?,EPA
United States
Environmental Protection
Agency
EPA/690/R-19/001F | April 23,2019 | FINAL
Provisional Peer-Reviewed Toxicity Values for
2-Ethylhexanol
(CASRN 104-76-7)
supERFUND
U.S. EPA Office of Research and Development
National Center for Environmental Assessment, Superfund Health Risk Technical Support Center (Cincinnati, OH)
-------
United States
I I Environmental Protection
Lb I m m Agency
EPA/690/R-19/00 IF
FINAL
04-23-2019
Provisional Peer-Reviewed Toxicity Values for
2-Ethylhexanol
(CASRN 104-76-7)
Superfund Health Risk Technical Support Center
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
-------
AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGERS
J. Phillip Kaiser, PhD, DABT
National Center for Environmental Assessment, Cincinnati, OH
Elizabeth Oesterling Owens, PhD
National Center for Environmental Assessment, Cincinnati, OH
DRAFT DOCUMENT PREPARED BY
SRC, Inc.
7502 Round Pond Road
North Syracuse, NY 13212
PRIMARY INTERNAL REVIEWERS
J. Allen Davis, MSPH
National Center for Environmental Assessment, Cincinnati, OH
Lucina E. Lizarraga, PhD
National Center for Environmental Assessment, Cincinnati, OH
Jon Reid, 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 content of this PPRTV assessment should 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 AND ACRONYMS iv
BACKGROUND 1
DISCLAIMERS 1
QUESTIONS REGARDING PPRTVs 1
INTRODUCTION 2
REVIEW OF POTENTIALLY RELEVANT DATA (NONCANCER AND CANCER) 6
HUMAN STUDIES 12
Oral Exposures 12
Inhalation Exposures 12
ANIMAL STUDIES 18
Oral Exposures 18
Inhalation Exposures 27
OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS) 31
Tests Evaluating Genotoxicity and/or Mutagenicity 31
Supporting Animal Studies 32
Metabolism/Toxicokinetic Studies 32
Mode-of-Action/Mechanistic Studies 33
DERIVATION 01 PROVISIONAL VALUES 48
DERIVATION OF ORAL REFERENCE DOSES 49
Derivation of a Subchronic Provisional Reference Dose 49
Derivation of a Chronic Provisional Reference Dose 54
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS 58
Derivation of a Subchronic Provisional Reference Concentration 58
Derivation of a Chronic Provisional Reference Concentration 63
CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR 65
MODE-OF-ACTION DISCI SSION 66
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES 67
Derivation of a Provisional Oral Slope Factor 67
Derivation of a Provisional Inhalation Unit Risk 69
APPENDIX A. PROVISIONAL SCREENING VALUES 70
APPENDIX B. DATA TABLES 71
APPENDIX C. BENCHMARK DOSE MODELING RESULTS 90
APPENDIX D. BENCHMARK DOSE CALCULATIONS FOR THE PROVISIONAL ORAL
SLOPE FACTOR 137
APPENDIX E. REFERENCES 151
in
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COMMONLY USED ABBREVIATIONS AND ACRONYMS1
a2u-g
alpha 2u-globulin
MN
micronuclei
ACGIH
American Conference of Governmental
MNPCE
micronucleated polychromatic
Industrial Hygienists
erythrocyte
AIC
Akaike's information criterion
MOA
mode of action
ALD
approximate lethal dosage
MTD
maximum tolerated dose
ALT
alanine aminotransferase
NAG
Y-acctyl-(}-D-glucosaminidasc
AR
androgen receptor
NCEA
National Center for Environmental
AST
aspartate aminotransferase
Assessment
atm
atmosphere
NCI
National Cancer Institute
ATSDR
Agency for Toxic Substances and
NOAEL
no-observed-adverse-effect level
Disease Registry
NTP
National Toxicology Program
BMD
benchmark dose
NZW
New Zealand White (rabbit breed)
BMDL
benchmark dose lower confidence limit
OCT
ornithine carbamoyl transferase
BMDS
Benchmark Dose Software
ORD
Office of Research and Development
BMR
benchmark response
PBPK
physiologically based pharmacokinetic
BUN
blood urea nitrogen
PCNA
proliferating cell nuclear antigen
BW
body weight
PND
postnatal day
CA
chromosomal aberration
POD
point of departure
CAS
Chemical Abstracts Service
PODadj
duration-adjusted POD
CASRN
Chemical Abstracts Service registry
QSAR
quantitative structure-activity
number
relationship
CBI
covalent binding index
RBC
red blood cell
CHO
Chinese hamster ovary (cell line cells)
RDS
replicative DNA synthesis
CL
confidence limit
RfC
inhalation reference concentration
CNS
central nervous system
RfD
oral reference dose
CPN
chronic progressive nephropathy
RGDR
regional gas dose ratio
CYP450
cytochrome P450
RNA
ribonucleic acid
DAF
dosimetric adjustment factor
SAR
structure activity relationship
DEN
diethylnitrosamine
SCE
sister chromatid exchange
DMSO
dimethylsulfoxide
SD
standard deviation
DNA
deoxyribonucleic acid
SDH
sorbitol dehydrogenase
EPA
Environmental Protection Agency
SE
standard error
ER
estrogen receptor
SGOT
serum glutamic oxaloacetic
FDA
Food and Drug Administration
transaminase, also known as AST
FEVi
forced expiratory volume of 1 second
SGPT
serum glutamic pyruvic transaminase,
GD
gestation day
also known as ALT
GDH
glutamate dehydrogenase
SSD
systemic scleroderma
GGT
y-glutamyl transferase
TCA
trichloroacetic acid
GSH
glutathione
TCE
trichloroethylene
GST
glutathione-S-transferase
TWA
time-weighted average
Hb/g-A
animal blood-gas partition coefficient
UF
uncertainty factor
Hb/g-H
human blood-gas partition coefficient
UFa
interspecies uncertainty factor
HEC
human equivalent concentration
UFc
composite uncertainty factor
HED
human equivalent dose
UFd
database uncertainty factor
i.p.
intraperitoneal
UFh
intraspecies uncertainty factor
IRIS
Integrated Risk Information System
UFl
LOAEL-to-NOAEL uncertainty factor
IVF
in vitro fertilization
UFs
subchronic-to-chronic uncertainty factor
LC50
median lethal concentration
U.S.
United States of America
LD50
median lethal dose
WBC
white blood cell
LOAEL
lowest-observed-adverse-effect level
Abbreviations and acronyms not listed on this page are defined upon first use in the PPRTV document.
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PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR
2-ETHYLHEXANOL (CASRN 104-76-7)
BACKGROUND
A Provisional Peer-Reviewed Toxicity Value (PPRTV) is defined as a toxicity value
derived for use in the Superfund Program. PPRTVs are derived after a review of the relevant
scientific literature using established Agency guidance on human health toxicity value
derivations. All PPRTV assessments receive internal review by at least two National Center for
Environment Assessment (NCEA) scientists and an independent external peer review by at least
three scientific experts.
The purpose of this document is to provide support for the hazard and dose-response
assessment pertaining to chronic and subchronic exposures to substances of concern, to present
the major conclusions reached in the hazard identification and derivation of the PPRTVs, and to
characterize the overall confidence in these conclusions and toxicity values. It is not intended to
be a comprehensive treatise on the chemical or toxicological nature of this substance.
Currently available PPRTV assessments can be accessed on the U.S. Environmental
Protection Agency's (EPA's) PPRTV website at https://www.epa.gov/pprtv. PPRTV
assessments are eligible to be updated on a 5-year cycle to incorporate new data or
methodologies that might impact the toxicity values or characterization of potential for adverse
human-health effects and are revised as appropriate. Questions regarding nomination of
chemicals for update can be sent to the appropriate U.S. EPA Superfund and Technology Liaison
(https://www.epa.gov/research/fact-sheets-regional-science).
DISCLAIMERS
The PPRTV document provides toxicity values and information about the adverse effects
of the chemical and the evidence on which the value is based, including the strengths and
limitations of the data. All users are advised to review the information provided in this
document to ensure that the PPRTV used is appropriate for the types of exposures and
circumstances at the site in question and the risk management decision that would be supported
by the risk assessment.
Other U.S. EPA programs or external parties who may choose to use PPRTVs are
advised that Superfund resources will not generally be used to respond to challenges, if any, of
PPRTVs used in a context outside of the Superfund program.
This document has been reviewed in accordance with U.S. EPA policy and approved for
publication. Mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
QUESTIONS REGARDING PPRTVS
Questions regarding the content of this PPRTV assessment should be directed to the
U.S. EPA Office of Research and Development's (ORD's) NCEA, Superfund Health Risk
Technical Support Center (513-569-7300).
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INTRODUCTION
2-Ethylhexanol (2-EH; also called 2-ethyl-l-hexanol), CASRN 104-76-7, belongs to the
class of compounds known as aliphatic alcohols. 2-EH is a volatile organic compound (VOC)
that has been identified as a metabolite and degradation product of diethylhexyl phthalate
(DEHP) (ChemlDplus. 2018). It is mainly used in the manufacture of ester plasticizers for soft
poly(vinyl chloride) (PVC), and the second largest application is in the production of
2-ethylhexyl aery late (Bahrmann ct al.. 2013). It can also be used as a penetrant in mercerizing
textiles; as a solvent for dyes, resins, oils, paints, lacquers, baking finishes, and nitrocellulose; as
a wetting agent; as a defoaming agent; and in textile finishing compounds, inks, rubber, paper,
lubricants, photography, and dry cleaning (HSDB. 2014). 2-EH is listed on the U.S. EPA's
Toxic Substances Control Act (TSCA) public inventory (U.S. HP A. 2018b); it is registered with
Europe's Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)
program (HCHA. 2018) and is also listed as a high production volume (HPV) chemical in the
U.S. and Europe under the U.S. EPA's HPV Challenge Program and Organisation for Economic
Co-operation and Development (OECD) (U.S. EPA, 2016; QECD, 1995). 2-EH is subject to the
Section 4 Test Rule under TSCA Flag T (U.S. EPA. 2015).
Commercial production of 2-EH occurs via a four-step process: (1) aldolization of
butyraldehyde and subsequent dehydration, (2) separation of the aldolization solution,
(3) hydrogenation of unsaturated 2-ethyl-2-hexenal as an intermediate product, and
(4) fractionation of 2-EH (Bahrmann ct al .. 2013). As an HPV chemical, 2-EH has an annual
production volume of over 2 million pounds in Europe and 1 million pounds in the United States
(U.S. EPA. 2016; OECD. 1995). 2-EH is also a naturally occurring plant volatile, specifically
identified in a variety of fruits (ChemlDplus. 2018).
The empirical formula for 2-EH is CsHisO. Its chemical structure is shown in Figure 1,
and Table 1 summarizes its physicochemical properties. 2-EH is a combustible, colorless liquid
at room temperature (ChemlDplus. 2018). Its moderate vapor pressure indicates that it will exist
almost entirely as a vapor in the atmosphere. The estimated half-life of vapor-phase 2-EH in air
by reaction with photochemically produced hydroxyl radicals is 9.7 hours. 2-EH's moderate
Henry's law constant indicates that it may volatilize from moist surfaces, but its vapor pressure
suggests that it is not expected to volatilize from dry soils. The water solubility and low soil
adsorption coefficient for 2-EH indicate that it may leach to groundwater or undergo runoff after
a rain event. 2-EH may also undergo ready biodegradation in the environment, based on
screening tests (HCHA. 2018).
Figure 1. 2-Ethylhexanol (CASRN 104-76-7) Structure
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Table 1. Physicochemical Properties of 2-Ethylhexanol (CASRN 104-76-7)
Property (unit)
Value
Physical state
Liquid
Boiling point (°C)
184a
Melting point (°C)
-74a
Density (g/cm3 at 20°C)
0.833°
Vapor pressure (mm Hg at 25 °C)
0.1363
pH (unitless)
NA
pKa (unitless)
15.75 (estimated)0
Solubility in water (mol/L at 25°C)
6.02 x l0-3a
Octanol-water partition coefficient (log Kow)
2.9°
Henry's law constant (atm-m3/mol at 25°C)
3.01 x 10 " (estimated)3
Soil adsorption coefficient log Koc (L/kg)
83.7 (estimated)3
Atmospheric OH rate constant (cm3/molecule-sec at 25°C)
1.84 x 10 11 (estimated)3
Atmospheric half-life (hours)
9.7 (estimated)13
Relative vapor density (air = 1)
4.49d
Molecular weight (g/mol)
1303
Flash point (closed cup in °C)
75°
aData were extracted from the U.S. EPA CompTox Chemicals Dashboard (2-Ethyl-l-hexanol, CASRN 104-76-
7. https://comptox.epa.gov/dashboard/DTXSID50206Q5. Accessed 24 April 2019).
''U.S. HP A (2012c).
°ECHA (2018).
dLewis (2012).
NA = not applicable.
A summary of available toxicity values for 2-EH from U.S. EPA and other
agencies/organizations is provided in Table 2.
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Table 2. Summary of Available Toxicity Values for 2-Ethylhexanol (CASRN 104-76-7)
Source (parameter)3'b
Value (applicability)
Notes
Reference
Noncancer
IRIS
NV
NA
U.S. EPA (2018a)
HEAST
NV
NA
U.S. EPA (2011a)
DWSHA
NV
NA
U.S. EPA (2012a)
ATSDR
NV
NA
ATSDR (2018)
IPCS
NV
NA
IPCS (2018)
CalFPA
NV
NA
CalEPA (2016);
CalEPA (2018a):
CalEPA (2018b)
OSHA
NV
NA
OSHA (2017a):
OSHA (2017b)
NIOSH
NV
NA
NIOSH (2016)
ACGIH
NV
NA
ACGIH (2018)
AIHA (ERPG)
ERPG-3:227 ppm
(1,209 mg/m3)
ERPG-2: 120 ppm
(639 mg/m3)
ERPG-1: 0.1 ppm
(0.53 mg/m3)
ERPG-3: Based on the absence of
life-threatening health effects in mice,
rats, and guinea pigs exposed at 227 ppm
for 6 hr.
ERPG-2: Based on a NOAEL of 120 ppm
in rats exposed 6 hr/d, 5 d/wk for 90 d.
ERPG-1: Based on perception of an
objectionable odor at 0.1 ppm.
AIHA (2007)
Cancer
IRIS
NV
NA
U.S. EPA (2018a)
HEAST
NV
NA
U.S. EPA (2011a)
DWSHA
NV
NA
U.S. EPA (2012a)
NTP
NV
NA
NTP (2016)
IARC
NV
NA
IARC (2018)
CalEPA
NV
NA
CalEPA (2011):
CalEPA (2018a):
CalEPA (2018b)
ACGIH
NV
NA
ACGIH (2018)
aSources: ACGIH = American Conference of Governmental Industrial Hygienists; AIHA = American Industrial
Hygiene Association; ATSDR = Agency for Toxic Substances and Disease Registry; CalEPA = California
Environmental Protection Agency; DWSHA = Drinking Water Standards and Health Advisories; HEAST = Health
Effects Assessment Summary Tables; IARC = International Agency for Research on Cancer; IPCS = International
Programme on Chemical Safety; IRIS = Integrated Risk Information System; NIOSH = National Institute for
Occupational Safety and Health; NTP = National Toxicology Program; OSHA = Occupational Safety and Health
Administration.
Parameters: ERPG = emergency response planning guideline.
NA = not applicable; NOAEL = no-observed-adverse-effect level; NV = not available.
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Literature searches were conducted in October 2015 and updated in August 2018 for
studies relevant to the derivation of provisional toxicity values for 2-EH, CASRN 104-76-7.
Searches were conducted using U.S. EPA's Health and Environmental Research Online (HERO)
database of scientific literature. HERO searches the following databases: PubMed, TOXLINE
(including TSCATS1), and Web of Science. The following databases were searched outside of
HERO for health-related values: American Conference of Governmental Industrial Hygienists
(ACGIH), American Industrial Hygiene Association (AIHA), Agency for Toxic Substances and
Disease Registry (ATSDR), California Environmental Protection Agency (CalEPA), European
Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC), European Chemicals
Agency (ECHA), International Agency for Research on Cancer (IARC), Japan Existing
Chemical Data Base (JECDB), National Institute for Occupational Safety and Health (NIOSH),
National Toxicology Program (NTP), OECD HPV, OECD International Uniform Chemical
Information Database (IUCLID), OECD Screening Information Data Sets (SIDS), Occupational
Safety and Health Administration (OSHA), U.S. EPA Health Effects Assessment Summary
Tables (HEAST), U.S. EPA HPV, U.S. EPA Integrated Risk Information System (IRIS),
U.S. EPA Office of Water (OW), U.S. EPA Toxic Substances Control Act Test Submissions
(TSCATS), and World Health Organization (WHO).
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REVIEW OF POTENTIALLY RELEVANT DATA
(NONCANCER AND CANCER)
Tables 3A and 3B provide overviews of the relevant noncancer and cancer databases,
respectively, for 2-EH and include all potentially relevant repeated-dose subchronic- and
chronic-duration studies, as well as reproductive and developmental toxicity studies. Principal
studies are identified in bold. The phrase "statistical significance," used throughout the
document, indicates ap-walue of < 0.05, unless otherwise noted.
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Table 3A. Summary of Potentially Relevant Noncancer Data for 2-Ethylhexanol (CASRN 104-76-7)
Category"
Number of
Male/Female, Strain,
Species, Study Type,
Reported Doses, Study
Duration
Dosimetryb
Critical Effects
NOAELb
LOAELb
Reference
(comments)
Notes'
Human
1. Oral (mg/kg-d)
ND
2. Inhalation (mg/m3)
Numerous studies in humans have examined the relationship between indoor air quality and the adverse health effects of 2-EH and VOCs (Kishi et at. 2018: Wieslander
et at. 2010: Putus et at. 2004: Tuomainen et at. 2004: Walinder et at. 2001: Norback et at. 2000: Wieslander et at. 1999: Norback et ai. 19901. These studies generally
observed eye, nose, and throat irritation. In an experimental study of short-term exposure to 2-EH in humans, the subjective ratings of eye and smell discomfort were
significantly increased compared to clean air exposure and nasal and throat irritation ratings were also increased, albeit not significantly (p > 0.08) (Emstgard et at.
20101. van Thrift et at (2003) reported that the highest average subjective ratings were observed for annoyance and olfactory symptoms in male volunteers exposed to
2-EH for 4 hours as well as significantly decreased nasal flow and increased concentrations of the neuropeptide substance P in nasal fluid (an indicator of nasal
che mo sensory irritation) (van 1'hricl et at. 20031. In two follow-up studies by the same authors, significant increases in the perception and intensity of acute symptoms
(olfactory symptoms, nasal irritation, eye irritation, odor, and/or annoyance) were observed with increasing exposure to 2-EH in male volunteers exposed for 4 hr (van
'1'hricl et at. 2007: van '1'hricl et at. 20051. Another study conducted by the same group concluded that 2-EH was irritating to the eyes in participants exposed for 4 hr
(Kiesswetter et at. 20051.
Animal
1. Oral (mg/kg-d)
Subchronic
10 M/10 F, Dow Wistar
albino rat, diet, 0, 0.01,
0.05,0.25, or 1.25% for
89 d (M) or 90 d (F)
0, 7, 36, 170,
840 (M);
0, 7,41, 190,
940 (F)
Increased liver weight; diffuse cloudy
swelling in the liver and the kidneys
(statistically significant in females only)
170 (M);
190 (F)
840 (M);
940 (F)
Mellon Institute of
Industrial Research
(19601
(mortality in control
and some treated
groups from lung
infection or peritonitis,
limited toxicological
endpoints evaluated,
and poor data
reporting)
NPR
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Table 3A. Summary of Potentially Relevant Noncancer Data for 2-Ethylhexanol (CASRN 104-76-7)
Category"
Number of
Male/Female, Strain,
Species, Study Type,
Reported Doses, Study
Duration
Dosimetryb
Critical Effects
NOAELb
LOAELb
Reference
(comments)
Notes0
Subchronic
10 M/10 F, F344 rat,
gavage, 0, 25, 125, 250,
or 500 mg/kg-d, 5 d/wk,
3 mo
0, 18, 89.3, 179,
357
Increased relative stomach, liver, and
kidney (males only) weights; increased
absolute liver (males only) and stomach
(females only) weights; forestomach
lesions (acanthosis)
89.3
179
Astill et al. (1996a):
BASF (1991a)
PR
Subchronic
10 M/10 F, B6C3Fi
mouse, gavage, 0, 25,
125, 250, or 500 mg/kg,
5 d/wk, 3 mo
0, 18, 89.3, 179,
357
Increased relative stomach weight in
males
89.3
179
Astill et al. (1996a):
BASF (1991c): BASF
(1991e)
PR
Chronic
50 M/50 F, F344 rat,
gavage, 0 (water), 0
(vehicle), 50, 150, or
500 mg/kg-d, 5 d/wk,
24 mo
0, 36, 107, 357
Decreased body weight in M.
Mortality, clinical signs, marked weight
reductions and histological lesions in F
at 357 mg/kg-d
36
107 (FEL = 357)
Astill et al. (1996b):
BASF (1992a)
PR
Chronic
50 M/50 F, B6C3Fi
mouse, gavage, 0
(water), 0 (vehicle), 50,
200, or 750 mg/kg-d,
5 d/wk, 18 mo
0, 36, 143, 536
Increased early mortality, decreased
body weight, increased
histopathological lesions (fatty
infiltration of the liver)
143
536 (FEL)
Astill et al. (1996b):
BASF (1991b)
PR
Reproductive/
Developmental
0 M/10 F, pregnant
Wistar rat, gavage, 0
(water), 0 (vehicle), 1,
5,10 mmol/kg on
GDs 6-15
0,130, 650,
1,300
Maternal: Severe toxicity (mortality,
clinical signs, body-weight loss) at
1,300 mg/kg-d
Fetal: Increased fetal incidences of
skeletal variations, skeletal
malformations, and skeletal
retardations; decreased fetal body
weight
Maternal:
650
Fetal: 130
Maternal:
1,300 (FEL)
Fetal: 650
Hellwis and JacHi
(1997): Confidential
(1991)
PS,
PR
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Table 3A. Summary of Potentially Relevant Noncancer Data for 2-Ethylhexanol (CASRN 104-76-7)
Category"
Number of
Male/Female, Strain,
Species, Study Type,
Reported Doses, Study
Duration
Dosimetryb
Critical Effects
NOAELb
LOAELb
Reference
(comments)
Notes0
Reproductive/
Developmental
0 M/28 F, CD-I mouse,
microencapsulated in
diet, 0, 0.009, 0.03, or
0.09% on GDs 0-17
0, 17,59, 191
No significant effects
Maternal: 191
Fetal: 191
Maternal: NDr
Fetal: NDr
NTP (1991)
NPR
Reproductive/
Developmental
0 M/50 F, pregnant
CD-I mouse, gavage, 0
or 1,525 mg/kg-d on
GDs 6-13
0, 1,525
Maternal: Death, decreased body
weight/body-weight gain
Developmental: Decreased survival and
growth of pups (PNDs 1-3)
Maternal:
NDr
Developmental:
NDr
Maternal:
1,525 (FEL)
Developmental:
1,525 (FEL)
Hardin etal. (1987):
Hazleton Laboratories
(1983)
PR
Reproductive/
Developmental
0 M/7 F, pregnant
Wistar rat, gavage, 0,
6.25, or 12.5 mmol/kg
on GD 12
0, 830, 1,700
Maternal: No effects reported
Fetal: Decreased fetal body weight and
increased number of surviving fetuses
with malformations
Maternal:
NDr
Developmental:
NDr
Maternal:
NDr
Developmental:
NDr
Ritter et al. (1987)
PR
(limited toxicological
endpoints evaluated,
and poor data
reporting)
Reproductive/
Developmental
4-5 M/0 F, neonatal CD
(S-D) rat pups, gavage,
167 mg/kg
0, 167
No significant effects
NDr
NDr
Li et al. (2000)
(limited scope of
study)
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Table 3A. Summary of Potentially Relevant Noncancer Data for 2-Ethylhexanol (CASRN 104-76-7)
Category"
Number of
Male/Female, Strain,
Species, Study Type,
Reported Doses, Study
Duration
Dosimetryb
Critical Effects
NOAELb
LOAELb
Reference
(comments)
Notes0
2. Inhalation (mg/m3)
Subchronic
10 M/10 F, Wistar rat,
whole-body inhalation,
0, 15, 40, or 120 ppm,
6 hr/d, 5 d/wk, 90 d
HECexresp: 0,
14, 38, 114
No significant, treatment-related effects
114
NDr
Klimisch et al. (1998);
BASF (1992b)
PR
Subchronic
5-7 M, ICR mouse,
whole-body inhalation,
0, 21.9, 65.8, or
153.2 ppm, 8 hr/d,
5-7 d/wk, up to 3 mo
HECet: 0, 4.17,
12.5,29.20
Morphological changes
(e.g., inflammation) in the olfactory
epithelium; leukocyte infiltration in
the olfactory epithelium at 1 wk and
3 mo; altered expression of olfactory
nerve-related markers in the
olfactory epithelium and bulb at 1 wk
and/or 3 mo; decreased glomerular
diameter in the olfactory bulb at 3 mo
NDr
4.17
Mivake et al. (2016)
PS,
PR
Reproductive/
Developmental
15 F, S-D rat, 0 or
850 mg/m3, 7 hr/d on
GDs 1-19
HECexresp: 0,
248
No effects reported
248
NDr
Nelson et al. (1989)
PR
aDuration categories are defined as follows: Acute = exposure for <24 hours; short term = repeated exposure for 24 hours to <30 days; long term (subchronic) = repeated
exposure for >30 days <10% lifespan for humans (>30 days up to approximately 90 days in typically used laboratory animal species); and chronic = repeated exposure
for >10% lifespan for humans (>~90 days to 2 years in typically used laboratory animal species) (U.S. EPA. 20021.
bDosimetry: Values are presented as ADDs (mg/kg-day) for oral effects and HECs (mg/m3) for inhalation effects. HECexresp = (ppm x molecular
weight ^ 24.45) x (hours per day exposed ^ 24) x (days per week exposed ^ 7) x ratio of blood-gas partition coefficients (animal:human).
HECet = (ppm x MW ^ 24.45) x (hours per day exposed ^ 24) x (days per week exposed ^ 7) x RGDRet (animal:human) (U.S. EPA. 1994).
°Notes: PS = principal study; PR = peer reviewed; NPR = not peer reviewed.
ADD = adjusted daily dose; ET = extrathoracic; EXRESP = extrarespiratory effects; F = female(s); FEL = frank effect level; GD = gestation day; HEC = human
equivalent concentration; LOAEL = lowest-observed-adverse-effect level; M = male(s); PND = postnatal day; ND = no data; NDr = not determined;
NOAEL = no-observed-adverse-effect level; RGDR = regional gas dose ratio; S-D = Sprague-Dawley.
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Table 3B. Summary of Potentially Relevant Cancer Data for 2-Ethylhexanol (CASRN 104-76-7)
Category
Number of Male/Female, Strain, Species, Study
Type, Reported Doses, Study Duration
Dosimetry"
Critical Effects
Reference (comments)
Notesb
Human
1. Oral (mg/kg-d)
ND
2. Inhalation (mg/m3)
ND
Animal
1. Oral (mg/kg-d)
Carcinogenicity
50 M/50 F, F344 rat, gavage, 0, 50, 150, or
500 mg/kg, 5 d/wk, 24 mo
0, 9.5, 27.9, 90.9 (M)
0, 8.4, 24.8, 81.2 (F)
No evidence of carcinogenicity
Astill et al. (1996b):
BASF (1992a)
PR
Carcinogenicity
50 M/50 F, B6C3Fi mouse, gavage, 0,50,200,
or 750 mg/kg, 5 d/wk, 18 mo
0, 5.5, 21.8, 79.9 (M)
0, 5.3, 21.1, 77.3 (F)
Hepatocellular carcinomas and
adenomas
Astill et al. f1996b):
BASF (1991b)
PS, PR
2. Inhalation (mg/m3)
ND
'Dosimetry: The units for oral exposures are expressed as HEDs (mg/kg-day); HED = adjusted daily animal dose (mg/kg-day) x (BWa ^ BWh)"4 (U.S. EPA. 2005).
using TWA body weights calculated from study reported body-weight data for rats and mice, and 70 kg for humans (U.S. EPA. 2011b).
bNotes: PR = peer reviewed; PS = principal study.
BW = body weight; F = female(s); HED = human equivalent dose; M = male(s); ND = no data; TWA = time-weighted average.
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HUMAN STUDIES
Oral Exposures
No studies have been identified.
Inhalation Exposures
Although there were no human studies suitable for reference value derivation, there are
several published human studies available that are briefly summarized in the text below.
Data for inhalation exposure in humans include experimental short-term exposures to 2-EH
(Emstgard et al.. 2010; van Thriet ct al.. 2007; Kiesswetter et al.. 2005; van Thriet et al.. 2005; van
Thriet et al., 2003), as well as several studies that evaluated adverse health effects (such as "sick
building syndrome" [SBS]) from exposures to volatile organic compounds (VOCs; including 2-EH)
and other substances (Kishi et al.. 2018; Wieslander et al.. 2010; Putus et al.. 2004; Tuomainen et
al.. 2004; Walinder et al.. 2001; Norback et al.. 2000; Wieslander et al.. 1999; Norback ct al .. 1990).
Supporting data include case reports of 2-EH exposure (Kondo et al.. 2007; Kamijima et al.. 2002)
and human health evaluations at sites of potential 2-EH exposure (Shell Oil. 1987; NIOSH. 1984.
1983).
Numerous studies have examined the relationship between indoor air quality and the adverse
health effects of 2-EH and VOCs (Kishi et al .. 2018; Wieslander et al., 2010; Putus et al., 2004;
Tuomainen et al.. 2004; Walinder et al.. 2001; Norback et al.. 2000; Wieslander et al.. 1999;
Norback et al.. 1990). In the subset of studies that evaluated health effects in buildings where
dampness is specifically a problem (e.g., those with concrete or masonry floors, etc.), 2-EH and
1-butanol (by-products of dampness-mediated degradation of diethylhexyl phthalate [DEHP] in
PVC flooring) were the focus of the investigation, but the levels of respirable dust, molds, and/or
bacteria were also considered. The levels of 2-EH in these settings typically ranged from about
<1-100 |ig/m3 (although one study reported levels as high as 1,556 |ig/m3). The types of health
effects assessed in these studies included subjective symptoms of SBS (characterized by irritation of
the eyes, upper airways, and/or skin; headache; cough; and/or fatigue), symptoms of asthma, nasal
lavage parameters, tear film stability, and respiratory function (measured using rhinometry and/or
spirometry). Effects attributed to poor indoor air quality (and 2-EH exposure) were ocular and
nasal irritation (in multiple studies), airway constriction, decreased tear film stability, changes in the
levels of some biomarkers in the lavage fluid (indicative of inflammation), symptoms of asthma
(but not doctor-diagnosed asthma), and/or an increased occurrence of viral (but not bacterial)
respiratory infections. However, considering that the subjects were exposed to multiple
contaminants, it is not possible to attribute these effects to 2-EH exposure alone.
Similar types of effects were reported in occupational case reports of 2-EH exposure (Kondo
et al.. 2007; Kamijima et al.. 2002). A college professor with multiple chemical sensitivity (MCS)
from exposure to the VOCs in campus buildings showed symptoms consistent with SBS, including
eye and throat irritation, cough, headache, blurred vision, and a slight fever. These symptoms were
most apparent in a faculty meeting room where 2-EH was the predominant VOC (469 |ig/m3,
compared to <30 |ig/m3 for other VOCs). In contrast, symptoms subsided in her office, where 2-EH
levels were substantially lower (85 |ig/m3; the concentrations of all other VOCs varied by
<10 |ig/m3 among these areas). Subsequent blood analyses showed a high serum concentration of
2-EH in the affected individual (4.6 ng/mL vs. <0.1 ng/mL for other VOCs) compared to other
patients with an onset of SBS (Kondo et al.. 2007).
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Symptoms associated with effects on the central nervous system (CNS) and irritation of the
respiratory tract were also identified in NIOSH Human Health Evaluations at sites of potential 2-EH
exposure. In a survey of office workers with complaints of SBS-like symptoms (including
headache, nausea, dizziness, and numbness), numerous indoor air contaminants in addition to 2-EH
were identified at detectable levels (e.g., metals, solvents, terpinene, and dimethyloctane), but the
levels of 2-EH were nonquantifiable. Furthermore, the levels of these contaminants were <1% of
their respective OSHA standards (if available), likely because corrective measures had been
implemented. Therefore, the compound(s) responsible for these symptoms was not clear (NIOSH.
1984). In another case, some rescue workers responding to an explosion from the nitration of 2-EH,
as well as subjects in the surrounding area, showed symptoms that included ocular and respiratory
irritation, headache, and cough (NIOSH, 1983). Although these effects are consistent with those
observed in other studies, the results are confounded by exposures to multiple contaminants, and no
estimates of exposure were reported.
Another industrial survey evaluated pregnancy-related morbidity in Shell Oil Company
employees assigned to jobs with potential 2-EH exposure based on their job descriptions (groups of
female workers assigned to jobs with potential 2-EH exposure and those ever in a job with a
description that listed 2-EH). No specific estimates of 2-EH exposure were reported. Pregnancy
outcomes were evaluated based on a query of the company's health surveillance system. No
significant effects on reproductive outcomes were observed in workers assigned to jobs with 2-EH
exposure. The rate of spontaneous abortions were similar among women assigned to jobs with
potential 2-EH exposure and in all female employees with potential exposure to 2-EH (past or
present) (Shell Oil. 1987). However, various study limitations were apparent (including the small
number of pregnancies evaluated, the lack of reliable estimates of 2-EH exposure, and the absence
of a control group).
Experimental studies of short-term exposure to 2-EH evaluated a comprehensive set of
subjective symptoms and compared the results with physiological measurements of eye, nose,
and/or lung irritation (Ernstgard et al.. 2010; van Thriel et al.. 2007; Kiesswetter et al.. 2005; van
Thriel et al.. 2005; van Thriel ct al.. 2003). One of the studies (van Thriel et al.. 2007) also
examined the neurotoxic potential of 2-EH.
Ernsteard et al. (2010)
Healthy volunteers (16 males and 14 females; aged 22-49 years with a mean age of
31 years) were exposed in random order to clean air or 2-EH as a vapor at 1 mg/m3 for 2 hours
while at rest. Twelve (40%) of the volunteers had laboratory-verified atopy (genetic predisposition
to develop allergic diseases), representing a possible sensitive subpopulation. Exposure periods
were at least 2 weeks apart. Symptom ratings were recorded at six time points (prior to exposure,
after 3, 60, and 118 minutes of exposure, and 15 and 200 minutes postexposure); the questionnaire
(encompassing the following 10 symptoms: smell, eye, nose, and throat irritation, dyspnea,
headache, fatigue, dizziness, nausea, and intoxication) required subjects to grade responses on a
0-100 mm visual analog scale (VAS) with 0 mm corresponding to "not at all" and 100 mm
corresponding to "almost unbearable." Blinking frequency (defined as blinks per minute for
2-minute intervals) was measured 2 minutes before exposure and throughout the entire exposure
period. Precorneal film stability, measured as tear film break-up time (BUT) in each eye, was
assessed using a biomicroscope just prior to and following exposure and 3 hours postexposure. To
evaluate epithelial damage to the cornea and/or conjunctivae, lissamine green was instilled into the
lower conjunctival sac of the eye 4 hours after exposure and the eye was examined using a
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binocular microscope with a slit lamp. Nasal lavage was performed immediately before and after
exposure, and at 3 hours postexposure. The lavage fluid was analyzed for the following
biomarkers: eosinophilic cationic protein (ECP), myeloperoxidase (MPO), lysozyme, and albumin.
At the same time points, acoustic rhinometry was performed to determine nasal volume (from the
nostril to 7 cm into the nasal cavity) and minimal cross-section area (based on an average of
3 measurements/nostril). Irritation in the airways and lungs was evaluated by dynamic spirometry
(pre- and postexposure, 3 hours after exposure); the endpoints evaluated were vital capacity, forced
vital capacity (FVC), forced expiratory volume, peak expiratory flow, and forced expiratory flows
in the middle half of FVC (FEF25, FEF50, and FEF75). Using a single-breath technique, a transfer
test was conducted to assess the diffusion capacity of carbon dioxide (prior to exposure and
20 minutes after exposure).
Numerical data for symptoms ratings were provided only for the 60- and 118-minute time
points in the study report, but data for some symptoms at additional time points were presented
graphically (Emstgard et al.. 2010). The scores for two symptoms, eye discomfort and solvent
smell, were significantly increased during 2-EH exposure relative to clean air exposure. After
118 minutes of 2-EH exposure, the median eye irritation score reached 7 mm compared to 3 mm for
clean air. The perception of solvent smell reached a maximum within 1 hour of exposure; the
median score was 26 mm (corresponding to a "somewhat" or "rather" strong smell) compared to
7 mm for clean air (p < 0.0001). The median score for solvent smell at 2 hours was about half the
value observed at 1 hour, owing to adaptation; there was also evidence for a "chamber effect" (odor
perception in clean air) throughout the exposure period. Nasal and throat irritation ratings following
almost 2 hours of 2-EH exposure were increased, albeit not quite significantly (p > 0.08). In
addition, ratings of irritation (of the nose, eyes, or throat) were not significantly associated with
ratings of smell (based on Spearman correlation tests). Scores for all other symptoms were at or
near 0 throughout the duration of the 2-EH exposure. There were no significant effects on any of
the other endpoints evaluated. Differences were not observed based on gender or atopy status.
van Thriel et al (2003)
Male volunteers (n = 24, with a mean age of 24 years) were exposed to 2-EH at mean
concentrations of 1.53, 10.63, and 21.88 ppm for 4 hours (with 2 days between sessions, 3-4
sessions total). These concentrations are equivalent to 8.15, 56.62, and 116.5 mg/m3, respectively.
Minimum and maximum exposure levels for the low-, mid-, and high-exposure groups were
1.39-1.58 ppm (7.40-8.42 mg/m3), 1.23-20.20 ppm (6.55-107.6 mg/m3), and 1.76-42.07 ppm
(9.37-224.1 mg/m3), respectively. Oscillations in solvent concentration (c) during exposure were
described by these functions: exposure duration (t), average solvent concentration (Ao), and the
value of the difference from the average to intended maximum concentration (a), assuming a cycle
duration of 60 minutes and phasing of 1.5 (c = Ao + a sin [271/ ^ 60 + 1.5]). Ratings of well-being
(tenseness, tiredness, and annoyance) and acute health symptoms ratings (29 symptoms, not
individually specified) were recorded at nine time points during exposure (50 minutes prior to
exposure [as baseline], 1, 26, 59, 85, 129, 145, 173, 199, and 232 minutes after the initiation of
exposure, and 52 minutes postexposure); the former were assessed using a validated seven-point
visual rating scale, the latter were assessed using an extended version of the "acute symptoms" test
from the Swedish Performance Evaluation System (SPES). The initial SPES (for 12 symptoms
associated with the olfactory system and nasal/eye irritation) was expanded to encompass
29 symptoms; severity was ranked on a scale of 0 ("not at all") to 5 ("very, very much").
Like-symptoms were grouped together for analyses (nonlinear regression fitting). Symptom groups
were prenarcotic (four symptoms), olfactory (four symptoms), taste (three symptoms), respiratory
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(three symptoms), nasal irritation (five symptoms), eye irritation (seven symptoms), and other
irritation (three symptoms). For the purposes of this study (which focused on chemosensory
exposure effects), analyses were limited to olfactory symptoms, annoyance, nasal irritation, eye
irritation, and nasal and eye irritation combined ("sensory irritation"). Anterior active
rhinomanometry (AAR) was performed immediately before and after exposure to evaluate measures
of nasal airway resistance (airflow, transnasal pressure gradient between nostrils and nasopharynx,
and anterior pressure). Nasal lavage was done 30 minutes prior to exposure and immediately after
exposure to measure neuropeptide substance P levels (as an indicator of nasal chemosensory
irritation). Additional analyses (using Spearman rank correlation) were performed to evaluate
potential associations between subjective chemosensory ratings and objective physiological
variables.
Chemosensory ratings for the low-exposure group were not shown (van Thriet et al.. 2003).
With respect to analyses at 56.62 and 116.5 mg/m3, the highest average ratings were observed for
annoyance and olfactory symptoms; ratings for sensory irritation were low (<1). However, the
scores for all chemosensory endpoints evaluated (including sensory irritation) varied consistently
with oscillations in exposure concentration (i.e., the goodness-of-fit [i?2] values were >0.50).
Overall, >67% of the variance in these ratings at the mid- and high-exposure levels could be
explained by nonlinear regression analyses indicating a positive dose-response relationship. When
sensory irritation endpoints (i.e., nasal irritation and eye irritation) were analyzed separately, the
symptoms associated with nasal irritation varied most consistently with exposure (R2 = 0.92 and
0.91 at the moderate and high-exposure levels, respectively); eye irritation scores were more
variable (R2 = 0.17 and 0.54 at the same exposure concentrations). Changes in nasal and eye
irritation scores as a function of time of exposure were presented graphically in the study report;
overall ratings for these individual endpoints were not shown. AAR measurements showed
decreased nasal flow (postexposure relative to pre-exposure) in subjects exposed to 2-EH at an
average concentration of 116.5 mg/m3 (p < 0.01); there were no significant effects at the low- or
moderate-exposure concentrations, and the reduction in nasal flow at the highest exposure
concentration was not significantly different than that at other exposure levels. Analyses of
substance P levels in nasal lavage (presented graphically in the study report) showed increased
substance P postexposure relative to pre-exposure at the high-exposure concentration (p = 0.01).
The difference between post- and pre-exposure values was significantly increased at the
high-exposure concentration relative to the low-exposure concentration (p = 0.03). Additional
analyses conducted to evaluate correlations between chemosensory ratings and physiological
variables did not reveal any clear, exposure-related associations.
van Thriel et al (2007); van Thriel et al (2005)
In two follow-up studies by the same authors, male volunteers were exposed to 2-EH as a
vapor for 4 hours at variable (Experiment A) or constant (Experiment B) time-weighted average
(TWA) exposure concentrations of 1.5, 10, and 20 ppm (in succession and at least 2 days apart).
