c/EPA
United States
Environmental Protection
Agency
EPA/690/R-24/003F | May 2024 | FINAL
Provisional Peer-Reviewed Toxicity Values for
p-lsopropyltoluene
(CASRN 99-87-6)
SUPERFUND
U.S. EPA Office of Research and Development
Center for Public Health and Environmental Assessment
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A mA United States
Environmental Protection
* ^ ^1 M % Agency
EPA 690 R-24 003F
May 2024
https://www.epa.gov/pprtv
Provisional Peer-Reviewed Toxicity Values for
/>-Isopropyltoluene
(CASRN 99-87-6)
Center for Public Health and Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
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AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGER
Lucina E Lizarraga, PhD
Center for Public Health and Environmental Assessment, Cincinnati, OH
CONTRIBUTOR
J. Allen Davis, MSPH
Center for Public Health and Environmental Assessment, Washington, DC
SCIENTIFIC TECHNICAL LEAD
Lucina E Lizarraga, PhD
Center for Public Health and Environmental Assessment, Cincinnati, OH
DRAFT DOCUMENT PREPARED BY
SRC, Inc.
7502 Round Pond Road
North Syracuse, NY 13212
PRIMARY INTERNAL REVIEWERS
Kyoungju Choi, PhD
Center for Public Health and Environmental Assessment, Washington, DC
Roman Mecenzev, PhD
Center for Public Health and Environmental Assessment, Washington, DC
PRIMARY EXTERNAL REVIEWERS
Organized by Eastern Research Group, Inc.
110 Hartwell Avenue
Lexington, MA 02421-3136
PPRTV PROGRAM MANAGEMENT
Teresa L. Shannon
Center for Public Health and Environmental Assessment, Cincinnati, OH
Allison L. Phillips, PhD
Center for Public Health and Environmental Assessment, Cincinnati, OH
J. Phillip Kaiser, PhD, DABT
Center for Public Health and Environmental Assessment, Cincinnati, OH
Questions regarding the contents of this PPRTV document may be directed to the U.S. EPA
Office of Research and Development (ORD) Center for Public Health and Environmental
Assessment (CPHEA) website at https://ecomments.epa.gov/pprtv.
in
/Msopropyltoluene
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TABLE OF CONTENTS
COMMONLY USED ABBREVIATIONS AND ACRONYMS v
BACKGROUND 1
QUALITY ASSURANCE 1
DISCLAIMERS 2
QUESTIONS REGARDING PPRTVs 2
1. INTRODUCTION 3
2. REVIEW OF POTENTIALLY RELEVANT DATA (NONCANCER AND
CANCER) 7
2.1. HUMAN STUDIES 11
2.2. ANIMAL STUDIES 11
2.2.1. Oral Exposures 11
2.2.2. Inhalation Exposures 15
2.3. OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS) 15
2.3.1. Genotoxicity 16
2.3.2. Supporting Animal Studies 20
2.3.3. Mode-of-Action/Mechanistic Studies 32
2.3.4. Metabolism/Toxicokinetic Studies 32
3. DERIVATION 01 PROVISIONAL VALUES 35
3.1. DERIVATION OF ORAL REFERENCE DOSES 35
3 .2. DERIVATION OF INHALATION REFERENCE CONCENTRATIONS 35
3.3. SUMMARY OF NONCANCER PROVISIONAL REFERENCE VALUES 35
3.4. CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR 36
3.5. DERIVATION OF PROVISIONAL CANCER RISK ESTIMATES 37
APPENDIX A. NONCANCER SCREENING PROVISIONAL VALUES 38
APPENDIX B. DATA TABLES 90
APPENDIX C. BENCHMARK DOSE MODELING RESULSTS 99
APPENDIX D. PARAMETERS OF TOOLS USED FOR READ-ACROSS 115
APPENDIX E. REFERENCES 117
iv
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COMMONLY USED ABBREVIATIONS AND ACRONYMS
a2u-g
alpha 2u-globulin
ACGIH
American Conference of Governmental
Industrial Hygienists
AIC
Akaike's information criterion
ALD
approximate lethal dosage
ALT
alanine aminotransferase
AR
androgen receptor
AST
aspartate aminotransferase
atm
atmosphere
