
death
Horton et al. (1985)
A gavage study in pregnant CD-I mice
exposed on GD 9, 10, 11, 12, or 13
Maternal: ND
Maternal: ND
Maternal: Reduced body weight gain in
unspecified groups

Daily doses: 0, 100, 175, 250, 350, 400,
450, or 500 mg/kg-day
Fetal: 100
Fetal: 175
Fetal: Dose-related increase in skeletal
(particularly digital) malformations
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Table 33. Summary of Oral Toxicity Studies of 2-Methoxyethanol and 2-Methoxyethanol Acetate in Animals
Reference
Description
NOAEL
(mg/kg-day)
LOAEL
(mg/kg-day)
Observed Responses
Hardin and
Eisenmann (1987)
A gavage study in pregnant CD-I mice
exposed on GD 11
Daily doses: 0 or 304 mg/kg-day
Maternal: ND
Fetal: ND
Maternal: ND
Fetal: 304
Maternal: Not reported
Fetal: Paw development defects,
predominantly syndactyly
Scott et al. (1989)
A gavage study in pregnant M fascicularis
(macaques) exposed on GDs 20-45
Maternal: ND
Maternal: 12
Maternal: Dose-related anorexia and maternal
body weight loss

Daily doses: 0, 12, 24, or 36 mg/kg-day
Fetal: ND
Fetal: 12
Fetal: Dose-related embryo lethality
"Calculated by study authors based on measurements of body weight and average daily drinking water or food consumption.
ND = not determined.
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Based on the above studies, the most sensitive endpoints appear to be reproductive,
including alterations in testicular histology in parents and embryo lethality. A number of studies
(Berndtson and Foote, 1997; NTP, 1993; Smialowicz et al., 1991; NTP, 1990, 1989, 1988a,b;
Hong et al., 1988; Chapin et al., 1985a,b) have reported testicular degeneration following oral
exposure to 2-ME in rats, mice, and rabbits. The study by NTP (1990) identified the lowest
LOAEL in available animal studies (9.07 mg/kg-day for testicular degeneration and decreased
sperm density in F1 male rats in a two-generation reproduction study) but did not identify a
NOAEL. This LOAEL is lower than the majority of the NOAELs identified by other studies,
suggesting that the juvenile male is more sensitive than the adult to reproductive effects. The
next lowest LOAEL in the database was 12 mg/kg-day for embryo lethality in macaques exposed
during gestation, also without an accompanying NOAEL (Scott et al., 1989). The data from
NTP (1990) and Scott et al. (1989), as well as data from other studies identifying LOAELs of
<25 mg/kg-day (Foote et al., 1995; Nelson et al., 1989; Toraason et al., 1986) and NTP (1993),
were evaluated as candidates for benchmark dose (BMD) modeling to estimate a point of
departure (POD) for RfD derivation. Three of the these data sets (Foote et al., 1995;
Nelson et al., 1989; Toraason et al., 1986) are not amenable to BMD modeling, either because
the effects were not dose-related, or data for variability about the mean were not reported for
continuous endpoints. These studies are not considered further for p-RfD derivation because the
LOAELs identified in each study (16-25 mg/kg-day) were higher than the LOAELs of
9.07 mg/kg-day identified by NTP (1990) and 12 mg/kg-day from Scott et al. (1989).
BMD modeling (BMDS version 2.1) was conducted using the dichotomous data of NTP
(1993, 1990) for seminiferous tubule degeneration in rats, and Scott et al. (1989) for embryo
lethality in macaques, as well as the continuous data of NTP (1990) for reduced sperm density in
F1 rats. Appendix A contains details of the modeling. The 95% lower confidence limit on the
BMD (BMDL) estimates obtained are as follows:
•	0.75 mg/kg-day for seminiferous tubule degeneration in F1 male rats (NTP, 1993)
•	1.64 mg/kg-day for embryo lethality in macaques (Scott et al., 1989)
•	14.3 mg/kg-day for reduced sperm density in F1 rats (NTP, 1990)
BMD modeling of the testicular degeneration data from NTP (1990) was not successful because
the models would not run.
The NTP (1993) data for seminiferous tubule degeneration in F1 male rats provided the
lowest BMDL. However, these results were considered unreliable due to weaknesses in the data
set (see Appendix A), which provided insufficient data to inform the dose-response curve near
the calculated BMDL. These weaknesses included no NOAEL, a LOAEL 95 times higher than
the BMDLio, and a 70% response rate at the LOAEL.
The BMDLisd of 14.3 mg/kg-day from the NTP (1990) sperm density data also is
considered unreliable because it is higher than the LOAEL of 9.07 mg/kg-day identified for this
data set. In addition, the sperm density in controls predicted by the BMD model is lower than
observed, while the prediction for sperm density at the LOAEL dose is considerably higher than
observed. These discrepencies appear to result from the large variability in control sperm
density data and the shallow slope of the fitted dose-response curve.
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The BMDL5 of 1.64 mg/kg-day based on embryo lethality in macaques is lower than the
LOAEL of 12 mg/kg-day in this study and is lower than either the NOAELs or LOAELs
identified in any of the studies, or the BMDL for effects on sperm density from NTP (1990). In
addition, BMD curve for these data fits the data well. Scott et al. (1989) was selected as the
critical study for the derivation of a provisional subchronic and chronic RfDs, because it
provides the lowest reliable POD and because it is based on a response in macaques, a species
more closely related to humans than rodents.
Although PBPK models for 2-ME have been developed (see above), they are not suitable
for extrapolating internal dosimetry for testicular effects in rats because they do not predict
internal doses relevant to male reproductive endpoints, such as testicular concentrations of 2-ME
or 2-MAA, and they have not been evaluated for their ability to predict internal dosimetry in
macaques.
Data from occupational studies indicate that the dermal route is the major route of
absorption in the workplace (Sparer et al., 1988; Piacitelli et al., 1990; Chang et al., 2004;
Kezic et al., 1997). In addition, physicochemical properties of 2-ME and 2-ME acetate indicate
the likelihood of substantial dermal absorption of the liquid (Dugard, 1984; Johanson, 1988).
Studies done in volunteers exposed to vapors and liquid 2-ME showed extensive dermal
absorption of both the vapor and the liquid. (Kezic et al., 1997). Because data indicate that
dermal absorption occurs readily, the potential for dermal exposure must be considered when
applying the p-RfDs and p-RfCs for 2-ME and its acetate.
The subchronic p-RfD is calculated as follows:
Subchronic p-RfD = BMDL5 UF
= 1.64 mg/kg-day/100
= 0.0164 or 2 x \{f2 mg/kg-day
The composite UF of 100 is composed of the following:
•	A default UFA of 10 is applied for interspecies extrapolation to account for
potential pharmacokinetic and pharmacodynamic differences between macaques
and humans.
•	A default UFh of 10 for intraspecies differences is applied to account for
potentially susceptible individuals in the absence of information on the variability
of response in humans.
•	An UFl is not needed because a BMDL5 is used as the POD for derivation of the
p-RfD.
•	An UFS is not needed because the POD is from a 26-day developmental toxicity
study with exposure during organogenesis and because subchronic studies
resulted in effects only at higher doses.
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•	An UFd is not needed because because the database for this chemical includes
numerous supporting developmental, reproductive, immunotoxicity, and
subchronic toxicity studies in rats, mice, and rabbits.
Confidence in the critical study is high. The study evaluated appropriate endpoints of
reproductive toxicity in adequately sized groups of female macaques exposed to three
appropriately chosen gavage doses. Confidence in the database also is high; high-quality
subchronic studies are available, while the reproductive and developmental toxicity of 2-ME has
been tested in numerous studies. Therefore, confidence in the subchronic p-RfD is high.
The chronic p-RfD is calculated as follows:
Chronic p-RfD = BMDLio UF
= 1.64 mg/kg-day/3 00
= 0.00547 or 5 x 10~3 mg/kg-day
The composite UF of 300 is composed of the following:
•	A default UFA of 10 is applied for interspecies extrapolation to account for
potential pharmacokinetic and pharmacodynamic differences between macaques
and humans.
•	A default UFh of 10 for intraspecies differences is applied to account for
potentially susceptible individuals in the absence of information on the variability
of response in humans.
•	An UFl is not needed because a BMDLio is used as the POD for derivation of the
p-RfD.