These concentrations are equivalent to 8.0, 53, and 110 mg/m3, respectively. The maximum
exposure concentration under variable conditions was 42 ppm (220 mg/m3). Both experiments used
healthy participants with self-reported multiple chemical sensitivity (sMCS); these individuals were
identified using a standardized questionnaire on chemical and general environmental sensitivity
(CGES) and age-matched controls. Subjects diagnosed with asthma, allergic rhinitis, or chronic
diseases (diabetes, liver disease, etc.) were excluded. Experiment A comprised 12 sMCS subjects
and 12 age-matched controls (mean age = 24 years); Experiment B used 7 sMCS subjects and
15 age-matched controls (mean age = 23 years). In both experiments, half of the subjects (a mix of
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controls and sMCS subjects) were exposed in the morning, and half were exposed in the afternoon.
Steady-state concentrations were used for the 8.0 mg/m3 exposure level. As in the 2003 study, the
2005 study evaluated the severity of acute health symptoms (29 in total) as ranked on a scale of 0-5
at various time points: 50 minutes prior to exposure, during exposure (at 1, 26, 59, 85, 120, 145,
173, 199, and 232 minutes) and 52 minutes following exposure; time points were selected to
correspond with minima and maxima exposure concentrations (for Experiment A). Given the focus
of the study (chemosensory effects), subsequent analyses were confined to olfactory symptoms,
nasal irritation, and eye irritation. The other focus of the 2007 study was on neurobehavioral tests:
namely, "divided attention" (DA), working memory (WM), and vigilance tasks (VT). For the DA
test, subjects were required to process and respond accurately to multiple stimuli (i.e., visual and
auditory) simultaneously. The WM test asked subjects to memorize a series of two-digit numbers
that appeared on the screen for 1.5 seconds and to compare these values to an earlier number
(2-back; delayed comparison test). For the VT test, subjects viewed the movement of a yellow dot
within a circular display of 24 red dots and were asked to document when the yellow dot crossed
over two red dots (rather than one). Each neurobehavioral test (5-30 minutes in duration) was
performed twice (following 5 and 175 minutes of exposure) in order (DA, WM, VT); the following
variables were measured: reaction times (RTs), detection rates (HITs), and false alarms (FAs).
Subjects were trained in the DA and WM tasks on the day of the medical examination to avoid
learning effects across exposure sessions. In both studies, subjects also used the labeled magnitude
scale (LMS) tool (Green ct al.. 1996) to estimate the intensity of odor (2005 study only) or
annoyance (2007 study only), nasal irritation, and eye irritation. Using this tool, the subject
indicated the intensity of each of the three symptoms using a slider positioned alongside six
categorical ratings (ranging from barely detectable to strongest imaginable). Ratings were collected
three times during each exposure period (at 65, 128, and 160 minutes; time points selected to
approximate TWA concentrations).
Results from the 2005 study showed that, in both Experiment A and Experiment B, acute
symptoms were significantly (p < 0.05 by analysis of variance [ANOVA]) affected by the following
factors: the TWA concentration of 2-EH, the time course of exposure, and the interaction of these
two factors [data presented graphically; van Thriel ct al. (2005)1. With respect to Experiment A,
variation in mean ratings for olfactory symptoms, nasal irritation, and eye irritation were typically
high, because responses mimicked the time course of concentration. In Experiment B (using
constant exposure concentrations), a significant exposure-response was also seen, but the variation
in response over time was low. In general, ratings for olfactory symptoms decreased, nasal
irritation remained unchanged, and eye irritation increased throughout exposure. There was a
significant difference for Experiment B only among the responses of control and sMCS subjects,
and the sMCS subjects showed heightened chemosensory responses (especially for olfactory
symptoms). With respect to olfactory symptoms, control subjects exposed to 2-EH at a constant
concentration of 53 or 110 mg/m3 showed decreased ratings over time (the effect was less profound
at 8.0 mg/m3). In contrast, sMCS subjects initially showed the highest ratings at 53 mg/m3, but
those ratings tended to decrease over time, whereas ratings at 110 mg/m3 increased and stabilized
throughout exposure.
With the exception of the DA task, no significant exposure-related effects were reported in
neurobehavioral tests (van Thriel et al.. 2007). In the DA task, HITs significantly decreased
(p = 0.03 based on ANOVA analysis) as the exposure concentration increased. Based on the data
shown graphically in the study report for control and sMCS subjects exposed under variable or
constant conditions in the morning or in the afternoon (eight exposure conditions in total),
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decreased HITs could be explained mainly by concentration-related reductions in HITs in sMCS
subjects (especially for the group exposed to variable concentrations in the morning, but also those
exposed to constant concentrations in the afternoon). A significant increase in RTs at the end of
exposure relative to the beginning of exposure (681 vs. 672 ms; p = 0.05) regardless of exposure
condition was also noted (HITs and FAs were unaffected). There were no significant findings with
respect to the WM task. While subjects' performance on the VT task was not affected by
exposure-related factors, the study authors reported an "observable vigilance decrement"
(significance not reported) during the VT task, as evidenced by increased RTs and decreased HITs
from the first 6-minute interval (487 ms and 92%, respectively) of the task to the last 6-minute
interval (521 ms and 85%); the number of FAs was too small for analyses. A significant interaction
was also noted between exposure duration and the "time of task" (p = 0.01; no further details
provided).
In both studies, the intensity of chemosensory effects (ranked using the LMS) was
significantly affected by 2-EH exposure (van Thriel et al.. 2007; van Thriel et al.. 2005). In the
earlier study, 2-EH exposure (in Experiments A and B) significantly increased the average intensity
of odor, nasal irritation, and eye irritation (p < 0.01 based on ANOVA) in a concentration-related
manner, from weak or barely detectable at 8.0 mg/m3 to strong or very strong at 110 mg/m3 (data
presented graphically in the study report; average values with no evaluation of variation over time).
In general, the intensity of these effects (especially for sensory irritation) was higher at 53 and
110 mg/m3 under variable exposure conditions (Experiment A) compared to constant exposure
conditions (Experiment B). Intensity ratings were not significantly different among control and
sMCS subjects. Similarly, the second study reported that the intensity of annoyance, nasal
irritation, and eye irritation significantly increased (p< 0.01 by multivariate analysis of variance
[MANOVA]) in a concentration-related manner; however, there was also a significant interaction
between exposure concentration, experiment (A vs. B), and sensitivity (control vs. sMCS subjects)
(van Thriel et al .. 2005). The ratings of sMCS subjects were higher than controls for Experiment B;
however, control responses were higher for Experiment A. Ratings for subj ects in Experiment A
reflected the variable exposure concentrations, whereas ratings for Experiment B were relatively
stable. There was evidence of adaptation (i.e., a reduction in ratings over time) in annoyance and
nasal irritation ratings only in subjects exposed to a constant concentration of 53 mg/m3 (but not
110 mg/m3). In general (regardless of exposure condition), ratings for annoyance (on average very
strong) were higher than those for eye and nasal irritation (on average strong).
Taken together, the data from these two studies showed significant increases in the
perception and intensity of acute symptoms (olfactory symptoms, nasal irritation, eye irritation,
odor, and/or annoyance) with increasing exposure to 2-EH. Other than a decrement in accuracy on
the DA task in a subset of chemically sensitive individuals (sMCS subjects), no significant effects
on neurobehavioral tests (including WM and VT tasks) were observed at concentrations up to
110 mg/m3.
Kiesswetter et al. (2005)
Another study conducted by the same group specifically evaluated eye blinks as an indicator
of sensory irritation in non-sMCS and sMCS participants exposed during constant (Experiment C)
and variable (Experiment V) exposures under the same experimental conditions. As in the previous
studies, the variable experiment used 12 non-sMCS subjects and 12 sMCS subjects, but
12 non-sMCS subjects and 8 sMCS subjects were used for constant exposure conditions.
Demographical information for these subjects (and the degree of overlap with the subjects used in
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the preceding experiments) was not provided. A half-hour vigilance test was performed to examine
blink frequency. The test was carried out twice (once near the start and once near the end of
exposure). Two 5-minute sections (i.e., Section A and Section B) within each test were selected for
blink analysis; these sections corresponded to the highest and lowest exposure levels during variable
test conditions. Based on ANOVA analyses, blink rates were significantly affected by exposure
concentration under both constant and variable conditions (p < 0.01); the strongest effects were seen
during variable exposure (i.e., the difference in blink rate in Section 1 [trough exposure] compared
to Section 2 [peak exposure]). Blink rates also increased significantly over time (from the start of
the experiment to the end; p < 0.05), suggesting that adaptation did not occur. There was little to no
difference in blink rates among non-sMCS and sMCS subjects (one significant difference seen
under constant conditions at the start only). The study authors concluded that 2-EH was irritating to
the eyes in both groups of subjects under both constant and variable exposure conditions; the
response was concentration-related with no evidence of adaptation.
ANIMAL STUDIES
Oral Exposures
The database for oral exposure in animals consists of two subchronic-duration studies [one
gavage study in rats and mice (Astill et al.. 1996a; BASF. 1991a. c, e) and one non-peer-reviewed
dietary study in rats (Mellon Institute of Industrial Research. 1960)1. There is also one
chronic-duration gavage study in rats and mice (Astiii et al., 1996b; BASF, 1992a, 1991b). The
available reproductive and developmental toxicity studies include one neonatal (Li et al., 2000) and
several gestational studies in rats and mice (Hellwig and Jackh. 1997; Confidential. 1991; NTP.
1991; Hardin et al.. 1987; Rittcr et al.. 1987; Hazleton Laboratories. 1983).
Subchronic-Duration Studies
Mellon Institute of Industrial Research (1960) (non-peer-reviewed study)
In a non-peer-reviewed study, groups of Dow Wistar albino rats (10/sex) were administered
diets containing 0, 0.01, 0.05, 0.25, or 1.25% 2-EH for 89 days (males) or 90 days (females). The
study authors calculated equivalent doses based on food consumption and body weight to be 0, 7,
36, 170, and 840 mg/kg-day for males and 0, 7, 41, 190, and 940 mg/kg-day for females. Mortality
was presumably monitored regularly (time points not specified). Body weights were measured
three times in the first week and weekly thereafter. At study termination, all rats were subjected to
necropsy, and liver and kidney weights were recorded. Urinary bladders were examined for
concretions. Histopathological examinations (using hematoxylin-eosin staining) of the following
tissues were performed on all surviving control and high-dose animals, as well as treated animals
that died during the study: lung, kidney, liver, heart, spleen, pancreas, stomach, duodenum,
descending colon, testes or ovary, esophagus, trachea, thyroid, adrenal, and urinary bladder. A
subset of these tissues (i.e., lung, liver, kidney, and urinary bladder) were examined in animals
exposed at 0.05 and 0.25%; tissues from the 0.01% group were not examined histologically.
Data for effects in rats treated with 2-EH, when available, are shown in Table B-l (Mellon
Institute of Industrial Research, 1960). Most of the data provided were presented as individual
animal data; means and standard deviations (SDs) were calculated for this PPRTV assessment when
necessary. However, for terminal body weight and body-weight gain, there is uncertainty
associated with the data values at 7 mg/kg-day in males and at 41 mg/kg-day in females
(see Table B-l) owing to the illegibility of individual data values in the study report. Mortality
(0-3 animals/group, including controls) was attributed to lung infection or peritonitis (not to
treatment). No statistically significant, adverse effects on food consumption were reported
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(36 mg/kg-day [males] and 41 mg/kg-day [females] consistently ate more food than controls). With
respect to body weights, in-life data (presented graphically in the study report), body-weight gains,
and terminal body weights were provided. Although overall body-weight gains varied by >10%
relative to controls in some dose groups, terminal body weights in all groups of treated rats were
similar to controls (i.e., >90% of control values). At necropsy, there were no significant,
treatment-related effects on kidney weights. However, absolute and relative (as percentage of body
weight) liver weights were significantly increased by 10-14%) in 840-mg/kg-day males and
940-mg/kg-day females (note a >10% increase in absolute and relative liver and kidney weight is
considered biologically significant by the U.S. EPA for the purposes of this PPRTV assessment).
The study authors reported significantly increased (p < 0.05) incidences of gross lesions in males
(cortical degeneration of the kidney) at 840 mg/kg-day and females (congestion and/or swelling of
the liver) at 940 mg/kg-day; although incidence data for these effects were provided in the study
report, these values are completely illegible at all doses and thus are not shown in Table B-l.
Histopathological examinations revealed significant increases in the incidence of diffuse cloudy
swelling in the kidneys (more specifically, the proximal convoluted tubules) and livers of females
exposed at 940 mg/kg-day. These lesions were also increased in males at 840 mg/kg-day, but the
differences from controls were not significant. Although there are several study limitations
(mortality in control and some treated groups, limited toxicological endpoints evaluated, and poor
data reporting), these limitations do not preclude identification of no-observed-adverse-effect levels
(NOAELs) of 170 mg/kg-day (males) and 190 mg/kg-day (females) and
lowest-observed-adverse-effect levels (LOAELs) of 840 mg/kg-day (males) and 940 mg/kg-day
(females) based on statistically and/or biologically significant increases in absolute and relative
liver weights in both sexes and increased incidences of microscopic liver and kidney lesions
(significant in females only).
Astilletal. (1996a) (published report); BASF (1991a), BASF (1991c), and BASF (199le)
(non-peer-reviewed studies)
Groups of F344 rats and B6C3Fi mice (10/sex/group) were administered 2-EH
(99.8%) purity) in aqueous Cremophor EL (polyoxyl-35 castor oil) via gavage at 0 (vehicle-only
control), 25, 125, 250, or 500 mg/kg-day 5 days/week for 3 months (about 93-94 days for male and
female rats and 96-97 days for male and female mice, respectively) (Astill et al.. 1996a; BASF.
1991a, c, e). Additional groups (three/sex) were exposed to 2-EH at the same dose levels for
3 months to specifically evaluate hepatic peroxisome proliferation. Doses of 0, 25, 125, 250, and
500 mg/kg-day were adjusted for continuous exposure to 0, 18, 89.3, 179, and 357 mg/kg-day by
multiplying the administered gavage dose by (5/7) days per week. Animals were monitored once
daily (on weekends and holidays) or twice daily (on weekdays) for mortality, and at least once daily
for clinical signs of toxicity. Body weights were measured prior to study initiation, weekly
thereafter, and at study termination. Average daily food consumption was determined weekly.
Hematology (hematocrit [Hct]; hemoglobin [Hb]; red blood cells [RBCs], total and differential
white blood cells [WBCs], reticulocyte, and platelet counts; mean corpuscular volume [MCV],
mean corpuscular hemoglobin [MCH], and mean corpuscular hemoglobin concentration [MCHC];
and thromboplastin time) and clinical chemistry (blood urea nitrogen [BUN], creatinine [rats only],
total protein, albumin, globulin, total bilirubin [rats only], glucose, cholesterol, triglycerides,
sodium, potassium, chloride, inorganic phosphate, calcium [rats only], the activities of alanine
aminotransferase [ALT], alkaline phosphatase [ALP], y-glutamyl transferase [GGT], and aspartate
aminotransferase [AST, rats only]) evaluations were performed on Days 29 and 84 (rats) or
Days 96-97 (mice). Rats (but not mice) from the control and 357-mg/kg-day groups were
subjected to ophthalmological evaluations at study initiation and at study termination. The animals
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from all exposure groups were subjected to necropsy; weights of the adrenals, brain, stomach,
kidneys, liver, testes, and ovaries were recorded. Complete histopathological examinations (of
approximately 40 tissues and including all gross lesions; using hematoxylin-eosin staining) were
conducted in rats and mice administered 0 or 357 mg/kg-day. The liver (including gall bladders in
mice), lung, spleen, kidney, stomach, sternum, femur, and bone marrow (from the femur) were
microscopically examined in all groups of rats and mice. The liver was also stained with oil red to
evaluate lipid content. The livers of animals in peroxisome proliferation-only groups were
weighed; cyanide-insensitive palmitoyl-Coenzyme A (pCoA) oxidation activity and protein
concentration were determined.
Effects in rats related to 2-EH exposure are presented in Table B-2 (males) and Table B-3
(females) (Astill et al.. 1996a; BASF. 1991a). All rats survived until study termination. No
significant clinical signs of toxicity were reported. Although the body weights of rats treated at
357 mg/kg-day were statistically significantly less than controls (starting at Week 4 in males and
Week 11 in females, 7-8% at study termination), body weights remained within 10% of their
respective controls throughout the study. There were no significant effects on food consumption.
Hematology and clinical chemistry findings included significant increases in Day 84 reticulocyte
counts in rats of both sexes at 357 mg/kg-day (21-25%) higher than controls) and in serum total
protein (13%>) and albumin (16%>) in male (but not female) rats at 357 mg/kg-day. Other
statistically significant changes in hematology and clinical chemistry endpoints were sporadic and
of questionable toxicological significance (decreased activities of ALP and ALT, a transient
reduction in glucose at Day 29, decreased serum cholesterol). There were no significant
ophthalmological findings. At 357 mg/kg-day, relative (percentage of body weight) and absolute
liver and relative kidney weights were statistically and biologically significantly increased in males.
Absolute kidney weight was statistically but not biologically significantly increased in males at
357 mg/kg-day. Relative stomach and testes weights were also statistically significantly increased
in males at 357 mg/kg-day. In females treated with 357 mg/kg-day, relative liver weight was
statistically and biologically significantly increased. Absolute liver and relative kidney weights
were statistically but not biologically significantly increased in females at 357 mg/kg-day. Relative
and absolute (only at 357 mg/kg-day) stomach weights were also statistically significantly increased
in females at >179 mg/kg-day. In both sexes, statistically but not biologically (< 10%) significant
increases in relative organ weights (liver and kidney) were also seen at 179 mg/kg-day. At gross
necropsy, the presence of single or multiple elevated foci in the forestomach was noted in
2/10 males and 4/10 females at 357 mg/kg-day. Microscopic examinations of the forestomach
revealed acanthosis in 2/10 males and 5/10 females at 357 mg/kg-day (compared to 0/10 in controls
and in other dose groups); the difference from controls was statistically significant in females
(see Tables B-2 and B-3). No statistically significant histopathological effects were reported in the
glandular stomach, liver, kidney, or other organs. Rats in the 357 mg/kg-day peroxisome
proliferation-only group showed increased pCoA activity (6.5- and 3.4-fold higher than control
males and females, respectively); there were no significant effects at <179 mg/kg-day.
Body-weight gains in the peroxisome proliferation group were decreased to a similar extent as in
the main study (no further details were provided).
A NOAEL and LOAEL of 89.3 and 179 mg/kg-day, respectively, are identified in female
rats based on statistically significantly increased relative stomach weight. Further, increased
relative stomach weights, observed at 357 mg/kg-day (11 and 15% higher than controls in males
and females, respectively), occurred in conjunction with an increase in the incidence of acanthosis
in the forestomach of treated rats (reaching statistical significance in females only).
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Effects in mice related to 2-EH exposure are presented in Table B-4 (Astill et al.. 1996a;
BASF. 1991e). One spontaneous death was reported in a female mouse treated at 179 mg/kg-day
(after 90 days of exposure). The cause of death was determined to be liver damage (after
hemorrhage into an ovarian pouch); death was not attributed to treatment. There were no
consistent, dose-related effects on the incidence of clinical signs, food consumption, body weights,
or hematology and clinical chemistry parameters. The only noteworthy change in organ weights
was statistically significantly increased relative (percentage of body weight) stomach weight in
male mice at 179 and 357 mg/kg-day; the increases were of similar magnitude (13-14% increase) in
both dose groups. Absolute stomach weights were not statistically significantly increased in males,
and there was no effect on absolute or relative stomach weight in females. Gross pathology
findings were limited to the observation of dark red foci in the glandular stomach of 2/10 females
treated at 357 mg/kg-day (not statistically significant). Histopathological examinations showed
evidence for forestomach effects (namely slight focal or multifocal acanthosis) at 357 mg/kg-day in
2/10 males and 1/10 females (compared to 0/10 in controls and all other dose groups). Liver
necrosis, noted in one 179-mg/kg-day female (as focal necrosis) and one 357-mg/kg-day female (as
single cell necrosis), was considered incidental by the study authors. In the peroxisome
proliferation-only group, no significant effects on clinical signs, food consumption, body weights,
or pCoA. activity were observed in male or female mice (BASF. 1991c).
A LOAEL of 179 mg/kg-day with a corresponding NOAEL of 89.3 mg/kg-day is identified
for this study based on increased relative stomach weight in male mice.
Chronic-Duration/Carcinogenicity Studies
Astill et al. (1996b) (publishedreport); BASF (1992a); BASF (1991b) (non-peer-reviewed
studies)
Groups of F344 rats (50/sex/group) were administered 2-EH in 0.005% aqueous
Cremophor EL via gavage at 0 (vehicle-only control), 0 (distilled water control), 50, 150, or
500 mg/kg-day, 5 days/week for 24 months. Doses of 0, 50, 150, and 500 mg/kg-day were adjusted
for continuous exposure to 0, 36, 107, and 357 mg/kg-day by multiplying the administered gavage
dose by (5/7) days per week. Groups of B6C3Fi mice (50/sex/group) were similarly treated at 0
(vehicle-only control), 0 (distilled water control), 50, 200, or 750 mg/kg-day, 5 days/week for
18 months (until Weeks 79-81). Doses of 0, 50, 200, and 750 mg/kg-day were adjusted for
continuous exposure to 0, 36, 143, and 536 mg/kg-day by multiplying the administered gavage dose
by (5/7) days per week. The animals were monitored twice daily (on weekdays) or once daily (on
weekends) for mortality and clinical signs of toxicity. Detailed clinical examinations were
performed weekly. Weekly food consumption was determined every 4 weeks. Body weights were
measured prior to study initiation, weekly for the first 13 weeks, and monthly thereafter.
Hematology parameters (differential RBC and WBC counts, including morphology evaluations)
were evaluated at 12, 18, and 24 months (rats only). All animals (whether sacrificed at study
termination or sacrificed moribund) were subjected to necropsy. Organ weights (of the stomach,
liver, kidneys, spleen, brain, and testes) were recorded for animals sacrificed on schedule.
Complete histopathological examinations (of approximately 40 tissues and including all gross
lesions; using hematoxylin-eosin staining) were performed.
Significant effects in rats related to 2-EH exposure are presented in Tables B-5 and B-6
(males and females, respectively) (Astill et al.. 1996b; BASF. 1992a). Except for body weight and
body-weight gain in males (slightly decreased [by 7%] in water controls relative to vehicle-only
controls), there were no significant differences among the two control groups. The results discussed
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herein focus on differences between the 2-EH-treated rats and the vehicle-only control group;
however, for comparison purposes, data for both control groups are shown in Tables B-5 and B-6.
Mortality was markedly increased in females at 357 mg/kg-day (52% at 357 mg/kg-day compared
to 28% in controls). An increased incidence of some clinical signs of toxicity (specifically, poor
general condition [characterized by lethargy and unkemptness] and/or labored breathing) were
noted in both sexes at 357 mg/kg-day; however, these clinical signs were statistically significantly
increased in female rats only (based on statistical analyses performed for this review;
see Table B-6). No consistent, dose-related effects on food consumption were reported. Body
weights and body-weight gain were statistically significantly decreased at doses as low as
36 mg/kg-day (in males), and were decreased >10% compared to their respective control groups in
males at >107 mg/kg-day and females at >357 mg/kg-day. For the purposes of this assessment, a
>10% decrease in body weight is considered biologically significant. There were no consistent,
treatment-related effects on hematology parameters. At gross necropsy, a significantly increased
incidence of focal lung lesions was observed in both high-dose males and females (see Tables B-5
and B-6).
In general, the absolute weights of most organs were decreased, whereas their relative
(percentage of body weight) weights were significantly increased. Statistically significant
reductions in absolute organ weights were seen for the stomach (males only at >36 mg/kg-day),
liver (-16%), 357-mg/kg-day males only), kidney (-7%, 357-mg/kg-day females only), and brain
(males at >107 mg/kg-day and females at 357 mg/kg-day). The relative weights of these organs
were statistically significantly increased compared to controls as follows: relative stomach weights
were increased in males at >107 mg/kg-day and in all groups of treated females (by 6—21%),
relative liver weights were increased in females at >107 mg/kg-day (by 11—13%; no dose-related
response was observed in males), and relative kidney and brain weights were increased in both
sexes at >107 mg/kg-day (by 7-22 and 9-20%, respectively). Male rats also showed significantly
increased relative (but not absolute) testes weights at 357 mg/kg-day. The toxicological
significance of these organ-weight changes is unclear; the opposing direction of absolute and
relative organ-weight changes suggests a confounding effect of body-weight changes at the same
doses.
With respect to histopathological changes observed, the incidences of bronchopneumonia
and liver and lung congestion were significantly increased at 357 mg/kg-day in both sexes.
However, congestion was diagnosed in decedents only. Furthermore, the study authors indicated
that the diagnosis of congestion was complicated by a high incidence of animals with malignant
lymphomas in the liver and lungs, particularly in the vehicle control group (13 of 17 male decedents
and 8 of 13 female decedents). Other lesions that were increased in 357 mg/kg-day females relative
to vehicle controls were hemosiderin in the spleen, kidney congestion, and hyperplasia of the
mesenteric and mandibular lymph nodes (see Tables B-5 and B-6). There was no evidence of
treatment-related carcinogenicity in male or female rats at up to 357 mg/kg-day (i.e., the incidence
of tumors was comparable among all treatment groups, including controls).
Based on decreased body weight in male rats (>10% compared to water- and
vehicle-controls), NOAEL and LOAEL values of 36 and 107 mg/kg-day (respectively) are
identified. The 357-mg/kg-day dose level is considered a frank effect level (FEL) for female rats
based on mortality, significantly increased incidences of clinical signs (including poor general
condition), and marked reductions in body weight (21%).
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Effects in mice related to 2-EH exposure are presented in Tables B-7 and B-8 (males and
females, respectively) (Astill ct al.. 1996b; BASF. 1991b). Except for a few types of
histopathological lesions (considered to be incidental), there were no significant differences
between the water and vehicle-only control groups; comparisons discussed here are based on
differences between 2-EH-treated mice and vehicle-only controls (data for both control groups are
shown in Tables B-7 and B-8). Mortality was 4-8% in all dose groups (including both control
groups); the 536-mg/kg-day group, however, showed a mortality rate of 30% (p < 0.01 in both
sexes). The increase in mortality was evident within 8 weeks of study initiation in females and
28 weeks in males. No treatment-related clinical signs of toxicity were reported. Food
consumption was significantly decreased at 536 mg/kg-day (by 9% in males and 13% in females
relative to controls) over the course of the study. Statistically significant reductions in body weight
and body-weight gain were seen at >143 mg/kg-day (in males), but this reduction was greater than
>10% compared to controls only at 536 mg/kg-day in both sexes (a >10% decrease in body weight
is considered to be biologically significant by the U.S. EPA for the purposes of this PPRTV
assessment). Significant changes in hematology parameters (increased polymorphonuclear
neutrophils and decreased lymphocyte counts at 12 and/or 18 months) were noted in 536-mg/kg-day
males only (see Tables B-7 and B-8). No significant findings were reported at gross necropsy.
Numerous organ weights were statistically significantly affected at 536 mg/kg-day. At this dose,
relative (percentage of body weight) stomach and liver weights were increased 16—18% and
11-21%) in both sexes, respectively; absolute liver and stomach weights were unaffected. Absolute
brain weights were statistically significantly decreased in both sexes, whereas relative brain weights
were statistically significantly increased. Similarly, absolute kidney weights were decreased
(males, —18%; females, -6%), whereas relative kidney weights were decreased (males, -8%) or
increased (females, 13%). Although relative testes weights were significantly increased (5—13%) in
all groups of treated males, these changes were not dose related, and there was no effect on absolute
testes weights. The observed organ-weight changes (predominantly unchanged or decreased
absolute weights coupled to increased relative organ weights) are consistent with, and likely
secondary to, the observed decreases in body weight. Histopathological examinations revealed
significantly increased incidences of liver and lung congestion in mice treated at 536 mg/kg-day;
however, these effects occurred mostly (with respect to the lung) or entirely (with respect to the
liver) in decedents (i.e., likely post mortem effects). Peripheral fatty infiltration of the liver was
significantly increased in rats of both sexes at 536 mg/kg-day, primarily in survivors. Other lesions
reported in survivors by the study authors were basophilic foci and focal hyperplasia in the liver.
Small (not statistically significant) increases in the incidence of focal hyperplasia of the epithelium
of the forestomach were noted in both males and females at 536 mg/kg-day (see Tables B-7 and
B-8).
A NOAEL of 143 mg/kg-day is identified for this study but a LOAEL cannot be identified
because the next highest dose, 536 mg/kg-day, is considered a FEL in both sexes based on increased
early onset mortality and markedly decreased body weight (30%).
Carcinogenicity data in mice are shown in Table B-9 (Astill et al.. 1996b; BASF. 1991b).
Both male and female mice showed statistically significant trends for increased hepatocellular
carcinoma with dose when tested by the time-dependent Peto test performed by the study authors
that considers the relatively high mortality in these groups (although the only pairwise increase was
in high-dose females). The study authors did not report the results of combined statistical analysis
of hepatocellular adenoma or carcinoma. An adenoma was detected in one 536-mg/kg-day male
(and no females). Statistical analysis of the combined adenoma or carcinoma male data performed
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by the U.S. EPA for this PPRTV assessment did not find a significant pairwise increase in males
treated at 536 mg/kg-day compared to vehicle-only controls. The study authors noted that (1) no
metastases were observed (indicative of a low grade of malignancy) and (2) the observed incidence
of hepatocellular carcinomas was within the historical control range at the testing facility (0-22% in
males, 0-14% in females). The study authors concluded that 2-EH is an equivocal hepatocellular
carcinogen in male mice and is a weak or equivocal hepatocellular carcinogen in female mice.
Reproductive/Developmental Studies
Hellwis and Jdckh (1997) (publishedstudy); Confidential (1991) (non-peer-reviewed)
In a study designed to compare the developmental toxicity of various alcohols, pregnant
Wistar rats (10/group) were administered 2-EH (>99.5% purity) via gavage (in distilled water and
0.005%) Cremophor EL as an emulsifier) at 1, 5, or 10 mmol/kg-day (the study authors calculated
dose equivalents of 130, 650, or 1,300 mg/kg-day) on Gestation Days (GDs) 6-15 and sacrificed on
GD 20. Distilled water and vehicle-only control groups were used. Mortality and clinical signs of
toxicity were monitored at least once daily. Food consumption and body weights were recorded
every 2-3 days (GDs 0, 1, 3, 6, 8, 10, 13, 15, 17, and 20) in pregnant dams only. At sacrifice on
GD 20, maternal animals were subjected to gross pathology examinations, and uterine weights were
recorded (pregnant dams at scheduled sacrifice). The uterus and ovaries were evaluated for
numbers of corpora lutea and implantations (including live fetuses and dead implantations, defined
as the sum of early resorptions, late resorptions, and dead fetuses). Placental weights were
determined. Conception rate and pre- and postimplantation losses were calculated. All fetuses
were sexed, weighed, and examined for external abnormalities. Half of the fetuses were fixed in
Bouin's solution and examined for visceral variations or malformations; the remaining half were
fixed in ethyl alcohol and examined for skeletal retardations, variations, or malformations.
Statistics were performed using both the litter and the fetus as units of analysis. For the purposes of
this PPRTV assessment, fetal incidence data are the preferred unit of analysis over litter incidence
data because the sample numbers are larger for fetuses than litters.
Significant effects are shown in Table B-10 (Hellwig and Jackh, 1997; Confidential 1991).
For the purposes of this review, the effects discussed here are based on comparisons between
treated rats and the vehicle-only control group; however, data for both control groups are shown in
Table B-10. Marked maternal toxicity, as evidenced by a 60%> mortality rate by GD 13, severe
clinical signs of toxicity (including abnormal position, unsteady gait, apathy, nasal discharge,
piloerection, and others), a marked reduction in food consumption, a 15%> reduction in body weight
(by GD 15), body-weight loss during the treatment period, and decrease in maternal net weight
change from GD 6 were observed at 1,300 mg/kg-day. Animals that died showed discoloration of
the liver and/or lung edema at necropsy. Significant developmental effects reported at this dose
included a decreased pregnancy rate at time of cesarean section (40%> compared to 100%> in
controls; associated with maternal mortality), and in surviving animals, seven- to eightfold increases
in percent postimplantation loss and resorptions (mainly early), a 50%> reduction in the percentage
of pregnant dams that produced viable fetuses, and decreased mean fetal body weights. Only two
litters were produced in the 1,300-mg/kg-day group. The fetal incidences of visceral variations
(dilated renal pelvis, hydroureter) and skeletal malformations (absent thoracic vertebrae, and
sternebrae bipartite with ossification centers dislocated) were statistically significantly increased at
1,300 mg/kg-day (see Table B-10). The fetal incidences of skeletal variations (accessory lumbar
vertebrae and 14th ribs, rudimentary cervical ribs, and 13th ribs absent or shortened) and
retardations (thoracic vertebral body/bodies and sternebrae not ossified, sternebrae incompletely
ossified or reduced in size) were significantly increased at >650 mg/kg-day (see Table B-10). Litter
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incidences of all abnormalities in the 1,300-mg/kg-day group were 100%, but not statistically
significant due to the small number available and high control incidences.
Other than one 650 mg/kg-day-animal that died from gavage error (on GD 10), no mortality
was observed at <650 mg/kg-day. Piloerection was noted in two dams treated at 650 mg/kg-day.
Maternal body weight measured on GD 15 or 20, maternal carcass weight (terminal body weight
minus uterine weight), or maternal net weight change from GD 6 (carcass weight - GD 6 body
weight) were not statistically significantly changed at <650 mg/kg-day. The body-weight gains of
rats treated at 130 and 650 mg/kg-day were 15-16% lower than controls during the dosing period
(GDs 6-15); rats treated at 650 mg/kg-day also gained 8% less than controls throughout gestation
(GDs 0-20). However, these body-weight measures do not represent only maternal body weight
but also fetal body weight. In fetuses, decreased fetal body weights were observed at
650 mg/kg-day (10% lower than controls based on the combined sexes; 11% for males and 9% for
females when considered separately). For the purposes of this PPRTV assessment, a >5% decrease
in fetal body weight is considered biologically significant by the U.S. EPA. No effects on
conception rate, numbers of corpora lutea, rates of pre- and postimplantation loss, resorptions, or
numbers of live/dead fetuses were seen at <650 mg/kg-day.
A FEL of 1,300 mg/kg-day is identified for this study based on severe toxicity (mortality,
clinical signs, and body-weight loss) in maternal animals. The next highest dose (650 mg/kg-day) is
a NOAEL for maternal toxicity. Based on the statistically significant increases in fetal incidences
of skeletal variations and retardations and statistically and biologically significantly decreased fetal
body weight, a developmental LOAEL of 650 mg/kg-day is identified for this study with a
corresponding NOAEL of 130 mg/kg-day.
NTP (1991)
Time-mated CD-I Swiss mice (28/group) were treated with microencapsulated 2-EH
(>99% purity) at 0, 0.009, 0.03, or 0.09% in the diet on GDs 0-17. The study authors calculated
equivalent doses that correspond to average intakes of 0, 17, 59, and 191 mg/kg-day. Maternal
animals were monitored daily for mortality and clinical signs of toxicity. Food consumption and
body weights were measured on GDs 0, 3, 6, 9, 12, 15, and 17. At sacrifice on GD 17, body, liver,
and uterine weights were recorded; uterine contents were examined for numbers of implantation
sites, resorptions, and live and dead fetuses. Corpora lutea (ovaries) were counted. Endpoints
evaluated in all fetuses included body weight and sex (determined by a fresh tissue dissection
technique) and presence/absence of external morphological abnormalities and visceral and skeletal
variations and malformations. The heads of half of the fetuses were fixed in Bouin's solution and
examined using a free-hand sectioning technique. The litter was the unit for statistical analyses.
No mortality occurred in maternal animals (NTP. 1991). The only clinical sign of toxicity
reported was hyperactivity, observed in one dam treated at 59 mg/kg-day (GD 6) and one dam
treated at 191 mg/kg-day (GDs 6, 9, and 12). There were no significant effects on food
consumption. Maternal body weight and body-weight gain (including weight gain corrected for
gravid uterine weight) of treated mice were >90% of control values throughout the study; gravid
uterine weights were also comparable among treated mice and controls. No significant effects on
absolute or relative liver weights were observed. The pregnancy rate at sacrifice was 93-96%
across all treatment groups (including controls). Numbers of corpora lutea (per dam) and
resorptions and implantations (per litter) were unaffected by treatment. Furthermore, there were no
significant effects on the numbers of live/dead fetuses, litter size, fetal body weights, sex of fetuses,
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or the incidences of variations or malformations (total, visceral, or skeletal). The highest dose of
191 mg/kg-day is identified as a NOAEL for maternal and developmental toxicity. No LOAEL was
identified.
Hardin et al. (1987) (publishedreport); Hazleton Laboratories (1983) (non-peer-reviewed
report)
In a study employing a short-term in vivo testing methodology to screen various chemicals,
pregnant CD-I mice (50/group) were administered 2-EH at 1,525 mg/kg-day (the approximate
lethal dose of 10% of the population [LDio] based on a preliminary dose range-finding experiment)
via gavage in corn oil on GDs 6-13 and were permitted to deliver their litters. The mice were
monitored at least once daily for mortality and clinical signs of toxicity. Maternal body weights
were recorded (for pregnant females only) on GDs 6 and 17, and Postnatal Day (PND) 3 [according
to Hardin et al. (1987)1 or on GDs 7, 14, and 18, and PND 3 (Hazleton Laboratories. 1983).
Developmental endpoints evaluated included mean numbers of live and dead pups and pup body
weights (on PNDs 1 and 3), and pup viability and pup body-weight gain (from PNDs 1-3), Pups
(whether alive or dead) were not examined for external malformations. Females that failed to
deliver a litter by GD 22 were sacrificed and their uteri examined for gross evidence of failed
pregnancy. If no evidence was found, uteri were stained with sodium sulfide to identify
implantation sites.
Significant effects in mice treated with 2-EH are shown in Table B-1 1 (Hardin et al.. 1987;
Hazleton Laboratories. 1983). In some cases, data in the two reports were similar in magnitude but
different in value (owing, at least in part, to variations in the times at which specific endpoints were
measured); in these cases, data from the more complete report (Hazleton 1 .aboratories. 1983) are
discussed here. Mortality was observed in 35% of treated mice compared to 0% controls (Hardin et
al.. 1987; Hazleton Laboratories. 1983). One treated animal that died (on GD 8) due to a dosing
error was omitted from analyses. All other deaths (one on GD 17; all others on GDs 8-13) were
attributed to treatment (no cause of death was specified). Clinical signs of toxicity (languidness,
ataxia, coldness to touch, wet stains, oily coat, and/or dark red discharge) were noted in treated
mice, predominantly during GDs 7-14 (with some signs being observed in fewer animals on
GDs 15-18). The body weights of 2-EH-treated dams were significantly decreased by 9—15%
relative to controls at all time points except GD 7 (see Table B-l 1). Similarly, maternal
body-weight gains encompassing treatment (GDs 7-14) and pregnancy (GDs 7-18) were markedly
reduced in treated mice (33 and 37% lower than controls, respectively). No data were provided
with respect to gravid uterine weights. However, pregnancy was likewise affected: the number of
pregnant females was lower in treated mice (20 compared to 34 for controls). No data for
nonpregnant females (with respect to implantation sites) were provided. In addition, a smaller
percentage of pregnant mice in the 2-EH group produced a viable litter (defined as a litter with at
least one live pup on PND 1); the rate was 97% in controls compared to 55% in 2-EH-treated mice.
With respect to pups, the number of live pups per litter was significantly decreased (by
35-50%)) and (consequently) the number of dead pups per litter was significantly increased (by
about 15-fold) in 2-EH-treated mice on PNDs 1 and 3 rHardin. et al. (1987); Hazleton I .aboratories
(1983) and Table B-l 1], Pup weights (expressed as mean pup weight/litter) were also significantly
lower for treated mice than controls at these time points (by 13% on PND 1 and 23% on PND 3).
Pup survival and growth from PNDs 1-3 were also affected; viability per litter was reduced from
98%) in controls to 73% in the 2-EH group, and surviving animals gained substantially less weight
than their untreated counterparts (33% less than controls). These data identify a FEL of
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1,525 mg/kg-day (the only dose tested) based on severe maternal toxicity (mortality and decreased
body-weight gain) and developmental toxicity (decreased survival and growth from PNDs 1-3).
Because 1,525 mg/kg-day is the only dose tested, identification of aNOAEL is precluded.
Hitter et al. (1987)
In a study designed to evaluate the teratogenicity of DEHP and its metabolites, pregnant
Wistar rats (7 litters/group) were administered 2-EH (unknown purity) via gavage, undiluted, at 0,
6.25, or 12.5 mmol/kg (0, 1.0, or 2.0 mL/kg) on GD 12. Based on a density of 0.833 g/mL for
2-EH, these doses are equivalent to about 0, 830, and 1,700 mg/kg as calculated by the U.S. EPA
for the purposes of this PPRTV assessment. The rats were sacrificed on GD 20 and cesarean
sections performed. Numbers of implantation sites and resorbed or dead fetuses were counted.
Live fetuses were weighed and examined for external malformations. Half of the fetuses were
evaluated for skeletal anomalies (by staining with alcian blue and alizarin red S); the remaining half
were evaluated for soft tissue anomalies (by staining with Bouin's fluid preparatory). Statistical
analyses were not performed.