ATSDR
Agency for Toxic Substances and
Disease Registry
BMC
benchmark concentration
BMCL
benchmark concentration lower
confidence limit
BMD
benchmark dose
BMDL
benchmark dose lower confidence limit
BMDS
Benchmark Dose Software
BMR
benchmark response
BUN
blood urea nitrogen
BW
body weight
CA
chromosomal aberration
CAS
Chemical Abstracts Service
CASRN
Chemical Abstracts Service Registry
Number
CBI
covalent binding index
CHO
Chinese hamster ovary (cell line cells)
CL
confidence limit
CNS
central nervous system
CPHEA
Center for Public Health and
Environmental Assessment
CPN
chronic progressive nephropathy
CYP
cytochrome P450
DAF
dosimetric adjustment factor
DEN
diethylnitrosamine
DMSO
dimethyl sulfoxide
DNA
deoxyribonucleic acid
EPA
Environmental Protection Agency
ER
estrogen receptor
FDA
Food and Drug Administration
FEVi
forced expiratory volume of 1 second
GD
gestation day
GDH
glutamate dehydrogenase
GGT
y-glutamyl transferase
GSH
glutathione
GST
glutathione S transferase
Hb/g A
animal blood gas partition coefficient
Hb/gH
human blood gas partition coefficient
HEC
human equivalent concentration
HED
human equivalent dose
i.p.
intraperitoneal
IRIS
Integrated Risk Information System
Abbreviations and acronyms not listed on
PPRTV assessment.
IVF
in vitro fertilization
LC50
median lethal concentration
LD50
median lethal dose
LOAEL
lowest-observed-adverse-effect level
MN
micronuclei
MNPCE
micronucleated polychromatic
erythrocyte
MOA
mode of action
MTD
maximum tolerated dose
NAG
7V-acetyl-P-D-glucosaminidase
NCI
National Cancer Institute
NOAEL
no-observed-adverse-effect level
NTP
National Toxicology Program
NZW
New Zealand White (rabbit breed)
OCT
ornithine carbamoyl transferase
ORD
Office of Research and Development
PBPK
physiologically based pharmacokinetic
PCNA
proliferating cell nuclear antigen
PND
postnatal day
POD
point of departure
PODadj
duration adjusted POD
QSAR
quantitative structure-activity
relationship
RBC
red blood cell
RDS
replicative DNA synthesis
RfC
inhalation reference concentration
RfD
oral reference dose
RGDR
regional gas dose ratio
RNA
ribonucleic acid
SAR
structure-activity relationship
SCE
sister chromatid exchange
SD
standard deviation
SDH
sorbitol dehydrogenase
SE
standard error
SGOT
glutamic oxaloacetic transaminase, also
known as AST
SGPT
glutamic pyruvic transaminase, also
known as ALT
SSD
systemic scleroderma
TCA
trichloroacetic acid
TCE
trichloroethylene
TWA
time-weighted average
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
U.S.
United States of America
WBC
white blood cell
this page are defined upon first use in the
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PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR
/7-ISOPROPYLTOLUENE (CASRN 99-87-6)
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 U.S. Environmental Protection Agency (U.S. EPA)
guidance on human health toxicity value derivations.
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. EPA's PPRTV
website at https://www.epa.gov/pprtv. PPRTV assessments are eligible to be updated on a 5-year
cycle and revised as appropriate to incorporate new data or methodologies that might impact the
toxicity values or affect the characterization of chemical's potential for causing toxicologically
relevant human-health effects. Questions regarding nomination of chemicals for update can be
sent to the appropriate U.S. EPA eComments Chemical Safety website at
https://ecomments.epa.gov/chemicalsafetv/.