•	An UFS of 3 (10°'5) is applied for use of a 26-day developmental toxicity study
with exposure during organogenesis as the source of the POD, for derivation of
the chronic p-RfD. Although subchronic studies resulted in effects only at higher
doses, there were no toxicity data from studies longer than 13 weeks in rodents to
assure that lifetime exposure might result in effects at lower doses.
•	An UFd is not needed for database insufficiencies. The database contains
adequate developmental toxicity and multigeneration reproduction studies, as well
as subchronic systemic toxicity studies; although no chronic oral exposure studies
for 2-ME are available.
Confidence in the critical study is high. The study evaluated the appropriate endpoints of
reproductive toxicity in adequately sized groups of female macaques exposed to three
appropriately chosen gavage doses. Confidence in the database is medium; although the
reproductive and developmental toxicity of 2-ME have been appropriately tested in numerous
studies, an adequate chronic study was not located. Therefore, confidence in the chronic p-RfD
is medium.
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DERIVATION OF SUBCHRONIC AND CHRONIC
p-RfDs FOR 2-METHOX YE THAN OL ACETATE
Nagano et al. (1979) showed that, when daily doses were converted from mg/kg-day to
mmol/kg-day, the reduction in relative testes weights were identical for mice treated with 2-ME
acetate or its hydrolysis product, 2-ME. Further, similar testicular histopathology was observed
in mice administered daily doses of 2-ME that were half that of 2-ME acetate, which corresponds
with the 0.64 ratio of molecular weights. This suggests that the internal dosimetry of the putative
toxicant for the acetate or glycol ether is equivalent, and equivalent external exposures may be
estimated by adjusting doses by molar ratios of the two compounds. Thus, the subchronic and
chronic p-RfDs for 2-ME acetate are derived by multiplying the subchronic and chronic RfDs for
2-ME by the 2-ME acetate-to-2-ME molecular weight ratio of 118.13 76.09 = 1.55.
Data from occupational studies indicate that the dermal route is the major route of
absorption in the workplace (Sparer et al., 1988; Piacitelli et al., 1990; Chang et al., 2004;
Kezic et al., 1997). In addition, physicochemical properties of 2-ME and 2-ME acetate indicate
the likelihood of substantial dermal absorption of the liquid (Dugard, 1984; Johanson, 1988).
Because data indicate that dermal absorption occurs readily, the potential for dermal exposure
must be considered when applying the p-RfDs and p-RfCs for 2-ME and its acetate.
The subchronic p-RfD is calculated as follows:
Subchronic p-RfD = 2-ME subchronic p-RfD x 1.55
= 0.0165 mg/kg-day x 1.55
= 0.025 or 3 x 10~2 mg/kg-day
The chronic p-RfD is calculated as follows:
Chronic p-RfD = 2-ME chronic p-RfD x 1.55
= 0.00547 mg/kg-day x 1.55
= 0.00848 or 8 x 10~3 mg/kg-day
Because the p-RfDs for 2-ME acetate were derived explicitly from those of 2-ME, the
uncertainties associated with the PODs for 2-ME and confidence in the critical studies and
database for 2-ME also apply to 2-ME acetate.
DERIVATION OF A SUBCHRONIC p-RfC FOR 2-METHOXYETHANOL
IRIS (U.S. EPA,2010a) includes a chronic RfC of 2 x 10 2 mg/m3 for 2-ME based on a
NOAEL of 30 ppm (93 mg/m ) for testicular effects in male rabbits exposed for 13 weeks
(Miller et al., 1983a). A duration-adjusted HEC of 17 mg/m3 was derived using a dosimetric
adjustment and was modified by an UF of 1000, which included an UFs of 10 for use of a
subchronic NOAEL, an UFH of 10 for sensitive human populations, and a combined factor of 10
for both interspecies extrapolation (UFa) and database deficiencies (UFd).
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Several occupational studies of 2-ME inhalation exposure have reported statistically
significant changes in hematological parameters and subjective neurological symptoms among
workers (Cohen, 1984; Cook et al., 1982; Greenburg et al., 1938; Parsons and Parsons, 1938;
Donley, 1936; Shih 2003). With the exception of Shih (2003), these data are limited by design
(cross-sectional sampling vs. cohort), observational bias, low numbers of subjects, and
confounding exposures.
Animal studies have identified multiple toxicological effects from subchronic inhalation
exposures to 2-ME (see Table 34). These include reduction in body weights, changes in
hematological and immunological parameters, and changes in organ weights (Hanley et al.,
1984; Doe et al., 1983; Miller et al., 1983a; Rao et al., 1983). However, like oral exposures, the
most consistently observed effects in multiple species are reproductive and developmental.
Induction of fetal soft tissue and skeletal abnormalities has been observed in rats (Hanley et al.,
1984; Nelson et al., 1984a; Doe et al., 1983) and mice and rabbits (Hanley et al., 1984). Adverse
responses of male reproductive tissues have been exhibited in both short-term (Doe et al., 1983)
and subchronic (Miller et al., 1983a; Rao et al., 1983) studies in rats and rabbits, including
decreased testicle size, testicular atrophy, degeneration of the seminiferous tubules, spermatocyte
degeneration, and reduced ability to successfully sire pups.
The NOAELs and LOAELs for the studies given in Table 34 have been adjusted for
continuous exposure and converted to an HEC (NOAELhec or LOAELhec) as follows, using the
NOAEL from a 6 hours/day, 5 days/week exposure, as an example:
NOAELadj = NOAEL x (6 hours ^ 24 hours) x (5 days ^ 7 days)
NOAELhec = NOAELadj x [(Hb/g) ^ (Hb/g)n]
where NOAELhec is calculated as the dosimetric adjustment from the NOAELadj in animals to a
NOAEL in humans based on the treatment of 2-ME as a Category 3 gas exhibiting
extrarespiratory effects. This is accomplished by multiplying the NOAELadj by the ratio of
animal and human blood:gas (air) partition coefficients (Hb/gA:Hb/gH) (U.S. EPA, 1994b). For
NOAELrecs based on rat data, rat and human blood:air partition coefficients of 31,300 and
32,836, respectively (Hays et al., 2000 and Johanson and Dynesius, 1988, respectively), yielded
an animal:human blood:gas(air) partition coefficient ratio of 0.95. No blood:gas(air) partition
coefficients were located for other species. Therefore, a default value of 1 was used for the ratio
of animal to human blood:gas(air) partition coefficients for species other than the rat. For studies
reporting exposures that were uninterrupted by weekends (7 days/week exposures), duration
adjustment was made only for hours of exposure per day. Current EPA (2002) policy, which
differs from the policy in place at the time of the IRIS RfC derivation, is to duration-adjust
exposures in developmental toxicity studies, as is done for other types of studies. As a result, the
HECs reported here for developmental toxicity studies differ from those reported on IRIS for the
same studies.