Effects in 2-EH treated rats are shown in Table B-12 (Ritter et al., 1987). The number of
implantations (91-113 per dose group, including controls) and the percentage of resorbed/dead
fetuses (about 8.5-10% for each dose group) were not affected by treatment. However, mean fetal
body weights were decreased in treated rats by 5% relative to controls at 830 mg/kg and by 15% at
1,700 mg/kg (no measure of variance for these data was provided). Furthermore, there was a
significant, dose-related increase in the percentage of surviving fetuses with malformations (0% in
controls compared to 2% at 830 mg/kg and 22% at 1,700 mg/kg). The malformations reported in
rats treated with 2-EH at 1,700 mg/kg were hydronephrosis (8%), tail and limb defects (5 and 10%,
respectively), and other (1%). No further information was provided. Owing to study limitations
(limited number of evaluated endpoints and inadequate data reporting), no NOAEL or LOAEL
values are identified.
Li et al. (2000)
Neonatal male CD Sprague-Dawley (S-D) rat pups (3 days old; 4-5/group) were
administered 2-EH as a single dose via gavage at 167 mg/kg and sacrificed 24 hours after dosing.
The right testis of each pup was fixed in 2% glutaraldehyde (in phosphate-buffered saline),
embedded in glycol methacrylate, sectioned, and stained with hematoxylin for morphological
examinations (using bright-field microscopy). The left testis was fixed in 10% buffered formalin,
sectioned, and visualized for 5-bromo-2'-deoxyuridine (BrdU) immunostaining to evaluate the rate
of Sertoli cell proliferation. No morphological changes were detected in the 2-EH-treated rats
compared to controls. The rate of Sertoli cell proliferation was not significantly affected by
treatment. Owing to the limited scope of this study, no NOAEL or LOAEL values can be
identified.
Inhalation Exposures
The database for inhalation exposure in animals is limited to two subchronic-duration
studies, one in rats (Klimisch et al.. 1998; BASF. 1992b) and one in mice (Mivake et al.. 2016). and
one developmental toxicity study in rats (Nelson et at.. 1989). Study deficiencies (limited study
details and poor reporting) were apparent in the Nelson et al. (1989) study.
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Subchronic-Duration Studies
Klimischetal (1998) (published study); BASF (1992b) (non-peer-reviewed study)
Wistar rats (10/sex/group) were exposed to 2-EH as a vapor by whole-body exposure at
mean measured concentrations of 0, 15, 40, or 120 ppm (with the highest concentration
corresponding to vapor saturation), 6 hours/day, 5 days/week for 90 days (65 exposures). These
concentrations are equivalent to 0, 80, 213, and 638 mg/m3. Mortality and clinical signs of toxicity
were monitored daily. Body weights were recorded 1 day prior to study initiation and weekly
thereafter. Hematology (WBC, RBC, platelet, and differential blood cell counts; Hb; Hct; MCV,
MCH, and MCHC; thromboplastin time) and clinical chemistry (activities of ALT, AST, and ALP;
urea, creatinine, total bilirubin, total protein, albumins, and globulins; glucose, cholesterol, and
triglycerides; levels of sodium, potassium, chloride, inorganic phosphate, and calcium) endpoints
were evaluated on study Day 94. Ophthalmological examinations were performed at study
initiation and at study termination. All animals were subjected to necropsy; select organ weights
(lungs, liver, kidneys, adrenal glands, and testes) were recorded. Complete histopathological
examinations (>30 tissues and including gross lesions) were performed in all control and
640-mg/m3 animals using hematoxylin-eosin staining. A subset of these tissues (including all gross
lesions, the nasal cavity [three levels], trachea with larynx and bifurcation, lungs, liver, and
mediastinal lymph node) were evaluated histologically in the 80- and 213-mg/m3 groups. Liver
homogenates were evaluated for cyanide-insensitive pCoA oxidation (as a marker for peroxisome
proliferation).
No significant treatment-related effects were observed on any of the parameters evaluated.
The highest dose of 638 mg/m3 (HEC =114 mg/m3) is identified as a NOAEL. No LOAEL is
identified. In accordance with U.S. EPA (1994) methodology, the concentrations of 0, 80, 213, and
638 mg/m3 were converted to human equivalent concentrations (HECs) of 0, 14, 38, and 114 mg/m3
for extrarespiratory effects from a Category 3 gas.2
Miyake et al. (2016)
Male ICR mice (5-7/group) were exposed to 2-EH by whole-body exposure at target
concentrations of 0, 20, 60, or 150 ppm, 8 hours/day, 5 days/week for 1 or 3 months, or for
8 hours/day, 7 days/week for 1 week. Mean analytical concentrations for 3 months were measured
at 0, 21.9, 65.8, and 153.2 ppm. The study authors reported that the mean concentration of the
20-ppm group differed by less than 5 ppm between the different time periods. These concentrations
are equivalent to 0, 27.7, 83.3, and 193.9 mg/m3. Body weights were recorded weekly throughout
the exposure period. Organ weights including liver were also measured but it is unclear from the
study which other organ weights were examined. For the 1-week exposure groups, mice were
decapitated one day after the last exposure. For the 1- and 3-month exposure groups, mice were
anesthetized the day after the last exposure and transcardially perfused with 4% (w/v)
paraformaldehyde phosphate buffer to facilitate the histopathological examination of the olfactory
bulb. After sacrifice, brain and nasal cavities were removed and examined histologically. The
olfactory epithelium of the nasal cavity from all exposure groups were analyzed via
immunohistochemical staining for the following parameters: CD45, CD3, neutrophil elastase (NE),
olfactory marker protein (OMP), and proliferating cell nuclear antigen (PCNA). The olfactory bulb
in the brain from only the 3-month exposure groups were analyzed via immunohistochemical
2CONC (HEC) = CONC (mg/m3) x (hours exposed ^ 24 hours) x (days exposed ^ 7 days) x blood-air partition
coefficient ratio U.S. EPA (1994). The value for the rat blood-air partition coefficient for 2-EH is unknown, so the
default ratio of 1 was applied.
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staining for the following parameters: OMP, tyrosine hydroxylase (TH), ionized calcium-binding
adapter molecule 1 (Ibal), and doublecortin (Dcx). The glomerular diameter of the olfactory bulb
was also measured.
One mouse from the control group died at the start of Week 5 from unknown causes. No
other mortalities were reported. The study authors stated that body weights were significantly
altered in mice exposed to 83.3 and 193.9 mg/m3 as follows: increased at the end of Weeks 1, 4, and
5 (both concentrations), decreased at the end of Week 3 (83.3 mg/m3 only), decreased at the end of
Week 6 (193.9 mg/m3 only), and increased at the end of Week 2 (27.7 mg/m3 only). However, the
biological significance of these weight changes could not be ascertained because the study authors
did not include sufficient quantitative data (i.e., magnitude of change). No effects on body weight
were reported after Week 7 of the exposure period at any concentration tested. The study authors
reported increased relative liver weight at 193.9 mg/m3 after 3 months of exposure; the biological
significance of this change is also unknown due to the lack of quantitative data reported. No other
changes in other organ weights were observed. The study authors suggested that decreased body
weight and increased relative liver weight could be due to increased lipid metabolism via the
2-EH-induced activation of peroxisome proliferator-activated receptors. After 1 week of exposure,
the study authors qualitatively reported the following morphological changes in the olfactory
epithelium of the nasal cavity: inflammation, degeneration, deciliation, decreased thickness,
reduced number of olfactory cells, infiltration of inflammatory cells in the epithelium and lamina
propria, and indistinct basement membrane. The study authors reported these morphological
alterations to be concentration dependent and statistically significant at 83.3. and 193.9 mg/m3.
Also at 1 week of exposure, the high iron diamine-alcian blue staining of the Bowman's gland was
qualitatively reported to decrease with increased exposure concentrations. This change could have
been due to a decreased number of Bowman's glands or reduced secretion of sulfomucin. No
morphological changes were observed in the 1-month exposure groups; the effects observed at
1 week of exposure had been repaired by regeneration of cell components in the olfactory
epithelium. After 3 months of exposure, morphological changes in the olfactory epithelium were
again statistically significant based on pathology scoring at the two highest concentrations and
consisted of inflammatory cell infiltration and expansion of the Bowman's glands (see Table B-13).
No 2-EH-induced lesions were reported in the respiratory epithelium.
The study authors also evaluated the infiltration of leukocytes in the olfactory epithelium via
immunostaining for CD45, CD3, and NE. The number of CD45-positive cells was qualitatively
reported to be increased at 1 week and 3 months at the two highest concentrations, but the study
authors did not report the statistical significance of this effect. The NE-positive cell count was
qualitatively stated to be significantly increased at >83.3 mg/m3 at 1 week of exposure but not at
3 months. The number of CD3-positive cells was not increased at 1 week but was significantly
increased at 3 months at the two highest concentrations (see Table B-13). The study authors
qualitatively stated that no change in these markers of leukocyte infiltration occurred in the 1-month
exposure groups. The effect of 2-EH exposure on olfactory nerve-related markers (i.e., OMP and
PCNA) via immunostaining was also examined. The expression of OMP was significantly
decreased at all concentrations after 1 week and 3 months (see Table B-13) of exposure; no change
was observed at 1 month. The expression of PCNA was significantly decreased at all
concentrations at 1 week and only at 193.9 mg/m3 at 3 months (see Table B-13). The decreased
expression of OMP and PCNA are indicative of a loss of olfactory neurons. At 1 month of
exposure, PCNA expression was qualitatively reported to be significantly increased at >83.3 mg/m3.
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In the olfactory bulb, the glomerular diameter and the expression of TH, an inhibitory
synapse marker, were both significantly decreased after 3 months of exposure to 2-EH at
193.9 mg/m3 (see Table B-13). The decreased expression of TH is indicative of a reduction in the
number of inhibitory interneurons and altered olfactory function. The expression of OMP was
significantly decreased in the olfactory bulb at >83.3 mg/m3 (see Table B-13). 2-EH exposure at
193.9 mg/m3 significantly increased the expression of Ibal and Dcx, which are both markers of
migrating cells during neuronal regeneration, indicating that inflammation occurred. Based on
these observed changes, the study authors concluded that 3 months of exposure to 2-EH caused
damage to the olfactory bulb, including inflammation and reduction in the number of olfactory
neurons.
A LOAEL of 27.7 mg/m3 (HEC = 4.17 mg/m3) is identified for this study based on the
decreased number of OMP-positive cells in the olfactory epithelium of the nasal cavity. Because
27.7 mg/m3 is the lowest concentration tested, identification of a NOAEL is precluded. In
accordance with U.S. EPA (1994) methodology, the concentrations of 0, 27.7, 83.3, and
193.9 mg/m3 were converted to human equivalent concentrations (HECs) of 0, 4.17, 12.5, and
29.20 mg/m3 for male mice for extrathoracic respiratory effects.3
Chronic-Duration/Carcinogenicity Studies
No studies have been identified.
Reproductive/Developmental Studies
Kelson et al. (1989)
In a study designed to compare the inhalation developmental toxicity of various alcohols,
pregnant female S-D rats (approximately 15/group) were exposed to 2-EH as a vapor at mean
measured concentrations of 0 or 850 mg/m3 (saturation) 7 hours/day on GDs 1-19. Maternal
animals were weighed daily during the first week and weekly thereafter; total food and water
consumption was measured weekly (i.e., GDs 7, 14, and 20). At sacrifice on GD 20, uteri (with
ovaries) were removed; numbers of corpora lutea, implantations, resorption sites, and live fetuses
were recorded. All fetuses were weighed, sexed, and examined for external malformations; half
were evaluated for skeletal malformations (using alizarin red S staining) and the remaining half
were evaluated for visceral abnormalities (using Bouin's solution).
Effects in dams exposed to 2-EH are shown in Table B-14 (Nelson et al.. 1989). Mortality
and clinical signs of toxicity (if they occurred) were not reported. Dams exposed to 2-EH showed
statistically significantly decreased food consumption (9% lower than controls). Although there
were no statistically significant effects on maternal body weights, rats allocated to the 2-EH group
weighed on average 16% more than controls at study initiation; by Day 20, the weights of 2-EH
dams were only 5% higher than controls (the study report does not indicate the methods by which
the animals were allocated to specific exposure groups). During the study (i.e., from GDs 0-20),
2-EH-exposed dams gained 21% less weight than controls. No exposure-related effects on the
numbers of corpora lutea, implantations, resorptions, or live fetuses or sex of fetuses were reported
'HEC calculated by treating 2-ethylhexanol as a Category 1 gas and using the following equation from U.S. EPA (1994)
methodology: HECet = exposure level (mg/m3) x (hours/day exposed ^ 24 hours) x (days/week
exposed ^ 7 days) x RGDRet, where RGDRet for all exposure groups was calculated to be 0.1505 using Equation 4-28
in U.S. EPA (1994) and minute volume (Ve) values of 0.03116 L/minute based on the mean reference body weight of
0.02695 kg for male BAFi and B6C3Fi mice in a subchronic-duration study U.S. EPA (1994) and the following default
values from U.S. EPA (1994): Ve of 13.8 L/ininute for humans and SAet of 33 cm2 for mice and 200 cm2 for humans.
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(data for implantations and total numbers of live fetuses per litter were not shown; no measure of
variance was provided for resorption data). The fetal body weights of males and females were 97
and 95% of controls, respectively (see Table B-14). No malformations were observed. Small (not
statistically significant) numbers of fetuses from 2-EH-exposed dams showed skeletal abnormalities
characterized by reversible delays in ossification of the caudal vertebrae, sternum, metacarpals,
and/or hind paw phalanges (data not presented by the study authors). Although the study report
contained deficiencies (unclear number of animals/group, limited study details, and poor data
reporting), they did not preclude identifying the tested exposure concentration of 850 mg/m3
(HEC = 248 mg/m3) as a NOAEL for developmental effects.4
OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS)
Tests Evaluating Genotoxicity and/or Mutagenicity
A number of genotoxicity tests have been conducted (see Table 4 A); data for several of
these tests were available only from non-peer-reviewed sources. Genotoxicity tests in vitro
(mutation, clastogenicity, deoxyribonucleic acid [DNA] repair, cell transformation, and
unscheduled DNA synthesis) produced predominantly negative results. Reverse mutations were not
induced in Salmonella typhimurium or Escherichia coli strains at concentrations up to
5,000 |ig/plate in multiple studies (Agarwai et al.. 1985; Shimizu et al.. 1985; Zeiger et al.. 1985;
Kirbv et al.. 1983; Litton Bionctics. 1983b; Zeiger et al.. 1982; Tenneco. 1980). Although Seed
(1982) reported positive results in strain TA100 (under unspecified activation conditions), weak
mutagenicity was observed only at concentrations associated with high levels of cytotoxicity (about
1 mM and above). Results were considered equivocal in a DNA repair assay in E. coli since
positive and negative results were obtained depending on the vehicle used (ethanol or
dimethylsulfoxide [DMSO]); the study authors suggested that there was a synergistic effect between
ethanol and 2-EH (Tenneco. 1980). In mammalian cells, studies of mutation (in mouse lymphoma
and Chinese hamster ovary [CHO] cells), chromosomal aberrations (CAs) (in CHO cells), cell
transformation (in mouse BALB/3T3 cells), and unscheduled DNA synthesis (in rat hepatocytes) all
yielded negative results, typically up to concentrations that elicited cytotoxicity (Litton Bionctics.
1987. 1985a. b; Kirbv et al.. 1983; Litton Bionctics. 1983a; Phillips et al.. 1982; Tenneco. 1980).
A limited number of genotoxicity tests in vivo were available; results were mostly negative.
There was no evidence for dominant lethal mutations in mice treated with 2-EH via gavage at up to
1,000 mg/kg-day (SRI International. 1981). CAs were not observed in rats following short-term
oral exposure (Putman et al.. 1983; Tenneco. 1980). However, there was a statistically significant
increase in the incidence of micronuclei (MN) in the bone marrow of male mice dosed twice with
2-EH via intraperitoneal (i.p.) injection (Litton Bionctics. 1982). No increased induction of MN
was observed in similarly treated female mice or in male or female mice treated via a single i.p.
injection. The study authors attributed the significant increase in males to an unusually low
spontaneous incidence of MN in the corresponding control group. Taken together, studies
evaluating genotoxicity/mutagenicity for 2-EH produced mostly negative results.
'In accordance with U.S. EPA (1994) methodology, the NOAEL of 850 mg/m3 was converted to a NOAEL (HEC) of
248 mg/m3 for extrarespiratory effects from a Category 3 gas, based on the following equation for gestational exposure:
CONC (HEC) = CONC (mg/m3) x (hours exposed ^ 24 hours) x blood-air partition coefficient ratio (default = 1) (U.S.
EPA. 1994).
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Supporting Animal Studies
A number of studies have evaluated the acute inhalation and oral toxicity of 2-EH
(see Table 4B):
• No mortality was observed in rats or guinea pigs exposed to a saturated vapor of 2-EH for
8 hours (Bio/dynamics. 1989; Mellon Institute of Industrial Research. 1951. 1940);
• The 4-hour median lethal concentration (LCso) in S-D CD rats exposed to 2-EH as a
vapor/aerosol is <5,000 mg/m3 (Bio/dynamics. 1989);
• Oral median lethal dose (LD50) values in male rats range from 2,830-7,000 mg/kg (Mellon
Institute of Industrial Research. 1962. 1956. 1940); and
• The oral LD50 values in male rabbits and in mixed guinea pigs are -1,470 and 600 mg/kg,
respectively (Mellon Institute of Industrial Research. 1962. 1940).
Numerous short-term-duration (generally 9-12 days in duration) oral toxicity studies have
been conducted using different vehicles (propylene glycol, corn oil, Cremophor EL, and
microencapsulated 2-EH) in F344 rats and B6C3Fi mice (Astill et at.. 1996a; BASF. 199Id. £ g, h,
1, k, 1, m). These studies consistently identified the stomach and liver (also spleen and kidney,
especially in animals that died) as the targets of 2-EH-induced toxicity (see Table 4B). In a
developmental study in F344 rats conducted via the dermal route, there was no evidence of
fetotoxicity or teratogenicity at doses that caused maternal toxicity (i.e., decreased body weight)
(Tvl et at.. 1992; BushvRun. 1989).
Metabolism/Toxicokinetic Studies
Evidence from oral studies conducted in rodents indicates that 2-EH is readily absorbed
from the gastrointestinal (GI) tract (Deisinger et at.. 1994; Eastman Kodak. 1992; Atbro. 1975).
Based on the observation that 2-EH was not detected in the blood of dosed male rats, metabolism
(leading to the formation of 2-ethylhexanoic acid) is considered rapid (Deisinger et at.. 1994;
Eastman Kodak, 1992). Gavage studies in male and female rats (at doses ranging from
50-500 mg/kg; single and/or repeated exposures) showed that, regardless of single or repeated
exposures, about 70-80% of the oxidative and conjugated metabolites of 2-EH were eliminated in
the urine; lesser amounts of oxidative and conjugated metabolites of 2-EH were detected in expired
air (6-14%) and the feces (8—15%). Elimination was rapid, occurring within about 24-28 hours of
dosing (Deisinger et at.. 1994; Eastman Kodak. 1992; Atbro. 1975). The urinary metabolites
identified in these studies (and in an additional rabbit study) were predominantly glucuronides of
oxidized metabolites of 2-EH, including 2-ethyladipic acid, 2-ethylhexanoic acid,
5-hydroxy-2-ethylhexanoic acid, 6-hydroxy-2-ethylhexanoic acid, 2-ethyl-5-ketohexanoic acid,
and/or 2-ethyl-l,6-hexanedioic acid; only small amounts of unchanged 2-EH were detected
(Deisinger et at.. 1994; Eastman Kodak. 1992; Atbro. 1975; Kamit et at.. 1953). Based on
differences in the profiles of metabolites detected in urine at 50 and 500 mg/kg, there was some
evidence of metabolic saturation at the high dose (Deisinger et at., 1994; Eastman Kodak, 1992).
However, there was no evidence of metabolic induction after repeated low-dose exposure
[compared to single low-dose exposures; Deisinger et at. (1994); Eastman Kodak (1992)1.
Dermal studies showed that neat 2-EH (applied at 1,000 mg/kg for 6 hours) is not readily
absorbed through the skin of male rats. Less than 5% of 2-EH was absorbed; the rest was recovered
from the skin surface. As in the oral studies, absorbed 2-EH was mainly excreted via the urine as
glucuronide conjugates of 2-EH metabolites, with lesser amounts being detected in expired air and
the feces (Deisinger et at., 1994; Eastman Kodak, 1992). Based on data from in vitro percutaneous
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absorption studies, the rate of absorption of 2-EH through rat skin is approximately six times higher
than that of the human stratum corneum (Barber et al.. 1992; Eastman Kodak. 1990).
Several studies that evaluated the metabolites of DEHP have shown that 2-EH alters liver
metabolism, as seen by data showing that 2-EH inhibits mouse alcohol dehydrogenase and rat
ketone body production in vitro (Badr et al.. 1990; Agarwai et al.. 1982) and induces mouse
cytosolic epoxide hydrolase in vivo (Hammock and Ota. 1983).
Mode-of-Action/Mechanistic Studies
A number of studies have evaluated the major metabolites of DEHP, namely
mono(2-ethylhexyl) phthalate (MEHP) and 2-EH, to elucidate their roles (if any) in the mode of
action (MO A) for DEHP-induced toxicity (particularly in the liver and the testes). In various
studies, 2-EH was investigated for its ability to alter fatty acid metabolism (Boies and Thurman.
1996. 1994; Cornu et al.. 1992; Moody and Reddv. 1982). intracellular Ca2 levels (Hijioka et al..
1991). cellular respiration (Keller et al.. 1992b; Keller ct al.. 1992a; Keller et al.. 1991; Keller et al..
1990). peroxisome proliferation (Dirven et al.. 1992; Keith et al.. 1992; Pollack et al.. 1989; Keith et
al.. 1988; Hodgson, 1987; Mitchell et al., 1985; Gray et al., 1983; Gray et al .. 1982; Moody and
Reddv. 1978). and cell proliferation in the liver, which precedes tumor formation. Similarly, 2-EH
was tested for its effects on Sertoli cell function to determine whether it is involved in
DEHP-induced testicular toxicity (Pidie et al., 2012; Williams and Foster, 1988; Gray and
Beamand, 1984). In general, these studies found that 2-EH was a weak inducer of liver and
testicular toxicity.
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Table 4A. Summary of 2-Ethylhexanol (CASRN 104-76-7) Genotoxicity
Endpoint
Test System
Dose/
Concentration3
Results
without
Activationb
Results
with
Activationb
Comments
References
Genotoxicity studies in prokaryotic organisms
Mutation
Salmonella typhimurium
strains TA98, TA100,
TA1535, TA1537
3.3-220 ng/plate
Positive and negative controls responded
appropriately. Slight clearing of the
background lawn was noted at 220 |ig/platc.
Zeieer et al.
(1985); Zeieer et
al. (1982)
Mutation
S. typhimurium strains
TA98, TA100, TA1535,
TA1537, TA1538;
Escherichia coli WP2uvrA
1-1,000 ng/plate
Positive and negative controls responded
appropriately.
Shimizu et al.
(1985)
Mutation
S. typhimurium strains
TA98, TA100, TA1535,
TA1538
100-2,000 |ig/platc
These strains (and strains TA1537 and
TA2637) were not mutagenic in spot tests.
Cytotoxicity was noted in all strains and was
enhanced in the presence of activation.
Agarwal et al.
(1985)
Mutation
S. typhimurium strains
TA98, TA100, TA1535,
TA1537, TA1538
10-5,000 |ig/platc
Cytotoxicity and/or a precipitate was noted at
1,000 |ig/platc (TA1535 strain only without
activation) and 5,000 |ig/platc.
Termeco (1980)
Mutation
S. typhimurium strains
TA98, TA100, TA1535,
TA1537, TA1538
0.01-1.0 nL/plate
Positive and negative controls responded
appropriately.
Kirbv et al.
(1983)
Mutation
S. typhimurium strains
TA98, TA100, TA1535,
TA1537, TA1538
0.002-1.8 nL/plate
Study report provided data tables only.
Results interpreted as negative in the absence
of a twofold increase in the number of
revertants.
Litton Bionetics
(1983b)
Mutation
S. typhimurium strain TA100
0,0.5, 1.0, 1.5 mM
(+)
NR
Significant cytotoxicity was associated with a
weak mutagenic response. The report
indicated that activation inhibited the
mutagenic response but does not explicitly
state whether the results were considered
weak positive or negative in the presence of
activation.
Seed (1982)
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Table 4A. Summary of 2-Ethylhexanol (CASRN 104-76-7) Genotoxicity
Endpoint
Test System
Dose/
Concentration3
Results
without
Activationb
Results
with
Activationb
Comments
References
DNA repair
E. coli strains W3110
(pol A+) and p3478 (pol A-)
0, 10, 50, 100, 250,
500 |ig/mL
(in EtOH or DMSO)
±
±
Overall results were considered equivocal;
the results were positive using EtOH as the
test vehicle, and negative using DMSO.
Termeco (1980)
Genotoxicity studies in mammalian cells—in vitro
Mutation
L5178YTK± mouse
lymphoma cells
0.013-1.0 nL/mL
Cytotoxicity was noted at >0.24 |iL/mL:
complete toxicity occurred at 1.0 |iL/mL.
Kirbv et al.
(1983); Termeco
(1980)
Mutation
(HGPRT locus)
CHO cells
0, 20, 50, 100, 200, 250,
300 nL/mL (-S9);
0, 100, 200, 250, 300,
350, 400 nL/mL (+S9)
Increases in mutant frequency at 200 nL/mL
(-S9) and 250 and 400 nL/mL (+S9) were
not considered indicative of mutagenicity
because responses were small, without
dose-response, and not confirmed in
duplicate cultures.
Litton Bionetics
(1985b): Litton
Bionetics (1985a)
CAs
CHO cells
0, 1.5, 1.9,2.2,2.4,
2.8 mM
NA
A slight (not statistically significant)
response was noted at 2.4 mM. No mitoses
occurred at 2.8 mM.
Phillips et al.
(1982)
Cell transformation
Mouse BALB/3T3 cells
0, 96, 144, 180 nL/mL
NA
—
Positive and negative controls responded
appropriately.
Litton Bionetics
(1983a)
Cell transformation
Mouse BALB/3T3 cells
0.188,0.375,0.75,
1.125, 1.5 nL/mL
(Trial 1);
0.011,0.043,0.086,
0.129,0.162 nL/mL
(Trial 2)
NA
No evidence of transforming activity was
observed under open-vessel (Trial 1) or
closed-vessel conditions (Trial 2).
Litton Bionetics
(1987)
Cell transformation
Mouse BALB/3T3 cells
0,0.03,0.1,0.3 nL/mL
—
—
Positive and negative controls responded
appropriately.
Tenneco (1980)
Unscheduled DNA
synthesis
Rat hepatocytes
2.5-1,000 nL/mL
—
NA
Complete toxicity was observed at
>500 nL/mL.
Termeco (1980)
35
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Table 4A. Summary of 2-Ethylhexanol (CASRN 104-76-7) Genotoxicity
Endpoint
Test System
Dose/
Concentration3
Results
without
Activationb
Results
with
Activationb
Comments
References
Genotoxicity studies in mammals—in vivo
Dominant lethal
assay
ICR/SIM mouse
(20 M/group); dosed via
gavage for 5 d; mated with
2 F/wk for 8 wk
0, 250, 500,
1,000 mg/kg-d
No significant effects on fertility indices or
the average number of dead and total
implants per pregnancy were observed.
SRI International
(1981)
CAs (oral)
F344 rat (5 M/group); dosed
via gavage for 5 d; sacrifice
6 hr after the last dose
0, 0.02, 0.07,
0.21 mL/kg-d
Positive and negative controls responded
appropriately.
Putman et al.
(1983); Tenneco
(1980)
MN assay (i.p.)
B6C3Fi mouse
(4/sex/group); dosed via i.p.
injection once or multiple
times (two doses 24 hr
apart); sacrifice 24-30 hr
after the last dose
0, 456 mg/kg-d
(+)
(+)
An increased incidence of MN was observed
in male mice dosed multiple times only; the
positive response was considered an artifact
of an unusually low incidence in the
corresponding control group. Clinical signs
(shallow breathing, hunched backs, eye
irritation) were noted following dosing; all
animals recovered within 24 hr.
Litton Bionetics
(1982)
aHighest dose tested for negative results.
b(+) = weak positive; + = positive; - = negative; ± = equivocal; NA = not applicable; NR = not reported.
CA = chromosomal aberration; CHO = Chinese hamster ovary; DMSO = dimethylsulfoxide; DNA = deoxyribonucleic acid; EtOH = ethyl alcohol; F = female(s);
i.p. = intraperitoneal; M = male(s); MN = micronuclei; NA = not applicable; NR = not reported.
36
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Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Supporting evidence—noncancer effects in animals
Acute toxicity
(oral)
M albino rat (4-12/group, strain not
specified) were administered 5,000, 6,000,
8,000, 10,000, 12,000, 15,000, or
18,000 mg/kg via gavage and observed for
14 d.
Mortality was 0, 45, 70, 70, 83, 75, and 100%,
respectively. Death was preceded by narcosis and
hypothermia. Congestion of the liver and spleen and
pale kidneys were noted at necropsy.
LD5o = 7,000 mg/kg
Mellon Institute
of Industrial
Research (1940)
Acute toxicity
(oral)
M rat (strain, number, doses administered
not reported) dosed via gavage.
No supporting study information.
LD50 = 2,830 mg/kg
Mellon Institute
of Industrial
Research (1956)
Acute toxicity
(oral)
M rat (strain, number, doses administered
not reported) dosed via gavage.
No supporting study information.
LD50 = 4.46 mL/kg
(-3,720 mg/kg)
Mellon Institute
of Industrial
Research (1962)
Acute toxicity
(oral)
M rabbit (strain, number, doses administered
not reported) dosed via gavage.
No supporting study information.
LD50 = 1.77 mL/kg
(-1,470 mg/kg)
Mellon Institute
of Industrial
Research (1962)
Acute toxicity
(oral)
Mixed guinea pig (5-10/group, sex not
specified) were administered 500, 630, 795,
or 1,260 mg/kg via gavage observed for
14 d.
Mortality was 40, 25, 30, and 100%, respectively. Death
was preceded by narcosis and hypothermia. Congestion
of the liver and spleen and pale kidneys were noted at
necropsy.
LD50 = 600 mg/kg
Mellon Institute
of Industrial
Research (1940)
Short-term
toxicity (oral)
F344 rat (5/sex/group) were administered 0,
100, 320, or 950 mg/kg-d via gavage (in
corn oil) for 21 d. Endpoints evaluated
included food consumption and body
weights, serum cholesterol and triglycerides,
and selected organ weights (liver, kidneys,
and testes). Samples of the liver were
viewed using electron microscopy to
evaluate peroxisomes, for histological
examinations of neutral fat, and for
biochemical evaluations (determinations of
cyanide-insensitive pCoA oxidation,
microsomal lauric acid 11- and
12-hydroxylation, and total microsomal
protein levels).
Decreased body-weight gain was noted in 950-mg/kg-d
females. Triglycerides were significantly increased at
950 mg/kg-d (M only).
At necropsy, absolute and relative liver weights were
increased at 950 mg/kg-d (M) and >320 mg/kg-d (F).
Relative kidney weights were significantly increased at
950 mg/kg-d (both sexes). Also at 950 mg/kg-d,
increased lauric 11- and 12-hydroxylase activities (both
sexes) and cyanide-insensitive pCoA oxidation (F only)
were observed. Cyanide-insensitive pCoA oxidation
was increased at >320 mg/kg-d (M). A dose-related
increase in neutral lipids was noted in all treated animals.
Hepatic peroxisomes were increased at 950 mg/kg-d
(both sexes).
A NOAEL of 320 mg/kg-d
and a LOAEL of 950 mg/kg-d
are identified based on
statistically and biologically
significantly increased
absolute and relative liver
weights in male and female
rats and biologically
significantly increased
relative kidney weight in
female rats.
BIBRA (1987)
37
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Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Short-term
toxicity (oral)
F344 rat (10/sex/group) were administered
0, 0.1, 0.33, 1.0, or 1.5 mL/kg-d (0, 83, 275,
834, or 1,250 mg/kg-d) via gavage (in water)
for 5 d followed by 2 d without treatment,
and 4 additional days of treatment (a total of
9 d of treatment in 12 d). Endpoints
evaluated included mortality and clinical
signs of toxicity, food and water
consumption, body weights, hematology and
clinical chemistry parameters, organ weights
(brain, liver, kidneys, lungs, stomach,
spleen, adrenals, and testes), and gross and
microscopic pathology.
One high-dose male died. Clinical signs (hypoactivity,
ataxia, prostration, delayed righting reflex, muscle
twitch, lacrimation and/or urine stains) were noted at
>834 mg/kg-d; signs (unkempt appearance and
urine-stained fur) were irreversible at 1,250 mg/kg-d.
Food consumption was significantly decreased at
>275 mg/kg-d (M) (not strictly dose related) and at
>834 mg/kg-d (F). Although body weights were
decreased at the same doses, they remained within 10%
of controls except in the 1,250-mg/kg-d male (decreased
by 17% on study D 11). Males treated at 1,250 mg/kg-d
and females treated at >834 mg/kg-d showed
significantly decreased numbers of total leukocytes and
lymphocytes (20-45% lower than their respective
control groups).
At necropsy, increased absolute and relative liver and
stomach weights (in both sexes at >834 mg/kg-d) and
decreased absolute and/or relative spleen weights (at
1,250 mg/kg-d [M] and at >834 mg/kg-d [F]) were
observed. Statistically significant and/or dose-related
increases in the incidence or severity of microscopic
effects were observed at >275 mg/kg-d (both sexes),
including lesions of the forestomach (hyperkeratosis,
mucosal hyperplasia, edema, gastritis, and exocytosis),
thymus (lymphoid cell degeneration), and spleen
(lymphoid cell degeneration and decreased amount of
extramedullary hematopoiesis).
A NOAEL of 83 mg/kg-d and
a LOAEL of 275 mg/kg-d are
identified for this study based
on a significantly increased
incidence of stomach lesions
in male and female rats.
BushvRun
(1988)
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Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Short-term
toxicity (oral)
In the study described above, F344 rat
(10/sex/group) were administered nominal
concentrations of 0, 350, and 700 ppm
(maximum attainable concentration) in
drinking water for 9 d. The same endpoints
were evaluated. Calculated doses on D 4
and 9 were 68.0 and 54.2 mg/kg-d for
350-ppmM (mean= 61.1 mg/kg-d), 159.1
and 143.3 mg/kg-d for 700-ppm M
(mean= 151.2 mg/kg-d), 80.8 and
65.9 mg/kg-d (mean = 73.4 mg/kg-d) for
350-ppmF, and 181.3 and 166.0 mg/kg-d
(mean = 173.7 mg/kg-d) for 700-ppm F.
No significant, treatment-related effects were observed.
A NOAEL of 750 ppm
(-174 mg/kg-d) is identified
for this study based on the
lack of significant,
treatment-related effects.
BushvRun
(1988)
39
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Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Short-term
toxicity (oral)
F344 rat (10/sex/group) were administered
0, 100, 330, 1,000, or 1,500 mg/kg-dvia
gavage (in propylene glycol) 9 times in 11 d.
Endpoints evaluated included mortality and
clinical signs of toxicity, food and water
consumption, body weights, hematology and
clinical chemistry parameters, organ weights
(brain, liver, kidneys, lungs, stomach,
spleen, adrenals, and testes), and gross (but
not microscopic) pathology.
Mortality occurred at 1,500 mg/kg-d only (all F and
6/10 M). Clinical signs (lethargy, ataxia, piloerection,
uncoordinated movements of the fore- and hindlimbs,
abdominal or lateral position, loss of consciousness,
urine-stained fur, hypothermia, and/or salivation) were
noted at >330 mg/kg-d. Food consumption and body
weights were decreased significantly at >1,000 mg/kg-d.
Females exhibited decreased ALT at >100 mg/kg-d;
hematological changes (decreased Hb, Hct, mean cell
volume, and numbers of neutrophilic polymorphonuclear
granulocytes) were observed at 330 mg/kg-d. Changes
in clinical pathology (those mentioned above and
including decreased numbers of leukocytes,
lymphocytes, and monocytes) were also observed in both
sexes at 1,000 mg/kg-d and in surviving males at
1,500 mg/kg-d.
At necropsy, low-dose M showed decreased relative (but
not absolute) liver weights. At all other doses
(>330 mg/kg-d), absolute and relative stomach weights
were increased. Absolute and relative spleen weights
were decreased at >1,000 mg/kg-d. Relative kidney
weight was increased and relative testes weight was
decreased in surviving 1,500 mg/kg-d M. Foci were
observed in the forestomach (but not glandular stomach)
of rats treated at >330 mg/kg-d (both sexes).
A FEL of 1,500 mg/kg-d is
identified based on high
mortality at this dose. A
NOAEL of 100 mg/kg-d and
a LOAEL of 330 mg/kg-d are
identified based on changes in
clinical pathology and
increased absolute/relative
stomach weights
accompanied by evidence of
gross pathology (i.e., foci) in
both sexes.
BASF (19911)
40
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Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Short-term
toxicity (oral)
F344 rat (10/sex/group) were administered
0, 100, 330, 1,000, or 1,500 mg/kg-dvia
gavage (in corn oil) 9 times in 11 d.
Endpoints evaluated were the same as the
gavage study described above.
One 1,500-mg/kg-d female and one 1,000-mg/kg-d male
died. Clinical signs (piloerection, ataxia, urine stains,
lethargy, and/or salivation) were seen in a few rats of
each sex at 330 mg/kg-d, and in most/all animals at
>1,000 mg/kg-d. Food consumption and body weights
were significantly decreased at >1,000 mg/kg-d; the
body weights of treated rats were <90% of controls at
1,500 mg/kg-d. Changes in hematology (decreased
MCV and/or MCH, lymphocytes, leukocytes,
monocytes, and reticulocytes) and/or clinical chemistry
(decreased ALT activity, cholesterol, and glucose;
increased total protein) were observed only at 1,000 and
1,500 mg/kg-d.
At necropsy, only relative testes weight was affected
(increased) at 330 mg/kg-d. At 1,000 and
1,500 mg/kg-d, increased absolute and/or relative
stomach and liver weights and decreased absolute and/or
relative spleen weights were observed. Forestomach
changes were noted at doses as low as 330 mg/kg-d
(thickening of the wall in 3 M and foci in 1 M); the
incidence of these effects increased in a dose-related
manner (in both sexes) at 1,000 and 1,500 mg/kg-d.
A NOAEL of 330 mg/kg-d
and a LOAEL of
1,000 mg/kg-d are identified
based on clinical signs of
toxicity, decreased body
weight, changes in clinical
pathology, organ-weight
changes (increased liver and
stomach weights and
decreased spleen weights),
and an increased incidence of
forestomach effects in both
sexes.
BASF (199 Ik)
41
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Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Short-term
toxicity (oral)
F344 rat (10/sex/group) were administered
0, 100, 330, 1,000, or 1,500 mg/kg-dvia
aqueous gavage (in Cremophor EL) 9 times
in 11 d. Endpoints evaluated were the same
as the gavage studies described above,
except that microscopic pathology was
performed.
No mortality was reported. Clinical signs (including
ataxia, lethargy, abdominal or lateral position,
piloerection, apathy, and/or urine stains) were seen in
several animals treated at 1,000 mg/kg-d and all animals
treated at 1,500 mg/kg-d. Food consumption was
significantly decreased in both sexes at 1,000 mg/kg-d.
Although body weights were significantly reduced at
1,000 mg/kg-d (F), the body weights of treated animals
remained within 10% of controls except at
1,500 mg/kg-d (decreased 17% in M and 13% in F by
D 10). Changes in hematology (decreased reticulocytes)
and/or clinical chemistry (decreased cholesterol and
glucose, increased ALT activity) were observed only at
1,000 and 1,500 mg/kg-d.
At necropsy, only relative kidney weight in females was
alfected (increased) at 330 mg/kg-d. At 1,000 and
1,500 mg/kg-d, increased absolute and/or relative
stomach and liver weights and decreased absolute and/or
relative spleen weights were observed. Some additional
relative (but not absolute) organ-weight changes were
also noted (increased relative kidney weight in both
sexes, increased relative adrenal, lung, or brain weights
in one sex). Gross pathology examinations revealed the
presence of foci in the forestomach of a few rats treated
at 1,000 mg/kg-d and in many rats (4 M and 7 F) treated
at 1,000 mg/kg-d. Increased incidences of
histopathological lesions occurred at 1,000 and
1,500 mg/kg-d, and included effects on the forestomach
(hyperkeratosis, focal or multifocal acanthosis, and/or
inflammatory edema), spleen (parenchymal involution of
lymphoreticular tissue), thymus (decreased size and/or
lymphocyte depletion), and/or liver (hypertrophy of
hepatocytes). Inflammatory edema of the forestomach
(IF) and decreased thymus size (2 M and 1 F) were
observed at 330 mg/kg-d.
A NOAEL of 330 mg/kg-d
and a LOAEL of
1,000 mg/kg-d are identified
based on clinical signs of
toxicity, decreased food
consumption, changes in
clinical pathology parameters,
organ-weight effects
(increased stomach and liver
weights and decreased spleen
weights), and
histopathological findings
(specifically of the
forestomach) in both sexes.
Astill et al.
(1996a): BASF
(1991i)
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Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Short-term
toxicity (oral)
F344 rat (10/sex/group) were administered
microencapsulated 2-EH in the diet at 0,
0.46, 0.92, 1.38, or 2.57% (w/w) (about 0,
500, 980, 1,430, or 2,590 mg/kg-d for M and
0, 540, 1,060, 1,580, or 2,820 mg/kg-d for
F) for lid. Endpoints evaluated were the
same as the gavage studies described above,
except that microscopic pathology was
performed.
No mortality occurred, and no clinical signs of toxicity
were reported. Food consumption was significantly
decreased on D 4 and/or D 10 at >500 mg/kg-d (M) and
>1,060 mg/kg-d (F). Although body weights were
significantly decreased at doses as low as 1,430 mg/kg-d
(M), they remained within 10% of controls except at
2,590-mg/kg-d (M) and 2,820-mg/kg-d (F) (decreased by
23 and 18%, respectively, on D 10). Changes in clinical
pathology seen at >500 mg/kg-d (M) included decreased
cholesterol and triglyceride levels and decreased ALT
activity; females treated at >540 mg/kg-d also showed
decreased cholesterol levels. Additional effects at higher
doses included increased total protein (at >980 mg/kg-d
[M] and at 2,820 mg/kg-d [F]), decreased platelets (at
2,590 mg/kg-d [M] and at >1,580 mg/kg-d [F]), and
decreased glucose, reticulocytes, MCV, and/or MCH and
increased RBCs (at 2,590 mg/kg-d [M] and/or at
2,820 mg/kg-d [F]).