QUALITY ASSURANCE
This work was conducted under the U.S. EPA Quality Assurance (QA) program to ensure
data are of known and acceptable quality to support their intended use. Surveillance of the work
by the assessment managers and programmatic scientific leads ensured adherence to QA
processes and criteria, as well as quick and effective resolution of any problems. The QA
manager, assessment managers, and programmatic scientific leads have determined under the
QA program that this work meets all U.S. EPA quality requirements. This PPRTV assessment
was written with guidance from the CPHEA Program Quality Assurance Project Plan (PQAPP),
the QAPP titled Program Quality Assurance Project Plan (POAPP) for the Provisional
Peer-Reviewed Toxicity Values (PPRTVs) and Related Assessments Documents
(L-CPAD-0032718-OP), and the PPRTV assessment development contractor QAPP titled
Quality Assurance Project Plan—Preparation of Provisional Toxicity Value (PIT) Documents
(L-CPAD-0031971-OP). As part of the QA system, a quality product review is done prior to
management clearance. A Technical Systems Audit may be performed at the discretion of the
QA staff.
All PPRTV assessments receive internal peer review by at least two CPHEA scientists
and an independent external peer review by at least three scientific experts. The reviews focus on
whether all studies have been correctly selected, interpreted, and adequately described for the
purposes of deriving a provisional reference value. The reviews also cover quantitative and
qualitative aspects of the provisional value development and address whether uncertainties
associated with the assessment have been adequately characterized.
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DISCLAIMERS
The PPRTV document provides toxicity values and information about the toxicologically
relevant 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 ORD CPHEA website at https://ecomments.epa.gov/pprtv.
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1. INTRODUCTION
/Msopropyltoluene, CASRN 99-87-6, is a naturally occurring monoterpene also known as
/;-cymene. Its structure consists of a benzene ring, substituted with one methyl group and one
isopropyl group in the para- (1 and 4) positions on the aromatic ring. /Msopropyltoluene is the
most common isomer; the other geometric isomers are o-isopropyltoluene (CASRN 527-84-4)
and w-isopropyltoluene (CASRN 535-77-3), with alkyl groups substituted in the ortho- and
meta- positions, respectively (NLM. 2022a. c; U.S. EPA 201 Id).
/Msopropyltoluene is preregistered with Europe's Registration, Evaluation, Authorisation
and Restriction of Chemicals (REACH) program (ECHA 2022) and is listed on the U.S. EPA's
Toxic Substances Control Act (TSCA) public inventory (U.S. EPA. 2024c). p-Isopropyltoluene
is identified as a plant metabolite and human urinary metabolite (NLM. 2022b). It has a sweet
aromatic, weak citrus odor, and is used to mask and improve the odor of products.
/?-Isopropyltoluene is also used in lacquers and varnishes as a thinner and in solvents as a starting
material and heat-transfer fluid (NLM. 2022b). p-Isopropyltoluene occurs naturally in foods
including butter, carrots, nutmeg, orange juice, oregano, raspberries, and lemon oil, as well as a
in number of essential oils (FFHPVC. 2005).
The empirical formula forp-isopropyltoluene is C10H14. The chemical structure is shown
in Figure 1. Table 1 summarizes the physicochemical properties for^-isopropyltoluene.
/Msopropyltoluene is a colorless, transparent liquid. Its low water solubility and high vapor
pressure indicate that this substance is hydrophobic and volatile and will exist predominantly in
the vapor phase in air. Additionally, volatilization from water surfaces or moist soil surfaces is
expected, based upon an estimated Henry's law constant of 1.13 x io~2 atm-m3/mole at 25°C. In
the atmosphere, /?-isopropyltoluene has an estimated half-life of 1 day, calculated from an
estimated rate constant of 1.45 x 10 " cm3/molecule-second at 25°C for reaction with
photochemically produced hydroxyl radicals (NLM. 2022b; Atkinson and Arev. 2003). The
estimated soil adsorption coefficient (Koc) for p-isopropyltoluene indicates moderate potential for
mobility in soil; therefore, p-isopropyltoluene has the potential for migration into groundwater
(U.S. EPA. 2012b). /Msopropyltoluene is not expected to undergo hydrolysis due to its lack of
hydrolysable functional groups.