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Table 34. Summary of Inhalation (Whole-Body) Toxicity Studies of 2-Methoxyethanol in Animals
Reference
Description
NOAEL
(mg/m3)
LOAEL
(mg/m3)
Observed Responses
Systemic and reproductive studies
Doe et al. (1983)
An inhalation study in male Wistar rats
exposed 6 hours/day for 10 consecutive
days
Inhalation concentrations:
0, 100, or 300 ppm
310
(100 ppm)
HECa: 74
930
(300 ppm)
HEC: 222
Decreases in hematological parameters,
testicular atrophy, and spermatocytic
degeneration
Rao et al. (1983)
An inhalation study in F344/N rats
exposed for 6 hours/day, 5 days/week for
13 weeks
Inhalation concentrations:
0, 50, 100, or 300 ppm
310
(100 ppm)
HEC: 53
930
(300 ppm)
HEC: 158
Reduced parental body weight; reduced
fertility, decreased testicular size, and atrophy
of the seminiferous tubules in males
Miller et al. (1983a)
An inhalation study in Sprague-Dawley
rats exposed for 6 hours/day, 5 days/week
for 13 weeks
Inhalation concentrations:
0, 30, 100, or 300 ppm
310
(100 ppm)
HEC: 53
930
(300 ppm)
HEC: 158
Testicular degeneration and decreased testicular
weight in males; also pancytopenia, lymphoid
atrophy, and reduced body weight
Miller et al. (1983a)
An inhalation study in New Zealand white
rabbits exposed 6 hours/day, 5 days/week
for 13 weeks
Inhalation concentrations:
0, 30, 100, or 300 ppm
93
(30 ppm)
HEC: 17
310
(100 ppm)
HEC: 56
Testicular degeneration and reduced testes size
(Data shown in Table 27)
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Table 34. Summary of Inhalation (Whole-Body) Toxicity Studies of 2-Methoxyethanol in Animals
Reference
Description
NOAEL
(mg/m3)
LOAEL
(mg/m3)
Observed Responses
Miller etal. (1982)
An inhalation study in New Zealand white
rabbits exposed 6 hours/day, 5 days/week
for 13 weeks
Inhalation concentrations:
0, 3, 10, or 30 ppm
93
(30 ppm)
HEC: 17
ND
ND
No treatment-related effects were identified
Developmental studies
Hanley et al. (1984)
An inhalation study in pregnant F344/N
rats exposed for 6 hours/day on GDs 6-15
Inhalation concentrations:
0, 3, 10, or 50 ppm
31
(10 ppm)
HEC: 7
155
(50 ppm)
HEC: 37
Minor skeletal abnormalities in fetuses; no
maternal effects
(Data shown in Table 29)
Hanley et al. (1984)
An inhalation study in pregnant CF-1 mice
exposed for 6 hours/day on GDs 6-15
Inhalation concentrations:
0, 10, or 50 ppm
31
(10 ppm)
HEC: 8
155
(50 ppm)
HEC: 39
Fetal skeletal and soft tissue abnormalities;
transient decreases in maternal body weight
gain
(Data shown in Table 30)
Hanley et al. (1984)
An inhalation study in pregnant New
Zealand white rabbits exposed for 6
hours/day on GDs 6-18
Inhalation concentrations:
0, 3, 10, or 50 ppm
31
(10 ppm)
HEC: 8
155
(50 ppm)
HEC: 39
Numerous fetal skeletal and soft tissue
abnormalities; transient decreases maternal
body weight gain
(Data shown in Table 31)
Nelson et al. (1984a)
An inhalation study in pregnant
Sprague-Dawley rats exposed for 7
hours/day on GDs 7-15
Inhalation concentrations:
0, 50, 100, or 200 ppm
ND
155
(50 ppm)
HEC: 43
Fetal resorptions, decreased fetal body weight,
and skeletal malformations
(Data shown in Table 28)
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Table 34. Summary of Inhalation (Whole-Body) Toxicity Studies of 2-Methoxyethanol in Animals
Reference
Description
NOAEL
(mg/m3)
LOAEL
(mg/m3)
Observed Responses
Nelson et al. (1984b)
An inhalation study in which (1) male
Sprague-Dawley rats were exposed
7 hours/day, 7 days/week, for 6 weeks and
then mated to unexposed females; or
(2) pregnant females were exposed for
7 hours/day on GDs 7-13 or 14-20
Inhalation concentrations:
0 (females only) or 25 ppm
ND
78
(25 ppm)
HEC: 21
Altered avoidance behavior in pups (maternal
exposure on GDs 7-13 only); neurochemical
changes of uncertain toxicological significance
in 21-day-old pups (maternal or paternal
exposure)
Doe et al. (1983)
An inhalation study in pregnant Wistar rats
exposed for 6 hours/day on GDs 6-17
Inhalation concentrations:
0, 100, or 300 ppm
ND
310
(100 ppm)
HEC: 53
Decreased live-pup birth and pup survival
aHEC = N(L)OAELadj x ((Hb/g)A/(Hb/g)H); where N(L)OAELadj = N(L)OAEL x (6 hours/24 hours) x (5 days/7 days), and (Hb/g)A/(Hb/g)H, the ratio of animal to human
blood:air partition coefficients, is 0.95 for rats based on measured data and 1 for all other species by default.
ND = not determined.
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"3
LOAELrecs were 158-222 mg/m for testicular and fertility effects in male rats
(Doe et al., 1983; Miller et al., 1983a; Rao et al., 1983). Rabbits were more sensitive than rats,
3	3
with testicular effects occurring with a LOAELhec of 56 mg/m and NOAELhec of 17 mg/m
(Miller et al., 1983a, 1982). Developmental effects were seen at LOAELhecs ranging from 21 to
3	3
53 mg/m in rats, mice, and rabbits, with corresponding NOAELhecs of 7-8 mg/m
(Hanley et al., 1984; Nelson et al., 1984a,b; Doe et al., 1983). The data sets for testicular effects
in rabbits from Miller et al. (1983a) and developmental effects in rats, mice, and rabbits from
Hanley et al. (1984) were considered further for dose-response modeling because these studies
identified the most sensitive effects and included ranges of exposure concentrations, giving both
NOAELs and LOAELs.
BMC modeling was performed using the most sensitive endpoints from these studies
(gross reduction in testes size and seminiferous tubule degeneration in rabbits [Miller et al.,
1983a]; delayed ossification of the vertebral centra in rat pups; extra lumbar ribs and unilateral
testicular hypoplasia in mouse pups; and limb and kidney defects in rabbit kits [Hanley et al.,
1984]). Appendix B provides details of the modeling. Table 35 shows the resulting benchmark
concentration (BMC) and 95% lower confidence limit on the BMCs and BMCLs as HECs.
BMCLiohecS for these endpoints ranged from 0.73 to 9.9 mg/m3, well below all LOAELs in the
database. The low BMCLiohec of 0.73 mg/m derived from the data for gross reduction in testes
size in male rabbits (Miller et al., 1983a) was selected as the POD for derivation of the
subchronic p-RfC.
Table 35. BMCs Calculated for the Most Sensitive Endpoints from Inhalation
(Whole-Body) Studies of 2-Methoxyethanol
Endpoint
BMCiohec
(mg/m3)
BMCLiohec
(mg/m3)
Gross reduction in testes size in rabbits (Miller et al., 1983a)
6.8
0.73
Seminiferous tubule degeneration in rabbits (Miller et al.,
1983a)
6.1
3.1
Delayed ossification of vertebral centra in rats (Hanley et al.,
1984)
13.3
9.6
Extra lumbar ribs in mice (Hanley et al., 1984)
4.3
2.9
Unilateral testicular hypoplasia in mice (Hanley et al., 1984)
18.6
7.5
Limb defects in rabbits (Hanley et al., 1984)
13.7
9.9
Renal defects in rabbits (Hanley et al., 1984)
4.8
3.3
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The subchronic p-RfC is calculated as follows:
Subchronic p-RfC = BMCLiohec ^ UF
0.73 mg/m3 - 100
0.0073 or 7 x 10"3 mg/m3
The composite UF of 100 is composed of the following:
•	An UFh of 10 for intraspecies differences is applied to account for potentially
susceptible individuals in the absence of information on the variability of
response in humans.
•	An UFa of 3 (10°5) is applied for interspecies extrapolation to account for
potential pharmacodynamic differences not quantified by the dosimetric
adjustment between rabbits and humans.
•	An UFl is not needed because a BMCLiohec is used as the POD for derivation of
the p-RfC.
•	An UFs is not needed because a subchronic toxicity study is used as the source of
the POD for derivation of the subchronic p-RfC.
•	An UFd of 3 (10°5) is applied for database insufficiencies (lack of a
multigeneration reproductive toxicity study by inhalation exposure and minimal
evaluation of respiratory effects).
Confidence in the critical study is medium. Miller et al. (1983a) is a well-designed
toxicity study in which data for multiple endpoints of toxicity, including the results of
histological examination of reproductive tissues, were evaluated for rats and rabbits. However,
group sizes in the rabbit study were small, and reporting of pathology findings was incomplete.
Confidence in the database is medium. Several high-quality subchronic systemic toxicity and
developmental toxicity studies in multiple animal species are available; however, the database is
lacking a multigeneration reproduction study by inhalation exposure. Neurotoxicity endpoints
were not reported in the subchronic studies, and respiratory tract toxicity was only indirectly
reported (lung histology). Given medium confidence in the critical study and in the database,
confidence in the subchronic p-RfC is medium.
Data from occupational studies indicate that the dermal route is the major route of
absorption in the workplace (Sparer et al., 1988; Piacitelli et al., 1990; Chang et al., 2004;
Kezic et al., 1997). In addition, physicochemical properties of 2-ME and 2-ME acetate indicate
the likelihood of substantial dermal absorption of the liquid (Dugard, 1984; Johanson, 1988).