At necropsy, relative (but not absolute) stomach weights
were increased in 540-mg/kg-d females only. Increased
absolute and/or relative stomach and liver weights were
consistently seen at >980 mg/kg-d (M) and
1,060 mg/kg-d (F). Histopathological examinations
revealed liver effects (hypertrophy of hepatocytes) and
forestomach effects (focal or multifocal acanthosis);
these lesions were observed in "most" animals at
1,430/1,580 and 2,590/2,820 mg/kg-d, respectively.
NOAEL values of
500 mg/kg-d (M) and
540 mg/kg-d (F) and LOAEL
values of 980 mg/kg-d (M)
and 1,060 mg/kg-d (F) are
identified based on increased
stomach and liver weights in
both sexes.
BASF (T991m)
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Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Short-term
toxicity (oral)
B6C3Fi mouse (10/sex/group) were
administered 0, 100, 330, 1,000, or
1,500 mg/kg-d via gavage (in propylene
glycol) 9 times in 11 d. Endpoints evaluated
included mortality and clinical signs of
toxicity, food and water consumption, body
weights, hematology and clinical chemistry
parameters, organ weights (brain, liver,
kidneys, lungs, stomach, spleen, adrenals,
and testes), and gross (but not microscopic)
pathology.
Mortality occurred at 1,000 mg/kg-d (1 M and 1 F) and
1,500 mg/kg-d (4 M and 6 F). Clinical signs (including
ataxia, lethargy, piloerection, dyspnea, hypothermia,
abdominal or lateral position, and/or loss of
consciousness) were noted at >1,000 mg/kg-d. Food
consumption was significantly decreased in
1,500-mg/kg-d male only (on D 4).
At necropsy, increased absolute and/or relative stomach
and liver weights were observed in both sexes at
>330 mg/kg-d. Changes in absolute or relative spleen
weight occurred at >1,000 mg/kg-d, but only in one sex.
Decreased absolute/relative testes weights were seen at
1,500 mg/kg-d. Foci were observed in the forestomach
of mice treated at >330 mg/kg-d.
A FEL of 1,000 mg/kg-d is
identified owing to mortality.
A NOAEL of 100 mg/kg-d
and a LOAEL of 330 mg/kg-d
are identified based on
organ-weight changes and
macroscopic forestomach
effects, which were
significant (M) at
330 mg/kg-d (with a
dose-related trend [F]).
BASF fl991d)
Short-term
toxicity (oral)
B6C3Fi mouse (10/sex/group) were
administered 0, 100, 330, 1,000, or
1,500 mg/kg-d via gavage (in corn oil)
9 times in 11 d. Endpoints evaluated were
the same as the gavage study described
above.
One male treated at 1,500 mg/kg-d died. Clinical signs
of toxicity (including ataxia, lethargy, piloerection,
abdominal position, and/or unconsciousness) were noted
at 1,500 mg/kg-d only.
At necropsy, absolute and relative testes weights were
decreased at >1,000 mg/kg-d. At 1,500 mg/kg-d,
additional organ-weight changes were observed
(increased absolute and relative stomach weights in both
sexes; increased relative liver and kidney weights [F
only]). Gross pathology examinations showed the
presence of foci in the forestomach of 7 M and 2 F mice
treated at 1,500 mg/kg-d.
A NOAEL of 330 mg/kg-d
and a LOAEL of
1,000 mg/kg-d are identified
based on decreased testes
weight in male mice.
BASF fl99le)
44
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Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Short-term
toxicity (oral)
B6C3Fi mouse (10/sex/group) were
administered 0, 100, 330, 1,000, or
1,500 mg/kg-d via aqueous gavage (in
Cremophor EL) 9 times in 11 d. Endpoints
evaluated were the same as the gavage
studies described above, except that
microscopic pathology was performed.
Mortality occurred at 1,000 mg/kg-d (IF) and
1,500 mg/kg-d (1 M and 4 F). Clinical signs (including
ataxia, piloerection, abdominal or lateral position, loss of
consciousness, and/or lethargy) were observed at 330
and 1,000 mg/kg-d (in 1 M and 1 F, respectively); effects
were pronounced at 1,500 mg/kg-d.
At necropsy, increased absolute and/or relative stomach
weights were observed at >1,000 mg/kg-d. Gross
pathology examinations revealed forestomach foci in
mice treated at 1,000 mg/kg-d (3 M and 2 F) and
1,500 mg/kg-d (7 M and 5 F). Microscopic forestomach
effects (acanthosis, hyperkeratosis, ulceration, and/or
inflammatory edema) were noted at >330 mg/kg-d. At
1,000 and/or 1,500 mg/kg-d, changes in liver
(hypertrophy of hepatocytes) and testes (tubular giant
cells) pathology were reported. Mice that died showed
histopathological changed in the kidney (tubular dilation
and nephrosis of renal cortex) and liver (centrilobular
fatty infiltration).
A FEL of 1,000 mg/kg-d is
identified based on mortality
(F). NOAEL and LOAEL
values of 330 and
1,000 mg/kg-d are identified
based on increased absolute
and/or relative stomach
weights accompanied by
histopathological changes.
Astill et al.
fl996a): BASF
(199 If)
Short-term
toxicity (oral)
B6C3Fi mouse (10/sex/group) were
administered microencapsulated 2-EH in the
diet at 0, 0.22, 0.44, 0.66, or 1.32% (w/w)
(about 0, 550, 1,150, 1,800, or
4,450 mg/kg-d [M] and 0, 750, 1,750, 2,650,
or 5,750 mg/kg-d [F]) for lid. Endpoints
evaluated were the same as the gavage
studies described above, except that
microscopic pathology was performed.
The significant, treatment-related effect reported was
decreased body weight in males treated at
>1,800 mg/kg-d and in females treated at 5,750 mg/kg-d.
The body weights of treated mice stayed within 90% of
controls throughout the study.
Since body weights remained
within 90% of controls
throughout the study, a
NO A F.I, of 5,750 mg/kg-d (F)
is identified.
BASF (199111)
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Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Acute toxicity
(inhalation)
Rat and guinea pig (6/group, strain and sex
not specified) were exposed to a saturated
mist for 8 hr and observed for 14 d. Gross
and microscopic examinations were
performed.
No mortality occurred. Irritation of the eyes and nose
was noted. No abnormalities were seen in rats. Guinea
pigs showed slight lung congestion and light, cloudy
swelling of the kidney (one animal).
No mortality was observed in
rats and guinea pigs exposed
to a saturated vapor.
Mellon Institute
of Industrial
Research (1940)
Acute toxicity
(inhalation)
Rat (6/group, strain and sex not specified)
were exposed to saturated vapor exposure
for 8 hr.
No supporting study information.
No mortality observed in rats
exposed to a saturated vapor.
Mellon Institute
of Industrial
Research (1951)
Acute toxicity
(inhalation)
S-D CD rat (3/sex/group) were exposed
whole-body as a vapor/aerosol at
5,000 mg/m3 or as a saturated vapor
(890 mg/m3) for 4 hr and observed for 7 d.
Necropsies were not performed.
Mortality was 0/6 and 6/6 in the 890- and 5,000-mg/m3
groups, respectively. Clinical signs (including labored
breathing, nasal discharge, prostration, closed eyes,
chromodacryorrhea) were noted in the 5,000-mg/m3
group.
LC50 = >890 mg/m3 (vapor);
<5,000 mg/m3 (vapor/aerosol)
Bio dynamics
(1989)
46
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Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Other
routes-develop
mental toxicity
F344 rat (8 or 25/group) were administered
0, 0.5, 1.0, 2.0, or 3.0 mL/kg-d (equivalent
to 0, 420, 840, 1,680, or 2,520 mg/kg-d) in a
range-finding study or 0, 0.3, 1.0, or
3.0 mL/kg-d (equivalent to 0, 252, 840, or
2,520 mg/kg-d) in a main study via the
dermal route under occluded conditions for
6 hr/d on GDs 6-15 and sacrificed on
GD21. Maternal animals were monitored
daily for clinical signs and skin irritation.
Maternal body weights were recorded on
GDs 0, 6, 9, 12, 15, and 21; food
consumption was measured for 3-d intervals
through GD 21. Maternal uterine and liver
weights were recorded; spleen, adrenals,
kidneys, and thymus weights were weighed
in the main study only. Numbers of corpora
lutea were counted; the ovaries, cervices,
vaginas, and abdominal and thoracic cavities
were examined grossly. The uteri were
examined for numbers of live and dead
fetuses; nonpregnant F were examined for
resorption sites. Fetuses were weighed,
sexed, and examined for external
malformations and variations. In the main
study, about half of the fetuses from each
litter were examined for visceral and
craniofacial abnormalities; the other half
were examined for skeletal malformations
and variations.
No mortality occurred. Clinical signs included nasal and
ocular effects, as well as a high incidence of exfoliation
and encrustation (all treatment levels). Skin irritation
(erythema, with no edema) was observed at
>840 mg/kg-d. Statistically significant effects on
maternal body-weight gains were occasionally seen;
body-weight gain during the treatment period
(GDs 6-15) was decreased at 1,680 and 2,520 mg/kg-d
in the range-finding study only (decreased 47 and 43%
relative to sham controls, respectively). There was no
effect on body-weight gain on GDs 0-21 in either study.
At necropsy, the only finding was residual exfoliation
and crusting at the application site at the mid- and
high-treatment levels (doses not further specified).
No significant, treatment-related effects on gestational
endpoints (total and nonviable implants, early or late
resorptions, live or dead fetuses, fetal sex ratio, or fetal
body weights) were observed. There were no
significantly increased incidences of external, visceral,
or skeletal malformations or variations in treated rats
compared to controls.
A NOAEL of 252 mg/kg-d
and a LOAEL of 840 mg/kg-d
is identified for skin irritation
(erythema). The NOAEL for
developmental toxicity is
2,520 mg/kg-d. 2-EH did not
induce developmental toxicity
at doses that caused maternal
toxicity.
Tvl et al.
(1992);
BushvRun
(1989)
ALT = alanine aminotransferase; 2-EH = 2-ethylhexanol; F = female(s); FEL = frank effect level; GD = gestation day; Hb = hemoglobin; Hct = hematocrit;
LC50 = median lethal concentration; LD50 = median lethal dose; LOAEL = lowest-observed-adverse-effect level; LOEL = lowest-observed-effect level; M = male(s);
MCH = mean corpuscular hemoglobin; MCV = mean corpuscular volume; NOAEL = no-observed-adverse-effect level; NOEL = no-observed-effect level;
pCoA = palmitoyl-Coenzyme A; RBC = red blood cell; S-D = Sprague-Dawley.
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DERIVATION OF PROVISIONAL VALUES
Tables 5 and 6 present summaries of noncancer and cancer references values,
respectively.
Table 5. Summary of Noncancer Reference Values for 2-Ethylhexanol (CASRN 104-76-7)
Toxicity Type
(units)
Species/
Sex
Critical
Effect
p-Reference
Value
POD
Method
POD
(HED/HEC)
UFc
Principal
Study
Subchronic
p-RfD (mg/kg-d)
Rat/
MandF
Increased fetal
skeletal variations
7 x 10-2
BMDL
7.37
100
Hellwig and
Jackh (1997):
Confidential
(1991)
Chronic p-RfD
(mg/kg-d)
Rat/
MandF
Increased fetal
skeletal variations
7 x 10-2
BMDL
7.37
100
Hellwig and
Jackh (1997):
Confidential
(1991)
Subchronic
p-RfC (mg/m3)
Mouse/
M
Increased diameter
of Bowman's
glands in the
olfactory
epithelium of the
nasal cavity
4 x icr3
BMCL
1.11
300
Mivake et al.
(2016)
Chronic p-RfC
(mg/m3)
Mouse/
M
Increased diameter
of Bowman's
glands in the
olfactory
epithelium of the
nasal cavity
4 x 1(T4
BMCL
1.11
3,000
Mivake et al.
(2016)
BMCL = benchmark concentration lower confidence limit; BMDL = benchmark dose lower confidence limit;
F = female(s); HEC = human equivalent concentration; HED = human equivalent dose; M = male(s);
p-RfC = provisional reference concentration; p-RfD = provisional reference dose; POD = point of departure;
UFC = composite uncertainty factor.
Table 6. Summary of Cancer Reference Values for 2-Ethylhexanol (CASRN 104-76-7)
Toxicity Type (units)
Species/Sex
Tumor Type
Cancer Value
Principal Study
p-OSF (mg/kg-d) 1
(adjusted)
Mouse/M
Hepatocellular (carcinoma
or adenoma)
9.5 x 10-3
Astill et al. (1996b):
BASF (1991b).
p-IUR (mg/m3)-1
NDr
M = male(s); NDr = not determined; p-IUR = provisional inhalation unit risk; p-OSF = provisional oral slope
factor.
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DERIVATION OF ORAL REFERENCE DOSES
Studies that are potentially relevant to the derivation of provisional reference dose
(p-RfD) values include two subchronic-duration studies [one gavage study in rats and mice
(Astill ct al.. 1996a; BASF. 1991a. c, e) and one non-peer-reviewed dietary study in rats (Mellon
Institute of Industrial Research, 1960)1. There is also one chronic-duration gavage study in rats
and mice (Astill et al.. 1996b; BASF. 1992a. 1991b). one limited neonatal exposure study in rats
(Li et al.. 2000). and several developmental studies of gestational exposure in rats and mice
(Hellwig and Jackh. 1997; Confidential. 1991; NTP. 1991; Hardin et al.. 1987; Rittcr ct al.. 1987;
Hazleton Laboratories. 1983). Subchronic and chronic p-RfDs are derived based on the
available studies.
Derivation of a Subchronic Provisional Reference Dose
The laboratory animal oral toxicity database as a whole identifies the liver, kidney,
stomach, and fetus as sensitive noncancer toxicity targets of repeated exposure to 2-EH. The
developmental study in rats administered 2-EH via gavage on GDs 6-15 is selected as the
principal study for deriving the subchronic p-RfD (Hellwig and Jackh. 1997; Confidential.
1991). with rationale provided below. Increased fetal incidence of skeletal variations is
identified as the critical effect. The study was reported in both a peer-reviewed publication and
technical report and was conducted according to Good Laboratory Practice (GLP) standards with
an adequate number of dose groups, sufficient group sizes and litters, and quantitation of results
to describe dose-response relationships for the critical effects. Details of the study are provided
in the "Review of Potentially Relevant Data" section.
With respect to 2-EH-induced liver toxicity, increased relative liver weight (as much as
29% higher than controls) was observed in male and female rats treated at an adjusted daily dose
(ADD) of 357 mg/kg-day in a subchronic-duration study in rats (Astill et al.. 1996a; BASF.
1991a). In rats administered 2-EH in the diet at 840 mg/kg-day (males) or 940 mg/kg-day
(females) for up to 90 days, relative liver weight was also increased and was accompanied by
cloudy swelling (diffuse) of hepatocytes (statistically significant in females only) (Mellon
Institute of Industrial Research. 1960). Numerous short-term-duration oral toxicity studies in
rats and mice administered 2-EH in the diet or via gavage for 9-21 days also identified the liver
as a target of 2-EH-induced toxicity. In these studies, increased liver weights were observed.
Histopathological examinations (when performed) frequently showed liver changes, including
hypertrophy of hepatocytes and/or evidence of peroxisome proliferation (Astill et al .. 1996a;
BASF. 199Id. f, g, h, i, k, 1, m; BIBRA, 1987).
Increased relative kidney weights were observed in male rats treated at an ADD of
357 mg/kg-day in the Astill et al. (1996a) and BASF (1991b) study. Female rats treated with
2-EH in the diet for 90 days at 940 mg/kg-day showed tubular cloudy swelling (diffuse) of the
kidneys. Kidney effects (increased weight; histopathological effects in animals that died only)
were also frequently identified in short-term-duration toxicity studies (Astill et al.. 1996a; BASF.
1991d. f, g, h, i, k, 1, m).
Increased relative stomach weights (>179 mg/kg-day) and histopathological changes in
the forestomach (acanthosis) were noted in rats at 357 mg/kg-day (Astill et al.. 1996a; BASF.
1991a). Increased relative stomach weight was also observed in male mice at > 179 mg/kg-day
(Astill et al.. 1996a; BASF. 1991e. 1). Several of the short-term-duration gavage studies
identified increased stomach weights, gross observations of foci in the forestomach, and/or
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histopathological forestomach effects (hyperkeratosis, acanthosis, and/or inflammatory edema)
(BASF. 199Id. £ g, h, 1, k, 1, m).
Identification of developmental toxicity as a health hazard from repeated oral exposure to
2-EH comes from several reports. Hardin et al. (1987) reported maternal (i.e., mortality,
decreased body weight and body-weight gain) and developmental (i.e., decreased survival and
growth of pups) toxicity at the only dose tested (1,525 mg/kg-day) in mice, which is identified as
a FEL in mice. Ritter et al. (1987) reported decreased fetal-body weight and increased fetal
malformations following maternal exposure to 2-EH on GD 12; however, inadequate reporting
did not allow for the identification of a NOAEL or LOAEL for this study. In a development
study in rats (Hellwig and Jackh, 1997; Confidential. 1991). decreased fetal body weight and
increased fetal incidences of skeletal effects (e.g., malformations) were observed at
>650 mg/kg-day. The fetal incidences of visceral variations (dilated renal pelvis, and
hydroureter), and skeletal malformations (absent thoracic vertebrae and sternebrae bipartite with
ossification centers dislocated) were statistically significantly increased at 1,300 mg/kg-day
(see Table B-10). The fetal incidences of skeletal variations (accessory lumbar vertebrae and
14th ribs, rudimentary cervical ribs, and 13th ribs absent or shortened) and retardations (thoracic
vertebral body/bodies and sternebrae not ossified, sternebrae incompletely ossified or reduced in
size) were significantly increased at >650 mg/kg-day (see Table B-10).
To provide a common basis for comparing potential points of departure (PODs) and
critical effects for deriving a subchronic p-RfD for 2-EH, data sets representing the most
sensitive endpoints (e.g., liver, kidney, stomach, and developmental effects) were selected for
benchmark dose (BMD) analysis. Based on a comparison of the PODs, the most sensitive
treatment-related changes from the oral toxicity database for 2-EH, were reported in the
developmental study conducted by Hellwig and Jackh (1997) and Confidential (1991) and the
subchronic-duration gavage study in rats and mice (Astill et al.. 1996a; BASF. 1991a). All
available continuous or dichotomous-variable models in the Benchmark Dose Software (BMDS,
Version 2.7) were fit to the data sets for the most sensitive endpoints presented in Table C-l.
Appendix C contains details of the modeling results for these data sets. The HED, in mg/kg-day,
was used as the dose metric except for relative stomach weight (see below). Because increased
fetal incidence of skeletal malformations in rats (Hellwig and Jackh. 1997; Confidential. 1991)
was statistically significant only at 325 mg/kg-day (HED) where severe maternal toxicity was
also observed, the effect was not considered as a potential POD because the interpretation of this
effect is confounded by the overt maternal toxicity. For this same reason, the data for decreased
fetal body weight and increased fetal incidence of skeletal variations and retardations in rats were
BMD modeled without the highest dose tested (325 mg/kg-day [HED]). The benchmark
response (BMR) for changes in liver or kidney weight used was a 10% relative deviation (RD)
change from control means, which is considered a biologically significant response. The BMR
for changes in stomach weight used was 1 standard deviation (SD) change from control means,
because no information is available regarding the change in this response that would be
considered biologically significant. The BMR for decreased fetal body weight used was 5% RD
change from control means, which is considered a biologically significant response and 5% extra
risk for dichotomous developmental data. One or more of the models provided adequate fit for
each data set except increased absolute liver weight in male rats (Astill et al.. 1996a; BASF.
1991a) and decreased fetal body weight and increased fetal incidence of skeletal retardations in
rats (Hellwig and Jackh, 1997; Confidential, 1991). Candidate PODs, including the benchmark
dose lower confidence limits (BMDLs) from the selected models, are presented in Table 7.
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In U.S. EPA's Recommended Use of Body Weight4 as the Default Method in Derivation
of the Oral Reference Dose (U.S. EPA. 201 lb), the Agency endorses a hierarchy of approaches
to derive human equivalent oral exposures from data from laboratory animal species, with the
preferred approach being physiologically based toxicokinetic modeling. Other approaches may
include using some chemical-specific information without a complete physiologically based
toxicokinetic model. In lieu of chemical-specific models or data to inform the derivation of
human equivalent oral exposures, U.S. EPA endorses body-weight scaling to the 3/4 power
(i.e., BW3/4) to extrapolate toxicologically equivalent doses of orally administered agents from
all laboratory animals to humans for deriving an oral reference dose (RfD) under certain
exposure conditions. More specifically, the use of BW3 4 scaling for deriving an RfD is
recommended when the observed effects are associated with the parent compound or a stable
metabolite, but not for portal-of-entry effects. A validated human physiologically based
toxicokinetic model for 2-EH is not available for use in extrapolating doses from animals to
humans. Furthermore, the most sensitive endpoints being considered are not portal-of-entry
effects, except changes in stomach weight in rats. The BW3/4 scaling factor was not applied to
the stomach-weight changes because allometric scaling has not been extensively evaluated with
portal-of-entry effects. However, scaling by BW3/4 is relevant for deriving HEDs for effects
other than stomach changes.
Following U.S. EPA (2011b) guidance, the doses administered resulting in the most
sensitive endpoints are converted to an HED through application of a dosimetric adjustment
factor (DAF) derived as follows:
DAF = (BWa1/4 - BWh1/4)
where
DAF = dosimetric adjustment factor
BWa = animal body weight
BWh = human body weight
Study-specific body weight is used to calculate the DAF for each dose group (U.S. EPA.
2011b). Calculated HEDs are presented in Table C-l for male and female rats and mice exposed
subchronically to 2-EH (Astill et at., 1996a; BASF, 1991a) and female rats exposed to 2-EH
during pregnancy (Hellwig and Jackh. 1997; Confidential 1991).
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Table 7. Candidate PODs in Rodents Administered 2-Ethylhexanol (CASRN 104-76-7) for
the Derivation of the Subchronic p-RfD
Endpoint
NOAEL (HED)
(mg/kg-d)
LOAEL (HED)
(mg/kg-d)
BMDL (HED)a
(mg/kg-d)
POD (HED)
(mg/kg-d)
Rats Astill et al. (1996a): BASF (1991a)
Absolute liver weight in males
43.0
85.7b
NDr
43.0 (NOAEL)
Relative liver weight in males
43.0
85.7b
45
45 (BMDLio)
Relative kidney weight in males
43.0
85.7b
48
48 (BMDLio)
Relative stomach weight in males0
179
357d
130
130 (BMDLisd)
Relative liver weight in females
39.4
75.0b
45
45 (BMDLio)
Relative stomach weight in females0
89.3
179d
87
87 (BMDLisd)
Mice Astill et al. (1996a): BASF (1991e): BASF (1991c)
Relative stomach weight in males0
89.3
179d
62
62 (BMDLisd)
Rats Hellwig and Jackh (1997); Confidential (1991)
Fetal body weight
32.5
163°
NDr
32.5 (NOAEL)
Fetal skeletal variationsf
32.5
163d
7.37
7.37 (BMDLos)
Fetal skeletal retardations
32.5
163d
NDr
32.5 (NOAEL)
"Modeling results are described in more detail in Appendix C.
increase was >10% compared to control values.
°As discussed above, doses for stomach-weight changes were not converted to HEDs.
dChange was statistically significantly increased compared to control values.
"Change was >5% compared to control values.
fChosen as the critical effect for deriving the subchronic p-RfD.
BMDL = benchmark dose lower confidence limit; HED = human equivalent dose;
LOAEL = lowest-observed-adverse-effect level; NDr = not determined; NOAEL = no-observed-adverse-effect
level; POD = point of departure; p-RfD = provisional reference dose.
Among all the sensitive endpoints evaluated, the lowest POD (HED) following oral
exposure to 2-EH is for increased fetal incidence of skeletal variations (i.e., accessory lumbar
vertebrae and 14th ribs, rudimentary cervical ribs, and 13th ribs, absent or shortened). The
BMDLos (HED) for fetal skeletal variations is expected to be protective of all developmental
effects during a susceptible life stage, as well as any potential liver, kidney, and stomach
systemic effects observed following subchronic 2-EH exposure. Thus, the BMDLos (HED) for
fetal skeletal variations (7.37 mg/kg-day) is selected as the POD for derivation of the subchronic
p-RfD.
Subchronic p-RfD = BMDLos (HED) UFc
= 7.37 mg/kg-day -MOO
= 7 x 10"2 mg/kg-day
Table 8 summarizes the uncertainty factors for the subchronic p-RfD for 2-EH.
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Table 8. Uncertainty Factors for the Subchronic p-RfD for
2-Ethylhexanol (CASRN 104-76-7)
UF
Value
Justification
UFa
3
A UFa of 3 (10°5) is applied to account for uncertainty in characterizing the toxicokinetic or
toxicodynamic differences between rats and humans following 2-EH exposure. The toxicokinetic
uncertainty has been accounted for by calculating an HED through application of a DAF as outlined
in the U.S. EPA's Recommended Use of Body Weight3/4 as the Default Method in Derivation of the
Oral Reference Dose (U.S. EPA, 1988).
UFd
3
A UFd of 3 (10°5) is applied to account for deficiencies and uncertainties in the database.
Well-conducted oral subchronic- and chronic-duration animal toxicity studies of 2-EH are available in
rats and mice CAstill et al.. 1996a: BASF. 1991a. c. o). Developmental toxicity studies are available
in rats and mice. Severe maternal effects were noted at high doses. There are no two-generation
reproductive toxicity studies of 2-EH.
UFh
10
A UFh of 10 is applied for interindividual variability to account for human-to-human variability in
susceptibility in the absence of quantitative information to assess the toxicokinetics and
toxicodynamics of 2-EH in humans.
UFl
1
A UFl of 1 is applied for LOAEL-to-NOAEL extrapolation because the POD is a BMDL.
UFS
1
A UFS of 1 is applied because developmental toxicity resulting from a narrow period of exposure was
used as the critical effect. The developmental period is recognized as a susceptible life stage when
exposure during a time window of development is more relevant to the induction of developmental
effects than lifetime exposure (U.S. EPA. 1991).
UFC
100
Composite UF = UFA x UFD x UFH x UFL x UFS.
BMDL = benchmark dose lower confidence limit; DAF = dosimetric adjustment factor; 2-EH = 2-ethylhexanol;
HED = human equivalent dose; LOAEL = lowest-observed-adverse-effect level;
NOAEL = no-observed-adverse-effect level; POD = point of departure; p-RfD = provisional reference dose;
UF = uncertainty factor; UFA = interspecies uncertainty factor; UFC = composite uncertainty factor;
UFd = database uncertainty factor; UFH = intraspecies uncertainty factor; UFL = LOAEL-to-NOAEL uncertainty
factor; UFS = subchronic-to-chronic uncertainty factor.
Confidence in the subchronic p-RfD for 2-EH is medium as explained in Table 9.
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Table 9. Confidence Descriptors for the Subchronic p-RfD for
2-Ethylhexanol (CASRN 104-76-7)
Confidence Categories
Designation
Discussion
Confidence in study
M
Confidence in the study is medium. Although the study by
Heliu m and Jackh (1997) and Confidential (1991) is a
peer-reviewed and GLP-conducted study, it tested a limited
number of animals (8-10 maternal animals per dose group).
Furthermore, there was a large degree of maternal toxicity at the
high dose, resulting in that dose being excluded from the
dose-response assessment.
Confidence in database
M
Confidence in the database is medium. The database includes
two subchronic-duration studies (one gavage study in rats and
mice and one dietary study in rats), a chronic-duration study in
rats and mice, one limited neonatal exposure study in rats, and
several developmental studies of gestational exposure in rats and
mice. There are no two-generation reproductive toxicity studies
of 2-EH. Furthermore, there are no oral toxicity studies that
assessed CNS effects, which were observed in humans exposed to
2-EH via inhalation.
Confidence in subchronic p-RfD'
M
Overall confidence in the subchronic p-RfD is medium.
aThe overall confidence cannot be greater than the lowest entry in the table (medium).
CNS = central nervous system; 2-EH = 2-ethylhexanol; GLP = Good Laboratory Practice; H = high; M = medium;
p-RfD = provisional reference dose.
Derivation of a Chronic Provisional Reference Dose
The developmental study in rats administered 2-EH via gavage on GDs 6-15 is also
selected as the principal study for deriving the chronic p-RfD (Hellwig and Jackh. 1997;
Confidential 1991). Increased fetal incidence of skeletal variations is identified as the critical
effect.
In addition to the subchronic-duration and developmental studies considered for the
derivation of the subchronic p-RfD described above, there was one chronic-duration gavage
study in rats and mice (Astill et al.. 1996b; BASF. 1992a). The chronic-duration study in rats
(Astill et al.. 1996b; BASF. 1992a) is well designed and provides data that adequately describe a
dose-response relationship for body-weight following exposure to 2-EH for 5 days/week for
24 months (see Tables B-5, B-6, and C-2). The most sensitive endpoint reported in the
chronic-duration study by Astill et al. (1996b) and BASF (1992a) was decreased body weight in
male (at >107 mg/kg-day) and female rats (at >357 mg/kg-day) (see Table C-2). The companion
chronic-duration toxicity/carcinogenicity study in mice noted increased mortality, decreased
body weight, and changes in relative organ weights and liver histopathology, but these effects
occurred at higher doses (Astill et al.. 1996b; BASF. 1991b). Developmental toxicity studies in
rats also reported decreased body weights in maternal animals at >650 mg/kg-day (Hellwig and
Jackh. 1997; Confidential. 1991; Hardin et al.. 1987; Hazleton Laboratories. 1983).
Following U.S. EPA (2011b) guidance, the doses administered resulting in the most
sensitive endpoints are converted to an HED through application of a DAF derived as follows:
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DAF = (BWa1/4 - BWh1/4)
where
DAF = dosimetric adjustment factor
BWa = animal body weight
BWh = human body weight
Study-specific body weight is used to calculate the DAF for each dose group (U.S. HP A.
2011b). Calculated HEDs are presented in Tables C-l and C-2 for female rats exposed to 2-EH
during pregnancy (Hellwig and Jackh. 1997; Confidential 1991) and for male and female rats
exposed chronically to 2-EH (Astill et al.. 1996b; BASF. 1992a). respectively.
To determine potential PODs for the chronic p-RfD, data sets for the most sensitive
treatment-related endpoints following chronic exposure to 2-EH were modeled using BMD
analysis and compared to the most sensitive treatment-related change reported in the
developmental study conducted by Hellwig and Jackh (1997) and Confidential (1991)
(see Table C-l). All available continuous-variable models in the BMDS (Version 2.7) were fit to
the data sets for decreased body weights in males and females (Astill et al.. 1996b; BASF.
1992a). In Appendix C, modeling results for these data sets are summarized in Tables C-l3 and
C-14. The HED in mg/kg-day was used as the dose metric. The BMR for decreased body
weight used was 10% RD change from control means, which is considered a biologically
significant response. As stated above, the BMR for decreased fetal body weight used was 5%
RD change from control means for continuous data and 5% extra risk for dichotomous
developmental data. For the developmental effects, the data were modeled without the highest
dose of 325 mg/kg-day (HED) because there was severe maternal toxicity (i.e., 60% mortality
and a 20% decrease in body weight) at that dose that confounds the interpretation of fetal
changes. One or more of the models provided adequate fit for each data set except decreased
body weight in females (see Table C-14); models with the lowest AIC were selected in each
case. Candidate PODs, including the BMDLs from the selected models, are presented in
Table 10.
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Table 10. Candidate PODs in Rats Administered 2-Ethylhexanol (CASRN 104-76-7) for
the Derivation of the Chronic p
-RfD
NOAEL (HED)
LOAEL (HED)
BMDL (HED)a
POD (HED)
Endpoint
(mg/kg-d)
(mg/kg-d)
(mg/kg-d)
(mg/kg-d)
Astill et al. (1996b): BASF (1992a)
Terminal body weight in males
9.5
27.9b
19
19 (BMDLio)
Terminal body weight in
24.8
81.2b
NDr
24.8 (NOAEL)
females
Hellwig and Jackh (1997); Confidential (1991)
Fetal skeletal variations0
32.5
163d
7.37
7.37 (BMDLos)
"Modeling results are described in more detail in Appendix C.
bChange was >10% compared to control values.
°Chosen as the critical effect for deriving the chronic p-RfD.
dChange was statistically significantly increased compared to control values.
BMDL = benchmark dose lower confidence limit; HED = human equivalent dose;
LOAEL = lowest-observed-adverse-effect level; NDr = not determined; NOAEL = no-observed-adverse-effect
level; POD = point of departure; p-RfD = provisional reference dose.
When the BMD results in Table 10 are compared, the lowest POD (HED) is for increased
fetal incidence of skeletal variations. The BMD Los (HED) for fetal skeletal variations is
expected to be protective of all developmental effects during a susceptible life stage, as well as
any potential systemic effects observed following chronic 2-EH exposure. Thus, the BMD Los
(HED) for fetal skeletal variations (7.37 mg/kg-day) is again selected as the POD for deriving the
chronic p-RfD which is derived as follows:
Chronic p-RfD = BMDLos (HED) UFc
= 7.37 mg/kg-day -MOO
= 7 x 10"2 mg/kg-day
Table 11 summarizes the uncertainty factors for the chronic p-RfD for 2-EH.
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Table 11. Uncertainty Factors for the Chronic p-RfD for
2-Ethylhexanol (CASRN 104-76-7)
UF
Value
Justification
UFa
3
A UFa of 3 (10°5) is applied to account for uncertainty in characterizing the toxicokinetic or
toxicodynamic differences between rats and humans following 2-EH exposure. The toxicokinetic
uncertainty has been accounted for by calculating an HED through application of a DAF as outlined
in the U.S. EPA's Recommended Use of Body Weight3/4 as the Default Method in Derivation of the
Oral Reference Dose (U.S. EPA, 1988).
UFd
3
A UFd of 3 (10°5) is applied to account for deficiencies and uncertainties in the database.
Well-conducted oral subchronic- and chronic-duration animal toxicity studies of 2-EH are available in
rats and mice CAstill et al.. 1996a: BASF. 1991a. c. o). Developmental toxicity studies are available
in rats and mice. Severe maternal effects were noted at high doses. There are no two-generation
reproductive toxicity studies of 2-EH.
UFh
10
A UFh of 10 is applied for interindividual variability to account for human-to-human variability in
susceptibility in the absence of quantitative information to assess the toxicokinetics and
toxicodynamics of 2-EH in humans.
UFl
1
A UFl of 1 is applied for LOAEL-to-NOAEL extrapolation because the POD is a BMDL.
UFS
1
A UFS of 1 is applied because developmental toxicity resulting from a narrow period of exposure was
used as the critical effect. The developmental period is recognized as a susceptible life stage when
exposure during a time window of development is more relevant to the induction of developmental
effects than lifetime exposure (U.S. EPA. 1991).
UFC
100
Composite UF = UFA x UFD x UFH x UFL x UFS.
BMDL = benchmark dose lower confidence limit; DAF = dosimetric adjustment factor; 2-EH = 2-ethylhexanol;
HED = human equivalent dose; LOAEL = lowest-observed-adverse-effect level;
NOAEL = no-observed-adverse-effect level; POD = point of departure; p-RfD = provisional reference dose;
UF = uncertainty factor; UFA = interspecies uncertainty factor; UFC = composite uncertainty factor;
UFd = database uncertainty factor; UFH = intraspecies uncertainty factor; UFL = LOAEL-to-NOAEL uncertainty
factor; UFS = subchronic-to-chronic uncertainty factor.
Confidence in the chronic p-RfD for 2-EH is medium as explained in Table 12.
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Table 12. Confidence Descriptors for the Chronic p-RfD for
2-Ethylhexanol (CASRN 104-76-7)
Confidence Categories
Designation
Discussion
Confidence in study
M
Confidence in the study is medium. Although the study by
Heliu m and Jackh (1997) and Confidential (1991) is a
peer-reviewed and GLP-conducted study, it tested a limited
number of animals (8-10 maternal animals per dose group).
Furthermore, there was a large degree of maternal toxicity at the
high dose, resulting in that dose being excluded from the
dose-response assessment.
Confidence in database
M
Confidence in the database is medium. The database includes two
subchronic-duration studies, a chronic-duration study in mice and
rats, one limited neonatal exposure study in rats, and several
developmental studies of gestational exposure in rats and mice.
There are no two-generation reproductive toxicity studies of 2-EH.
Furthermore, there are no oral toxicity studies that assessed CNS
effects, which were observed in humans exposed to 2-EH via
inhalation.
Confidence in chronic p-RfDa
M
Overall confidence in the chronic p-RfD is medium.
aThe overall confidence cannot be greater than the lowest entry in the table (medium).
CNS = central nervous system; 2-EH = 2-ethylhexanol; GLP = Good Laboratory Practice; H = high; M = medium;
p-RfD = provisional reference dose.
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS
Derivation of a Subchronic Provisional Reference Concentration
The database of potentially relevant studies for deriving a subchronic inhalation reference
value for 2-EH includes a subchronic-duration study in Wistar rats (Klimisch et al.. 1998; BASF.
1992b). a subchroni c-durati on study in male ICR mice (Mivake et al.. 2016). and a
developmental study in S-D rats (Nelson ct al.. 1989). The subchroni c-durati on inhalation study
in male ICR mice (Mivake et al.. 2016) is selected as the principal study, and increased diameter
of Bowman's glands in the olfactory epithelium of the nasal cavity is identified as the critical
effect. The Bowman's glands play a major role in the ability to smell by releasing nasal fluid
that trap odorants and binds them to receptors located on olfactory sensory neurons. The nasal
secretions from the Bowman's gland also protect the olfactory mucosa from infection and
dehydration (Hellier. 2016). Morphological changes in the Bowman's glands could affect its
function and impact the sense of smell. As discussed above, exposure to 2-EH has been shown
to cause "sick building syndrome" in humans with one of the symptoms being an increased sense
of smell. Therefore, it is possible that 2-EH-induced effects on the Bowman's glands could be
related to effects observed in humans.
The Mivake et al. (2016) study was published in a peer-reviewed journal. The study is
adequate regarding design (e.g., inclusion of controls and several exposure levels) and
performance pertaining to examination of potential toxicity endpoints, and presentation of
materials, methods, and results. Regarding the histopathology performed in the study, additional
caudal nasal cross sections beyond the more anterior (rostral) region of the nose were not taken,
but the study did perform a very complete morphological assessment of the changes in the
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olfactory bulb size. Details of the study are provided in the "Review of Potentially Relevant
Data" section.
No significant treatment-related effects were reported in either the subchronic-duration
study (Klimisch et al.. 1998; BASF. 1992b) or developmental study (Nelson et al.. 1989) in rats.
Potential PODs for these studies are a NOAEL (HEC) of 114 mg/m3 for the subchronic-duration
study and a NOAEL (HEC) of 248 mg/m3 for the developmental study. In the
subchronic-duration study by Miyake et al. (2016), the effects of 2-EH on the olfactory system
(i.e., the olfactory epithelium of the nasal cavity and the olfactory bulb of the brain) were
examined in mice exposed to 2-EH via inhalation for up to 3 months. Severity scores for
morphological alterations in the olfactory epithelium were significantly increased at
>12.5 mg/m3 (HEC) in mice exposed to 2-EH for 1 week and 3 months. The study authors also
reported changes in the expression of nerve-related markers (e.g., OMP, TH, etc.) in the
olfactory epithelium and bulb at >4.17 mg/m3 (HEC). In accordance with U.S. EPA (1994)
methodology, the following dosimetric adjustments are made for male mice with a NOAEL for
respiratory effects in the extrathoracic (ET) region:
Exposure concentration adjustment for continuous exposure:
CONCadj
Exposure CONC x (MW - 24.45) x
(hours exposed ^ 24) x (days exposed ^ 7 days per week)5
21.9 ppm x (130 -h 24.45) x (8 hours 24 hours) x
(5 days ^ 7 days)
27.7 mg/m3
HEC conversion for respiratory effects:
CONC (HEC) = CONCadj x RGDRet
where
where
RGDRet
VE[mouse]
- (Ye SAet) n
Ve [human] =
SAET[mouse] =
SAET[human] —
Male mouse RGDRet =
(Ve SAet)ii uman
Mouse minute volume [mouse = 0.03116 L/min, based on
the mean reference body weight of 0.02695 kg for male
BAFi and B6C3Fi mice in a subchronic-duration study
(U.S. EPA. 1994)1
13.8 L/min
Mouse default surface area of the ET region (3 cm2)
Human default surface area of the ET region (200 cm2)
(0.03116 L/min 3 cm2) (13.8 L/min 200 cm2)
0.1505
CONCresp (HEC) =
CONCadj x RGDRet
27.7 mg/m3 x 0.1505
4.17 mg/m3
5CONC = concentration from the Miyake et al. (2016) study.
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All available continuous models in the BMDS (Version 2.7) were fit to the data sets
(see Table B-13) from the inhalation study in mice by Mivake et al. (2016). which was the only
inhalation study that reported significant treatment-related effects. Appendix C contains details
of the modeling results for these data sets. The HEC, in mg/m3, was used as the dose metric.
The BMR for all changes observed in the Mivake et al. (2016) study was 1 SD change from
control means, because no information is available regarding the change in response that would
be considered biologically significant. One or more of the models provided adequate fit for each
data set except increased number of CD3-, Ibal-, and Dcx-positive cells. Candidate PODs,
including the benchmark concentration lower confidence limits (BMCLs) from the selected
models, are presented in Table 13.
Table 13. Candidate PODs in Rodents Administered 2-Ethylhexanol (CASRN 104-76-7)
for the Derivation of the Subchronic p-RfCa
Endpoint
NOAEL (HEC)
(mg/m3)
LOAEL (HEC)
(mg/m3)
BMCL (HEC)b
(mg/m3)
POD (HEC)
(mg/m3)
Morphological alterations in the OE
4.17
12.5°
NDr
4.17 (NOAEL)
Diameter of Bowman's glands in the
OEd
4.17
12.5C
1.11
1.11
(BMCLisd)
CD3 -positive cells in the OE
4.17
12.5°
NDr
4.17 (NOAEL)
OMP-positive cells (ratio of OMP[+]
cells to olfactory epithelium cells) in
the OE
NDr
4.17°
3.14
3.14 (BMCLisd)
PCNA-positive cells in the OE
12.5
29.20°
11.5
11.5 (BMCLisd)
Glomerular diameter in the OB
12.5
29.20°
8.67
8.67 (BMCLisd)
OMP-positive cells in the OB
4.17
12.5°
5.72
5.72 (BMCLisd)
TH-positive cells in the OB
12.5
29.20°
3.59
3.59 (BMCLisd)
Ibal-positive cells in the OB
12.5
29.20°
NDr
12.5 (NOAEL)
Dcx-positive cells in the OB
12.5
29.20°
NDr
12.5 (NOAEL)
aMivake et al. (2016).
bModeling results are described in more detail in Appendix C.
°Change was statistically significantly increased compared to control values.
dChosen as the critical effect for deriving the subchronic p-RfC.
BMCL = benchmark concentration lower confidence limit; Dcx = doublecortin; HEC = human equivalent
concentration; Ibal = ionized calcium-binding adapter molecule 1; LOAEL = lowest-observed-adverse-effect level;
NDr = not determined; NOAEL = no-observed-adverse-effect level; OE = olfactory epithelium; OB = olfactory
bulb; OMP = olfactory marker protein; PCNA = proliferating cell nuclear antigen; POD = point of departure;
p-RfC = provisional reference concentration; PCNA = proliferating cell nuclear antigen; SD = standard deviation;
TH = tyrosine hydroxylase.
The potential PODs for deriving the subchronic p-RfC for 2-EH include a NOAEL
(HEC) of 114 mg/m3 and NOAEL (HEC) of 248 mg/m3, both for the lack of significant,
treatment-related effects in rats from the subchronic-duration (Klimisch et al.. 1998; BASF.
1992b) and developmental (Nelson et al.. 1989) studies, respectively. The most sensitive POD
from the Mivake et al. (2016) study is a BMCLisd of 1.11 mg/m3 (HEC) for increased diameter
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of the Bowman's glands in the olfactory epithelium of the nasal cavity in male mice exposed to
2-EH via inhalation for 3 months (Mivake et al.. 2016). Among the candidate endpoints for
potential critical effect, the lowest POD is the BMCLisd of 1.11 mg/m3 (HEC) for increased
diameter of the Bowman's glands in the olfactory epithelium of the nasal cavity in male mice.
Effects on the olfactory system were also reported in humans that were acutely exposed to 2-EH
via inhalation, providing further support that the olfactory system is indeed a target for inhalation
exposure to 2-EH (Ernstgard et al.. 2010; van Thriel et al.. 2007; Kiesswetter et al.. 2005; van
Thriet et al.. 2005; van Thriel et al.. 2003).
The subchronic-duration inhalation study by Mivake et al. (2016) with a LOAEL (HEC)
of 4.17 mg/m3 and no corresponding NOAEL is selected as the principal study for deriving the
subchronic p-RfC. The critical effect is increased diameter of the Bowman's glands in the
olfactory epithelium of the nasal cavity in male ICR mice exposed to 2-EH via inhalation for
3 months (Mivake et al.. 2016) with a BMCLisd of 1.11 mg/m3 (HEC); this BMCLisd is selected
as the POD for deriving the subchronic p-RfC. The subchronic p-RfC for 2-EH is derived as
follows:
Subchronic p-RfC = BMCLisd (HEC) - UFC
1.11 mg/m3-300
= 4 x 10 3 mg/m3
Table 14 summarizes the uncertainty factors for the subchronic p-RfC for 2-EH.
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Table 14. Uncertainty Factors for the Subchronic p-RfC for
2-Ethylhexanol (CASRN 104-76-7)
UF
Value
Justification
UFa
3
A UFa of 3 (10°5) is applied to account for uncertainty associated with extrapolating from animals to
humans using toxicokinetic cross-species dosimetric adjustment for respiratory effects from a
Cateeorv 1 sas. as specified in U.S. EPA (1994) guidelines for deriving RfCs.
UFd
10
A UFd of 10 is applied to account for deficiencies and uncertainties in the database.
Repeated-exposure inhalation toxicity data for 2-EH are limited to two subchronic-duration studies,
one in mice and one in rats, and a developmental studv in rats (Mivake et al.. 2016; Klimiseh et al..
1998; BASF. 1992b; Nelson et al.. 1989). The limited developmental toxicity studv in rats bv
inhalation exposure found no effects at concentrations ud to saturation (Nelson et al.. 1989).
However, developmental effects were found to be the most sensitive endpoint for subchronic or
chronic oral 2-EH exposure and thus more comprehensive studies would be warranted. There are no
two-generation reproductive toxicity studies of 2-EH.
UFh
10
A UFh of 10 is applied to account for human variability in susceptibility, in the absence of
information to assess toxicokinetic and toxicodynamic variability of 2-EH in humans.
UFl
1
A UFl of 1 is applied because the POD is a BMCL.
UFS
1
A UFS of 1 is applied because the POD comes from a subchronic-duration study of mice.
UFC
300
Composite UF = UFA x UFD x UFH x UFL x UFS.
BMCL = benchmark concentration lower confidence limit; 2-EH = 2-ethylhexanol;
LOAEL = lowest-observed-adverse-effect level; NOAEL = no-observed-adverse-effect level; POD = point of
departure; p-RfC = provisional reference concentration; RfC = inhalation reference concentration; UF = uncertainty
factor; UFA = interspecies uncertainty factor; UFC = composite uncertainty factor; UFD = database uncertainty
factor; UFH = intraspecies uncertainty factor; UFL = LOAEL-to-NOAEL uncertainty factor;
UFS = subchronic-to-chronic uncertainty factor.
The confidence descriptors for the subchronic p-RfC are described in Table 15.
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Table 15. Confidence Descriptors for the Subchronic p-RfC for
2-Ethylhexanol (CASRN 104-76-7)
Confidence Categories
Designation
Discussion
Confidence in study
M
Confidence in the principal study is medium. The study was
peer reviewed, tested multiple exposure levels in groups of
adequate size in male mice, and measured exposure
concentrations in all groups. However, the study is not
comprehensive, focusing primarily on the olfactory system, and
only tested one sex of mice (males).
Confidence in database
L
Confidence in the database is low. Repeated-exposure inhalation
toxicity data for 2-EH are limited to two subchronic-duration
studies, one in mice and one in rats, and a developmental study
in rats (Mivake et al. 2016; Klimisch et al.. 1998; BASF. 1992b;
Nelson et al.. 1989). The limited developmental toxicity studv in
rats by inhalation exposure found no effects at concentrations up
to saturation (Nelson et al.. 1989). However, developmental
effects were found to be the most sensitive endpoint for oral
2-EH exposure and thus more comprehensive studies would be
warranted. There are no two-generation reproductive toxicity
studies of 2-EH.
Confidence in subchronic p-RfCa
L
Overall confidence in the subchronic p-RfC is low.
aThe overall confidence cannot be greater than the lowest entry in the table (low).
2-EH = 2-ethylhexanol; L = low; M = medium; p-RfC = provisional reference concentration.
Derivation of a Chronic Provisional Reference Concentration
In the absence of toxicity studies in humans or animals chronically exposed to 2-EH by
inhalation, a chronic p-RfC for 2-EH is derived using the subchronic POD (HEC). Justification
for selecting the critical effect and principal study are described in the previous section of this
document.
The chronic p-RfC for 2-EH, based on a BMCLisd (HEC) of 1.11 mg/m3 in male mice
exposed to 2-EH for 3 months, is derived as follows:
Chronic p-RfC = BMCLisd (HEC) - UFC
1.11 mg/m3-3,000
= 4 x 10 4 mg/m3
Table 16 summarizes the uncertainty factors for the chronic p-RfC for 2-EH.
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Table 16. Uncertainty Factors for the Chronic p-RfC for
2-Ethylhexanol (CASRN 104-76-7)
UF
Value
Justification
UFa
3
A UFa of 3 (10°5) is applied to account for uncertainty associated with extrapolating from animals to
humans, using toxicokinetic cross-species dosimetric adjustment for respiratory effects from a
Cateeorv 1 sas. as specified in U.S. EPA (1994) guidelines for deriving RfCs.
UFd
10
A UFd of 10 is applied to account for deficiencies and uncertainties in the database.
Repeated-exposure inhalation toxicity data for 2-EH are limited to two subchronic-duration studies,
one in mice and one in rats, and a developmental studv in rats (Mivake et al.. 2016; Klimiseh et al..
1998; BASF. 1992b; Nelson et al.. 1989). The limited developmental toxicity studv in rats bv
inhalation exposure found no effects at concentrations ud to saturation (Nelson et al.. 1989).
However, developmental effects were found to be the most sensitive endpoint for subchronic or
chronic oral 2-EH exposure and thus more comprehensive studies would be warranted. There are no
two-generation reproductive toxicity studies of 2-EH.
UFh
10
A UFh of 10 is applied to account for human variability in susceptibility, in the absence of
information to assess toxicokinetic and toxicodynamic variability of 2-EH in humans.
UFl
1
A UFl of 1 is applied because the POD is a BMCL.
UFS
10
A UFS of 10 is applied because the POD comes from a subchronic-duration study of mice.
UFC
3,000
Composite UF = UFA x UFD x UFH x UFL x UFS.
BMCL = benchmark concentration lower confidence limit; 2-EH = 2-ethylhexanol;
LOAEL = lowest-observed-adverse-effect level; NOAEL = no-observed-adverse-effect level; POD = point of
departure; p-RfC = provisional reference concentration; RfC = inhalation reference concentration; UF = uncertainty
factor; UFA = interspecies uncertainty factor; UFC = composite uncertainty factor; UFD = database uncertainty
factor; UFH = intraspecies uncertainty factor; UFL = LOAEL-to-NOAEL uncertainty factor;
UFS = subchronic-to-chronic uncertainty factor.
The confidence descriptors for the chronic p-RfC are described in Table 17.
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Table 17. Confidence Descriptors for the Chronic p-RfC for
2-Ethylhexanol (CASRN 104-76-7)
Confidence Categories
Designation
Discussion
Confidence in study
M
Confidence in the principal study is medium. The study was peer
reviewed, tested multiple exposure levels in groups of adequate size
in male mice, and measured exposure concentrations in all groups.
However, the study is not comprehensive, focusing primarily on the
olfactory system, and only tested one sex of mice (males).
Confidence in database
L
Confidence in the database is low. Repeated-exposure inhalation
toxicity data for 2-EH are limited to two subchronic-duration
studies, one in mice and one in rats, and a developmental study in
rats (Mivake et al.. 2016; Klimisch et al.. 1998; BASF. 1992b;
Nelson et al.. 1989). The limited developmental toxicity studv in
rats by inhalation exposure found no effects at concentrations up to
saturation (Nelson et al.. 1989). However, developmental effects
were found to be the most sensitive endpoint for oral 2-EH exposure
and thus more comprehensive studies would be warranted. There
are no two-generation reproductive toxicity studies of 2-EH.
Confidence in chronic p-RfC'
L
Overall confidence in the chronic p-RfC is low.
aThe overall confidence cannot be greater than the lowest entry in the table (low).
2-EH = 2-ethylhexanol; L = low; M = medium; p-RfC = provisional reference concentration.
CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR
Following U.S. EPA (2005) Guidelines for Carcinogen Risk Assessment, the cancer
weight of evidence (W OE) for 2-EH is "Suggestive Evidence of Carcinogenic Potential" for oral
exposure (see Table 18). In the chronic-duration cancer bioassays in rats and mice following
oral exposure to 2-EH (Astill et al.. 1996b; BASF. 1992a. 1991b). there were significant
dose-related trends for hepatocellular carcinoma in male and female mice. A single adenoma
was detected in one 536-mg/kg-day male (and no females), but the study authors did not report
the results of combined statistical analysis of hepatocellular adenoma or carcinoma in male mice.
The incidence of hepatocellular carcinoma was significantly increased in female mice at the
highest dose tested based on pairwise comparison to vehicle controls. There was no evidence of
treatment-related carcinogenicity in male or female rats. Although these data are consistent with
one of the examples provided in the U.S. EPA's Cancer Guidelines (U.S. EPA. 2005) for the
descriptor "Likely to Be Carcinogenic to Humans " (which states that supporting data for this
descriptor include "an agent that has tested positive in animal experiments in more than one
species, sex, strain, site, or exposure route, with or without evidence of carcinogenicity in
humans"), the incidence of hepatocellular tumors was statistically significantly increased only in
female mice at the highest dose tested, and there was no evidence of cancer in male or female
rats treated with 2-EH for 2 years. Furthermore, the data for hepatocellular tumors in male mice
only showed a significant dose-related trend and were not significant based on a pairwise
comparison test. As stated in the Cancer Guidelines (U.S. EPA. 2005). one of the examples for a
chemical to be considered to have "Suggestive Evidence of Carcinogenic Potential" is "a small,
and possibly not statistically significant, increase in tumor incidence observed in a single animal
or human study that does not reach the weight of evidence for the descriptor "Likely to Be
Carcinogenic to Humans. " The Cancer Guidelines (U.S. EPA. 2005) further state that the
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descriptor "Suggestive Evidence of Carcinogenic Potential" is appropriate when "the weight of
evidence is suggestive of carcinogenicity, a concern for potential carcinogenic effects is raised,
but the data are not judged sufficient for a stronger conclusion." Thus, based on these guidelines
and the carcinogenicity data from available animal studies, the WOE descriptor of "Suggestive
Evidence of Carcinogenic Potential" is appropriate for 2-EH for the oral route of exposure.
For the inhalation route of exposure, the cancer WOE for 2-EH is "Inadequate
Information to Assess Carcinogenic Potential" based on the lack of information on
carcinogenicity for this route, as described in Table 18.
Table 18. Cancer WOE Descriptor for 2-Ethylhexanol (CASRN 104-76-7) by Oral
Exposure
Possible WOE Descriptor
Designation
Route of Entry
(oral, inhalation, or both)
Comments
"Carcinogenic to Humans "
NS
NA
No human data are available.
"Likely to Be Carcinogenic to
Humans "
NS
NA
The available animal bioassays do not
support this.
"Suggestive Evidence of
Carcinogenic Potential"
Selected
Oral
In the chronic-duration oral cancer
bioassays of 2-EH in rats and mice
( Astill et aL. 1996b: BASF. 1992a.
1991b). there were significant
dose-related trends for
hepatocellular carcinoma in male
and female mice. The incidence of
hepatocellular carcinoma was
significantly increased in female
mice at the highest dose tested
based on pairwise comparison to
vehicle controls. There was no
evidence of treatment-related
carcinogenicity in male or female
rats.
"Inadequate Information to
Assess Carcinogenic
Potential"
Selected
Inhalation
This descriptor is selected due to
the lack of any information on the
carcinogenicity of 2-EH by
inhalation exposure.
"Not Likely to Be
Carcinogenic to Humans "
NS
NA
The available animal bioassays do not
support this.
2-EH = 2-ethylhexanol; F = female(s); M = male(s); NA = not applicable; NS = not selected; WOE = weight of
evidence.
MODE-OF-ACTION DISCUSSION
The Guidelines for Carcinogenic Risk Assessment (U.S. EPA. 2005) define MO A ".. .as a
sequence of key events and processes, starting with interaction of an agent with a cell,
proceeding through operational and anatomical changes, and resulting in cancer formation."
Examples of possible modes of carcinogenic action for any given chemical include
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"mutagenicity, mitogenesis, programmed cell death, cytotoxicity with reparative cell
proliferation, and immune suppression" (pp. 1-10).
Genotoxicity tests of 2-EH in vitro (mutation, clastogenicity, DNA repair, cell
transformation, and unscheduled DNA synthesis) have produced predominantly negative results
in both bacterial systems and in mammalian cells (see Table 4A). Results were also mostly
negative in a limited number of genotoxicity tests in vivo. No additional data regarding potential
mechanisms of carcinogenicity are available. Thus, a detailed MOA discussion for 2-EH is
precluded.
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES
Derivation of a Provisional Oral Slope Factor
The 18-month carcinogenicity study in mice conducted by Astill et al. (1996b) and BASF
(1991b) was selected as the principal study for the development of a provisional oral slope factor
(p-OSF). This study was conducted in accordance with GLP principles, the results are peer
reviewed, and the study meets the standards of study design and performance with respect to the
number of animals used, the examination of potential toxicity endpoints, and the presentation of
information.
Male and female mice (but not rats) exposed to 2-EH for 18 months (5 days/week)
showed evidence for a carcinogenic response via the oral route of exposure (Astill et al .. 1996b;
BASF. 1991b). Evidence for carcinogenicity in mice includes:
• Significant dose-related trends for hepatocellular carcinoma in female mice based on
time-independent tests (simple Peto and Cochran-Armitage tests performed by the study
authors; p < 0.01 and 0.05, respectively) and in male and female mice based on
time-dependent analyses that consider the relatively high mortality in these groups (Peto
test performed by the study authors; p < 0.05 in males andp < 0.01 in females).
• A significantly increased incidence of hepatocellular carcinomas in female mice at
536 mg/kg-day relative to vehicle controls based on Fisher's exact test performed by the
study authors. The incidence of carcinoma and carcinoma or adenoma (combined) was
not statistically significantly increased in male mice at any dose by pairwise comparison
to vehicle controls.
Because significant trends were observed in both sexes (see Table B-9), the incidences of
hepatocellular carcinomas in both male and female mice were modeled using BMD analysis to
determine the potential POD for the p-OSF. An adenoma was detected in one 536-mg/kg-day
male (and no females). The study authors did not report the results of combined statistical
analysis of hepatocellular adenoma or carcinoma, but the combined incidence in males was
modeled using BMD analysis.
Multistage Cancer models in the U.S. EPA BMDS (Version 2.7) were fit to the male and
female incidence data of the tumor types shown in Table B-9, and modeling results are
summarized in Appendix D. The BMR used was 10% extra risk. The HED in mg/kg-day was
used as the dose metric. To account for group differences in survival, data for tumor incidence
in male and female mice were also modeled using a Poly-3 survival-adjusted number at risk.
The Poly-k approach (Piegorsch and Bailer. 1997; Portier and Bailer. 1989; Bailer and Portier.
1988) is a survival-adjusted quantal-response method to assess neoplastic and non-neoplastic
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lesion prevalence when differences in survival are apparent across dose-groups. This method
scales the denominator (i.e., number of animals per dose group) according to the amount of time
on a study the animals survived. Animals that develop the lesion of interest or survive to the end
of the exposure period are assigned a weight of 1. Animals that do not develop the lesion and die
before the end of the study period are given a weight equal to the fraction of the study period for
which they survived raised to the third power. For each dose group, all the individual animal
weights are summed, and this new value is the survival-adjusted incidence for that dose group.
The Multistage Cancer model (1-degree) provided an adequate fit to the data sets. From the
Multistage Cancer models, predicted BMDs associated with 10% extra risk (BMDio) and their
95% lower confidence limits (BMDLio) are shown in Table 19 (also see Appendix D).
Table 19. BMDio and BMDLio Values from Best Fitting Models for Tumor Data in Mice
Treated Chronically with 2-Ethylhexanol (CASRN 104-76-7) via Gavagea'b
Endpoint
Best Fitting Model
BMDio
(mg/kg-d)
BMDLio
(mg/kg-d)
Potential p-OSF
(mg/kg-d)1
Hepatocellular carcinoma
or adenoma; M
Multistage Cancer (1-degree);
unadjusted
66
31
3.2 x lO-3
Multistage Cancer (1-degree);
Poly-3-adjustedc
49
25
4.0 x 10"3
Hepatocellular carcinoma; F
Multistage Cancer (1-degree);
unadjusted
63
35
2.9 x lO-3
Multistage Cancer (1-degree);
Poly-3-adjusted0
44
27
3.7 x 10-3
aAstill et at (1996b): BASF (1991b).
bModeling results are described in more detail in Appendix D.
°Due to group differences in survival, data for tumor incidence in M and F mice were also modeled using a Poly-3
survival-adjusted number at risk.
BMDio = benchmark dose 10% extra risk; BMDLio = 95% benchmark dose lower confidence limit; F = female(s);
M = male(s); p-OSF = provisional oral slope factor.
Among the modeled tumor types, the lowest POD (BMDLio [FLED] = 25 mg/kg-day) was
obtained in modeling of the observed incidence of hepatocellular carcinoma or adenoma and
Poly-3 weighted number at risk in male mice. The MOA by which 2-EH induces tumors is not
known. In the absence of definitive information, a linear approach is used to obtain the slope
from the POD. The p-OSF of 4.0 x 10 3 (mg/kg-day) 1 was derived as follows:
p-OSF (unadjusted) = 0.1 -^BMDLio (FLED)
= 0.1 ^ 25 mg/kg-day
= 4.0 x 10"3 (mg/kg-day)"1
An adjustment was applied to account for the less-than-lifetime observation period (U.S.
EPA. 1980). The Astill et at. (1996b)/BASF (1991b) bioassay was terminated after 18 months
(compared to the reference mouse lifespan of 2 years) due to early mortality. Thus, due to the
short duration of the study, it cannot be known how an increased duration (i.e., the full 2-year
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lifetime exposure) might have influenced the tumor incidence in the low-dose treated rats.
Therefore, an adjustment factor of (L Le)3 was applied to the unadjusted screening p-OSF,
where L = the lifetime of the animal and Le = the duration of experimental dosing (U.S. HP A.
1980). Using this adjustment, an adjusted screening p-OSF is derived as follows:
p-OSF (adjusted) = p-OSF (unadjusted) x (L Le)3
= 4.0 x 10 3 (mg/kg-day) 1 x (24 months 18 months)3
= 9.5 x 10"3 (mg/kg-day)"1
The adjusted p-OSF for 2-EH should not be used with exposure exceeding the POD
(BMDLio [HED] = 25 mg/kg-day) because above this level the fitted dose-response model better
characterizes what is known about the carcinogenicity of 2-EH.
Derivation of a Provisional Inhalation Unit Risk
There are no data available regarding the carcinogenicity of 2-EH by inhalation exposure,
precluding derivation of a provisional inhalation unit risk (p-IUR) for 2-EH.
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APPENDIX A. PROVISIONAL SCREENING VALUES
No provisional screening values are derived for 2-ethylhexanol.
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APPENDIX B. DATA TABLES
Table B-l. Effects in Dow Wistar Albino Rats Treated with
2-Ethylhexanol (CASRN 104-76-7) in the Diet for 89 or 90 Days3
Male
Parameter
Dose (mg/kg-d)
0
7
36
170
840
Number of animals
10
10
10
10
10
Mortality
2/10b
1/10
0/10
1/10
1/10
Body-weight gain0 (g)
127.8 ±25.30d
143.0 ±34.14°
(+12%)
141.0 ±26.41
(+10%)
113.1 + 42.76
(-12%)
114.6 + 23.15
(-10%)
Terminal body weight0 (g)
289.4 ±40.65
314.3 ±48.39°
(+9%)
309.1 ±29.81
(+7%)
279.1 + 39.37
(-4%)
284.2 + 23.42
(-2%)
Absolute organ weight0 (g)
Liver
9.95 ± 1.11
10.65 ± 1.42
(+7%)
10.43 ± 1.62
(+5%)
9.57+ 1.86
(-4%)
10.96+1.20
(+10%)
Kidney
2.06 ±0.25
2.09 ±0.34
(+1%)
2.15±0.35
(+4%)
2.06 + 0.20
(0%)
2.08 + 0.17
(+1%)
Relative organ weight0 (% BW)
Liver
3.39 ± 0.18
3.41 ± 0.15
(+1%)
3.36 + 0.30
(-1%)
3.41 + 0.24
(+1%)
3.85 + 0.24**
(+14%)
Kidney
0.70 ± 0.04
0.67 ±0.03
(-5%)
0.69 + 0.06
(-1%)
0.74 + 0.06
(+6%)
0.73 + 0.05
(+5%)
Histopathology
Kidney; tubular cloudy
swelling (diffuse)
0/8 (0%)
NE
1/10 (10%)
2/9 (22%)
4/9 (44%)
Liver; cloudy swelling
(diffuse)
0/8 (0%)
NE
1/10 (10%)
2/9 (22%)
3/9 (33%)
Liver; cloudy swelling
(totalf)
5/8 (63%)
NE
5/10 (50%)
6/9 (68%)
6/9 (68%)
Female
Parameter
Dose (mg/kg-d)
0
7
41
190
940
Number of animals
10
10
10
10
10
Mortality
3/10
0/10
2/10
0/10
1/10
Body-weight gain0 (g)
61.1 ± 12.06
63.8 ± 16.86
(+4%)
69.5 + 19.91°
(+14%)
60.1 + 14.75
(-2%)
59.0+18.87
(-3%)
Terminal body weight0 (g)
196.1 ± 13.75
196.4 ± 17.60
(0%)
202.4 + 17.48°
(+3%)
194.9+ 14.70
(-1%)
193.8+ 18.03
(-1%)
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Table B-l. Effects in Dow Wistar Albino Rats Treated with
2-Ethylhexanol (CASRN 104-76-7) in the Diet for 89 or 90 Days"
Absolute organ weight0 (g)
Liver
7.24 ±0.48
7.11 ± 0.55
(-2%)
7.40 ± 1.07
(+2%)
7.24 ±0.75
(0%)
7.93 ±0.63*
(+10%)
Kidney
1.51 ±0.13
1.42 ±0.10
(-6%)
1.42 ±0.39
(-6%)
1.52 ±0.15
(+1%)
1.60 ± 0.13s
(+6%)
Relative organ weight0 (% BW)
Liver
3.69 ±0.13
3.62 ±0.15
(-2%)
3.65 ±0.40
(-1%)
3.71 ±0.22
(+1%)
4.11 ±0.27**
(+11%)
Kidney
0.77 ± 0.04
0.72 ±0.05
(-6%)
0.76 ± 0.07
(-1%)
0.78 ±0.05
(+1%)
0.84 ±0.128
(+9%)
Histopathology
Kidney; tubular cloudy
swelling (diffuse)
0/7 (0%)
NE
1/8 (13%)
4/10 (40%)
6/9* (68%)
Liver; cloudy swelling
(diffuse)
0/7 (0%)
NE
0/8 (0%)
0/10 (0%)
6/9* (68%)
Liver; cloudy swelling
(totalf)
1/7 (14%)
NE
3/8 (38%)
3/10 (30%)
9/9* (100%)
"Mellon Institute of Industrial Research (1960).
bNumber affected/number examined.
°Means (for absolute weights and body-weight gain) and SDs (body-weight gain and all organ weights) were
calculated by the U.S. EPA for the purposes of this PPRTV assessment based on individual animal data provided in
the study report.
dValues represent means ± SD (surviving animals).
"Uncertainty is associated with this value owing to illegibility of individual data value(s) in the study report.
fTotal = sum of diffuse + focal.
g« = 8; 1 value beyond 2 SDs from the mean was excluded from analyses.
*p < 0.05 based on statistics performed by study authors.
**p < 0.01.
BW = body weight; NE = not examined; SD = standard deviation.
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Table B-2. Effects in Male F344 Rats Treated with 2-Ethylhexanol (CASRN 104-76-7) via
Gavage for 3 Months3
Parameter
Dose (mg/kg-d)
0
18
89.3
179
357
Number of animals
10
10
10
10
10
Terminal body weight (g)
279.4 ± 10.85b
281.0 ± 17.61
(+1%)
277.2 ± 16.32
(-1%)
270.9 ±6.32
(-3%)
256.4+ 11.21**
(-8%)
Reticulocytes (% 10 3 RBCs);
84 d
20 ±2
21 ± 3
(+5%)
20 ±3
(0%)
19 ±4
(-5%)
25 + 3**
(+25%)
Clinical chemistry; 84 d
Total protein (g/L)
71.90 ±5.23
74.21 ±4.58
(+3%)
73.40 ±6.32
(+2%)
73.28 ±4.70
(+2%)
81.34 + 6.52**
(+13%)
Albumin (g/L)
40.13 ±2.12
41.61 ±2.42
(+4%)
41.32 ±2.16
(+3%)
41.73 ±2.03
(+4%)
46.69 + 3.02**
(+16%)
Absolute organ weight (g)
Stomach
1.59 ±0.10
1.57 ±0.10
(-1%)
1.54 ±0.06
(-3%)
1.58 ±0.07
(-1%)
1.62 + 0.07
(+2%)
Liver
7.74 ±0.57
7.94 ±0.77
(+3%)
7.95 ± 0.66
(+3%)
8.07 ±0.27
(+4%)
9.17 + 0.85**
(+18%)
Kidney
1.94 ±0.08
1.99 ±0.13
(+3%)
1.98 ±0.10
(+2%)
2.04±0.07
(+5%)
2.07 + 0.13*
(+7%)
Testes
3.10 ± 0.10
3.07 ±0.16
(-1%)
3.11 ± 0.11
(0%)
3.13 + 0.07
(+1%)
3.00 + 0.17
(-3%)
Relative organ weight (% BW)
Stomach
0.57 ±0.04
0.56 ±0.03
(-2%)
0.56 ±0.04
(-2%)
0.59 + 0.03
(+4%)
0.63 + 0.02**
(+11%)
Liver
2.77 ±0.11
2.82 ±0.12
(+2%)
2.86 ±0.10
(+3%)
2.98 + 0.08**
(+8%)
3.57 + 0.22**
(+29%)
Kidney
0.70 ± 0.02
0.71 ±0.02
(+1%)
0.71 ±0.03
(+1%)
0.75 + 0.03*
(+7%)
0.81 + 0.04**
(+16%)
Testes
1.11 ±0.05
1.09 ±0.04
(-2%)
1.13 ±0.07
(+2%)
1.16 + 0.03
(+5%)
1.17 + 0.06*
(+5%)
Histopathology
Forestomach; acanthosis0
0/10d (0%)
0/10 (0%)
0/10 (0%)
0/10 (0%)
2/10 (20%)
Mean pCoA activity
(nmol/min-mg protein)6
3.38 ± 1.11
4.49 ±0.78
(+33%)
5.21 ±0.70
(+54%)
6.24+1.83
(+85%)
22.0 + 0.56*
(+551%)
"Astill et at (1996a): BASF (1991a).
bValues represent means ± SD.
°Sum acanthosis (focal or multifocal) and acanthosis (diffuse).
dNumber affected/number examined.
"Peroxisome proliferation-only group; n = 3.
*p < 0.05 based on statistics performed by the study authors.
**p < 0.01.
BW = body weight; pCoA = palmitoyl-Coenzyme A; RBC = red blood cell; SD = standard deviation.
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Table B-3. Effects in Female F344 Rats Treated with 2-Ethylhexanol (CASRN 104-76-7)
via Gavage for 3 Months"
Parameter
Dose (mg/kg-d)
0
18
89.3
179
357
Number of animals
10
10
10
10
10
Terminal body weight (g)
167.0 ± 4.96b
165.8 ±8.73c
(-1%)
162.2 ±9.91
(-3%)
158.7 ±8.41
(-5%)
155.1 + 6.61**
("7%)
Reticulocytes (% 10 3 RBCs); 84 d
14 ±2
14 ± 3 (0%)
14 ± 3 (0%)
14 ± 2 (0%)
17 + 5* (+21%)
Clinical chemistry; 84 d
Total protein (g/L)
69.37 ±4.03
73.10 ±6.03
(+5%)
68.59 ±3.40
(-1%)
69.77 ±4.52
(+1%)
71.33+2.73
(+3%)
Albumin (g/L)
41.17 ±2.22
43.11 ±3.12
(+5%)
41.05 ± 1.44
(0%)
41.78 ±2.06
(+1%)
43.14+1.44
(+5%)
Absolute organ weight (g)
Stomach
1.19 ±0.07
1.17 ±0.05
(-2%)
1.18 ±0.07
(-1%)
1.19 ±0.06
(0%)
1.27 + 0.04**
(+7%)
Liver
4.47 ±0.28
4.49 ±0.34
(0)
4.40 ± 0.27
(-2%)
4.57 ±0.22
(+2%)
4.76 + 0.16*
(+6%)
Kidney
1.29 ±0.06
1.28 ±0.04
(-1%)
1.26 ±0.07
(-2%)
1.28 ±0.05
(-1%)
1.26 + 0.05
(-2%)
Relative organ weight (% BW)
Stomach
0.71 ±0.03
0.71 ±0.02
(0%)
0.73 ± 0.04
(+3%)
0.75 ±0.03*
(+6%)
0.82 + 0.04**
(+15%)
Liver
2.67 ±0.11
2.71 ±0.09
(+1%)
2.72 ±0.10
(+2%)
2.88 ±0.08**
(+8%)
3.07 + 0.07**
(+15%)
Kidney
0.77 ±0.02
0.77 ±0.03
(0%)
0.78 ±0.03
(+1%)
0.81 ±0.03*
(+5%)
0.82 + 0.03**
(+6%)
Histopathology
Forestomach; acanthosis, focal or
multifocal
0/10d (0%)
0/10 (0%)
0/10 (0%)
0/10 (0%)
5/10f (50%)
Mean pCoA activity (nmol/min-mg
protein)6
2.95 ±2.36
4.54 ±0.69
(+54%)
5.01 ±0.74
(+70%)
6.6±1.43
(+124%)
9.92 + 2.26**
(+236%)
"Astill et at (1996a): BASF (1991a).
bValues represent means ± SD.
°SD not clearly legible in the study report.
dNumber affected/number examined.
"Peroxisome proliferation-only group; n = 3.
*p < 0.05 based on statistics performed by the study authors.
**p < 0.01.
tp < 0.05 based on Fisher's exact test performed by the U.S. EPA for the purposes of this PPRTV assessment.
BW = body weight; pCoA = palmitoyl-Coenzyme A; RBC = red blood cell; SD = standard deviation.
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Table B-4. Effects in B6C3Fi Mice Treated with 2-Ethylhexanol (CASRN 104-76-7) via
Gavage for 3 Months3
Parameter
Dose (mg/kg-d)
0
18
89.3
179
357
Male
Number of surviving animals
10
10
10
10
10
Terminal body weight (g)
26.95 ±2.02
26.26 ± 1.43
(-3%)
26.22 ± 1.58
(-3%)
25.80 ±2.31
(-4%)
26.13 ± 1.76
(-3%)
Absolute organ weight (g)
Liver
1.09 ± 0.06b
1.09 ±0.08
(0%)
1.14 ±0.08
(+5%)
1.12 ± 0.10
(+3%)
1.13 ±0.08
(+4%)
Kidney
0.48 ±0.05
0.50 ±0.03
(+4%)
0.51 ±0.03
(+6%)
0.49 ±0.04
(+2%)
0.46 ±0.03
(-4%)
Stomach
0.21 ±0.01
0.20 ± 0.02
(-5%)
0.21 ±0.02
(0%)
0.23 ±0.05
(+10%)
0.24 ±0.03
(+14%)
Relative organ weight (% BW)
Liver
4.07 ±0.16
4.15 ±0.14
(+2%)
4.36 ±0.33*
(+7%)
4.36 ±0.24*
(+7%)
4.31±0.24
(+6%)
Kidney
1.78 ±0.10
1.91 ±0.10*
(+7%)
1.93 ±0.11*
(+8%)
1.90 ±0.15
(+7%)
1.75 + 0.10
(-2%)
Stomach
0.79 ±0.07
0.77 ± 0.07
(-3%)
0.81 ±0.09
(+3%)
0.90 ±0.13*
(+14%)
0.89 + 0.10*
(+13%)
Histopathology
Forestomach; acanthosis, focal
or multifocal
0/10° (0%)
0/10 (0%)
0/10 (0%)
0/10 (0%)
2/10 (20%)
Female
Number of surviving animals
10
10
10
9d
10
Terminal body weight (g)
21.11 ± 1.50
21.92 ± 1.16
(+4%)
20.80 ± 1.26
(-1%)
21.42 ± 1.16
(+1%)
20.77+ 1.72
(-2%)
Absolute organ weight (g)
Liver
1.03 ±0.06
1.08 ±0.09
(+5%)
1.06 ±0.06
(+3%)
1.05 ±0.05
(+2%)
1.03 + 0.12
(0%)
Kidney
0.35 ±0.03
0.37 ±0.02
(+6%)
0.35 ±0.02
(0%)
0.35 ±0.02
(0%)
0.35 + 0.02
(0%)
Stomach
0.24 ±0.03
0.24 ±0.03
(0%)
0.23 ±0.03
(-4%)
0.23 ±0.02
(-4%)
0.23 + 0.02
(-4%)
Relative organ weight (% BW)
Liver
4.89 ±0.22
4.91 ±0.26
(0%)
5.09 ±0.24
(+4%)
4.90 ±0.21
(0%)
4.97 + 0.33
(+2%)
Kidney
1.67 ±0.09
1.66 ± 0.10e
(-1%)
1.69 ±0.07
(+1%)
1.66 ±0.05
(-1%)
1.71 + 0.08
(+2%)
Stomach
1.13 ± 0.13
1.07 ±0.12
(-5%)
1.13 ± 0.18
(0%)
1.11 ± 0.13
(-2%)
1.13 + 0.10
(0%)
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Table B-4. Effects in B6C3Fi Mice Treated with 2-Ethylhexanol (CASRN 104-76-7) via
Gavage for 3 Months3
Dose (mg/kg-d)
Parameter
0
18
89.3
179
357
Histopathology
Forestomach; acanthosis, focal
0/10 (0%)
0/10 (0%)
0/10 (0%)
0/10 (0%)
1/10 (10%)
or multifocal
aAstill et at (1996a): BASF (19916).
bValues represent means ± SD based on data for surviving animals.
°Number affected/number examined.
dOne female mouse died after 90 days of exposure from liver damage after hemorrhage into one ovarian pouch.
en = 9. No explanation was given but based on data for individual animals provided in the study report, 1 value
was likely regarded as an outlier (more than 2 SDs lower than the mean).
*p < 0.05 based on statistics performed by the study authors.
**p < 0.01.
BW = body weight; SD = standard deviation.
Table B-5. Select Non-neoplastic Effects in Male F344 Rats Treated with
2-Ethylhexanol (CASRN 104-76-7) via Gavage for 24 Months"
Parameter
Dose (mg/kg-d)
0 (water)
0 (vehicle)
36
107
357
Number of animals
50
50
50
50
50
Mortality
16/50b (32%)
17/50 (34%)
23/50 (46%)
16/50 (32%)
19/50 (38%)
Clinical signs
Poor general condition
7/50 (14%)
12/50 (24%)
14/50 (28%)
15/50 (30%)
14/50 (28%)
Labored breathing
2/50 (4%)
1/50 (2%)
1/50 (2%)
1/50 (2%)
4/50 (8%)
Terminal body weight (g)
349.7 ±27.5C
368.1 ±30.4
349.5 ±28.2*
(-5%)
328.8 ±26.9**
("11%)
282.9 ±24.0**
(-23%)
Body-weight gain (g)
247.6 ± 27.5
266.5 ± 30.7
245.2 ±28.0**
(-8%)
223.7 ±26.2**
(-16%)
178.7 ±23.4**
(-33%)
Gross lesions
Lung; foci
5/50 (10%)
4/50 (8%)
7/50 (14%)
6/50 (12%)
13/50"f* (26%)
Absolute organ weight (g)
Stomach
2.03 ±0.14
2.06 ±0.13
1.96 ±0.16*
(-5%)
1.96 ±0.13*
(-5%)
1.91 ± 0.13**
("7%)
Liver
12.43 ± 2.44
12.54 ±2.05
11.67 ±2.09
("7%)
12.53 ±3.03
(0%)
10.56 ± 1.57**
(-16%)
Kidney
2.98 ±2.48
2.67 ±0.25
2.66 ±0.23
(0%)
2.66 ±0.25
(0%)
2.54 ±0.24
(-5%)
Brain
2.08 ±0.04
2.10 ±0.04
2.07 ± 0.06
(-1%)
2.06 ±0.07*
(-2%)
1.97 ±0.06**
(-6%)
Testes
5.01 ± 1.90
4.85 ± 1.73
4.10 ± 1.41
4.40 ± 1.53
4.58 ± 1.35
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Table B-5. Select Non-neoplastic Effects in Male F344 Rats Treated with
2-Ethylhexanol (CASRN 104-76-7) via Gavage for 24 Months"
Parameter
Dose (mg/kg-d)
0 (water)
0 (vehicle)
36
107
357
(-15%)
(-9%)
(-6%)
Relative organ weight (to BW)
Stomach
0.60 ±0.04
0.58 ±0.04
0.59 ±0.05
(+2%)
0.62 ±0.04**
(+7%)
0.70 + 0.07**
(+21%)
Liver
3.69 ±0.83
3.53 ±0.42
3.47 ±0.50
(-2%)
3.96 ±0.95*
(+12%)
3.85 + 0.47
(+9%)
Kidney
0.89 ±0.78
0.76 ±0.05
0.80 ±0.09
(+5%)
0.85 ±0.10**
(+12%)
0.93 + 0.09**
(+22%)
Brain
0.62 ±0.06
0.60 ±0.05
0.62 ± 0.06
(+3%)
0.66 ±0.06**
(+10%)
0.72 + 0.06**
(+20%)
Testes
1.47 ±0.53
1.38 ±0.48
1.22 ±0.42
(-12%)
1.39±0.45
(+1%)
1.66 + 0.42*
(+20%)
Histopathology
Liver; congestion
6/50 (12%)
5/50 (10%)
7/50 (14%)
4/50 (8%)
14/50* (28%)
Lung; congestion
7/50 (14%)
5/50 (10%)
4/50 (8%)
4/50 (8%)
11/50* (22%)
Lung; bronchopneumonia
4/50 (8%)
5/50 (10%)
2/50 (4%)
2/50 (4%)
14/50* (28%)
Spleen; hemosiderin
30/50 (60%)
32/50 (64%)
12/50 (24%)
5/50 (10%)
36/50 (72%)
Mesenteric lymph nodes;
hyperplasia
37/50 (74%)
35/50 (70%)
9/50 (18%)
6/50 (12%)
39/50 (78%)
Mandibular lymph nodes;
hyperplasia
39/50 (78%)
38/50 (76%)
9/50 (18%)
8/50 (16%)
42/50 (84%)
Kidney; congestion
4/50 (8%)
5/50 (10%)
7/50 (14%)
4/50 (8%)
6/50 (12%)
Prostate; atrophy
35/50 (70%)
28/50 (52%)
34/50 (68%)
40/50 (80%)
36/50* (72%)
aAstill et at (1996b): BASF (1992a).
bNumber affected/number examined.