Because data indicate that dermal absorption occurs readily, the potential for dermal exposure
must be considered when applying the p-RfDs and p-RfCs for 2-ME and its acetate.
The subchronic p-RfC derived here for 2-ME is lower than the chronic RfC available for
this chemical on IRIS (U.S. EPA, 2010a). The subchronic p-RfC and chronic RfC are based on
the same endpoint (testicular effects in male rabbits studied by Miller et al., 1983a) but were
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derived independently. BMC modeling and duration adjustment in the HEC calculation
performed for the subchronic p-RfC (but not the older chronic RfC) lowered the POD from
3	3
17 mg/m to 0.73 mg/m . The lack of an UF to extrapolate across durations for the subchronic
p-RfC partially offsets this change so that (after rounding) the subchronic p-RfC is nearly a
factor of 3 lower than the chronic RfC on IRIS (U.S. EPA, 2010a).
DERIVATION OF SUBCHRONIC AND CHRONIC p-RfCs
FOR 2-METHOXYETHANOL ACETATE
No human or animal data for 2-ME acetate inhalation exposure were available for
derivation of a p-RfC. Chronic and subchronic p-RfDs for 2-ME acetate were derived by
analogy to 2-ME; however, the database to support a derivation of a p-RfC by analogy to 2-ME
is much more limited. Unlike oral exposures to 2-ME or its acetate, it is unclear if inhalation of
the acetate would result in substantial upper respiratory tract effects (i.e., because of hydrolytic
production of acetic acid) that were not observed in the inhaled 2-ME database. However,
because the potential acetic acid solution is unlikely to result in a pH sufficiently low to cause
substantial upper respiratory tract irritation (ACGIH, 2006b), p-RfCs are derived from the Miller
(1983a) subchronic data in rats used to derive the subchronic p-RfC for 2-ME acetate, based on
differences in molecular weight.
"3
The low BMCLiohec of 0.73 mg/m is derived from the data for gross reduction in testes
size in male rabbits (Miller et al., 1983a). This BMCLiohec for 2-ME is multiplied by the
molecular-weight difference between the alcohol and acetate of 1.55, to calculate the POD of
1.13 mg/m3 for derivation of the subchronic p-RfC for 2-ME acetate.
The subchronic p-RfC is calculated as follows:
Subchronic p-RfC = POD - UF
1.13 mg/m3-100
= 0.0113 or 1 x 10"2 mg/m3
As for the subchronic derivation for 2-ME, the composite UF of 100 is composed of the
following:
•	An UFh of 10 for intraspecies differences is applied to account for potentially
susceptible individuals in the absence of information on the variability of
response in humans.
•	An UFa of 3 (10°5) is applied for interspecies extrapolation to account for
potential pharmacodynamic differences not quantified by the dosimetric
adjustment between rabbits and humans.
•	An UFl is not needed because a BMCLiohec is used as the POD for derivation of
the p-RfC.
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•	An UFs is not needed because a subchronic toxicity study is the source of the
POD for derivation of the subchronic p-RfC.
•	An UFd of 3 (10°5) is applied for database insufficiencies (lack of a
multigeneration reproductive toxicity study by inhalation exposure and minimal
evaluation of respiratory effects).
Confidence in the critical study is medium. Miller et al. (1983a) is a well-designed
toxicity study in which data for multiple endpoints of toxicity, including the results of
histological examination of reproductive tissues, were evaluated for rats and rabbits. However,
group sizes in the rabbit study were small, reporting of pathology findings was incomplete, and
the data were for the alcohol rather than the acetate. Confidence in the database is medium.
Several high-quality subchronic systemic toxicity and developmental toxicity studies in multiple
animal species are available for the alcohol; however, the database is lacking a multigeneration
reproduction study by inhalation exposure. Neurotoxicity endpoints were not reported in the
subchronic studies, and respiratory tract toxicity was only indirectly reported (lung histology).
Given medium confidence in the critical study and in the database, confidence in the subchronic
p-RfC is medium.
Data from occupational studies indicate that the dermal route is the major route of
absorption in the workplace (Sparer et al., 1988; Piacitelli et al., 1990; Chang et al., 2004;
Kezic et al., 1997). In addition, physicochemical properties of 2-ME and 2-ME acetate indicate
the likelihood of substantial dermal absorption of the liquid (Dugard, 1984; Johanson, 1988).
Because data indicate that dermal absorption occurs readily, the potential for dermal exposure
must be considered when applying the p-RfDs and p-RfCs for 2-ME and its acetate.
The chronic p-RfC for 2-ME acetate was calculated similarly from the subchronic POD,
applying an additional UFS of 10 for extrapolation of the subchronic data to chronic exposure, as
follows:
The chronic p-RfC is calculated as follows:
Chronic p-RfC = POD - UF
1.13 mg/m3- 1000
= 0.00113 or 1 x 10"3 mg/m3
"3
This chronic p-RfC for 2-ME acetate is a factor of 30 lower than the value of 0.03 mg/m
that would be calculated from the IRIS RfC of 0.02 mg/m3 for the alcohol, because of the much
lower POD calculated by BMC modeling.
The discussion of confidence in the chronic p-RfC is identical to that for the subchronic
p-RfC, with the same conclusion of medium confidence in the chronic p-RfC for 2-ME acetate.
Data from occupational studies indicate that the dermal route is the major route of
absorption in the workplace (Sparer et al., 1988; Piacitelli et al., 1990; Chang et al., 2004;
Kezic et al., 1997). In addition, physicochemical properties of 2-ME and 2-ME acetate indicate
the likelihood of substantial dermal absorption of the liquid (Dugard, 1984; Johanson, 1988).
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Because data indicate that dermal absorption occurs readily, the potential for dermal exposure
must be considered when applying the p-RfDs and p-RfCs for 2-ME and its acetate.
PROVISIONAL CARCINOGENICITY ASSESSMENT
FOR 2-ME THOX YE THAN OL AND 2-ME THOX YE THAN OL ACETATE
WEIGHT-OF-EVIDENCE CLASSIFICATION
The human database for 2-ME exposure is limited to case reports of acute exposures.
The animal data do not include any chronic-exposure oral or inhalation bioassays; none of the
available studies are longer than ~3 months in duration. None of these data suggest the
development of precancerous lesions. The mutagenicity data for 2-ME (Guzzie et al., 1986;
McGregor et al., 1983; Ong, 1980) do not suggest substantial mutagenic activity leading to
carcinogenicity. No human or animal data are available on the carcinogenicity of 2-ME acetate.
Because 2-ME acetate is rapidly and extensively hydrolyzed to 2-ME, the carcinogenic potential
of the acetate is likely to be very similar, if not identical, to that of the glycol ether. Overall,
under the EPA (2005) Guidelines for Carcinogen Risk Assessment, these data are considered to
provide "Inadequate Information to Assess Carcinogenic Potential" for 2-ME or its acetate.
QUANTITATIVE ESTIMATES OF CARCINOGENIC RISK
Due to the lack of adequate human or animal data, no quantitative estimate for
carcinogenic risk from exposure to 2-ME or its acetate is derived.
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dysmorphogenesis in the rat embryo and near-term fetus. Toxicol Appl Pharmacol
145(2):415-424.
Smialowicz, RJ; Riddle, MM; Luebke, RW; et al. (1991) Immunotoxicity of 2-methoxyethanol
following oral administration in Fischer 344 rats. Toxicol Appl Pharmacol 109(3):494-506.
Smialowicz, RJ; Williams, WC; Riddle, MM; et al. (1992a) Comparative immunosuppression of
various glycol ethers orally administered to Fischer 344 rats. Fundam Appl Toxicol
18(4):621-627.
Smialowicz, RJ; Riddle, MM; Williams, WC; et al. (1992b) Differences between rats and mice
in the immunosuppressive activity of 2-methoxyethanol and 2-methoxyacetic acid. Toxicology
74(l):57-67.
Sparer J; Welch LS; McManus K; et al. (1988). Effects of exposure to ethylene glycol ethers on
shipyard painters: I. Evaluation of exposure. Am J Ind Med 14:497-507.