°Values represent means ± SD.
*p < 0.05 compared to the vehicle-only control group based on statistics performed by the study authors.
**p < 0.01.
tp < 0.05 compared to vehicle-only control based on Fisher's exact test performed by the U.S. EPA for the
purposes of this PPRTV assessment.
BW = body weight; SD = standard deviation.
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Table B-6. Select Non-neoplastic Effects in Female F344 Rats Treated with
2-Ethylhexanol (CASRN 104-76-7) via Gavage for 24 Months"
Parameter
Dose (mg/kg-d)
0 (water)
0 (vehicle)
36
107
357
Number of animals
50
50
50
50
50
Mortality
8/50 (16%)
14/50 (28%)
14/50 (28%)
13/50 (26%)
26/50f (52%)
Clinical signs
Poor general condition
3/50 (6%)
8/50 (16%)
7/50 (14%)
11/50 (22%)
21/50ff (42%)
Labored breathing
0/50 (0%)
3/50 (6%)
3/50 (6%)
5/50 (10%)
12/50f (24%)
Terminal body weight (g)
261.3 ± 19.8b
259.7 ±27.1
252.1 ± 15.1
(-3%)
236.2 ±24.7**
(-9%)
205.6 ±21.4**
(-21%)
Body-weight gain (g)
181.0 ± 18.7
177.5 ± 27.0
170.5 ± 14.8
(-4%)
155.9 ±23.6**
(-12%)
123.3 ±22.2**
(-31%)
Gross lesions
Lung; foci
3/50 (6%)
7/50 (14%)
4/50 (8%)
9/50 (18%)
15/50f (30%)
Absolute organ weight (g)
Stomach
1.66 ±0.10
1.64 ±0.13
1.71 ± 0.11
(+4%)
1.64 ±0.16
(0%)
1.59 ±0.09
(-3%)
Liver
9.27 ± 1.32
8.74 ± 1.46
9.00 ±0.99
(+3%)
8.93 ± 1.56
(+2%)
7.97 ± 1.07
(-9%)
Kidney
2.04 ±0.16
2.03 ±0.21
2.03 ±0.12
(0%)
2.01 ±0.18
(-1%)
1.89 ± 0.11**
("7%)
Brain
1.92 ±0.05
1.90 ±0.05
1.91 ±0.04
(+1%)
1.89 ±0.05
(-1%)
1.82 ±0.05**
(-4%)
Relative organ weight (to BW)
Stomach
0.66 ±0.05
0.66 ±0.06
0.70 ±0.05*
(+6%)
0.72 ±0.08**
(+9%)
0.79 ±0.08**
(+20%)
Liver
3.69 ±0.52
3.49 ±0.53
3.68 ±0.35
(+5%)
3.89 ±0.60**
(+11%)
3.94 ±0.28**
(+13%)
Kidney
0.81 ±0.07
0.82 ±0.13
0.83 ±0.05
(+1%)
0.88 ±0.08**
(+7%)
0.94 ±0.08**
(+15%)
Brain
0.77 ± 0.07
0.77 ±0.09
0.78 ±0.05
(+1%)
0.84 ±0.11**
(+9%)
0.91 ±0.09**
(+18%)
Histopathology
Liver; congestion
2/50 (4%)
4/50 (8%)
7/50 (14%)
6/50 (12%)
23/50* (46%)
Lung; congestion
1/50 (2%)
2/50 (4%)
4/50 (8%)
6/50 (12%)
18/50** (36%)
Lung; bronchopneumonia
0/50 (0%)
3/50 (6%)
2/50 (4%)
4/50 (8%)
15/50** (30%)
Spleen; hemosiderin
35/50(70%)
36/50 (72%)
7/50 (14%)
8/50 (16%)
44/50* (88%)
Mesenteric lymph nodes;
hyperplasia
38/50 (76%)
40/50 (80%)
11/50 (22%)
7/50 (14%)
46/50* (92%)
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Table B-6. Select Non-neoplastic Effects in Female F344 Rats Treated with
2-Ethylhexanol (CASRN 104-76-7) via Gavage for 24 Months"
Parameter
Dose (mg/kg-d)
0 (water)
0 (vehicle)
36
107
357
Mandibular lymph nodes;
hyperplasia
40/50° (80%)
42/50 (84%)
10/50 (20%)
10/50 (20%)
47/50* (94%)
Kidney; congestion
1/50 (2%)
4/50 (8%)
6/50 (12%)
4/50 (8%)
16/50** (32%)
aAstill et at (1996b): BASF (1992a).
bValues represent means ± SD.
°Number affected/number examined.
*p < 0.05 compared to the vehicle-only control group based on statistics performed by the study authors.
**p < 0.01.
tp < 0.05 compared to vehicle-only control based on Fisher's exact test performed by the U.S. EPA for the
purposes of this PPRTV assessment,
ff/? <0.01.
BW = body weight; SD = standard deviation.
Table B-7. Select Non-neoplastic Effects in Male B6C3Fi Mice Treated with
2-Ethylhexanol (CASRN 104-76-7) via Gavage for 18 Months"
Parameter
Dose (mg/kg-d)
0 (water)
0 (vehicle)
36
143
536
Number of animals
50
50
50
50
50
Mortality
2/50b (4%)
2/50 (4%)
2/50 (4%)
2/50 (4%)
15/50ff (30%)
Food consumption
(g/animal-d)°
5.6 ± 0.7d
5.6 ±0.7
5.4 ±0.6
("4%)
5.5 ±0.6
(-2%)
5.1 ±0.6f
(-9%)
Terminal body weight (g)
42.6 ±4.1
42.7 ±3.5
40.9 ±4.0
("4%)
40.7 ±4.5*
(-5%)
37.4 ± 3.0**
(-12%)
Body-weight gain (g)
18.9 ±4.0
19.4 ±3.4
17.8 ±4.0
(-8%)
17.6 ±4.0*
(-9%)
14.3 ±2.8**
(-26%)
Neutrophils (%); 18 mo
18.00 ±5.94
20.54 ±7.57
19.19 ±6.32
("7%)
22.29 ±9.12
(+9%)
26.86 ± 13.50ft
(+31%)
Lymphocytes (%); 18 mo
78.88 ±6.96
76.60 ±7.58
77.79 ± 6.24
(+2%)
74.19 ±8.14e
(-3%)
70.57 ± 13.19ff
(-8%)
Absolute organ weight (g)
Stomach
0.32 ±0.04
0.33 ±0.03
0.31 ±0.04
(-6%)
0.33 ±0.05
(0%)
0.34 ±0.04
(+3%)
Liver
1.59 ±0.60
1.61 ±0.60
1.47 ±0.40
(-9%)
1.61 ±0.46
(0%)
1.61 ±0.38
(0%)
Kidney
0.76 ±0.09
0.76 ±0.06
0.75 ±0.09
(-1%)
0.71 ±0.10**
("7%)
0.62 ±0.06**
(-18%)
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Table B-7. Select Non-neoplastic Effects in Male B6C3Fi Mice Treated with
2-Ethylhexanol (CASRN 104-76-7) via Gavage for 18 Months3
Parameter
Dose (mg/kg-d)
0 (water)
0 (vehicle)
36
143
536
Brain
0.50 ±0.02
0.50 ±0.02
0.50 ±0.02
(0%)
0.49 ±0.02*
(-2%)
0.48 ±0.02**
("4%)
Testes
0.24 ± 0.02
0.23 ±0.02
0.24 ±0.02
(+4%)
0.23 ± 0.02
(0%)
0.23 ±0.02
(0%)
Relative organ weight (to BW)
Stomach
0.85 ±0.08
0.86 ±0.09
0.84 ±0.09
(-2%)
0.89 ±0.13
(+3%)
1.0 ±0.13**
(+16%)
Liver
4.30 ±2.01
4.30 ± 1.87
4.01 ± 1.21
("7%)
4.42 ± 1.29
(+3%)
4.77 ± 1.25
(+11%)
Kidney
2.04 ±0.17
2.01 ±0.21
2.04 ±0.20
(+1%)
1.93 ±0.18
("4%)
1.84 ±0.15**
(-8%)
Brain
1.35 ±0.13
1.33 ±0.12
1.38 ± 0.14
(+4%)
1.36 ±0.13
(+2%)
1.43 ±0.11**
(+8%)
Testes
0.63 ± 0.06
0.61 ±0.06
0.65 ±0.05**
(+7%)
0.64 ±0.06*
(+5%)
0.69 ±0.04**
(+13%)
Histopathology
Lung; congestion
1/50 (2%)
1/50 (2%)
1/50 (2%)
2/50 (4%)
9/50** (18%)
Liver; congestion
1/50 (2%)
0/50 (0%)
0/50 (0%)
0/50 (0%)
7/50** (14%)
Liver; peripheral fatty
infiltration
0/50 (0%)
0/50 (0%)
0/50 (0%)
1/50 (2%)
31/50** (62%)
Liver; basophilic foci
4/50 (8%)
4/50 (8%)
5/50 (10%)
12/50* (24%)
6/50 (12%)
Liver; focal hyperplasia
2/50 (4%)
7/50 (14%)
4/50 (8%)
9/50 (18%)
10/50 (20%)
Forestomach; focal
hyperplasia
0/50 (0%)
1/50 (2%)
1/50 (2%)
1/50 (2%)
5/50 (10%)
aAstill et at (1996b): BASF (1991b).
bNumber affected/number examined.
Tor this endpoint, means and SDs for the entire study period were calculated by the U.S. EPA for the purposes of
this PPRTV assessment based on data for individual time points provided in the study report.
dValues represent means ± SD.
"Value not clearly legible in the study report (BASF. 1991b).
*p < 0.05 compared to the vehicle-only control group based on statistics performed by the study authors.
**p < 0.01.
tp < 0.05 compared to vehicle-only control based on Fisher's exact test (for categorical data) or Student's Mest (for
continuous data) performed by the U.S. EPA for the purposes of this PPRTV assessment,
ff/? < 0.01.
BW = body weight; SD = standard deviation.
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Table B-8. Select Non-neoplastic Effects in Female B6C3Fi Mice Treated with
2-Ethylhexanol (CASRN 104-76-7) via Gavage for 18 Months"
Parameter
Dose (mg/kg-d)
0 (water)
0 (vehicle)
36
143
536
Number of animals
50
50
50
50
50
Mortality
2/50b (4%)
4/50 (8%)
2/50 (4%)
4/50 (8%)
15/50ff (30%)
Food consumption (g/animal-d)°
6.6 ± 0.8d
6.3 ±0.7
6.6 ±0.7
(+5%)
6.2 ±0.8
(-2%)
5.5 ± 0.5ft
(-13%)
Terminal body weight (g)
42.0 ±6.9
41.1 ±5.6
40.6 ±5.8
(-1%)
39.9 ±5.4
(-3%)
35.5 ±4.2**
(-14%)
Body-weight gain (g)
22.6 ±6.8
21.8 ±5.3
21.1 ±5.4
(-3%)
20.4 ±5.4
(-6%)
16.5 ±4.0**
(-24%)
Neutrophils (%); 18 mo
20.40 ± 7.09
22.64 ± 10.74
22.65 ± 8.40
(0%)
21.85 ±7.60
(-3%)
25.11 ±8.96
(+11%)
Lymphocytes (%); 18 mo
77.10 ±7.48
74.87 ± 11.62
74.92 ± 8.64
(0%)
75.77 ±8.39
(+1%)
72.09 ± 9.68
(-4%)
Absolute organ weight (g)
Stomach
0.34 ±0.04
0.34 ±0.04
0.34 ±0.04
(0%)
0.34 ±0.04
(0%)
0.34 ±0.04
(0%)
Liver
1.38 ± 0.15
1.37 ± 0.15
1.37 ±0.21
(0%)
1.42 ±0.32
(+4%)
1.40 ±0.15
(+2%)
Kidney
0.48 ±0.04
0.47 ±0.04
0.46 ±0.03
(-2%)
0.45 ± 0.04
(-4%)
0.44 ±0.04**
(-6%)
Brain
0.51 ±0.02
0.51 ±0.02
0.51 ±0.01
(0%)
0.50 ±0.02**
(-2%)
0.48 ±0.02**
(-6%)
Relative organ weight (to BW)
Stomach
0.91 ±0.18
0.94 ±0.15
0.94 ±0.15
(0%)
0.97 ±0.15
(+3%)
1.11 ± 0.11**
(+18%)
Liver
3.67 ±0.49
3.75 ±0.54
3.79 ±0.53
(+1%)
4.08 ± 1.48
(+9%)
4.54 ±0.28**
(+21%)
Kidney
1.27 ±0.20
1.28 ±0.20
1.28 ±0.18
(0%)
1.28 ±0.17
(0%)
1.44 ±0.16**
(+13%)
Brain
1.37 ±0.25
1.41 ±0.22
1.43 ±0.23
(+1%)
1.42 ±0.19
(+1%)
1.57 ±0.21**
(+11%)
Histopathology
Lung; congestion
1/50 (2%)
2/50 (4%)
0/50 (0%)
1/50 (2%)
10/50* (20%)
Liver; congestion
1/50 (2%)
0/50 (0%)
0/50 (0%)
3/50 (6%)
2/50 (4%)
Liver; peripheral fatty
infiltration
0/50 (0%)
1/50 (2%)
0/50 (0%)
3/50 (6%)
22/50** (44%)
Liver; basophilic foci
2/50 (4%)
1/50 (2%)
2/50 (4%)
4/50 (8%)
6/50*-e (12%)
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Table B-8. Select Non-neoplastic Effects in Female B6C3Fi Mice Treated with
2-Ethylhexanol (CASRN 104-76-7) via Gavage for 18 Months"
Parameter
Dose (mg/kg-d)
0 (water)
0 (vehicle)
36
143
536
Liver; focal hyperplasia
1/50 (2%)
0/50 (0%)
3/50 (6%)
4/50*-e (8%)
1/50 (2%)
Forestomach; focal
hyperplasia
1/50 (2%)
1/50 (2%)
0/50 (0%)
1/50 (2%)
4/50 (8%)
aAstill et at (1996b): BASF (1991b).
bNumber affected/number examined.
Tor this endpoint, means and SDs for the entire study period were calculated by the U.S. EPA for the purposes of
this PPRTV assessment based on data for individual time points provided in the study report.
dValues represent means ± SD.
"Statistical significance reported by the study authors not confirmed by Fisher's exact test performed by the
U.S. EPA for the purposes of this PPRTV assessment (one-tailed, p > 0.05).
*p < 0.05 compared to the vehicle-only control group based on statistics performed by the study authors.
**p < 0.01.
tp < 0.05 compared to vehicle-only control based on Fisher's exact test (for categorical data) or Student's /-test (for
continuous data) performed by the U.S. EPA for the purposes of this PPRTV assessment,
ff/? < 0.01.
BW = body weight; PPRTV = provisional peer-reviewed toxicity value; SD = standard deviation.
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Table B-9. Cancer Effects in Male and Female B6C3Fi Mice Treated with
2-Ethylhexanol (CASRN 104-76-7) via Gavage for 18 Months"
Male
ADD (HED), mg/kg-d
0 (water)
0 (vehicle)
36 (5.5)
143 (21.8)
536 (79.9)
Number of animals
50
50
50
50
50
Hepatocellular adenoma
0/50b (0%)
0/50 (0%)
0/50 (0%)
0/50 (0%)
1/50 (2%)
Hepatocellular carcinoma
4/50 (8%)
6/50° (12%)
3/50 (6%)
7/50 (14%)
9/50 (18%)
Hepatocellular adenoma or carcinoma
4/50 (8%)
6/50 (12%)
3/50 (6%)
7/50 (14%)
10/50d (20%)
Female
ADD (HED), mg/kg-d
0 (water)
0 (vehicle)
36 (5.3)
143 (21.1)
536 (77.3)
Number of animals
50
50
50
50
50
Hepatocellular adenoma
0/50 (0%)
0/50 (0%)
0/50 (0%)
0/50 (0%)
0/50 (0%)
Hepatocellular carcinoma
1/50 (2%)
0/50°-e (0%)
1/50 (2%)
3/50 (6%)
5/50* (10%)
Hepatocellular adenoma or carcinoma
1/50 (2%)
0/50 (0%)
1/50 (2%)
3/50 (6%)
5/50* (10%)
"Astill et at (1996b): BASF (1991b).
bNumber affected/number examined.
Significant (p < 0.05) trend based on time-dependent test (Peto test) performed by the study authors.
dBased on statistical analyses (Fisher's exact test) performed by the U.S. EPA for the purposes of this PPRTV
assessment, the combined incidence of carcinoma or adenoma was not significantly increased in males treated at
536 mg/kg-day compared to vehicle-only controls.
"Significant (p < 0.05) trend based on time-independent tests (Cochran-Armitage and Peto tests) performed by the
study authors.
*p < 0.05 compared to the vehicle-only control group based on Fisher's exact test performed by the study authors.
ADD = adjusted daily dose; HED = human equivalent dose.
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Table B-10. Effects in Wistar Rats Exposed to 2-Ethylhexanol (CASRN 104-76-7) via
Gavage on GDs 6-15a
Parameter
Dose (mg/kg-d)
0 (water)
0 (vehicle)
130
650
1,300
Maternal Effects
Number of dams
10
10
10
10
10
Mortality
0/10b (0%)
0/10 (0%)
0/10 (0%)
1/10 (10%)
6/10 (60%) f
Food consumption;
GDs 6-15 (g/animal-d)
23.6 ± 1.51°
23.9 ± 1.98
23.2 ± 1.14
(-3%)
21.7 ± 1.91
(-9%)
13.3 ±4.42**
(-44%)
Maternal body weight;
GD 15 (g)
303.0 ± 16.34
309.0 ± 15.96
303.8 ± 17.32
(-2%)
296.3 ±7.11
("4%)
261.3 ±48.29*
(-15%)
Maternal body weight;
GD 20 (g)
375.0 ± 19.90
384.2 ±25.99
377.4 ±23.81
(-2%)
367.1 ± 12.84
("4%)
308.9 ±63.25**
(-20%)
Maternal body-weight gain;
GDs 6-15 (g)
48.6 ±3.33
51.6 ±3.40
44.1 ± 18.20
(-15%)
43.6±5.25ff
(-16%)
-3.1 ±50.26**
(-106%)
Maternal body-weight gain;
GDs 0-20 (g)
143.5 ±7.90
153.4 ± 13.70
147.8 ±24.14
("4%)
140.5 ± 15.lld
(-8%)
73.6 ±61.60**
(-52%)
Maternal net weight change
from GD 6e
42.9 ±3.8
44.7 ±6.19
45.4 ± 16.03
38.4 ±5.84
11.6 ±27.52*
Gravid uterine weight (g)
77.7 ±9.15
82.2 ± 10.87
72.2 ± 17.14
(-12%)
75.9 ±9.80
(-8%)
32.9 ± 37.64**
(-60%)
Dams with viable fetuses
10/10 (100%)
10/10 (100%)
10/10 (100%)
9/9 (100%)
2/4 (50%)
% Postimplantation loss
8.2 ±6.07
7.0 ±7.59
5.0 ±7.12
(-28%)
4.5 ±4.56
(-36%)
54.7 ± 52.88**
(+680%)
Resorptions
1.2 ±0.97
1.1 ± 1.20
0.6 ±0.94
(-45%)
0.7 ±0.71
(-36%)
7.8 ±7.50**
(+609%)
Fetal Effects
Number of litters
9
10
10
9
2
Fetal body weight on GD 20
(g)
3.80 ±0.324
3.82 ±0.177
3.80 ±0.249
(-1%)
3.44 ±0.227*
(-10%)
2.86± 0.333**
(-25%)
Fetal body weights; M (g)
3.88 ± 0.350f
3.92 ±0.211
3.90 ±0.273
(-1%)
3.50 ±0.222*
("11%)
2.87 ±0.314**
(-27%)
Fetal body weights; F (g)
3.70 ±0.297
3.70 ±0.137
3.70 ±0.228
(0%)
3.36 ±0.267
(-9%)
2.85 ±0.363**
(-23%)
Visceral variations
Fetal incidence
23/59 (39%)
29/71 (41%)
18/62 (29%)
22/62 (36%)
10/13 (77%)*
Litter incidence
6/9 (67%)
9/10 (90%)
8/10 (80%)
8/9 (89%)
2/2 (100%)
Skeletal malformations
Fetal incidence
1/65 (2%)
2/75 (3%)
2/68 (3%)
6/65 (9%)
4/15
(27%)**
Litter incidence
1/9(11%)
2/10 (20%)
2/10 (20%)
3/9 (33%)
2/2 (100%)
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Table B-10. Effects in Wistar Rats Exposed to 2-Ethylhexanol (CASRN 104-76-7) via
Gavage on GDs 6-15a
Dose (mg/kg-d)
Parameter
0 (water)
0 (vehicle)
130
650
1,300
Skeletal variations
Fetal incidence
23/65 (35%)
17/75 (23%)
23/68 (34%)
27/65*
10/15**
(42%)
(67%)
Litter incidence
7/9 (78%)
6/10 (60%)
8/10 (80%)
7/9 (78%)
2/2 (100%)
Skeletal retardations
Fetal incidence
28/65 (43%)
38/75 (51%)
31/68 (46%)
51/65**
15/15**
(79%)
(100%)
Litter incidence
8/9 (89%)
10/10 (100%)
8/10 (80%)
9/9 (100%)
2/2 (100%)
"Hellwig and Jackh (1997): Confidential Q99D.
bNumber affected/number examined.
°Values represent (litter) means ± SD.
dMarginally significant; p = 0.08 based on statistical analysis (t-test) performed by the U.S. EPA for the purposes
of this PPRTV assessment.
eNet weight change from GD 6 = (terminal body weight - gravid uterine weight) - GD 6 BW.
fValue(s) not clearly legible in the study report.
tp < 0.05 compared to the vehicle-only control group based on statistics performed by the U.S. EPA for the
purposes of this PPRTV assessment,
ff/? < 0.01.
*p < 0.05 compared to the vehicle-only control group based on statistics performed by study authors.
**p < 0.01.
BW = body weight; F = female(s); GD = gestation day; M = male(s); SD = standard deviation.
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Table B-ll. Significant Effects in CD-I Mice Administered
2-Ethylhexanol (CASRN 104-76-7) via Gavage on GDs 6-13a
Parameter
Dose (mg/kg-d)
0
1,525
Maternal Effects
Number of dams
50
50
Mortality
0/50b (0%)
17/49° f (35%)
Maternal body weight; GD 7 (g)
29.9 ± 2.35d
29.0 ± 2.07 (-3%)
Maternal body weight; GD 14 (g)
37.1 ±2.89
33.8 ± 1.58* (-9%)
Maternal body weight; GD 18 (g)
47.9 ±5.01
40.5 ± 6.38* (-15%)
Maternal body weight; PND 3 (g)
36.8 ±2.68
33.6 ±3.81* (-9)
Maternal body-weight gain; GDs 7-14 (g)
7.2 ±2.72
4.8 ±2.73* (-33%)
Maternal body-weight gain; GDs 7-18 (g)
18.0 ±5.18
11.4 ±5.86* (-37%)
Number of viable litters/number of pregnant Fe
33/34
11/20*
Reproductive index (%)f
97
55*
Pup Effects
Number of litters
33
11
Number of live pups/litter
PND 1
9.9 ±2.36
6.4 ± 3.23*-g (-35%)
PND 3
9.8 ±2.46
4.9 ± 3.70* (-50%)
Number of dead pups/litter
PND 1
0.1 ±0.29
1.5 ± 1.63*-h (+1,400%)
PND 3
0.1 ±0.24
1.5 ±2.02 (+1,400%)
Pup weights (g)
PND 1
1.6 ± 0.13h
1.4 ±0.17* (-13%)
PND 3
2.2 ±0.19
1.7 ±0.31* (-23%)
Pup viability/litter (%); PNDs 1-3
98.2 ±8.80
73.4 ± 32.20* (-25%)
Pup-weight change (%); PNDs 1-3
35.3 ±6.28
23.7 ± 13.15* (-12%)
aHardin et al. (1987): Hazleton Laboratories (1983).
bNumber affected/number examined.
°The denominator is not 50 because the death of 1 F (owing to a dosing error) was omitted from analysis.
dValues represent means ± SD.
eA viable litter was defined as a litter that had at least one live pup on Day 1.
Reproductive index = (number of females that produced viable litters + number of proven pregnant females) x 100.
'Value is not identical to that shown in the Hardin ct al. (1987) publication (6.8 ± 3.4*).
hValue(s) not clearly legible in the study report.
*p < 0.05 based on statistics performed by the study authors.
tp < 0.01 compared to the control group based on statistics (/-test) performed by the U.S. EPA for the purposes of
this PPRTV assessment.
F = female(s); GD = gestation day; PND = postnatal day; SD = standard deviation.
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Table B-12. Effects in Wistar Rats Exposed to 2-Ethylhexanol (CASRN 104-76-7) via
Gavage on GD 12a
Parameter
Dose (mg/kg-d)
0
830
1,700
Number of dams
7
7
7
Number of implantations
91
104 (+14%)
113 (+24%)
Resorbed/dead fetuses (%)
9.6 ± 4.1b
10.1 ±9.1 (+0.5%)
8.5 ± 1.7 (-1%)
Mean fetal body weight (g)
4.1°
3.9 (-5%)
3.5 (-15%)
Survivors malformed (%)
0.0 ±0.0
2.0 ± 1.3 (+2%)
22.2 ± 14.7d- f (+22%)
"Ritter et al. (1987).
bValues represent means ± SEM.
°Mean (no measure of variance was provided).
dMalformations reported included hydronephrosis (8%), tail and limb defects (5 and 10%), and other (1%).
tp < 0.01 compared to the control group based on statistics (/-test) performed by the U.S. EPA for the purposes of
this PPRTV assessment.
GD gestation day; SEM = standard error of the mean.
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Table B-13. Effects in ICR Mice Exposed to 2-Ethylhexanol (CASRN 104-76-7) as a Vapor
8 Hours/Day for 3 Months"
Parameter
Exposure Concentration (ppm) (HECet, mg/m3)b'c
0(0)
21.9 (4.17)
65.8 (12.5)
153.2 (29.20)
Olfactory Epithelium Effects
Morphological alterations (severity scores/
ld
1.6
2 9**
3.8**
Diameter of Bowman's glands (|im)
6.80 ± 1.3e
9.80 ±2.0
15.0 ± 3.9**
16.3 ± 1.6**
CD3 -positive cells (/mm2)
o±o
1.03 ± 1.4
10.3 ±9.9*
18.5 ± 12**
OMP-positive cells (ratio of OMP[+] cells
to olfactory epithelium cells)
0.791 ±0.16
0.473 ± 0.20**
0.411 ±0.086**
0.143 ±0.079**
PCNA-positive cells (/mm2)
12.5 ±3.6
11.3 ±4.5
9.55 ±4.8
5.67 ±4.8*
Olfactory Bulb Effects
Glomerular diameter (|im)
80 ±2.1
77.9 ±2.1
77.9 ±2.8
62.1 ±2.8**
OMP-positive cells (pixel)
28.7 ±4.0
29.0 ±2.4
24.4 ±3.8*
19.1 ±0.81**
TH-positive cells (pixel)
27.0 ±7.2
23.2 ±3.6
21.2 ±3.3
17.9 ±2.8**
Ibal-positive cells (/mm3)
28,237 ± 3,548
24,075 ± 4,838
25,723 ± 2,903
37,688 ±2,580*
Dcx-positive cells (/mm3)
8,588 ± 1,342
9,876 ±3,580
6,355 ± 1,006
12,007 ± 1,454*
"Mivake et al. (2016).
bAnalytical exposure concentrations were converted to HECs for extrathoracic respiratory effects using the
following equation: HECet = (ppm x MW ^ 24.45) x (hours/day exposed ^ 24) x (days/week
exposed ^ 7) x RGDRet. RGDRet is the extrathoracic regional gas-dose ratio (animal:human) (U.S. EPA. 1994).
Data were digitally extracted using Grablt! software.
dMean; no measure of variance was reported.
"Values represent means ± SD.
Severity scores: 1 (normal), 2 (slight), 3 (moderate), and 4 (severe).
*p < 0.05 based on statistics performed by the study authors.
**p < 0.01 based on statistics performed by the study authors.
Dcx = doublecortin; ET = extrathoracic respiratory effects; HEC = human equivalent concentration; Ibal = ionized
calcium-binding adapter molecule 1; MW = molecular weight; OMP = olfactory marker protein;
PCNA = proliferating cell nuclear antigen; RGDR = regional gas dose ratio; SD = standard deviation;
TH = tyrosine hydroxylase.
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Table B-14. Effects in S-D Rats Exposed to 2-Ethylhexanol (CASRN 104-76-7) as a Vapor
7 Hours/Day on GDs l-19a
Exposure Concentration (mg/m3)
Parameter
0
850
Maternal Effects
Number of dams (approximate)
15
15
Maternal body weight; GD 0 (g)
243 ± 25b
283 ± 18 (+16%)
Maternal body weight; GD 20 (g)
354 ±32
371 ± 20 (+5%)
Maternal body-weight gain; GDs 0-20 (g)
lllc
88 (-21%)
Feed consumption (g)
117± 13
106 ± 15* (-9%)
Resorptions per litter
0.4
0.3 (-25%)
Fetal Effects
Fetal body weight; males (g)
3.28 ±0.27
3.18 ±0.30 (-3%)
Fetal body weight; females (g)
3.19 ±0.20
3.02 ± 0.20 (-5%)
"Nelson et al. (1989).
bValues represent (litter) means ± SD.
°Mean; no measure of variance was reported.
*p < 0.05 based on statistics performed by the study authors.
GD = gestation day; S-D = Sprague-Dawley; SD = standard deviation.
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APPENDIX C. BENCHMARK DOSE MODELING RESULTS
MODELING OF NONCANCER ENDPOINTS
As discussed in the body of the report under "Derivation of Subchronic Provisional Oral
Reference Dose," the most sensitive treatment-related changes were reported in the
developmental study conducted by Hell wig and Jackh (1997) and Confidential (1991) and in the
subchronic-duration gavage study in rats and mice (Astill et al.. 1996a; BASF. 1991a); these
changes are presented in Table C-l. Endpoints selected to determine potential points of
departure (PODs) for the subchronic provisional reference dose (p-RfD) using benchmark dose
(BMD) analysis were as follows: (1) absolute liver weight in male rats; (2) relative liver, kidney,
and stomach weight in male rats; (3) relative liver and stomach weight in female rats (Astill et
al.. 1996a; BASF. 1991a); (4) relative stomach weight in male mice (Astill et al.. 1996a; BASF.
1991e. 1); (5) fetal body weight; and (6) fetal skeletal malformations, variations, and retardations
(Hellwig and Jackh. 1997; Confidential. 1991). Summaries of modeling approaches and results
(see Tables C-3 through C-12) for each data set follow.
The most sensitive endpoints showing treatment-related changes in the study of rats
administered 2-ethylhexanol (2-EH) via gavage 5 days/week for 24 months (Astill et al.. 1996b;
BASF, 1992a) were decreased body weight in males and females (see Tables B-5, B-6, and C-2).
Data sets for these endpoints were selected to determine potential PODs for the chronic p-RfD,
using BMD analysis. Summaries of modeling approaches and results (see Tables C-13 and
C-l4) for each data set follow.
As discussed in the body of the report under "Derivation of Subchronic Provisional
Inhalation Reference Concentration," the most sensitive treatment-related changes due to
inhalation exposure of 2-EH were reported in male mice from the Mivakc et al. (2016) study and
are presented in Table B-13. Endpoints selected to determine potential PODs for the subchronic
provisional reference concentration (p-RfC) using BMD analysis were as follows: (1) diameter
of Bowman's glands in the olfactory epithelium, (2) CD3-positive cells in the olfactory
epithelium, (3) olfactory marker protein (OMP)-positive cells in the olfactory epithelium,
(4) proliferating cell nuclear antigen (PNCA)-positive cells in the olfactory epithelium,
(5) glomerular diameter in the olfactory bulb, (6) protein (OMP)-positive cells in the olfactory
bulb (7) tyrosine hydroxylase (TH)-positive cells in the olfactory bulb, (8) ionized
calcium-binding adapter molecule 1 (Ibal)-positive cells in the olfactory bulb, and
(9) doublecortin (Dcx)-positive cells in the in the olfactory bulb. Summaries of modeling
approaches and results (see Tables C-l5 through C-23) for each data set follow.
MODELING PROCEDURE FOR DICHOTOMOUS NONCANCER DATA
BMD modeling of dichotomous noncancer data was conducted with the U.S. EPA's
Benchmark Dose Software (BMDS, Version 2.7). For these data, the Gamma, Logistic,
Log-Logistic, Log-Probit, Multistage, Probit, and Weibull dichotomous models available within
the software were fit using a benchmark response (BMR) of 10% extra risk. The Multistage
model is run for all polynomial degrees up to n - 1, where n is the number of dose groups
including control. Adequacy of model fit was judged based on the %2 goodness-of-fit />value
(p > 0.1), scaled residuals at the data point (except the control) closest to the predefined
benchmark response (absolute value <2.0), and visual inspection of the model fit. In the cases
where no best model was found to fit to the data, a reduced data set without the high-dose group
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was further attempted for modeling and the result was present along with that of the full data set.
Among the models providing adequate fit, the benchmark dose lower confidence limit (BMDL)
from the model with the lowest Akaike's information criterion (AIC) was selected as a potential
POD when BMDL values were sufficiently close. Otherwise, the lowest BMDL was selected as
a potential POD.
MODELING PROCEDURE FOR CONTINUOUS NONCANCER DATA
BMD modeling of continuous data is conducted with U.S. EPA's BMDS (Version 2.7).
All continuous models available within the software are fit using a BMR of 1 standard deviation
(SD) relative risk or 10% extra risk when a biologically determined BMR is available (e.g., BMR
10% relative deviation [RD] for body weight based on a biologically significant weight loss of
10%), as outlined in the Benchmark Dose Technical Guidance (U.S. EPA. 2012b). An adequate
fit is judged based on the x2 goodness-of-fitp-walue (p> 0.1), magnitude of the scaled residuals
near the BMR, and visual inspection of the model fit. In addition to these three criteria for
judging adequacy of model fit, a determination is made as to whether the variance across dose
groups is homogeneous. If a homogeneous variance model is deemed appropriate based on the
statistical test provided by BMDS (i.e., Test 2), the final BMD results are estimated from a
homogeneous variance model. If the test for homogeneity of variance is rejected (p < 0.1), the
model is run again while modeling the variance as a power function of the mean to account for
this nonhomogeneous variance. If this nonhomogeneous variance model does not adequately fit
the data (i.e., Test 3;p<0. 1), the data set is considered unsuitable for BMD modeling. Among
all models providing adequate fit, the lowest BMDL/benchmark concentration lower confidence
limit (BMCL) is selected if the BMDL/BMCL estimates from different models vary >threefold;
otherwise, the BMDL/BMCL from the model with the lowest AIC is selected as a potential POD
from which to derive the oral reference dose/inhalation reference concentration (RfD/RfC).
MODELING PROCEDURE FOR NESTED DICHOTOMOUS NONCANCER DATA
BMD modeling of nested dichotomous noncancer developmental effects was conducted
with the U.S. EPA's BMDS (Version 2.7). A BMR of 5% extra risk was used for developmental
effects, as an excess risk of 5% approximates the no-observed-adverse-effect level (NOAEL) for
most developmental studies. In addition, developmental studies provide increased statistical
power compared to regular toxicity studies (i.e., use of pups as the observational subject) and
developmental effects are often considered to be severe. The Nested Logistic model includes a
litter-specific covariate and intralitter correlation to address intralitter similarity. The
litter-specific covariate considers the condition of the exposed dam before the onset of exposure,
and the intralitter correlation statistically describes the similarity of responses among pups in the
same litter. For each data set, the Nested Logistic model was fit with and without each of the
selected model parameters (i.e., litter-specific covariate, intralitter covariate). Adequacy of the
model fit was judged based on the %2 goodness-of-fit p-w alue (p > 0.1), scaled residuals at the
data point (except the control) closest to the predefined benchmark response (absolute
value <2.0), and visual inspection of the model fit. Among the models providing adequate fit,
the BMDL from the model with the lowest AIC was selected as a potential POD when BMDL
values were sufficiently close.
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Table C-l. Selected Non-neoplastic Endpoints in Rats or Mice Exposed to 2-Ethylhexanol (CASRN 104-76-7)
Astill et al. (1996a): BASF (1991a)
Male Rats
Adjusted daily dose (mg/kg-d) (HED)a
0
18 (4.3)
89.3 (21.4)
179 (43.0)
357 (85.7)
Number of animals
10
10
10
10
10
Absolute liver weight
7.74 ± 0.57b
7.94 ± 0.77 (+3%)
7.95 ± 0.66 (+3%)
8.07 ± 0.27 (+4%)
9.17 ±0.85** (+18%)
Relative liver weight
2.77 ±0.11
2.82 ±0.12 (+2%)
2.86 ±0.10 (+3%)
2.98 ± 0.08** (+8%)
3.57 ±0.22** (+29%)
Relative kidney weight
0.70 ± 0.02
0.71 ±0.02 (+1%)
0.71 ±0.03 (+1%)
0.75 ±0.03* (+7%)
0.81 ±0.04** (+16%)
Relative stomach weight0
0.57 ±0.04
0.56 ± 0.03 (-2%)
0.56 ± 0.04 (-2%)
0.59 ± 0.03 (+4%)
0.63 ±0.02** (+11%)
Female Rats
Adjusted daily dose (mg/kg-d) (HED)a
0
18 (4.0)
89.3 (19.6)
179 (39.4)
357 (75.0)
Number of animals
10
10
10
10
10
Relative liver weight
2.67 ±0.11
2.71 ±0.09 (+1%)
2.72 ±0.10 (+2%)
2.88 ± 0.08** (+8%)
3.07 ±0.07** (+15%)
Relative stomach weight0
0.71 ±0.03
0.71 ± 0.02 (0%)
0.73 ± 0.04 (+3%)
0.75 ±0.03* (+6%)
0.82 ± 0.04** (+15%)
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Table C-l. Selected Non-neoplastic Endpoints in Rats or Mice Exposed to 2-Ethylhexanol (CASRN 104-76-7)
Astill et al. (1996a): BASF (1991e): BASF (1991c)
Male Mice
Adjusted daily dose (mg/kg-d)
0
18
89.3
179
357
Number of animals
10
10
10
10
10
Relative stomach weight0
0.79 ±0.07
0.77 ± 0.07 (-3%)
0.81 ±0.09 (+3%)
0.90 ±0.13* (+14%)
0.89 ±0.10* (+13%)
Heliu m and Jackh (1997); Confidential (1991)
Adjusted daily dose (mg/kg-d) (HED)a
0
130 (32.5)
650 (163)
1,300 (325)
Fetal body weight on GD 20 (g)
3.82 ±0.177
3.80 ±0.249 (-1%)
3.44 ±0.227* (-10%)
2.86 ± 0.333** (-25%)
Skeletal malformations—fetal incidence
2/75 (3%)
2/68 (3%)
6/65 (9%)
4/15** (27%)
Skeletal variations—fetal incidence
17/75 (23%)
23/68 (34%)
27/65* (42%)
10/15** (67%)
Skeletal retardations—fetal incidence
38/75 (51%)
31/68 (46%)
51/65** (79%)
15/15** (100%)
'HED = adjusted daily animal dose (mg/kg-day) x (BWa + BWh)1/4 (U.S. EPA. 20051. using TWA body weights calculated from study reported body-weight data for rats
and using 70 kg for humans (U.S. EPA. 2011b).
bValues expressed as mean ± SD (% change compared with control); % change control = ([treatment mean - control mean] + control mean) x 100.
°As discussed above, doses for stomach-weight changes were not converted to HEDs.
*p < 0.05 based on statistics performed by the study authors.
**p < 0.01.
BW = body weight; GD = gestation day; HED = human equivalent dose; SD = standard deviation; TWA = time-weighted average.
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Table C-2. Data for the Decreased Body Weight of Rats Exposed to
2-Ethylhexanol (CASRN 104-76-7) via Gavage 5 Days/Week for 24 Months3
Male
Parameter
ADD (HED)b, mg/kg-d
0
36 (9.5)
107 (27.9)
357 (90.9)
Number of animals
33
27
34
31
Body weight at study termination (g)
368.1 ±30.4C
349.5 ±28.2
(-5%)
328.8 ±26.9
("11%)
282.9 ±24.0
(-23%)
Female
Parameter
ADD (HED), mg/kg-d
0
36 (8.4)
107 (24.8)
357 (81.2)
Number of animals
36
37
37
24
Body weight at study termination (g)
259.7 ±27.1
252.1 ± 15.1
(-3%)
236.2 ±24.7
(-9%)
205.6 ±21.4
(-21%)
"Astill et at (1996b): BASF (1992a).
'HED = adjusted daily animal dose (mg/kg-day) x (BWa ^ BWh)"4 (U.S. EPA. 2005). using TWA body weights
calculated from study reported body-weight data for rats and using 70 kg for humans (U.S. EPA. 2011b).
°Mean± SD.
ADD = adjusted daily dose; B W = body weight; HED = human equivalent dose; SD = standard deviation;
TWA = time-weighted average.