Starek, A; Szymczak, W; Zapor, L. (2008). Hematological effects of four ethylene glycol
monoalkyl ethers in short-term repeated exposure in rats. Arch Toxicol 82:125-136.
Stott, WT; McKenna, MJ. (1985) Hydrolysis of several glycol ether acetates and acrylate esters
by nasal mucosal carboxylesterase in vitro. Fundam Appl Toxicol 5(2):399-404.
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2-ethoxyethanol exposure in male rats reveals abnormal expression of the actin binding protein
cortactin in degenerating spermatocytes. Toxicology Letters 190:193-201.
Toraason, M; Breitenstein, M. (1988) Prenatal ethylene glycol monomethyl ether (EGME)
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Toraason, M; Stringer, B; Stober, P; et al. (1985) Electrocardiographic study of rat fetuses
exposed to ethylene glycol monomethyl ether (EGME). Teratology 32:33-39.
Toraason, M; Stringer, B; Smith, R. (1986) Ornithine decarboxylase activity in the neonatal rat
heart following prenatal exposure to ethylene glycol monomethyl ether. Drug Chem Toxicol
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National Center for Environmental Assessment, Cincinnati, OH for the Office of Emergency and
Remedial Response, Washington, DC. EPA/540/R-97/036. NTIS PB 97-921199.
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III. Hematological effects. Am J Ind Med 14:527-536.
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on shipyard painters: II. Male reproduction. Am J Ind Med 14:509-526.
Welsch, F. (2005) The mechanism of ethylene glycol ether reproductive and developmental
toxicity and evidence for adverse effects in humans. Toxicol Lett 156:13-28.
Welsch, F; Blumenthal, GM; Conolly, RB. (1995) Physiologically based pharmacokinetic
models applicable to organogenesis: extrapolation between species and potential use in prenatal
toxicity risk assessments. Toxicol Lett 82:539-547.
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2-Methoxyethanol, 2-ethoxyethanol, and their acetates. Available online at
http://www.inchem.org/documents/ehc/ehc/ehcll5.htm (accessed May 2010).
Williams, WC; Riddle, MM; Copeland, CB; et al. (1995) Immunological effects of
2-methoxyethanol administered dermally or orally to Fischer 344 rats. Toxicology 98:215-223.
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Toxicol 28:267-268.
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APPENDIX A. DETAILS OF BENCHMARK DOSE MODELING FOR
SUBCHRONIC AND CHRONIC p-RfDs
MODEL-FITTING PROCEDURE FOR QUANTAL NONCANCER DATA
The model-fitting procedure for dichotomous noncancer data is as follows. All available
dichotomous models in the EPA BMDS (version 2.1) are fit to the incidence data using the extra
risk option. The multistage model is run for all polynomial degrees up to n - 1 (where n is the
number of dose groups including control). Adequate model fit is judged by three criteria:
goodness-of-fit p-value (p > 0.1), visual inspection of the dose-response curve, and scaled
residual at the data point (except the control) closest to the predefined benchmark response rate
(BMR). Among all of the models providing adequate fit to the data, the lowest BMDL is
selected as the POD when the difference between the BMDLs estimated from these models is
>3-fold (unless it appears to be an outlier); otherwise, the BMDL from the model with the lowest
Akaike Information Criterion (AIC) is chosen. In accordance with EPA (2000) guidance, BMDs
and BMDLs associated with an extra risk of 10% BMR generally are calculated for all models; a
5% BMR is used for developmental data.
Model Fitting for Incidence of Seminiferous Tubule Degeneration in Rats (NTP, 1993)
Following the above procedure, the quantal models in the EPA BMDS (version 2.1) were
fit to the incidence data for seminiferous tubule degeneration in rats from the NTP (1993) study
shown in Table 7. Table A-l shows the modeling results. Only the log logistic model produced
a nonzero p-v alue (p = 0.22). However, modeling of these data was problematic, based on
limitations in the data set for BMD modeling, including the lack of data points in the region of
the BMR, very high response rate at the lowest dose (70 vs. 0% in controls), fractional response
in only one dose group, and a plateau at the maximum response rate at higher doses and actual
decrease in response rate to 90% at the second highest dose. The models other than the log
logistic failed due to the drop in response at the second highest dose. When the models were run
again after dropping the two high dose groups, most models achieved perfect fit. This model
over-fit reflects the lack of information in the available data (fractional response at only one data
point).
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Table A-l. Model Predictions for the Incidence of Seminal Tubule
Degeneration in Male Rats
Model
Degrees
of
Freedom
2
1
X Goodness
of Fit
p-Value3
AIC
BMDio
(mg/kg-day)
BMDL10
(mg/kg-day)
All doses
Gammab
5
178.22
0.00
35.08
-
-
Logistic
4
99.57
0.00
46.69
-
-
Log logistic0
4
5.75
0.22
28.13
3.92
0.75
Log probitc
5
34.26
0.00
28.59
-
-
Multistage (degree = l)d
5
178.22
0.00
35.08
-
-
Multistage (degree = 2)d
5
178.22
0.00
35.08
-
-
Multistage (degree = 3)d
5
178.22
0.00
35.08
-
-
Multistage (degree = 4)d
5
178.22
0.00
35.08
-
-
Multistage (degree = 5)d
5
178.22
0.00
35.08
-
-
Probit
4
29.56
0.00
50.07
-
-
Weibullb
5
178.22
0.00
35.08
-
-
Quantal-linear
5
178.22
0.00
35.08
-
-
Two highest doses dropped
Gammab
3
0
1.00
14.22
45.61
3.42
Logistic
2
0
1.00
16.22
60.22
14.81
Log logistic0
3
0
1.00
14.22
59.95
4.83
Log probitc
2
0
1.00
16.22
53.06
6.20
Multistage (degree = l)d
3
0.61
0.89
15.17
2.50
1.51
Multistage (degree = 2)d
3
0.61
0.89
15.17
0.49
0.30
Multistage (degree = 3)d
2
0
1.00
16.22
31.52
3.42
Probit
2
0
1.00
16.22
50.56
13.44
Weibullb
2
0
1.00
16.22
32.14
3.42
Quantal-linear
3
0.61
0.89
15.17
5.13
3.10
7?-Values <0.10 fail to meet conventional goodness-of-fit criteria.
''Power restricted to >1.
°Slope restricted to >1.
dBetas restricted to >0.
Source: NTP (1993).
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Model Fitting for Incidence of Testicular Degeneration in Rats (NTP, 1993)
The quantal models in the EPA BMDS (version 2.1) were fit to the incidence data for
testicular degeneration in rats from the NTP (1990) study shown in Table 16. However, it was
not possible to model these data, as they caused unrecoverable software failures in the BMDS
system. This was probably due to the higher incidence in the mid-dose group than the high-dose
group. However, due to the availability of data for only three dose groups, it was not possible to
drop the high-dose group.
Model Fitting for Incidence of Embryo Lethality in Macaques (Scott et al., 1989)
Scott et al. (1989) found a dose-related increase in incidence of embryo lethality
following gavage exposures during GDs 20-45. The data of Scott et al. (1989), showing
increasing response (0/6, 3/13, 3/10, or 8/8) over four evenly-spaced doses (0, 12, 24, or
36 mg/kg-day), were subjected to BMD modeling, as described above, using a 5% BMR for
developmental data. Table A-2 shows the modeling results. Adequate fit (p > 0.01) is achieved
with several models; fit for the 1-degree polynomial model is marginal. Of these, the 2-degree
polynomial model had the lowest AIC, indicating best fit to the data, and one of the lowest
BMDL5S. Figure A-l shows a graph of the 2-degree polynomial model superimposed on the
data. Although model fit at the 2 higher-dose groups is marginal (scaled residuals of 1.3-1.6), fit
at the control and low-dose groups, which bracket the BMR of 5%, is reasonably good (scaled
residuals of 0-0.48). The BMDL5 estimated from the 2-degree multistage model for these data
is 1.64 mg/kg-day.