Model Predictions for Increased Absolute Liver Weight in Male Rats (Astill et at, 1996a;
BASF, 1991a)
The procedure outlined above for continuous data was applied to the data for increased
absolute liver weight in male F344 rats treated with 2-EH via gavage for 3 months (Astill et al..
1996a; BASF. 1991a) (see Table C-l). Table C-3 summarizes the BMD modeling results.
Neither the constant nor the nonconstant variance models provided adequate fit to the variance
data; thus, these data were not suitable for BMD modeling.
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Table C-3. Modeling Results for Increased Absolute Liver Weights in Male F344 Rats Administered
2-Ethylhexanol (CASRN 104-76-7) via Gavage for 3 Months"'b
Model
Test for Significant
Difference />-Valuec
Variance
/>-Valucd
Means
/>-Valucd
Scaled Residuals
for Dose Group®
AIC
BMD io
(mg/kg-d)
BMDLio
(mg/kg-d)
Constant variance
Exponential (Model 2)f
<0.0001
0.01742
0.3539
-1.357
11.76113
51.2915
39.2714
Exponential (Model 3)f
<0.0001
0.01742
0.7407
0.008858
11.10582
70.1208
47.6063
Exponential (Model 4)f
<0.0001
0.01742
0.162
-1.42
14.14542
50.277
37.3994
Exponential (Model 5)f
<0.0001
0.01742
0.436
0.009035
13.11232
69.8765
47.7935
Hillf
<0.0001
0.01742
0.4359
0.00905
13.112554
69.8668
47.7978
Linear6
<0.0001
0.01742
0.3031
-1.42
12.145307
50.2772
37.3999
Polynomial (2-degree)8
<0.0001
0.01742
0.8583
0.0979
9.268477
65.8413
46.5696
Polynomial (3-degree)8
<0.0001
0.01742
0.7779
0.0181
11.007912
69.505
47.7577
Power'
<0.0001
0.01742
0.7383
0.00903
11.112275
69.8781
47.793
Nonconstant variance
Linear8
<0.0001
0.01888
0.1627
-1.35
13.605381
52.181
37.9956
aAstill et at (1996a): BASF (1991b).
bNo model was selected. Neither the constant nor nonconstant variance models provide adequate fit to the variance data.
°Values >0.05 fail to meet conventional goodness-of-fit criteria.
dValues <0.10 fail to meet conventional goodness-of-fit criteria.
"Scaled residuals at dose closest to BMD.
fPower restricted to >1.
Coefficients restricted to be negative.
AIC = Akaike's information criterion; BMD = benchmark dose.
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Model Predictions for Increased Relative Liver Weight in Male Rats (Astill et at, 1996a;
BASF, 1991a)
The procedure outlined above for continuous data was applied to the data for increased
relative liver weight in male F344 rats treated with 2-EH via gavage for 3 months (Astill et al..
1996a; BASF, 1991a) (see Table C-l). The BMD modeling results are summarized in Table C-4
and Figure C-l. The constant variance model did not provide adequate fit to the variance data.
The nonconstant variance model provided adequate fit to the variance data, and adequate fit to
the means was provided by several of the included models. The BMDLs for the models
providing adequate fit are sufficiently close (i.e., differ by
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Table C-4. Modeling Results for Increased Relative Liver Weights in Male F344 Rats Administered
2-Ethylhexanol (CASRN 104-76-7) via Gavage for 3 Months"
Model
Test for Significant
Difference />-Valucb
Variance
/>-Valuce
Means
p-Value0
Scaled Residuals for
Dose Groupd
AIC
BMDio
(mg/kg-d)
BMDLio
(mg/kg-d)
Nonconstant variance
Exponential (Model 2)e
<0.0001
0.3089
0.001957
-2.101
-142.2335
36.2632
31.0952
Exponential (Model 3)e
<0.0001
0.3089
0.4633
-0.06315
-153.536
53.0526
44.8859
Exponential (Model 4)e
<0.0001
0.3089
0.0001697
-2.079
-137.7124
35.6373
29.5695
Exponential (Model 5)e
<0.0001
0.3089
0.2
-0.07957
-151.4325
52.8248
44.8682
Hille
<0.0001
0.3089
0.1998
-0.0797
-151.4313
52.8221
44.8677
Linear®
<0.0001
0.3089
0.0005952
-2.08
-139.7124
35.6373
29.5695
Polynomial (2-degree)8
<0.0001
0.3089
0.4251
-0.309
-153.3640
51.5971
43.9782
Polynomial (3-degree)f'g
<0.0001
0.3089
0.6541
0.0741
-154.2259
53.4491
45.0018
Power"
<0.0001
0.3089
0.4399
-0.0796
-153.4326
52.8249
44.8682
aAstill et at (1996a): BASF (1991i).
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dScaled residuals at dose closest to BMD.
Tower restricted to >1.
Selected model. Lowest AIC among models that provided an adequate fit.
gCoefficients restricted to be negative.
AIC = Akaike's information criterion; BMD = benchmark dose; BMDio = benchmark dose 10% extra risk; BMDLio = 95% benchmark dose lower confidence limit.
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Polynomial Model, with BMR of 0.1 Rel. Dev. for the BMDand 0.95 Lower Confidence Limit for the BMDL
3.8
Polynomial
3.6
3.4
3.2
3
2.8
BMDL
3MD
2.6
0
10
20
30
40
50
60
70
80
90
14:25 07/05 2017
Figure C-l. Polynomial 3-Degree Model for Increased Relative Liver Weight in Male F344
Rats Treated with 2-Ethylhexanol via Gavage for 3 Months (Astill et al., 1996a; BASF,
1991a)
Model Predictions for Increased Relative Kidney Weight in Male F344 Rats Treated with
2-Ethylhexanol via Gavage for 3 Months (Astill et al., 1996a; BASF, 1991a)
The procedure outlined above for continuous data was applied to the data for increased
relative kidney weight in male F344 rats treated with 2-EH via gavage for 3 months (Astill et al.,
1996a; BASF. 1991a) (see Table C-l). The BMD modeling results are summarized in Table C-5
and Figure C-2. The constant variance model provided adequate fit to the variance data, and
adequate fit to the means was provided by all included models. The BMDLs for the models
providing adequate fit are sufficiently close (i.e., differ by
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FINAL
04-23-2019
Table C-5. Modeling Results for Increased Relative Kidney Weights in Male F344 Rats Administered
2-Ethylhexanol (CASRN 104-76-7) via Gavage for 3 Months"
Model
Test for Significant
Difference />-Valucb
Variance />-Valuce
Means />-Valuce
Scaled Residuals
for Dose Groupd
AIC
BMDio
(mg/kg-d)
BMDLio
(mg/kg-d)
Constant variance
Exponential (Model 2)e f
<0.0001
0.1351
0.365
-0.03743
-300.1957
55.6598
47.8532
Exponential (Model 3)e
<0.0001
0.1351
0.3819
0.6324
-299.4481
62.3457
49.7456
Exponential (Model 4)e
<0.0001
0.1351
0.1614
-0.1682
-297.7261
54.4022
46.1082
Exponential (Model 5)e
<0.0001
0.1351
0.4185
0.0007963
-298.719
52.4009
43.6538
Hille
<0.0001
0.1351
0.4182
0.0007
-298.7180
54.3476
44.01
Linear6
<0.0001
0.1351
0.3022
-0.168
-299.7265
54.4033
46.1302
Polynomial (2-degree)8
<0.0001
0.1351
0.3578
0.676
-299.3178
62.3791
48.8468
Polynomial (3-degree)8
<0.0001
0.1351
0.3578
0.676
-299.3178
62.3791
48.8468
Power"
<0.0001
0.1351
0.3936
0.614
-299.5089
62.1489
49.3059
aAstill et at (1996a): BASF (1991b).
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dScaled residuals at dose closest to BMD.
Tower restricted to >1.
Selected model. Lowest AIC among models that provided an adequate fit.
gCoefficients restricted to be negative.
AIC = Akaike's information criterion; BMD = benchmark dose; BMDio = benchmark dose 10% extra risk; BMDLio = 95% benchmark dose lower confidence limit.
99
2-Ethylhexanol
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04-23-2019
Exponential 2 Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
Exponential 2
0.84
0.82
0.78
(L)
Cfl
£=
o
CL
(fl
CD
££
0.76
£Z
(C
(D
0.74
0.72
0.7
0.68
BMDL
BMD
0
10
20
30
40
50
60
70
80
90
14:27 07/05 2017
Figure C-2. Exponential 2 Model for Increased Relative Kidney Weight in Male F344 Rats
Treated with 2-Ethylhexanol via Gavage for 3 Months (Astill et al>, 1996a; BASF, 1991a)
Model Predictions for Increased Relative Stomach Weight in Male F344 Rats Treated with
2-Ethylhexanol via Gavage for 3 Months (Astill et al., 1996a; BASF, 1991a)
The procedure outlined above for continuous data was applied to the data for increased
relative stomach weight in male F344 rats treated with 2-EH via gavage for 3 months (Astill et
al., 1996a; BASF, 1991a) (see Table C-l). The BMD modeling results are summarized in
Table C-6 and Figure C-3. The constant variance model provided adequate fit to the variance
data, and adequate fit to the means was provided by all included models. The BMDLs for the
models providing adequate fit are sufficiently close (i.e., differ by
-------
FINAL
04-23-2019
Table C-6. Modeling Results for Increased Relative Stomach Weight in Male F344 Rats Administered
2-Ethylhexanol (CASRN 104-76-7) via Gavage for 3 Months"
Model
Test for Significant
Difference />-Valucb
Variance
p-Value0
Means
/>-Valuce
Scaled Residuals
for Dose Groupd
AIC
BMDisd
(mg/kg-d)
BMDLisd
(mg/kg-d)
Constant variance
Exponential (Model 2)e f
<0.0001
0.2212
0.3036
-0.103
-287.1724
173.525
131.152
Exponential (Model 3)e
<0.0001
0.2212
0.3833
0.6132
-286.8899
228.179
145.102
Exponential (Model 4)e
<0.0001
0.2212
0.1449
-0.1558
-284.9439
169.989
126.289
Exponential (Model 5)e
<0.0001
0.2212
0.4092
1.54 x 10-6
-286.1265
181.2
148.611
Hille
<0.0001
0.2212
0.7113
3.32 x 10-6
-288.1265
181.894
146.441
Linear6
<0.0001
0.2212
0.2766
-0.156
-286.9442
169.992
126.294
Polynomial (2-degree)8
<0.0001
0.2212
0.3626
0.751
-286.7788
237.018
142.126
Polynomial (3-degree)8
<0.0001
0.2212
0.3626
0.751
-286.7788
237.018
142.126
Power"
<0.0001
0.2212
0.3903
0.6
-286.9260
226.878
144.22
aAstill et at (1996a): BASF (1991b).
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dScaled residuals at dose closest to BMD.
Tower restricted to >1.
Selected model. Lowest AIC among models that provided an adequate fit.
gCoefficients restricted to be negative.
AIC = Akaike's information criterion; BMD = benchmark dose; BMDL = benchmark dose lower confidence limit; SD = standard deviation.
101
2-Ethylhexanol
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Exponential 2 Model, with BMR of 1 Std. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
Exponential 2
0.64
0.62
(L)
Cfl
£=
0.6
o
CL
(fl
CD
££
£Z
(C
(D
0.58
0.56
0.54
BMDL
BMD
0.52
0
50
100
150
200
250
300
350
14:21 09/06 2017
Figure C-3. Exponential 2 Model for Increased Relative Stomach Weight in Male F344
Rats Treated with 2-Ethylhexanol via Gavage for 3 Months (Astill et al>, 1996a; BASF,
1991a)
Model Predictions for Increased Relative Liver Weight in Female F344 Rats Treated with
2-Ethylhexanol via Gavage for 3 Months (Astill et al., 1996a; BASF, 1991a)
The procedure outlined above for continuous data was applied to the data for increased
relative liver weight in female F344 rats treated with 2-EH via gavage for 3 months (Astill et al.,
1996a; BASF, 1991a) (see Table C-l). The BMD modeling results are summarized in Table C-7
and Figure C-4. The constant variance model provided adequate fit to the variance data, and
adequate fit to the means was provided by all included models. The BMDLs for the models
providing adequate fit are sufficiently close (i.e., differ by
-------
FINAL
04-23-2019
Table C-7. Modeling Results for Increased Relative Liver Weight in Female F344 Rats Administered
2-Ethylhexanol (CASRN 104-76-7) via Gavage for 3 Months"
Model
Test for Significant
Difference />-Valucb
Variance />-Valuce
Means />-Valuce
Scaled Residuals
for Dose Groupd
AIC
BMDio
(mg/kg-d)
BMDLio
(mg/kg-d)
Constant variance
Exponential (Model 2)e f
<0.0001
0.6481
0.311
0.3949
-185.2671
51.0066
44.6836
Exponential (Model 3)e
<0.0001
0.6481
0.2303
0.8464
-183.9059
54.5788
45.4073
Exponential (Model 4)e
<0.0001
0.6481
0.1393
0.249
-182.9014
49.9416
43.2639
Exponential (Model 5)e
<0.0001
0.6481
0.3064
0.003548
-183.797
47.5836
41.2759
Hille
<0.0001
0.6481
0.3052
0.00321
-183.7918
49.0486
41.8255
Linear6
<0.0001
0.6481
0.2679
0.249
-184.9019
49.9428
43.2655
Polynomial (2-degree)8
<0.0001
0.6481
0.2143
0.857
-183.7627
54.3407
44.3267
Polynomial (3-degree)8
<0.0001
0.6481
0.2143
0.857
-183.7627
54.3407
44.3267
Power"
<0.0001
0.6481
0.2398
0.825
-183.9874
54.472
44.6499
aAstill et at (1996a): BASF (1991b).
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dScaled residuals at doses closest to BMD.
Tower restricted to >1.
Selected model. Lowest AIC among models that provided an adequate fit.
gCoefficients restricted to be negative.
AIC = Akaike's information criterion; BMD = benchmark dose; BMDio = benchmark dose 10% extra risk; BMDLio = 95% benchmark dose lower confidence limit.
103
2-Ethylhexanol
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Exponential 2 Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
Exponential 2
(L)
Cfl
£=
o
CL
(fl
CD
££
£Z
(C
(D
2.7
2.6
BMDL
BMD
0
10
20
30
40
50
60
70
14:11 07/05 2017
Figure C-4. Exponential 2 Model for Increased Relative Liver Weight in Female F344 Rats
Treated with 2-Ethylhexanol via Gavage for 3 Months (Astill et al., 1996a; BASF, 1991a)
Model Predictions for Increased Relative Stomach Weight in Female F344 Rats Treated
with 2-Ethylhexanol via Gavage for 3 Months (Astill et al., 1996a; BASF, 1991a)
The procedure outlined above for continuous data was applied to the data for increased
relative stomach weight in female F344 rats treated with 2-EH via gavage for 3 months (Astill et
al., 1996a; BASF, 1991a) (see Table C-l). The BMD modeling results are summarized in
Table C-8 and Figure C-5. The constant variance model provided adequate fit to the variance
data, and adequate fit to the means was provided by all included models. The BMDLs for the
models providing adequate fit are sufficiently close (i.e., differ by
-------
FINAL
04-23-2019
Table C-8. Modeling Results for Increased Relative Stomach Weight in Female F344 Rats Administered
2-Ethylhexanol (CASRN 104-76-7) via Gavage for 3 Months"
Model
Test for Significant
Difference />-Valucb
Variance
/>-Valuce
Means
p-Value0
Scaled Residuals
for Dose Groupd
AIC
BMDisd
(mg/kg-d)
BMDLisd
(mg/kg-d)
Constant variance
Exponential (Model 2)e f
<0.0001
0.2212
0.756
-0.1024
-289.6202
106.785
86.5786
Exponential (Model 3)e
<0.0001
0.2212
0.8998
-0.2348
-288.5965
142.132
90.0041
Exponential (Model 4)e
<0.0001
0.2212
0.4506
-0.1802
-287.2136
102.232
81.9437
Exponential (Model 5)e
<0.0001
0.2212
0.6279
-0.2545
-286.5728
142.657
85.271
Hille
<0.0001
0.2212
0.6262
-0.256
-286.5705
142.715
85.1918
Linear6
<0.0001
0.2212
0.6608
-0.18
-289.2140
102.236
81.9476
Polynomial (2-degree)8
<0.0001
0.2212
0.9165
-0.173
-288.6334
140.534
87.3605
Polynomial (3-degree)8
<0.0001
0.2212
0.9377
-0.0908
-288.6790
138.367
87.5901
Power"
<0.0001
0.2212
0.8892
-0.254
-288.5728
142.655
87.0642
aAstill et at (1996a): BASF (1991b).
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dScaled residuals at doses closest to BMD.
Tower restricted to >1.
Selected model. Lowest AIC among models that provided an adequate fit.
gCoefficients restricted to be negative.
AIC = Akaike's information criterion; BMD = benchmark dose; BMDL = benchmark dose lower confidence limit; SD = standard deviation.
105
2-Ethylhexanol
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Exponential 2 Model, with BMR of 1 Std. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
0.86
Exponential 2
0.84
0.82
(L)
Cfl
£=
0.78
o
CL
(fl
CD
££
£Z
(C
(D
0.76
0.74
0.72
0.7
0.68
BMDL
3MD
0
50
100
150
200
250
300
350
14:09 09/06 2017
Figure C-5. Exponential 2 Model for Increased Relative Stomach Weight in Female F344
Rats Treated with 2-Ethylhexanol via Gavage for 3 Months (Astill et al>, 1996a; BASF,
1991a)
Model Predictions for Increased Relative Stomach Weight in Male B6C3Fi Mice Treated
with 2-Ethylhexanol via Gavage for 3 Months (Astill et al„ 1996a; BASF, 1991a)
The procedure outlined above for continuous data was applied to the data for increased
relative stomach weight in male B6C3Fi mice treated with 2-EH via gavage for 3 months (Astill
et aL 1996a; BASF, 1991a) (see Table C-l). The BMD modeling results are summarized in
Table C-9 and Figure C-6. The constant variance model provided adequate fit to the variance
data, and adequate fit to the means was provided by all included models. The BMDLs for the
models providing adequate fit are not sufficiently close (i.e., differ by >threefold), so the model
with the lowest BMDL (Exponential 4) is selected. For relative stomach weight, the BMDLisd
of 62 mg/kg-day from this model is selected.
106
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Table C-9. Modeling Results for Increased Relative Stomach Weight in Male B6C3Fi Mice Administered
2-Ethylhexanol (CASRN 104-76-7) via Gavage for 3 Months"
Model
Test for Significant
Difference />-Valucb
Variance
/>-Valuce
Means
p-Value0
Scaled Residuals
for Dose Groupd
AIC
BMDisd
(mg/kg-d)
BMDLisd
(mg/kg-d)
Constant variance
Exponential (Model 2)e
0.008253
0.228
0.2098
-0.7226
-180.4891
277.528
189.93
Exponential (Model 3)e
0.008253
0.228
0.2098
-0.7226
-180.4891
277.528
189.93
Exponential (Model 4)e f
0.008253
0.228
0.2568
1.078
-180.2984
163.1
62.1258
Exponential (Model 5)e
0.008253
0.228
0.5783
1.19 x 10-7
-180.7082
105.713
89.5467
Hille
0.008253
0.228
0.8568
-8.53 x 10-6
-182.708201
101.62
89.7842
Linear6
0.008253
0.228
0.2291
1.7
-180.699138
267.534
177.731
Polynomial (2-degree)8
0.008253
0.228
0.2291
1.7
-180.699138
267.534
177.731
Polynomial (3-degree)8
0.008253
0.228
0.2291
1.7
-180.699138
267.534
177.731
Power"
0.008253
0.228
0.2291
1.7
-180.699138
267.534
177.731
aAstill et at (1996a): BASF (1991b).
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dScaled residuals at doses closest to BMD.
Tower restricted to >1.
Selected model. Lowest AIC among models that provided an adequate fit.
gCoefficients restricted to be negative.
AIC = Akaike's information criterion; BMD = benchmark dose; BMDL = benchmark dose lower confidence limit; SD = standard deviation.
107
2-Ethylhexanol
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Exponential 4 Model, with BMR of 1 Std. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
Exponential 4
0.95
(L)
Cfl
£=
o
CL
(fl
CD
££
0.85
£Z
(C
(D
0.75
BryiDL
50
BMD
0.7
0
100
150
200
250
300
350
dose
13:49 12/06 2018
Figure C-6. Exponential 2 Model for Increased Relative Stomach Weight in Male B6C3Fi
Treated with 2-Ethylhexanol via Gavage for 3 Months (Astill et al., 1996a; BASF, 1991a)
Model Predictions for Decreased Fetal Body Weight Following 2-Ethylhexanol Exposure
via Gavage on Gestation Days 6-15 in Female Wistar Rats (Hellwig and Jackh, 1997;
Confidential, 1991)
The procedure outlined above for continuous data was applied to the data for decreased
fetal body weight in pups of Wistar rats treated with 2-EH via gavage on Gestation Days
(GDs) 6-15 (Hellwig and Jackh. 1997; Confidential 1991) (see Table C-l). Table C-10
summarizes the BMD modeling results. For decreased fetal body weight, the data were modeled
without the highest dose of 325 mg/kg-day (HED) because there was severe maternal toxicity
(i.e., 60% mortality and a 20% decrease in body weight) at that dose that confounds the
interpretation of fetal body-weight changes. Therefore, only the BMD modeling results based on
data without the highest dose group are summarized in Table C-10. Neither the constant nor
nonconstant variance models provided adequate fit to the variance data using the full or reduced
data set. This data set was not amenable to BMD modeling.
108
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Table C-10. Modeling Results for Decreased Fetal Body Weight Following 2-Ethylhexanol (CASRN 104-76-7) Exposure via Gavage
on Gestation Days 6-15 in Female Wistar Rats3
Model
Test for Significant
Difference />-Valucb
Variance />-Valuce
Means />-Valuce
Scaled Residuals
for Dose Groupd
AIC
BMDos
(mg/kg-d)
BMDLos
(mg/kg-d)
Nonconstant variance
Exponential (Model 2)e
<0.0001
0.0002566
0.02605
1.998
-834.9316
77.0138
69.4236
Exponential (Model 3)e
<0.0001
0.0002566
NDr
-0.01302
-837.8841
106.7
82.0473
Exponential (Model 4)e
<0.0001
0.0002566
0.02605
1.998
-834.9316
77.0138
67.0081
Hille
NDr
NDr
NDr
NDr
NDr
NDr
NDr
Linearf
<0.0001
0.0002566
0.03736
1.87
-835.550055
79.1485
71.7972
Polynomial (2-degree/
<0.0001
0.0002566
NDr
-0.013
-837.884091
110.747
85.0661
Polynomial (3-degree/
<0.0001
0.0002566
NDr
-0.013
-835.884091
119.479
85.0661
Power"
<0.0001
0.0002566
NDr
-0.013
-837.884091
107.59
82.8021
aHellwig and Jackh (1997): Confidential (1991).
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dScaled residuals at doses closest to BMD.
Tower restricted to >1.
Coefficients restricted to be negative.
AIC = Akaike's information criterion; BMD = benchmark dose; BMDos = benchmark dose 5% extra risk; BMDLos = 95% benchmark dose lower confidence limit;
NDr = not determined.
109
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Model Predictions for Increased Fetal Variations Following 2-Ethylhexanol Exposure via
Gavage on Gestation Days 6-15 in Female Wistar Rats (Hellwig and Jackfa, 1997;
Confidential, 1991)
The procedure outlined above for nested dichotomous data was applied to the data for
increased fetal variations in Wistar rats treated with 2-EH via gavage on GDs 6-15 (Hellwig and
Jackh. 1997; Confidential 1991) (see Table C-l). Table C-l 1 summarizes the BMD modeling
results. For increased fetal incidence of skeletal variations, the data were modeled without the
highest dose of 325 mg/kg-day (HED) because there was severe maternal toxicity (i.e., 60%
mortality and a 20% decrease in body weight) at that dose that confounds the interpretation of
fetal skeletal changes. Therefore, only the BMD modeling results based on data without the
highest dose group are summarized in Table C-l 1. Including implantation sites as a covariate
and using intralitter correlations had significant effects on the %2 goodness-of-fit statistics, AIC
scores, and visual inspections. As assessed by the %2 goodness-of-fit statistic, AIC score, and
visual inspection, only the NLogistic model with estimating intralitter correlations and not
including implantation sites as a covariate provided an optimal fit (see Table C-l 1 and
Figure C-7). For increased fetal variations, the BMDLos of 7.37 mg/kg-day from this model is
selected.
Table C-ll. Modeling Results for Increased Fetal Variations Following
2-Ethylhexanol (CASRN 104-76-7) Exposure via Gavage on GDs 6-15 in Female Wistar
Rats"
Parameter
Litter-Specific
Covariate; Intralitter
Correlation11
No Litter-Specific
Covariate; Intralitter
Correlationb
Litter-Specific
Covariate; No
Intralitter Correlation
No Litter-Specific
Covariate; No
Intralitter Correlation
BMDLos
173.095
7.37139
234.062
13.0394
BMDos
346.19
21.9447
349.659
27.0881
/?-Value0
0.6717
0.501
0.0007
0.0007
AIC
242.535
249.404
252.69
260.431
aHellwig and JSckh (1997): Confidential (19911
bSelected model parameters. Lowest AIC among models that provided an adequate fit.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dModel failed visual inspection.
AIC = Akaike's information criterion; BMDos = benchmark dose 5% extra risk; BMDLos = 95% benchmark dose
lower confidence limit; GD = gestation day.
110
2-Ethylhexanol
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Nested Logistic Model, with BMR of 5% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL
0.7
Nested Logistic
BMD Lower Bound
0.6
0.5
o
oj
<
£Z
o
~o
0.4
u_
0.3
0.2
BMD
20
40
60
80
100
120
140
160
dose
08:39 04/18 2018
Figure C-7. Nested Logistic Model for Increased Fetal Variations Following 2-Ethylhexanol
Exposure via Gavage on Gestation Days 6-15 in Female Wistar Rats (Hellwig and Jackh,
1997: Confidential. 1991)
Text Output for Figure C-7:
NLogistic Model. (Version: 2.20; Date: 04/27/2015)
Input Data File: //Aa.ad.epa.gov/ord/CIN/Users/main/F-K/JKaiser/Net
MyDocuments/BMDS/BMDS27 04/Data/nln_Nested2EH_var_Nln-BMR10-Restrict.(d)
Wed Apr 18 08:39:54 2018
BMDS Model Run
The probability function is:
Prob. = alpha + thetal*Rij + [1 - alpha - thetal*Rij]/
[1+exp(-beta-theta2*Rij-rho*log(Dose))],
where Rij is the litter specific covariate.
Restrict Power rho >= 1.
Total number of observations = 2 9
Total number of records with missing values = 0
111
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Total number of parameters in model = 8
Total number of specified parameters = 2
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Number of Bootstrap Iterations per run: 1000
Bootstrap Seed: 1524055194
User specifies the following parameters:
thetal = 0
theta2 = 0
Default Initial Parameter Values
alpha =
beta =
thetal =
theta2 =
rho =
phil =
phi 2 =
phi 3 =
0.245569
-6.03296
0
0
1
0.183845
0.0680539
0.374869
Specified
Specified
Parameter Estimates
Variable
alpha
beta
rho
phil
phi 2
phi 3
Estimate
0.245569
-6.03296
1
0.183845
0.0680539
0.374869
Std. Err.
0.0631849
0.867426
Bounded
0.246128
NA
NA
Log-likelihood: -119.702 AIC: 249.404
Litter Data
Dose
Lit.-Spec.
Cov.
Est. Prob.
Litter
Size
Expected Observed
Scaled
Residual
0.0000
0.0000
0.0000
0. 0000
0.0000
0.0000
0. 0000
0.0000
0.0000
0. 0000
13.0000
13.0000
15.0000
15.0000
15.0000
16.0000
16.0000
16.0000
17.0000
21.0000
0.246
0.246
0.246
0.246
0.246
0.246
0.246
0.246
0.246
0.246
10
1. 473
1.719
1. 965
1.719
1.719
1. 473
1. 965
1. 965
1. 965
2.456
-1.0088
-1.0409
0.0193
1.3812
0.7757
-1.0088
-1.0671
1.1056
-0.5239
0.2454
32.5000
32.5000
32.5000
32.5000
32.5000
32.5000
7.0000
11.0000
11.0000
13.0000
13.0000
14.0000
0.300
0.300
0.300
0.300
0.300
0.300
0. 900
1.801
1.501
2.101
2.101
2.101
1.2996
-1.3855
-1.2982
-0.7650
-0.0701
1.3198
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32.5000
16.0000
0.300
8
2.401
3
0.3803
32.5000
17.0000
0.300
8
2.401
5
1.6501
32.5000
17.0000
0.300
8
2.401
2
-0.2546
32.5000
17.0000
0.300
9
2.701
4
0.7602
163.0000
11.0000
0. 458
5
2.288
5
1.5398
163.0000
13.0000
0. 458
7
3.203
0
-1.3482
163.0000
14.0000
0. 458
7
3.203
2
-0.5064
163.0000
15.0000
0. 458
7
3.203
4
0.3353
163.0000
15.0000
0. 458
8
3. 661
7
1.2448
163.0000
15.0000
0. 458
7
3.203
2
-0.5064
163.0000
16.0000
0. 458
8
3. 661
2
-0.6191
163.0000
17.0000
0. 458
8
3. 661
5
0.4992
163.0000
17.0000
0. 458
8
3. 661
0
-1.3647
Scaled Residual(s) for Dose Group Nearest the BMD
Minimum scaled residual for dose group nearest the BMD = 1.3198
Minimum ABS(scaled residual) for dose group nearest the BMD = 1.3198
Average scaled residual for dose group nearest the BMD = 1.3198
Average ABS(scaled residual) for dose group nearest the BMD = 1.3198
Maximum scaled residual for dose group nearest the BMD = 1.3198
Maximum ABS(scaled residual) for dose group nearest the BMD = 1.3198
Number of litters used for scaled residual for dose group nearest the BMD = 1
Observed Chi-sguare = 28.3186
Bootstrapping Results
Number of Bootstrap Iterations per run: 1000
Bootstrap Chi-sguare Percentiles
Bootstrap
Run P-value 5 0th 90th 95th 99th
1 0.5070 28.5329 37.7451 39.7631 44.7794
2 0.4920 28.1849 37.8921 41.3515 47.9404
3 0.5040 28.3576 37.8756 40.6406 47.3071
Combined 0.5010 28.3314 37.8319 40.6425 47.0264
The results for three separate runs are shown. If the estimated p-values are
sufficiently
stable (do not vary considerably from run to run), then then number of iterations is
considered adeguate. The p-value that should be reported is the one that combines
the results of the three runs. If sufficient stability is not evident (and especially
if the p-values are close to the critical level for determining adeguate fit, e.g.,
0.05) ,
then the user should consider increasing the number of iterations per run.
To calculate the BMD and BMDL, the litter specific covariate is fixed
at the mean litter specific covariate of all the data: 14.689655
Benchmark Dose Computation
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Specified effect
Risk Type
Confidence level
BMD
BMDL
0. 05
Extra risk
0. 95
21.9447
7.37139
Model Predictions for Increased Fetal Retardations Following 2-Ethylhexanol Exposure
via Gavage on Gestation Days 6-15 in Female Wistar Rats (Hellwig and Jackfa, 1997;
Confidential, 1991)
The procedure outlined above for nested dichotomous data was applied to the data for
increased fetal retardations in Wistar rats treated with 2-EH via gavage on GDs 6-15 (Hellwig
and Jackh, 1997; Confidential, 1991) (see Table C-l). Table C-12 summarizes the BMD
modeling results. For increased fetal incidence of skeletal retardations, the data were modeled
without the highest dose of 325 mg/kg-day (HED) because there was severe maternal toxicity
(i.e., 60% mortality and a 20% decrease in body weight) at that dose that confounds the
interpretation of fetal skeletal changes. Therefore, only the BMD modeling results based on data
without the highest dose group are summarized in Table C-12. Including implantation sites as a
covariate and using intralitter correlations had significant effects on the %2 goodness-of-fit
statistics, AIC scores, and visual inspections. As assessed by the %2 goodness-of-fit statistic, AIC
score, and visual inspection, none of the NLogistic models provided an optimal fit
(see Table C-12). This data set was not amenable to BMD modeling.
Table C-12. Modeling Results for Increased Fetal Retardations Following
2-Ethylhexanol (CASRN 104-76-7) Exposure via Gavage on GDs 6-15 in Female Wistar
Ratsa
Parameter
Litter-Specific
Covariate; Intralitter
Correlationb
No Litter-Specific
Covariate; Intralitter
Correlationb
Litter-Specific
Covariate; No
Intralitter Correlation
No Litter-Specific
Covariate; No
Intralitter Correlation
BMDLos
2.99784
4.52848
5.31025
10.6269
BMDos
115.268
115.965
52.8472
115.888
/?-Valuec
0.517
0.397
0.0007
0
AIC
243.692
247.109
254.613
271.796
"Hellwig and Jackh (1997): Confidential (1991).
bBMD:BMDL ratio is too high, signifying there is uncertainty in estimating the BMDL.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
AIC = Akaike's information criterion; BMDos = benchmark dose 5% extra risk; BMDL = benchmark dose lower
confidence limit; BMDLos = 95% benchmark dose lower confidence limit; GD = gestation day.
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Model Predictions for Decreased Body Weight at Study Termination in Male F344 Rats
Treated with 2-Ethylhexanol via Gavage for 24 Months (Astill et at, 1996b; BASF, 1992a)
The procedure outlined above for continuous data was applied to the data for decreased
body weight in male F344 rats treated with 2-EH via gavage for 24 months (Astill et al.. 1996b;
BASF, 1992a) (see Table C-2). Table C-13 summarizes the BMD modeling results. The
constant variance model provided adequate fit to the variance data, and adequate fit to the means
was provided by the Exponential 4 and 5 models and the Hill model. The BMDLs for the
models providing adequate fit are sufficiently close (i.e., differ by
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Table C-13. Modeling Results for Decreased Body Weight in Male F344 Rats Administered 2-Ethylhexanol (CASRN 104-76-7) via
Gavage for 24 Months"
Model
Test for Significant
Difference />-Valucb
Variance
/>-Valuce
Means
/>-Valuce
Scaled Residuals
for Dose Groupd
AIC
BMDio
(mg/kg-d)
BMDLio
(mg/kg-d)
Constant variance
Exponential (Model 2)e
<0.0001
0.4099
0.055
-1.492
1,533.038
37.5406
33.853
Exponential (Model 3)e
<0.0001
0.4099
0.055
-1.492
1,533.038
37.5406
33.853
Exponential (Model 4)e
<0.0001
0.4099
0.4925
0.2944
1,529.708
25.6039
19.5673
Exponential (Model 5)e
<0.0001
0.4099
0.4925
0.2944
1,529.708
25.6039
19.5673
Hill6 f
<0.0001
0.4099
0.5517
0.288
1,529.5918
25.3591
18.9584
Linear6
<0.0001
0.4099
0.01787
-1.84
1,535.28639
40.8555
37.3042
Polynomial (2-degree)8
<0.0001
0.4099
0.01787
-1.84
1,535.28639
40.8555
37.3042
Polynomial (3-degree)8
<0.0001
0.4099
0.01787
-1.84
1,535.28639
40.8555
37.3042
Power"
<0.0001
0.4099
0.01787
-1.84
1,535.28639
40.8555
37.3042
aAstill et at (1996b): BASF (1992a).
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dScaled residuals at doses closest to BMD.
Tower restricted to >1.
Selected model. Lowest AIC among models that provided an adequate fit.
gCoefficients restricted to be negative.
AIC = Akaike's information criterion; BMD = benchmark dose; BMDio = benchmark dose 10% extra risk; BMDLio = 95% benchmark dose lower confidence limit.
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Hill Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
Hill
380
360
340
320
300
280
BMDL
BMD
0
10
20
30
40
50
60
70
80
90
dose
10:47 12/12 2017
Figure C-8. Hill Model for Decreased Body Weight at Study Termination in Male F344
Rats Treated with 2-Ethylhexanol via Gavage for 24 Months (Astill et al., 1996b; BASF,
1992a)
Model Predictions for Decreased Body Weight at Study Termination in Female F344 Rats
Treated with 2-Ethylhexanol via Gavage for 24 Months (Astill et al., 1996b; BASF, 1992a)
The procedure outlined above for continuous data was applied to the data for decreased
body weight in female F344 rats treated with 2-EH via gavage for 24 months (Astill et al..
1996b; BASF. 1992a) (see Table C-2). Table C-14 summarizes the BMD modeling results.
Neither the constant nor nonconstant variance models provided adequate fit to the variance data
using the full or reduced data set. This data set was not amenable to BMD modeling.
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Table C-14. Modeling Results for Decreased Body Weight in Female F344 Rats Administered 2-Ethylhexanol (CASRN 104-76-7) via
Gavage for 24 Months"
Model
Test for Significant
Difference />-Valucb
Variance
/>-Valuce
Means
/>-Valuce
Scaled Residuals
for Dose Groupd
AIC
BMDio
(mg/kg-d)
BMDLio
(mg/kg-d)
Nonconstant variance
Exponential (Model 2)e
<0.0001
<0.0001
0.9486
-0.04382
1,094.236
27.4323
20.8617
Exponential (Model 3)e
<0.0001
<0.0001
N/A
-0.02024
1,096.232
27.3874
20.8655
Exponential (Model 4)e
<0.0001
<0.0001
0.9486
-0.04382
1,094.236
27.4323
18.9954
Exponential (Model 5)e
<0.0001
0.001795
NDr
-0.04085
977.3824
27.6968
18.9199
Hille
NDr
NDr
NDr
NDr
NDr
NDr
NDr
Lineal
<0.0001
<0.0001
0.9894
-0.0249
1,094.232088
27.3368
21.0994
Polynomial (2-degree/
<0.0001
<0.0001
NDr
-0.0202
1,096.231911
27.3145
21.0995
Polynomial (3-degree/
<0.0001
<0.0001
NDr
-0.0202
1,098.231911
27.3093
21.0995
Power"
<0.0001
<0.0001
NDr
-0.0202
1,096.231911
27.3288
21.0995
aAstill et at (1996b): BASF (1992a).
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dScaled residuals at doses closest to BMD.
Tower restricted to >1.
Coefficients restricted to be negative.
AIC = Akaike's information criterion; BMD = benchmark dose; BMDio = benchmark dose 10% extra risk; BMDLio = 95% benchmark dose lower confidence limit;
NDr = not determined.
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Model Predictions for Increased Diameter of Bowman's Glands in the Olfactory
Epithelium of the Nasal Cavity in Male ICR Mice Exposed to 2-Ethylhexanol via
Inhalation for 3 Months (Mivake et at, 2016)
The procedure outlined above for continuous data was applied to the data for increased
diameter in ICR mice exposed to 2-EH via inhalation for 3 months (Mivake et al.. 2016)
(see Table B-13). Table C-15 summarizes the BMC modeling results. The initial modeling of
these data including all dose groups failed to provide an adequate fit to the data, as assessed by
the x2 goodness-of-fit test. Therefore, only the BMC modeling results based on data without the
high-dose group included are summarized in Table C-15 and Figure C-9. The nonconstant
variance model provided adequate fit to the variance data, and adequate fit to the means was
provided by all included models except Exponential 4 and 5 and Hill models. The BMCLs for
the models providing adequate fit are sufficiently close (i.e., differ by
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Table C-15. Modeling Results for Increased Diameter of Bowman's Glands in the Olfactory Epithelium of the Nasal Cavity in Male
ICR Mice Exposed to 2-Ethylhexanol (CASRN 104-76-7) via Inhalation for 3 Months"
Model
Test for Significant
Difference />-Valucb
Variance
p-Value0
Means
/>-Valuce
Scaled Residuals
for Dose Groupd
AIC
BMCisd
(mg/kg-d)
BMCLisd
(mg/kg-d)
Nonconstant variance
Exponential (Model 2)e
<0.0001
0.8476
0.2689
0.8578
57.9133
2.66935
1.74839
Exponential (Model 3)e
<0.0001
0.8476
0.2689
0.8578
57.9133
2.66936
1.74839
Exponential (Model 4)e
<0.0001
0.8476
NDr
-0.0284
58.69101
1.60493
0.769781
Exponential (Model 5)e
NDr
NDr
NDr
NDr
NDr
NDr
NDr
Hille
NDr
NDr
NDr
NDr
NDr
NDr
NDr
Linear''g
<0.0001
0.8476
0.8106
-0.106
56.748452
1.75839
1.10808
Polynomial (2-degree)f'g
<0.0001
0.8476
0.8106
-0.106
56.748452
1.75839
1.10808
Polynomial (3-degree)f'g
<0.0001
0.8476
0.8106
-0.106
56.748452
1.75839
1.10808
Power6'f
<0.0001
0.8476
0.8106
-0.106
56.748452
1.75839
1.10808
aMivake et al. (2016).
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dScaled residuals at doses closest to BMC.
Tower restricted to >1.
Selected model. Lowest AIC among model that provided an adequate fit.
gCoefficients restricted to be negative.
AIC = Akaike's information criterion; BMC = benchmark concentration; BMCL = benchmark concentration lower confidence limit; NDr = not determined;
SD = standard deviation.
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Linear Model, with BMR of 1 Std. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
Linear
18
16
14
12
10
8
6
BMDL
Biyip
2
0
4
6
8
10
12
15:05 08/15 2018
Figure C-9. Linear Model for Increased Diameter of Bowman's Glands in the Olfactory
Epithelium of the Nasal Cavity in Male ICR Mice Exposed to 2-Ethylhexanol via
Inhalation for 3 Months (Miyake et al., 2016)
Model Predictions for Increased Number of CD3-Positive Cells in the Olfactory Epithelium
of the Nasal Cavity in Male ICR Mice Exposed to 2-Ethylhexanol via Inhalation for
3 Months (Miyake et al., 2016)
The procedure outlined above for continuous data was applied to the data for increased
number of CD3-positive cells in the olfactory epithelium of the nasal cavity in ICR mice exposed
to 2-EH via inhalation for 3 months (Miyake et al.. 2016) (see Table B-13). Table C-16
summarizes the BMC modeling results. Neither the constant nor nonconstant variance models
provided adequate fit to the variance data using the full or reduced data set. This data set was not
amenable to BMC modeling.