Table A-2. BMD5 Modeling Results for Incidence of Embryo
Lethality in Pregnant Macaques Given Gavage Doses of 2-Methoxyethanol
on GDs 20-45
Model
Degrees of
Freedom
2
1
2
1
Goodness
of Fit
p-Value3
AIC
BMD5
(mg/kg-day)
BMDL5
(mg/kg-day)
Gammab
2
4.89
0.09
36.45
-
-
Logistic
2
4.11
0.13
35.37
7.60
3.91
Log logistic0
2
5.31
0.07
37.13
-
-
Log probitc
2
5.38
0.07
37.13
-
-
Multistage (degree = l)d
3
6.30
0.10
37.09
2.73
1.09
Multistage (degree = 2)d
3
4.32
0.23
33.92
8.15
1.64
Multistage (degree = 3)d
2
3.64
0.16
34.85
5.01
1.70
Probit
2
4.03
0.13
35.19
7.17
3.59
Weibullb
1
1.64
0.20
34.79
21.1
10.0
"/^-Values <0.10 fail to meet conventional goodness-of-fit criteria.
bPower restricted to >1.
°Slope restricted to >1.
dBetas restricted to >0.
Sources: Scott etal. (1989).
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Multistage Model with 0.95 Confidence Level
Multistage
BMD Lower Bound
1
0.8
0.6
0.4
0.2
0
BMDL
BMD
0
5
10
15
20
25
30
35
Dose
11:36 05/06 2010
BMD and BMDL indicated are associated with an extra risk of 5% and are in units
of mg/kg-day.
Source: Scott et al. (1989).
Figure A-l. Fit of 2-Degree Multistage Model to Data on Incidence of Embryo
Lethality in Macaques
MODEL-FITTING PROCEDURE FOR CONTINUOUS DATA
The model-fitting procedure for continuous data using the EPA benchmark dose software
(BMDS) is as follows. The simplest model (linear) is first applied to the data while assuming
constant variance. If the data are consistent with the assumption of constant variance (p > 0.1),
then the fit of the linear model to the means is evaluated, and the polynomial, power, and Hill
models are fit to the data while assuming constant variance. Adequate model fit is judged by
three criteria: goodness-of-fit /rvalue (p > 0.1), visual inspection of the dose-response curve, and
scaled residual at the data point (except the control) closest to the predefined BMR. Among all
of the models providing adequate fit to the data, the lowest BMDL is selected as the POD when
the difference between the BMDLs estimated from these models is >3-fold (unless it appears to
be an outlier); otherwise, the BMDL from the model with the lowest AIC is chosen. If the test
for constant variance is negative, the linear model is run again while applying the power model
integrated into the BMDS to account for nonhomogenous variance. If the nonhomogenous
variance model provides an adequate fit (p > 0.1) to the variance data, then the fit of the linear
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model to the means is evaluated, and the polynomial, power, and Hill models are fit to the data
and evaluated while the variance model is applied. Model fit and POD selection proceed as
described earlier. If the test for constant variance is negative and the nonhomogenous variance
model does not provide an adequate fit to the variance data, then the data set is considered
unsuitable for modeling.
Model Fitting for Sperm Density in Rats (NTP, 1990)
All available continuous-variable models in the EPA BMDS (version 2.1) were fit to the
data of NTP (1990) for sperm density in rats treated with 2-ME (see Table 16). Data were not
available for the percent reduction in sperm density in rats or humans resulting in diminished
reproductive capacity. Therefore, a default BMR of one SD from the control means was used.
For the F1 sperm density data of NTP (1990), the calculated BMDisd and the BMDLisd are
estimates of the doses associated with a negative change of one SD from the control mean.
Table A-3 shows the modeling results. The assumption of constant variance did not hold, but the
nonhomogenous variance model provided adequate fit to the variance data. The linear model
adequately fit the means (p> 0.1). The higher order models, except the Hill model for which
there were insufficient data points to fit the model, defaulted back to the linear model. Thus, the
linear model was selected for BMD derivation. The resulting BMDisd and BMDLisd are 26.0
and 14.3 mg/kg-day, respectively. Figure A-2 shows a graph of the linear model fit.
Table A-3. Model Predictions for Reduced Sperm Density in F1 Rats
Model
Variance
p-Value3
Means
p-Value3
AIC
BMDisd
(mg/kg-day)
BMDLisd
(mg/kg-day)
Constant variance
Linearb
0.0738
0.2396
692.6674
38.1162
21.945
Nonconstant variance
Hilf
NA
Linearb
0.5608
0.2283
689.861
26.0378
14.3388
Polynomial (2-degree)b
0.5608
0.2283
689.861
26.0378
14.3388
Powerc
0.5608
0.2283
689.861
26.0378
14.3388
^-Values <0.10 fail to meet conventional goodness-of-fit criteria.
bCoefficients restricted to be negative.
°Power restricted to >1.
NA = Too many parameters in model for number of observations.
Source: NTP (1990).
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Linear Model with 0.95 Confidence Level
Linear
700
650
600
550
500
450
400
BMDL
BMD
0
5
10
15
20
25
14:31 10/03 2009
BMD and BMDL indicated are associated with a change of 1 SD from the control mean
and are in units of mg/kg-day.
Source: NTP (1990).
Figure A-2. Fit of Linear Model to Data for Sperm Density in Rats
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APPENDIX B. DETAILS OF BENCHMARK DOSE MODELING FOR
SUBCHRONIC p-RfC
MODEL-FITTING PROCEDURE FOR QUANTAL NONCANCER DATA
The model-fitting procedure for dichotomous noncancer data is as follows. All available
dichotomous models in the EPA BMDS (version 2.1) are fit to the incidence data using the extra
risk option. The multistage model is run for all polynomial degrees up to n - 1 (where n is the
number of dose groups including control). Adequate model fit is judged by three criteria:
goodness-of-fit p-value (p > 0.1), visual inspection of the dose-response curve, and scaled
residual at the data point (except the control) closest to the predefined BMR. Among all of the
models providing adequate fit to the data, the lowest BMCL is selected as the POD when the
difference between the BMCLs estimated from these models is >3-fold (unless it appears to be
an outlier); otherwise, the BMCL from the model with the lowest AIC is chosen. In accordance
with EPA (2000) guidance, BMCs and BMCLs associated with an extra risk of 10% BMR are
calculated for all models.
Model Fitting for Incidence of Gross Reduction in Testes Size in Rabbits (Miller et al.,
1983a)
Following the above procedure, the quantal models in the EPA BMDS (version 2.1) were
fit to the incidence data for gross reduction in testes size in rabbits from the Miller et al. (1983a)
study shown in Table 27. HECs, calculated as shown in Table 34, were used for modeling, so
results are reported as HECs as well. Table B-l shows the modeling results. All models
produced adequate fit (p > 0.01). With the exception of the BMCL from the log logistic model,
the BMCLs all were within a factor of 3. The best fitting model, with the lowest AIC, was the
quantal linear (1-degree multistage), giving BMCiohec and BMCLiohecs of 3.5 and 1.8 mg/m3,
respectively. However, because the log logistic model also provided excellent fit and the lowest
BMCL, its BMCiohec of 6.76 and BMCLiohec of 0.73 mg/m3 were selected for derivation of the
p-RfC. Figure B-l shows the fit of this model to the data.
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Table B-l. Model Predictions for the Incidence of Gross Reduction in
Testes Size in Rabbits
Model
Degrees of
Freedom
2
X
X Goodness
of Fit
p-Value3
AIC
BMCiohec
(mg/m3)
BMCLiohec
(mg/m3)
Gammab
2
0.04
0.9793
15.8006
4.0251
1.8352
Logistic
2
1.11
0.5734
17.282
10.9333
5.32525
Log logistic0
2
0.21
0.8996
16.0589
6.75982
0.727736
Log probitc
2
0.14
0.9321
15.9437
6.96769
3.03858
Multistage (degree = l)d
3
0.04
0.9979
13.8069
3.49396
1.83389
Multistage (degree = 2)d
2
0.03
0.9849
15.7739
3.86908
1.84076
Multistage (degree = 3)d
2
0.02
0.9921
15.7527
3.74913
1.84522
Probit
2
1.07
0.5868
17.1681
10.5005
5.53729
Weibullb
2
0.04
0.9797
15.7952
4.05573
1.83632
7?-Valucs <0.10 fail to meet conventional goodness-of-fit criteria.
bPower restricted to >1.
°Slope restricted to >1.
dBetas restricted to >0.
Source: Miller etal.. (1983a).
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Log-Logistic Model with 0.95 Confidence Level
Log-Logistic
1
0.8
0.6
0.4
0.2
0
i/IDL
BMD
0
20
40
60
80
100
120
140
160
Dose
06:48 10/09 2009
BMC and BMCL indicated are associated with an extra risk of 10% and are in units of
mg/m3 as HEC.
Source: Miller et al. (1983a).
Figure B-l. Fit of Log Logistic Model to Incidence Data for Gross Reduction
in Testes Size in Rabbits
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Model Fitting for Incidence of Seminiferous Tubule Degeneration in Rabbits (Miller et al.,
1983a)
Following the above procedure, the quantal models in the EPA BMDS (version 2.1) were
fit to the incidence data for seminiferous tubule degeneration in rabbits from the Miller et al.
(1983a) study shown in Table 27. HECs, calculated as shown in Table 34, were used for
modeling, so results are reported as HECs as well. Table B-2 shows the modeling results. All
models produced adequate fit (p > 0.01). With the exception of the BMCL from the log logistic
model, which appears to be an outlier, the other BMCLs were all within a factor of 3. The best
fitting model, with the lowest AIC, was the quantal linear (1-degree multistage), giving
BMCiohec and BMCLiohecS of 6.1 and 3.1 mg/m3, respectively. Figure B-2 shows the fit of this
model to the data.
Table B-2. Model Predictions for the Incidence of Seminiferous Tubule
Degeneration in Rabbits
Model
Degrees
of
Freedom
2
1
X Goodness
of Fit
p-Value3
AIC
BMCiohec
(mg/m3)
BMCLjqhec
(mg/m3)
Gammab
2
0.11
0.9468
15.8979
11.1269
3.20073
Logistic
2
0.5
0.7796
16.4504
19.1263
9.39131
Log logistic0
2
0.32
0.8524
16.2222
12.447
1.89119
Log probitc
2
0.27
0.872
16.1352
12.8247
5.57338
Multistage (degree = l)d
3
0.27
0.9661
14.1695
6.05074
3.09883
Multistage (degree = 2)d
2
0.03
0.9853
15.7776
9.52467
3.24906
Multistage (degree = 3)d
2
0
0.9992
15.7367
8.35829
3.266
Probit
2
0.43
0.8059
16.341
17.6572
9.04958
Weibullb
2
0.08
0.962
15.8418
10.9506
3.22303
Quantal-linear
3
0.27
0.9661
14.1695
6.0507
3.09883
7?-Valucs <0.10 fail to meet conventional goodness-of-fit criteria.
bPower restricted to >1.
°Slope restricted to >1.
dBetas restricted to >0.
Source: Miller etal. (1983a).
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Quantal Linear Model with 0.95 Confidence Level
Quantal Linear
1
0.8
0.6
0.4
0.2
0
ESMDL
BMD
0
20
40
60
80
100
120
140
160
09:35 10/09 2009
BMC and BMCL indicated are associated with an extra risk of 10% and are in units of
mg/m3 as HEC.
Source: Miller et al. (1983a).
Figure B-2. Fit of Quantal-Linear Model to Incidence Data for Seminiferous
Tubule Degeneration in Rabbits
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Model Fitting for Incidence of Delayed Ossification of Vertebral Centra in Rats
(Hanley et al., 1984)
Following the above procedure, the quantal models in the EPA BMDS (version 2.1) were
fit to the incidence data for delayed ossification of vertebral centra in rats from the Hanley et al.
(1984) study shown in Table 29. Because the litter is the preferred unit of statistical analysis for
developmental studies, the data modeled were those for incidence of litters affected. HECs,
calculated as shown in Table 34, were used for modeling, so results are reported as HECs as
well. Exposures for this gestational exposure study were duration-adjusted as part of the HEC
calculation, as currently recommended by EPA (2002). Table B-3 shows the modeling results.
All models produced adequate fit (p > 0.01). The BMCLs were all within a factor of 3. The best
fitting model, with the lowest AIC, was the probit, giving BMCiohec and BMCLiohecs of 13.3
and 9.6 mg/m3, respectively. Figure B-3 shows the fit of this model to the data.
Table B-3. Model Predictions for the Litter Incidence of Delayed
Ossification of Vertebral Centra in Rats
Model
Degrees of
Freedom
2
1
2
1
Goodness
of Fit
p-Value8
AIC
BMCiohec
(mg/m3)
BMCLiohec
(mg/m3)
Gammab
1
0.22
0.6364
115.897
12.4033
5.37379
Logistic
2
0.27
0.8736
113.946
14.0009
10.241
Log logistic0
1
0.22
0.6404
115.891
12.0016
4.18566
Log probitc
2
0.32
0.8538
113.987
14.9242
9.44885
Multistage (degree = l)d
2
0.38
0.8266
114.05
8.98591
5.30379
Multistage (degree = 2)d
1
0.26
0.608
115.938
13.0639
5.35445
Multistage (degree = 3)d
1
0.26
0.608
115.938
13.0639
5.35445
Probit
2
0.27
0.8751
113.942
13.2573
9.61316
Weibullb
1
0.23
0.6309
115.904
12.5187
5.3702
Quantal-linear
2
0.38
0.8266
114.05
8.98591
5.30379
aValues <0.10 fail to meet conventional goodness-of-fit criteria.
bPower restricted to >1.
°Slope restricted to >1.
dBetas restricted to >0.
Source: Hanley et al. (1984).
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FINAL
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Probit Model with 0.95 Confidence Level
0.6
0.5
"S 0.4
O
CD
o	0.3
o
2
LL
0.2
0.1
0
0	5	10	15	20	25	30	35
09:52 10/09 2009	Dose
BMC and BMCL indicated are associated with an extra risk of 10% and are in units of
mg/m3 as HEC.
Source: Hanley et al. (1984).
Figure B-3. Fit of Probit Model to Data on Litter Incidence of Delayed Ossification of
Vertebral Centra in Rats
Probit
BMDL
BMD
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Model Fitting for Incidence of Extra Lumbar Ribs in Mice (Hanley et al., 1984)
Following the above procedure, the quantal models in the EPA BMDS (version 2.1) were
fit to the incidence data for extra lumbar ribs in mice from the Hanley et al. (1984) study shown
in Table 30. Because the litter is the preferred unit of statistical analysis for developmental
studies, the data modeled were those for incidence of litters affected. HECs, calculated as shown
in Table 34, were used for modeling, so results are reported as HECs as well. Exposures for this
gestational exposure study were duration-adjusted as part of the HEC calculation, as currently
recommended by EPA (2002). Table B-4 shows the modeling results. There were insufficient
degrees of freedom available to fit several of the models. Among the models that produced
adequate fit (p > 0.01), the BMCLs were all within a factor of 3. The best fitting model, with the
"3
lowest AIC, was the probit, giving BMCiohec and BMCLiohecs of 4.3 and 2.9 mg/m ,
respectively. Figure B-4 shows the fit of this model to the data.
Table B-4. Model Predictions for the Litter Incidence of Extra Lumbar
Ribs in Mice
Model
Degrees of
Freedom
2
1
2
1
Goodness
of Fit
p-Value8
AIC
BMCiohec
(mg/m3)
BMCLiohec
(mg/m3)
Gammab
0
0
NA
90.7637
5.60675
1.77342
Logistic
1
0.02
0.8968
88.7805
4.10839
2.55048
Log logistic0
0
0
NA
90.7637
5.86561
0.842663
Log probitc
0
0
NA
90.7637
6.07437
2.989
Multistage (degree = l)d
1
0.08
0.7716
88.8476
3.19226
1.75927
Multistage (degree = 2)d
0
0
NA
90.7637
5.22928
1.77342
Probit
1
0.01
0.9257
88.7724
4.34628
2.85615
Weibullb
0
0
NA
90.7637
5.49992
1.77342
Quantal-linear
1
0.08
0.7716
88.8476
3.19226
1.75927
aValues <0.10 fail to meet conventional goodness-of-fit criteria.
bPower restricted to >1.
°Slope restricted to >1.
dBetas restricted to >0.
NA = not applicable.
Source: Hanley et al. (1984).
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FINAL
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Probit Model with 0.95 Confidence Level
Pro bit
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
BMDL
BMD
0
5
10
15
20
25
30
35
40
10:07 10/09 2009
BMC and BMCL indicated are associated with an extra risk of 10% and are in units of
mg/m3 as HEC.
Source: Hanley et al. (1984).