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Table C-16. Modeling Results for Increased Number of CD3-Positive Cells in the Olfactory Epithelium of the Nasal Cavity in Male
ICR Mice Exposed to 2-Ethylhexanol (CASRN 104-76-7) via Inhalation for 3 Months"
Model
Test for Significant
Difference />-Valucb
Variance
p-Value0
Means
/>-Valuce
Scaled Residuals
for Dose Groupd
AIC
BMCisd
(mg/kg-d)
BMCLisd
(mg/kg-d)
Nonconstant variance
Exponential (Model 2)e
<0.0001
<0.0001
0.8936
-0.001534
94.03584
10.4885
8.74097
Exponential (Model 3)e
<0.0001
<0.0001
NDr
4.976
121.0273
79.0861
NDr
Exponential (Model 4)e
<0.0001
<0.0001
NDr
0
NDr
NDr
NDr
Exponential (Model 5)e
NDr
NDr
NDr
NDr
NDr
NDr
NDr
Hille
NDr
NDr
NDr
NDr
NDr
NDr
NDr
Lineal
<0.0001
<0.0001
0.3674
-0.699
94.830496
6.37672
4.17971
Polynomial (2-degree/
<0.0001
<0.0001
0.9678
0.00324
94.019578
9.0903
4.42983
Polynomial (3-degree/
<0.0001
<0.0001
NDr
-1.87 x 10-6
98.017947
9.68929
5.54314
Power"
<0.0001
<0.0001
NDr
-2.11 x 10-9
96.017947
9.24935
5.54314
aMivake et al. (2016).
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dScaled residuals at doses closest to BMC.
Tower restricted to >1.
Coefficients restricted to be negative.
AIC = Akaike's information criterion; BMC = benchmark concentration; BMCL = benchmark concentration lower confidence limit; NDr = not determined;
SD = standard deviation.
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Model Predictions for Decreased Number of OMP-Positive Cells in the Olfactory
Epithelium of the Nasal Cavity in Male ICR Mice Exposed to 2-Ethylhexanol via
Inhalation for 3 Months (Mivake et at, 2016)
The procedure outlined above for continuous data was applied to the data for decreased
number of OMP-positive cells in the olfactory epithelium of the nasal cavity in ICR mice
exposed to 2-EH via inhalation for 3 months (Mivake et al.. 2016) (see Table B-13). The BMC
modeling results are summarized in Table C-17 and Figure C-10. The nonconstant variance
model provided adequate fit to the variance data, and adequate fit to the means was provided by
the Exponential 2, 3, and 4 models. The BMCLs for the models providing adequate fit are
sufficiently close (i.e., differ by
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04-23-2019
Table C-17. Modeling Results for Decreased Number of OMP-Positive Cells in the Olfactory Epithelium of the Nasal Cavity in Male
ICR Mice Exposed to 2-Ethylhexanol (CASRN 104-76-7) via Inhalation for 3 Months"
Model
Test for Significant
Difference />-Valucb
Variance
/>-Valuce
Means
/>-Valuce
Scaled Residuals
for Dose Groupd
AIC
BMCisd
(mg/kg-d)
BMCLisd
(mg/kg-d)
Nonconstant variance
Exponential (Model 2)e
<0.0001
0.1928
0.1891
-1.585
-75.57555
5.85444
3.68145
Exponential (Model 3)e
<0.0001
0.1928
0.1891
-1.585
-75.57555
5.85444
3.68145
Exponential (Model 4)e f
<0.0001
0.1928
0.1891
-1.585
-75.57555
5.85444
3.1366
Exponential (Model 5)e
<0.0001
0.1928
0.068
-1.585
-73.57555
5.85444
3.68145
Hille
<0.0001
0.1928
0.08033
-1.53
-73.847892
5.30912
2.38254
Linear6
<0.0001
0.1928
0.05275
-0.342
-73.021654
11.2189
8.0351
Polynomial (2-degree)8
<0.0001
0.1928
0.05275
-0.342
-73.021654
11.2189
8.0351
Polynomial (3-degree)8
<0.0001
0.1928
0.05275
-0.342
-73.021654
11.2189
8.0351
Power"
<0.0001
0.1928
0.05275
-0.342
-73.021654
11.2189
8.0351
aMivake et al. (2016).
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dScaled residuals at doses closest to BMC.
Tower restricted to >1.
Selected model. Lowest AIC among model that provided an adequate fit.
gCoefficients restricted to be negative.
AIC = Akaike's information criterion; BMC = benchmark concentration; BMCL = benchmark concentration lower confidence limit; OMP = olfactory marker protein;
SD = standard deviation.
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Exponential Model 4, with BMR of 1 Std. Dev. for the BMD and 0.95 Lower Confidence Level for BM[
Exponential
1
0.8
0.6
0.4
0.2
BMDL
BMD
0
0
5
10
15
20
25
30
dose
13:55 03/29 2018
Figure C-10. Exponential 4 Model for Decreased Number of OMP-Positive Cells in the
Olfactory Epithelium of the Nasal Cavity in Male ICR Mice Exposed to 2-Ethylhexanol via
Inhalation for 3 Months (Miyake et al., 2016)
Model Predictions for Decreased Number of PCNA-Positive Cells in the Olfactory
Epithelium of the Nasal Cavity in Male ICR Mice Exposed to 2-Ethylhexanol via
Inhalation for 3 Months (Miyake et al., 2016)
The procedure outlined above for continuous data was applied to the data for decreased
number of PCNA-positive cells in the olfactory epithelium of the nasal cavity in ICR mice
exposed to 2-EH via inhalation for 3 months (Miyake et al.. 2016) (see Table B-13). The BMC
modeling results are summarized in Table C-18 and Figure C-l 1. The constant variance model
provided adequate fit to the variance data, and adequate fit to the means was provided by all
included models except the Exponential 5 model. The BMCLs for the models providing
adequate fit are sufficiently close (i.e., differ by
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Table C-18. Modeling Results for Decreased Number of PCNA-Positive Cells in the Olfactory Epithelium of the Nasal Cavity in
Male ICR Mice Exposed to 2-Ethylhexanol (CASRN 104-76-7) via Inhalation for 3 Months"
Model
Test for Significant
Difference />-Valucb
Variance
p-Value0
Means
/>-Valuce
Scaled Residuals
for Dose Groupd
AIC
BMCisd
(mg/kg-d)
BMCLisd
(mg/kg-d)
Constant variance
Exponential (Model 2)e
0.1465
0.8733
0.9498
0.2597
109.8212
15.2964
7.92323
Exponential (Model 3)e
0.1465
0.8733
0.854
0.09973
111.7521
17.2237
7.98115
Exponential (Model 4)e
0.1465
0.8733
0.9498
0.2597
109.8212
15.2964
5.31474
Exponential (Model 5)e
0.1465
0.8733
NDr
0.09973
113.7521
17.2237
7.98115
Hille
0.1465
0.8733
0.9159
0.0419
111.729351
17.7558
4.77342
Lineal
0.1465
0.8733
0.03181
1.43
116.536305
-9,999
126.062
Polynomial (2-degree)f'g
0.1465
0.8733
0.9942
0.0234
109.729916
17.9784
11.5389
Polynomial (3-degree)f'g
0.1465
0.8733
0.9942
0.0234
109.729916
17.9784
11.5389
Power6'g
0.1465
0.8733
0.9942
0.0234
109.729916
17.9784
11.5389
aMivake et al. (2016).
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dScaled residuals at doses closest to BMC.
Tower restricted to >1.
Coefficients restricted to be negative.
gSelected models. Lowest AIC among models that provided an adequate fit.
AIC = Akaike's information criterion; BMC = benchmark concentration; BMCL = benchmark concentration lower confidence limit; NDr = not determined;
PCNA = proliferating cell nuclear antigen; SD = standard deviation.
126
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Polynomial Model, with BMR of 1 Std. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMD
Polynomial
16
14
12
10
8
6
4
2
BMDL
BMD
0
0
5
10
15
20
25
30
dose
14:07 03/29 2018
Figure C-ll. Polynomial 2-Degree for Decreased Number of PCNA-Positive Cells in the
Olfactory Epithelium of the Nasal Cavity in Male ICR Mice Exposed to 2-Ethylhexanol via
Inhalation for 3 Months (Miyake et al., 2016)
Model Predictions for Decreased Glomerular Diameter in the Olfactory Bulb of the Brain
in Male ICR Mice Exposed to 2-Ethylhexanol via Inhalation for 3 Months (Miyake et al.,
2016)
The procedure outlined above for continuous data was applied to the data for decreased
glomerular diameter in the olfactory bulb of the brain in ICR mice exposed to 2-EH via
inhalation for 3 months (Miyake et al.. 2016) (see Table B-13). The BMC modeling results are
summarized in Table C-19 and Figure C-12. The constant variance model provided adequate fit
to the variance data, and adequate fit to the means was provided by the Exponential 3,
Exponential 5, Polynomial 3-degree, and Power models. The BMCLs for the models providing
adequate fit are sufficiently close (i.e., differ by
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Table C-19. Modeling Results for Decreased Glomerular Diameter in the Olfactory Bulb of the Brain Cavity in Male ICR Mice
Exposed to 2-Ethylhexanol (CASRN 104-76-7) via Inhalation for 3 Months"
Model
Test for Significant
Difference />-Valucb
Variance
p-Value0
Means
/>-Valuce
Scaled Residuals
for Dose Groupd
AIC
BMCisd
(mg/kg-d)
BMCLisd
(mg/kg-d)
Constant variance
Exponential (Model 2)e
<0.0001
0.7667
<0.0001
-0.7433
99.93473
5.197
4.00168
Exponential (Model 3)e
<0.0001
0.7667
0.1127
0.06175
82.44754
15.9383
10.9087
Exponential (Model 4)e
<0.0001
0.7667
<0.0001
-0.7433
99.93473
5.197
4.00168
Exponential (Model 5)e
<0.0001
0.7667
0.1127
0.06175
82.44754
15.9383
10.9087
Hille
<0.0001
0.7667
NDr
0.0699
84.440034
15.9859
10.7732
Lineal
<0.0001
0.7667
<.0001
1.85
140.542612
-9,999
204.321
Polynomial (2-degree/
<0.0001
0.7667
0.08259
1.46
82.919374
11.1254
9.92596
Polynomial (3-degree)f'g
<0.0001
0.7667
0.1179
0.48
82.376899
14.245
8.66779
Power8, e
<0.0001
0.7667
0.1134
0.0719
82.438191
15.9958
10.7427
aMivake et al. (2016).
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dScaled residuals at doses closest to BMC.
Tower restricted to >1.
Coefficients restricted to be negative.
gSelected models. Lowest AIC among models that provided an adequate fit.
AIC = Akaike's information criterion; BMC = benchmark concentration; BMCL = benchmark concentration lower confidence limit; NDr = not determined;
SD = standard deviation.
128
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Polynomial Model, with BMR of 1 Std. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMD
Polynomial
BMDL
0 5 10 15 20 25 30
dose
14:24 03/29 2018
Figure C-12. Polynomial 3-Degree for Decreased Glomerular Diameter in the Olfactory
Bulb of the Brain Cavity in Male ICR Mice Exposed to 2-Ethylhexanol via Inhalation for
3 Months (Miyake et al., 2016)
Model Predictions for Decreased Number of OMP-Positive Cells in Olfactory Bulb of the
Brain Cavity in Male ICR Mice Exposed to 2-Ethylhexanol via Inhalation for 3 Months
(Miyake et al., 2016)
The procedure outlined above for continuous data was applied to the data for decreased
number of OMP-positive cells in the olfactory bulb of the brain in ICR mice exposed to 2-EH via
inhalation for 3 months (Miyake et al.. 2016) (see Table B-13). Table C-20 summarizes the
BMC modeling results. The initial modeling of these data including all dose groups failed to
provide an adequate fit to the data, as assessed by the %2 goodness-of-fit test. Therefore, only the
BMC modeling results based on data without the high-dose group included are summarized in
Table C-20 and Figure C-13. The constant variance model provided adequate fit to the variance
data, and adequate fit to the means was provided by the Exponential 2, Exponential 4,
Polynomial 2-degree, and Polynomial 3-degree models. The BMCLs for the models providing
adequate fit are sufficiently close (i.e., differ by
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Table C-20. Modeling Results for Decreased Number of OMP-Positive Cells in the Olfactory Bulb of the Brain Cavity in Male ICR
Mice Exposed to 2-Ethylhexanol (CASRN 104-76-7) via Inhalation for 3 Months"
Model
Test for Significant
Difference />-Valucb
Variance
p-Value0
Means
/>-Valuce
Scaled Residuals
for Dose Groupd
AIC
BMCisd
(mg/kg-d)
BMCLisd
(mg/kg-d)
Constant variance
Exponential (Model 2)e
0.05415
0.3983
0.2404
-0.3415
73.54965
8.4853
4.80775
Exponential (Model 3)e
0.05415
0.3983
NDr
-9.44 x 10-8
74.20023
12.1444
5.52921
Exponential (Model 4)e
0.05415
0.3983
0.2404
-0.3415
73.54965
8.4853
4.00758
Exponential (Model 5)e
NDr
NDr
NDr
NDr
NDr
NDr
NDr
Hille
NDr
NDr
NDr
NDr
NDr
NDr
NDr
Lineal
0.05415
0.3983
0.02407
0.897
77.62454
-9,999
43.4358
Polynomial (2-degree/
0.05415
0.3983
0.6414
-0.0372
72.388258
10.3266
5.6273
Polynomial (3-degree)f'g
0.05415
0.3983
0.7907
-0.00682
72.241793
11.0983
5.71511
Power"
0.05415
0.3983
NDr
-3.04 x 10-9
74.200226
12.2351
5.7413
aMivake et al. (2016).
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dScaled residuals at doses closest to BMC.
Tower restricted to >1.
Coefficients restricted to be negative.
gSelected models. Lowest AIC among models that provided an adequate fit.
AIC = Akaike's information criterion; BMC = benchmark concentration; BMCL = benchmark concentration lower confidence limit; NDr = not determined;
OMP = olfactory marker protein; SD = standard deviation.
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Polynomial Model, with BMR of 1 Std. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMD
34
Polynomial
32
30
28
26
24
22
BMDL
BMD
20
0
2
4
6
8
10
12
dose
14:33 03/29 2018
Figure C-13. Polynomial 3-Degree for Decreased Number of OMP-Positive Cells in the
Olfactory Bulb of the Brain Cavity in Male ICR Mice Exposed to 2-Ethylhexanol via
Inhalation for 3 Months (Miyake et al., 2016)
Model Predictions for Decreased Number of TH-Positive Cells in Olfactory Bulb of the
Brain Cavity in Male ICR Mice Exposed to 2-Ethylhexanol via Inhalation for 3 Months
(Miyake et al., 2016)
The procedure outlined above for continuous data was applied to the data for decreased
number of TH-positive cells in the olfactory bulb of the brain in ICR mice exposed to 2-EH via
inhalation for 3 months (Miyake et al.. 2016) (see Table B-13). The BMC modeling results are
summarized in Table C-21 and Figure C-14. The nonconstant variance model provided adequate
fit to the variance data, and adequate fit to the means was provided by all included models. The
BMCLs for the models providing adequate fit are not sufficiently close (i.e., differ by
>threefold), so the models with the lowest BMCL (Exponential 4 and 5 models) are selected.
For decreased number of TH-positive cells in the olfactory bulb of the brain, the BMCLisd of
3.59 mg/m3 from the Exponential 4 and 5 models is selected.
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Table C-21. Modeling Results for Decreased Number of TH-Positive Cells in the Olfactory Bulb of the Brain Cavity in Male ICR
Mice Exposed to 2-Ethylhexanol (CASRN 104-76-7) via Inhalation for 3 Months"
Model
Test for Significant
Difference />-Valucb
Variance
/>-Valuce
Means
/>-Valuce
Scaled Residuals
for Dose Groupd
AIC
BMCisd
(mg/kg-d)
BMCLisd
(mg/kg-d)
Nonconstant variance
Exponential (Model 2)e
0.00213
0.654
0.2442
-0.3891
107.0891
19.0429
11.1862
Exponential (Model 3)e
0.00213
0.654
0.2442
-0.3891
107.0891
19.0429
11.1862
Exponential (Model 4)e f
0.00213
0.654
0.311
0.548
107.2963
10.6561
3.58878
Exponential (Model 5)e f
0.00213
0.654
0.311
0.548
107.2963
10.6561
3.58878
Hille
0.00213
0.654
0.3952
0.61
106.992906
9.29205
NDr
Linear6
0.00213
0.654
0.1865
0.148
107.629108
20.9134
13.2696
Polynomial (2-degree)8
0.00213
0.654
0.1865
0.148
107.629108
20.9134
13.2696
Polynomial (3-degree)8
0.00213
0.654
0.1865
0.148
107.629108
20.9134
13.2696
Power''e
0.00213
0.654
0.1865
0.148
107.629108
20.9134
13.2696
aMivake et al. (2016).
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dScaled residuals at doses closest to BMC.
Tower restricted to >1.
Selected models. Lowest BMCL among models that provided an adequate fit.
gCoefficients restricted to be negative.
AIC = Akaike's information criterion; BMC = benchmark concentration; BMCL = benchmark concentration lower confidence limit; NDr = not determined;
SD = standard deviation; TH = tyrosine hydroxylase.
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Exponential Model 4, with BMR of 1 Std. Dev. for the BMD and 0.95 Lower Confidence Level for BMD
Exponential
35
30
25
20
BMD
BMDL
0
5
10
15
20
25
30
dose
14:39 03/29 2018
Figure C-14. Exponential 4 Model for Decreased Number of TH-Positive Cells in the
Olfactory Bulb of the Brain Cavity in Male ICR Mice Exposed to 2-Ethylhexanol via
Inhalation for 3 Months (Miyake et al., 2016)
Model Predictions for Increased Number of Ibal-Positive Cells in Olfactory Bulb of the
Brain Cavity in Male ICR Mice Exposed to 2-Ethylhexanol via Inhalation for 3 Months
(Miyake et al., 2016)
The procedure outlined above for continuous data was applied to the data for increased
number of Ibal-positive cells in the olfactory bulb of the brain in ICR mice exposed to 2-EH via
inhalation for 3 months (Miyake et al.. 2016) (see Table B-13). Table C-22 summarizes the
BMC modeling results. Neither the constant nor nonconstant variance models provided adequate
fit to the variance data using the full data set. This data set was not amenable to BMC modeling.
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Table C-22. Modeling Results for Increased Number of Ibal-Positive Cells in the Olfactory Bulb of the Brain Cavity in Male ICR
Mice Exposed to 2-Ethylhexanol (CASRN 104-76-7) via Inhalation for 3 Months"
Model
Test for Significant
Difference />-Valucb
Variance
/>-Valuce
Means
p-Value0
Scaled Residuals
for Dose Groupd
AIC
BMCisd
(mg/kg-d)
BMCLisd
(mg/kg-d)
Nonconstant variance
Exponential (Model 2)e
<0.0001
0.5336
0.003127
-1.77
481.9362
13.1059
9.94605
Exponential (Model 3)e
<0.0001
0.5336
0.03183
0.003192
477.0084
23.0931
15.345
Exponential (Model 4)e
<0.0001
0.5336
0.0001987
-2.081
486.2443
12.1118
8.80593
Exponential (Model 5)e
<0.0001
0.5336
NDr
0.003325
479.0071
22.6623
15.1094
Hille
<0.0001
0.5336
0.03197
-0.277
477.001284
14.5898
NDr
Lineal
<0.0001
0.5336
<0.0001
-0.28
505.383851
-9,999
52.6469
Polynomial (2-degree/
<0.0001
0.5336
<0.0001
2.33
589.055535
-9,999
51.9659
Polynomial (3-degree/
<0.0001
0.5336
<0.0001
2.33
589.075607
-9,999
14.97
Power"
<0.0001
0.5336
0.03186
0.00332
477.007142
22.6623
15.1094
aMivake et al. (2016).
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dScaled residuals at doses closest to BMC.
Tower restricted to >1.
Coefficients restricted to be negative.
AIC = Akaike's information criterion; BMC = benchmark concentration; BMCL = benchmark concentration lower confidence limit; Ibal = ionized calcium-binding
adapter molecule 1; NDr = not determined; SD = standard deviation.
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Model Predictions for Increased Number of Dcx-Positive Cells in Olfactory Bulb of the
Brain Cavity in Male ICR Mice Exposed to 2-Ethylhexanol via Inhalation for 3 Months
(Miyake et at, 2016)
The procedure outlined above for continuous data was applied to the data for increased
number of Dcx positive cells in the olfactory bulb of the brain in ICR mice exposed to 2-EH via
inhalation for 3 months (Miyake ct al.. 2016) (see Table B-13). Table C-23 summarizes the
BMC modeling results. Neither the constant nor nonconstant variance models provided adequate
fit to the variance data using the full data set. This data set was not amenable to BMC modeling.
135
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Table C-23. Modeling Results for Increased Number of Dcx-Positive Cells in the Olfactory Bulb of the Brain Cavity in Male ICR
Mice Exposed to 2-Ethylhexanol (CASRN 104-76-7) via Inhalation for 3 Months"
Model
Test for Significant
Difference />-Valucb
Variance
/>-Valuce
Means
/>-Valuce
Scaled Residuals
for Dose Groupd
AIC
BMCisd
(mg/kg-d)
BMCLisd
(mg/kg-d)
Nonconstant variance
Exponential (Model 2)e
<0.0001
0.0184
<0.0001
0.4599
457.4724
23.8777
17.3388
Exponential (Model 3)e
<0.0001
0.0184
0.0004841
1.62 x 10-7
452.5633
28.7021
23.5487
Exponential (Model 4)e
<0.0001
0.0184
<0.0001
0.7851
461.039
24.9115
16.014
Exponential (Model 5)e
<0.0001
0.0184
NDr
1.84 x 10^
454.5633
28.3721
13.7625
Hille
<0.0001
0.0184
NDr
6.63 x 10^
454.563332
28.4344
NDr
Lineal
<0.0001
0.0184
<0001
-0.548
462.890803
-9,999
36.982
Polynomial (2-degree/
<0.0001
0.0184
0.000597
0.219
453.234583
25.1026
20.8016
Polynomial (3-degree/
<0.0001
0.0184
0.0014
0.082
451.530498
26.0806
23.0285
Power"
<0.0001
0.0184
0.00227
4.29 x 10-'
450.563321
28.6184
23.1719
aMivake et al. (2016).
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dScaled residuals at doses closest to BMC.
Tower restricted to >1.
Coefficients restricted to be negative.
AIC = Akaike's information criterion; BMC = benchmark concentration; BMCL = benchmark concentration lower confidence limit; Dcx = doublecortin; SD = standard
deviation.
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APPENDIX D. BENCHMARK DOSE CALCULATIONS FOR THE PROVISIONAL
ORAL SLOPE FACTOR
BMD MODELING TO IDENTIFY POTENTIAL PODS FOR THE DERIVATION OF A
PROVISIONAL ORAL SLOPE FACTOR
Significant dose-related trends were found for hepatocellular carcinomas in male and
female mice (see Table B-9) in the principal study of mice administered 2-ethylhexanol (2-EH)
via gavage 5 days/week for 18 months (Astill et al.. 1996b; BASF. 1991b). Data for these
endpoints were selected to determine the potential point of departure (POD) for the provisional
oral slope factor (p-OSF), using benchmark dose (BMD) analysis. Summaries of modeling
approaches and results (see Tables D-l through D-4) for each data set follow.
MODEL-FITTING PROCEDURE FOR CANCER INCIDENCE DATA
The model-fitting procedure for dichotomous cancer incidence is as follows. The
Multistage Cancer model in the U.S. EPA's Benchmark Dose Software (BMDS, Version 2.7) is
fit to the incidence data using the extra risk option. The Multistage-Cancer model is run for all
polynomial degrees up to n - 1 (where n is the number of dose groups including control). An
adequate model fit is judged by three criteria: (1) goodness-of-fit p-value (p < 0.1), (2) visual
inspection of the dose-response curve, and (3) scaled residual at the data point (except the
control) closest to the predefined benchmark response (BMR). Among the models providing
adequate fit to the data, the BMDL/BMCL (benchmark dose/concentration lower confidence
limit) for the model with the lowest Akaike's information criterion (AIC) is selected as the POD.
In accordance with U.S. EPA (2012b) Benchmark Dose Technical Guidance and the U.S. EPA
(2005) Guidelines for Carcinogen Risk Assessment, BMD/BMC- (benchmark dose/concentration)
and BMDL/BMCL values associated with an extra risk of 10% are calculated.
Hepatocellular Carcinomas or Adenomas in Male B6C3Fi Mice Treated with
2-Ethylhexanol via Gavage for 18 Months
The procedure outlined above for cancer incidence data was applied to the incidence data
for hepatocellular carcinomas and adenomas in male B6C3Fi mice treated with 2-EH via gavage
for 18 months (Astill et al.. 1996b; BASF. 1991b) (see Table B-9). To account for group
differences in survival, data for tumor incidence in male mice were modeled using a Poly-3
survival-adjusted number at risk. Table D-l summarizes the BMD modeling results for the
unadjusted incidence of hepatocellular carcinomas or adenomas in males. Table D-2
summarizes the BMD modeling results for the Poly-3-weighted number at risk. BMDLs for
models were sufficiently close (differed by less than two- to threefold), so the model with the
lowest AIC was selected (Multistage 1-degree; see Figures D-l and D-2). Modeling the
observed incidence and Poly-3 weighted number at risk gives a more conservative BMDL than
unadjusted number, thus the Poly-3-adjusted BMDLio for the 1-degree Multistage Cancer model
of 25 mg/kg-day is selected.
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Table D-l. BMD Model Predictions for Unadjusted Incidence of Hepatocellular
Carcinomas or Adenomas in Male B6C3Fi Mice Treated with
2-Ethylhexanol (CASRN 104-76-7) via Gavage for 18 Months3
Model
DF
X2
X2 Goodness-of-Fit
/j-Valucb
Scaled Residual:
Dose Nearest BMDC
AIC
BMD io
(mg/kg-d)
BMDLio
(mg/kg-d)
Multistage Cancer
(l-degree)d'e
2
1.47
0.4795
-0.024
155.503
66.3299
30.9815
Multistage Cancer
(2-degree)d
1
1.47
0.2254
-0.028
157.503
66.6271
30.982
Multistage Cancer
(3-degree)d
1
1.47
0.2254
-0.028
157.503
66.6271
30.982
"Astill et at (1996b): BASF (1991b).
bValues <0.05 fail to meet conventional goodness-of-fit criteria.
°Scaled residuals for dose group nearest the BMD.
dPower restricted to >1.
"Selected model. All models provided adequate fit to the data.
AIC = Akaike's information criterion; BMD = benchmark dose; BMDL = benchmark dose lower confidence limit;
DF = degree(s) of freedom.
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Multistage Cancer Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL
Multistage Cancer
Linear extrapolation
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
BMDL
BMD,
0
10
20
30
40
50
60
70
80
dose
16:28 10/18 2017
Figure D-l. Multistage Cancer 1-Degree BMD Model for Unadjusted Incidence of
Hepatocellular Carcinomas or Adenomas in Male B6C3Fi Mice Treated with
2-Ethylhexanol via Gavage for 18 Months (Astill et al., 1996b; BASF, 1991b)
Text Output for Figure D-l:
Multistage Model. (Version: 3.4; Date: 05/02/2014)
Input Data File: //Aa.ad.epa.gov/ord/CIN/Users/main/A-E/bowens/Net
MyDocuments/BMDS/BMDS2704/Data/msc_Dichotomous_Opt.(d)
Gnuplot Plotting File: //Aa.ad.epa.gov/ord/CIN/Users/main/A-E/bowens/Net
MyDocuments/BMDS/BMDS2704/Data/msc_Dichotomous_Opt.pit
Wed Oct 18 16:28:03 2017
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl) ]
The parameter betas are restricted to be positive
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Dependent variable = Effect
Independent variable = Dose
Total number of observations = 4
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.0938431
Beta(1) = 0.00158119
Asymptotic Correlation Matrix of Parameter Estimates
Background Beta(l)
Background 1 -0.5 6
Beta(1) -0.56 1
Parameter Estimates
Interval
Variable
Limit
Background
0.148848
Beta(1)
0.00354491
95.0% Wald Confidence
Estimate Std. Err. Lower Conf. Limit Upper Conf.
0.0932971 0.028343 0.0377459
0.00158843 0.000998223 -0.000368048
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-74.9629
-75.7517
-77.2773
# Param's
4
2
1
Deviance Test d.f.
1.57766
4 . 62884
P-value
0.4544
0.2011
AIC:
155 .503
Dose
Goodness of Fit
Est._Prob. Expected Observed Size
Scaled
Residual
0.0000
5.5000
21.8000
79.9000
Chi^2 = 1.47
0.0933
0.1012
0.1242
0.2014
d.f. = 2
4.665 6.000 50.000 0.649
5.059 3.000 50.000 -0.966
6.208 7.000 50.000 0.340
10.068 10.000 50.000 -0.024
P-value = 0.4795
140
2-Ethylhexanol
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Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 66.32 99
BMDL = 30.9815
BMDU = 3.80333e+007
Taken together, (30.9815, 3.80333e+007) is a 90 % two-sided confidence
interval for the BMD
Cancer Slope Factor = 0.00322773
Table D-2. BMD Model Predictions for Poly-3-Adjusted Incidence of Hepatocellular
Carcinomas or Adenomas in Male B6C3Fi Mice Treated with
2-Ethylhexanol (CASRN 104-76-7) via Gavage for 18 Months3
Model
DF
x2
X2 Goodness-of-Fit
/>-Valucb
Scaled Residual:
Dose Nearest BMDC
AIC
BMDio
(mg/kg-d)
BMDLio
(mg/kg-d)
Multistage Cancer
(l-degree)d'e
2
1.47
0.4794
0.219
149.954
48.6288
24.5793
Multistage Cancer
(2-degree)d
1
1.44
0.2306
-0.033
151.928
53.5151
24.6449
Multistage Cancer
(3-degree)d
1
1.44
0.2306
-0.033
151.928
53.5149
24.6449
aAstill et at (1996b): BASF (1991b).
bValues <0.05 fail to meet conventional goodness-of-fit criteria.
°Scaled residuals for dose group nearest the BMD.
dPower restricted to >1.
"Selected model. All models provided adequate fit to the data.
AIC = Akaike's information criterion; BMD = benchmark dose; BMDL = benchmark dose lower confidence limit;
DF = degree(s) of freedom.
141
2-Ethylhexanol
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Multistage Cancer Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL
Multistage Cancer
Linear extrapolation
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
BMDL
0
10
20
30
40
60
70
80
dose
12:51 08/15 2018
Figure D-2. Multistage Cancer 1-Degree BMD Model for Poly-3-Adjusted Incidence of
Hepatocellular Carcinomas or Adenomas in Male B6C3Fi Mice Treated with
2-Ethylhexanol via Gavage for 18 Months (Astill et al., 1996b; BASF, 1991b)
Text Output for Figure D-2:
Multistage Model. (Version: 3.4; Date: 05/02/2014)
Input Data File: //Aa.ad.epa.gov/ord/CIN/Users/main/F-K/JKaiser/Net
MyDocuments/BMDS/BMDS2704/Data/msc_Dichotomouspol3adjmales_Mscl-BMR10.(d)
Gnuplot Plotting File: //Aa.ad.epa.gov/ord/CIN/Users/main/F-K/JKaiser/Net
MyDocuments/BMDS/BMDS2704/Data/msc_Dichotomouspol3adjmales_Mscl-BMR10.pit
Wed Aug 15 12:51:17 2018
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl) ]
The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Total number of observations = 4
Total number of records with missing values = 0
142
2-Ethylhexanol
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Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.0938868
Beta (1) = 0.00220263
Asymptotic Correlation Matrix of Parameter Estimates
Background Beta(l)
Background 1 -0.52
Beta (1) -0.52 1
Parameter Estimates
Interval
Variable
Limit
Background
0.151777
Beta(1)
0.00444597
Estimate
0.0943095
0.00216663
95.0% Wald Confidence
Std. Err. Lower Conf. Limit Upper Conf.
0.0293206 0.0368422
0.00116295 -0.000112708
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-72.1933
-72.9771
-75.188
# Param's
4
2
1
Deviance Test d.f.
1.5677
5.98943
P-value
0.4566
0.1121
AIC:
149.954
Dose
Goodness of Fit
Est._Prob. Expected Observed Size
Scaled
Residual
0.0000
5.5000
21.8000
79.9000
Chi^2 = 1.47
0.0943
0.1050
0.1361
0.2383
d.f. = 2
4.560 6.000 48.349
5.036 3.000 47.940
6.481 7.000 47.622
9.930 10.000 41.675
P-value = 0.4794
0.709
-0.959
0.219
0. 025
Benchmark Dose Computation
Specified effect = 0.1
143
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Risk Type
Extra risk
Confidence level
0. 95
BMD
48.6288
BMDL
24.5793
BMDU
244.468
Taken together, (24.5793, 244.468) is a 90 % two-sided confidence
interval for the BMD
Cancer Slope Factor
0.00406847
Hepatocellular Carcinomas in Female B6C3Fi Mice Treated with 2-Ethylhexanol via
Gavage for 18 Months
The procedure outlined above for cancer incidence data was applied to the incidence data
for hepatocellular carcinomas in female B6C3Fi mice treated with 2-EH via gavage for
18 months (Astill et al.. 1996b; BASF. 1991b) (see Table B-9). To account for group differences
in survival, data for tumor incidence in female mice were also modeled using a Poly-3
survival-adjusted number at risk. Table D-3 summarizes the BMD modeling results for the
unadjusted incidence of hepatocellular carcinomas in females. Table D-4 summarizes the BMD
modeling results for the Poly-3-weighted number at risk. All models provided adequate fit to the
data. The Multistage Cancer 2- and 3-degree models converged onto the Multistage
Cancer 1-degree model for both data sets (see Figures D-3 and D-4). Modeling the observed
incidence and Poly-3-weighted number at risk gives a more conservative BMDL than unadjusted
number, thus the Poly-3-adjusted BMDLio for the 1-degree Multistage Cancer model of
27 mg/kg-day is selected.
144
2-Ethylhexanol
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Table D-3. BMD Model Predictions for Unadjusted Incidence of Hepatocellular
Carcinomas in Female B6C3Fi Mice Treated with 2-Ethylhexanol (CASRN 104-76-7) via
Gavage for 18 Months3
Model
DF
X2
X2 Goodness-of-Fit
/>-Valucb
Scaled Residual:
Dose Nearest BMDC
AIC
BMDio
(mg/kg-d)
BMDLio
(mg/kg-d)
Multistage Cancer
(l-degree)d'e
2
1.35
0.5092
-0.534
70.4171
62.6923
35.2874
Multistage Cancer
(2-degree)d
2
1.35
0.5092
-0.534
70.4171
62.6923
35.2874
Multistage Cancer
(3-degree)d
2
1.35
0.5092
-0.534
70.4171
62.6923
35.2874
aAstill et al. (1996b): BASF (1991b).
bValues <0.05 fail to meet conventional goodness-of-fit criteria.
°Scaled residuals for dose group nearest the BMD.
dPower restricted to >1.
Selected model. All models provided adequate fit to the data. The Multistage Cancer 2- and 3-degree models
converged onto the Multistage Cancer 1-degree model.
AIC = Akaike's information criterion; BMD = benchmark dose; BMDL = benchmark dose lower confidence limit;
DF = degree(s) of freedom.
Multistage Cancer Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL
Multistage Cancer
Linear extrapolation
0.2
0.15
o
<
o
~o
LL
0.05
BMDL
3MD
0
10
20
30
40
50
60
70
80
dose
13:11 08/15 2018
Figure D-3. Multistage Cancer 1-Degree BMD Model for Unadjusted Incidence of
Hepatocellular Carcinomas in Female B6C3Fi Mice Treated with 2-Ethylhexanol via
Gavage for 18 Months (Astill et al., 1996b; BASF, 1991b)
145
2-Ethylhexanol
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Text Output for Figure D-3:
Multistage Model. (Version: 3.4; Date: 05/02/2014)
Input Data File: //Aa.ad.epa.gov/ord/CIN/Users/main/F-K/JKaiser/Net
MyDocuments/BMDS/BMDS2704/Data/msc_Dichotomousnopol3adjfemales_Mscl-BMR10.(d)
Gnuplot Plotting File: //Aa.ad.epa.gov/ord/CIN/Users/main/F-K/JKaiser/Net
MyDocuments/BMDS/BMDS2704/Data/msc_Dichotomousnopol3adjfemales_Mscl-BMR10.pit
Wed Aug 15 13:11:37 2018
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl) ]
The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Total number of observations = 4
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.0143667
Beta(1) = 0.00124932
Asymptotic Correlation Matrix of Parameter Estimates
Background Beta(l)
Background 1 -0.63
Beta (1) -0.63 1
Parameter Estimates
95.0% Wald Confidence
Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf.
Limit
Background 0.00345348 0.0137041 -0.0234061
0.0303131
Beta(1) 0.0016806 0.000778964 0.000153855
0.00320734
146
2-Ethylhexanol
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Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood) # Param'
-32.5045 4
-33.2085 2
-36.7042 1
Deviance Test d.f.
1.40813
8.39949
P-value
0.4946
0. 03844
AIC:
70.4171
Dose
Goodness of Fit
Est._Prob. Expected Observed Size
Scaled
Residual
0.0000
5.3000
21.1000
77.3000
0.0035
0.0123
0.0382
0.1249
0.173
0.615
1.909
6.243
0.000
1.000
3.000
5.000
50.000
50.000
50.000
50.000
-0.416
0. 495
0.806
-0.532
Chi^2 =1.35
d.f. = 2
P-value = 0.5092
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 62.6923
BMDL = 35.2874
BMDU = 188.051
Taken together, (35.2874, 188.051) is a 90 % two-sided confidence
interval for the BMD
Cancer Slope Factor = 0.00283387
147 2-Ethylhexanol
-------
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04-23-2019
Table D-4. BMD Model Predictions for Poly-3-Adjusted Incidence of Hepatocellular
Carcinomas in Female B6C3Fi Mice Treated with 2-Ethylhexanol (CASRN 104-76-7) via
Gavage for 18 Months3
Model
DF
X2
X2 Goodness-of-Fit
/>-Valucb
Scaled Residual:
Dose Nearest BMDC
AIC
BMDio
(mg/kg-d)
BMDLio
(mg/kg-d)
Multistage Cancer
(l-degree)d'e
3
0.70
0.8721
0.475
63.6119
44.2035
26.7312
Multistage Cancer
(2-degree)d
3
0.70
0.8721
0.475
63.6119
44.2035
26.7312
Multistage Cancer
(3-degree)d
3
0.70
0.8721
0.475
63.6119
44.2035
26.7312
aAstill et al. (1996b): BASF (1991b).
bValues <0.05 fail to meet conventional goodness-of-fit criteria.
°Scaled residuals for dose group nearest the BMD.
dPower restricted to >1.
Selected model. All models provided adequate fit to the data. The Multistage Cancer 2- and 3-degree models
converged onto the Multistage Cancer 1-degree model.
AIC = Akaike's information criterion; BMD = benchmark dose; BMDL = benchmark dose lower confidence limit;
DF = degree(s) of freedom.
Multistage Cancer Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL
Multistage Cancer
Linear extrapolation
0.3
0.25
0.2
o
<
o
~o
0.15
u_
0.05
BMDL
BMD
0
10
20
30
40
50
60
70
80
dose
13:25 08/15 2018
Figure D-4. Multistage Cancer 1-Degree BMD Model for Poly-3-adjusted Incidence of
Hepatocellular Carcinomas in Female B6C3Fi Mice Treated with 2-Ethylhexanol via
Gavage for 18 Months (Astill et al., 1996b; BASF, 1991b)
148
2-Ethylhexanol
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Text Output for Figure D-4:
Multistage Model. (Version: 3.4; Date: 05/02/2014)
Input Data File: //Aa.ad.epa.gov/ord/CIN/Users/main/F-K/JKaiser/Net
MyDocuments/BMDS/BMDS2704/Data/msc_Dichotomouspol3adjfemales_Mscl-BMR10.(d)
Gnuplot Plotting File: //Aa.ad.epa.gov/ord/CIN/Users/main/F-K/JKaiser/Net
MyDocuments/BMDS/BMDS2704/Data/msc_Dichotomouspol3adjfemales_Mscl-BMR10.pit
Wed Aug 15 13:25:00 2018
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl) ]
The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Total number of observations = 4
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.0112972
Beta(1) = 0.00185417
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background
have been estimated at a boundary point, or have been specified by
the user,
and do not appear in the correlation matrix )
Beta(1)
Beta (1) 1
Parameter Estimates
95.0% Wald Confidence
Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf.
Limit
Background 0 NA
149
2-Ethylhexanol
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04-23-2019
Beta(1) 0.00238353 0.000795164 0.000825041
0.00394203
NA - Indicates that this parameter has hit a bound
implied by some inequality constraint and thus
has no standard error.
Model
Full model
Fitted model
Reduced model
Analysis of Deviance Table
#
Log(likelihood)
-30.4802
-30. 8059
-35.5269
Param's
4
1
1
Deviance Test d.f.
0.651419
10. 0934
P-value
0.8846
0. 01779
AIC:
63.6119
Dose
Goodness of Fit
Est._Prob. Expected Observed Size
Scaled
Residual
0.0000
5.3000
21.1000
77.3000
Chi^2 = 0.70
0.0000
0.0126
0.0490
0.1683
d.f. = 3
0.000 0.000 45.551
0.601 1.000 47.837
2.297 3.000 46.839
6.025 5.000 35.806
P-value = 0.8721
0. 000
0.519
0. 475
-0.458
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 44.2035
BMDL = 2 6.7312
BMDU = 108.057
Taken together, (26.7312, 108.057) is a 90 % two-sided confidence
150 2-Ethylhexanol
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