Figure B-4. Fit of Probit Model to Data on Litter Incidence of
Extra Lumbar Ribs in Mice
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Model Fitting for Unilateral Testicular Hypoplasia in Mice (Hanley et al., 1984)
Following the above procedure, the quantal models in the EPA BMDS (version 2.1) were
fit to the incidence data for unilateral testicular hypoplasia in mice from the Hanley et al. (1984)
study shown in Table 30. Because the litter is the preferred unit of statistical analysis for
developmental studies, the data modeled were those for incidence of litters affected. HECs,
calculated as shown in Table 34, were used for modeling, so results are reported as HECs as
well. Exposures for this gestational exposure study were duration-adjusted as part of the HEC
calculation, as currently recommended by EPA (2002). Table B-5 shows the modeling results.
All models produced adequate fit (p > 0.01). The BMCLs were all within a factor of 3. The best
fitting model, with the lowest AIC, was the log logistic, giving BMCiohec and BMCLiohecS of
18.6 and 7.5 mg/m , respectively. Figure B-5 shows the fit of this model to the data.
Table B-5. Model Predictions for the Litter Incidence of Unilateral
Testicular Hypoplasia in Mice
Model
Degrees of
Freedom
2
1
2
1
Goodness
of Fit
p-Value3
AIC
BMCiohec
(mg/m3)
BMCLjqhec
(mg/m3)
Gammab
1
0.04
0.8457
62.9431
19.7721
8.91306
Logistic
1
0.13
0.717
63.0362
25.4106
15.9418
Log logistic0

0.02
0.8766
62.9296
18.6536
7.49865
Log probitc
1
0.32
0.5712
63.2242
27.954
15.2951
Multistage (degree = l)d
1
0.04
0.8457
62.9431
19.772
8.91306
Multistage (degree = 2)d
1
0.04
0.8457
62.9431
19.772
8.91306
Probit
1
0.12
0.7318
63.0222
24.6053
14.9646
Weibullb
1
0.04
0.8457
62.9431
19.7721
8.91306
Quantal-linear
1
0.04
0.8457
62.9431
19.772
8.91306
aValues <0.10 fail to meet conventional goodness-of-fit criteria.
bPower restricted to >1.
°Slope restricted to >1.
dBetas restricted to >0.
Source: Hanley et al. (1984).
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Log-Logistic Model with 0.95 Confidence Level
Log-Logistic
0.5
0.4
0.3
0.2
0.1
0
BMDL
BMD
0
5
10
15
20
25
30
35
40
10:16 10/09 2009
BMC and BMCL indicated are associated with an extra risk of 10% and are in units of
mg/m3 as HEC.
Source: Hanley et al. (1984).
Figure B-5. Fit of Log Logistic Model to Data on Litter Incidence of Unilateral Testicular
Hypoplasia in Mice
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Model Fitting for Limb Defects in Rabbits (Hanley et al., 1984)
Following the above procedure, the quantal models in the EPA BMDS (version 2.1) were
fit to the incidence data for limb defects in rabbits from the Hanley et al. (1984) study shown in
Table 31. Because the litter is the preferred unit of statistical analysis for developmental studies,
the data modeled were those for incidence of litters affected. HECs, calculated as shown in
Table 34, were used for modeling, so results are reported as HECs as well. Exposures for this
gestational exposure study were duration adjusted as part of the HEC calculation, as currently
recommended by EPA (2002). Table B-6 shows the modeling results. All models produced
adequate fit (p > 0.01). With the exception of the BMCL from the quantal linear/1-degree
multistage model, which was considered to be an outlier due to poor fit to the mid-dose group
(the point closest to the BMR; scaled residual = -1.7), the BMCLs were all within a factor of 3.
The best fitting model, with the lowest AIC, was the Probit, giving a BMCiohec and a
"3
BMCLiohecs of 13.7 and 9.9 mg/m , respectively. Figure B-6 shows the fit of this model to the
data.
Table B-6. Model Predictions for the Litter Incidence of
Limb Defects in Rabbits
Model
Degrees of
Freedom
2
1
X Goodness
of Fit
p-Value3
AIC
BMCiohec
(mg/m3)
BMCLiohec
(mg/m3)
Gammab
1
1.03
0.3093
49.717
13.2462
4.67047
Logistic
2
0.87
0.6475
47.4831
15.6353
11.085
Log logistic0
1
1.03
0.309
49.7061
13.1984
5.14219
Log probitc
1
1.02
0.3117
49.7303
12.429
6.14406
Multistage (degree = l)d

4.11
0.2498
49.5567
4.26928
2.92108
Multistage (degree = 2)d
1
1.47
0.225
49.7311
9.48507
4.38246
Multistage (degree = 3)d
1
1.12
0.2896
49.2537
10.5605
4.58557
Probit

0.96
0.6203
47.4635
13.7093
9.91939
Weibullb
1
1.05
0.3055
49.6823
13.7322
4.62413
Quantal-linear
3
4.11
0.2498
49.5567
4.26928
2.92108
aValues <0.10 fail to meet conventional goodness-of-fit criteria.
bPower restricted to >1.
°Slope restricted to >1.
dBetas restricted to >0.
Source: Hanley et al. (1984).
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Probit Model with 0.95 Confidence Level
Pro bit
0.8
0.6
0.4
0.2
0
BMDL
BMD
0
5
10
15
20
25
30
35
40
11:10 10/09 2009
BMC and BMCL indicated are associated with an extra risk of 10% and are in units of
mg/m3 as HEC.
Source: Hanley et al. (1984).
Figure B-6. Fit of Probit Model to Litter Data on Limb Defects in Rabbits
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Model Fitting for Renal Defects in Rabbits (Hanley et al., 1984)
Following the above procedure, the quantal models in the EPA BMDS (version 2.1) were
fit to the incidence data for limb defects in rabbits from the Hanley et al. (1984) study shown in
Table 31. Because the litter is the preferred unit of statistical analysis for developmental studies,
the data modeled were those for incidence of litters affected. HECs, calculated as shown in
Table 34, were used for modeling, so results are reported as HECs as well. Exposures for this
gestational exposure study were duration adjusted as part of the HEC calculation, as currently
recommended by EPA (2002). Table B-7 shows the modeling results. All models produced
adequate fit (p > 0.01) except the 2-degree multistage, although none fit especially well. BMCLs
from the adequate models were all within a factor of 3 (approximately). The best fitting model,
with the lowest AIC, was the quantal linear/1-degree multistage, giving a BMCiohec and a
BMCLiohecS of 4.8 and 3.3 mg/m3, respectively. Figure B-7 shows the fit of this model to the
data.
Table B-7. Model Predictions for the Litter Incidence of
Renal Defects in Rabbits
Model
Degrees of
Freedom
2
1
X Goodness
of Fit
p-Value3
AIC
BMCiohec
(mg/m3)
BMCLiohec
(mg/m3)
Gammab
1
2.12
0.1453
59.61
25.4759
3.67068
Logistic
2
2.55
0.2797
57.5421
15.0818
10.9677
Log logistic0
1
2.12
0.1453
59.61
29.2418
3.75507
Log probitc
1
2.12
0.1453
59.61
24.003
6.94654
Multistage (degree =
l)d
3
3.58
0.3101
57.0039
4.82348
3.28055
Multistage (degree = 2)d
1
2.92
0.0874
59.8557
11.5469
3.58575
Multistage (degree = 3)d
1
2.58
0.1081
59.4755
13.8241
3.72181
Probit
2
2.7
0.2592
57.5999
13.3542
9.86922
Weibullb
1
2.12
0.1453
59.61
30.6671
3.67068
Quantal-linear
3
3.58
0.3101
57.0039
4.82348
3.28055
aValues <0.10 fail to meet conventional goodness-of-fit criteria.
bPower restricted to >1.
°Slope restricted to >1.
dBetas restricted to >0.
Source: Hanley et al. (1984).
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Quantal Linear Model with 0.95 Confidence Level
0.9
Quantal Linear
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
BMDL
BMD
0
5
10
15
20
25
30
35
40
11:20 10/09 2009
BMC and BMCL indicated are associated with an extra risk of 10% and are in units of
mg/m3 as HEC.
Source: Hanley et al. (1984).
Figure B-7. Fit of Quantal-Linear Model to Litter Data on Renal Defects in Rabbits
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