H,C
Figure l./7-Isopropyltoluene (CASRN 99-87-6) Chemical Structure
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EPA 690 R-24 003F
Table 1. Physicochemical Properties of/>-Isopropyltoluene (CASRN 99-87-6)
Property (unit)
Value3
Physical state
Liquidb
Boiling point (°C)
177
Melting point (°C)
-68.2
Density (g/cm3 at 20°C)
0.8573b
Vapor pressure (mm Hg at 25°C)
0.772
pH (unitless)
NA
Acid dissociation constant (pKa) (unitless)
NA
Solubility in water (mg/L at 25°C)
23.2 (reported as 1.73 x 10~4mol/L)
Octanol-water partition coefficient (log Kow)
4.10
Henry's law constant (atm-m3/mol at 25°C)
7.94 x 10 3 (predicted)
Soil adsorption coefficient Koc (L/kg)
2.78 x 103 (predicted)
Atmospheric OH rate constant (cm3/molecule-sec at 25°C)
1.51 x 10-11
Atmospheric half-life (d)
lb
Relative vapor density (air = 1)
4.62b
Molecular weight (g/mol)
134.222
Flash point (°C)
47.2 (reported as 117°F open cup and closed cup)b
aUnless otherwise noted, average values were extracted from the U.S. EPA CompTox Chemicals Dashboard
(https://comptox.epa.gov/dashboard/chemical/details/DTXSID3026645. Accessed May 21, 2024); U.S. EPA
£2024a). Values are experimental unless otherwise specified.
' NLM (2022b): Values are experimental unless otherwise specified.
NA = not applicable; U.S. EPA = U.S. Enviromnental Protection Agency.
A summary of available toxicity values for p-isopropyltoluene from the U.S. EPA and
other agencies/organizations is provided in Table 2.
Table 2. Summary of Available Toxicity Values and Qualitive Conclusions
Regarding Carcinogenicity for />-Isopropyltoluene (CASRN 99-87-6)
Source (parameter)ab
Value (applicability)
Notes
Reference0
Noncancer
IRIS
NV
NA
U.S. EPA (2024b)
HEAST
NV
NA
U.S. EPA (2011c)
DWSHA
NV
NA
U.S. EPA (2018a)
ATSDR
NV
NA
ATSDR (2021)
WHO (safety evaluation)
No safety concern at the
estimated intake levels of
approximately
1,100 (ig/person in Europe
and 470 (ig/person in the
United States
Expected to be metabolized
to innocuous products
WHO (2022. 2006)
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Table 2. Summary of Available Toxicity Values and Qualitive Conclusions
Regarding Carcinogenicity for />-Isopropyltoluene (CASRN 99-87-6)
Source (parameter)ab
Value (applicability)
Notes
Reference0
CalEPA
NV
NA
CalEPA (2022. 2020)
OSHA
NV
NA
OSHA (2021a. 2021b.
2021c)
NIOSH
NV
NA
NIOSH (2018)
ACGIH
NV
NA
ACGIH (2021)
TCEQ (RfD)
0.1 mg/kg-d
Basis for RfD not specified;
value developed with
TCEQ's protocol
TCEO (2021.2015)
DOE (PAC)
PAC 1: 120 mg/m3
PAC 2: 1,300 mg/m3
PAC 3: 1,900 mg/m3
PAC-1 based on TEEL,
PAC-2 based on
unspecified rat 300-min
TClo, PAC-3 based on
unspecified rat 240-min
LClo
DOE (2018)
USAPHC (air-MEG)
1-h critical: 500 mg/m3
1-h marginal: 500 mg/m3
1-h negligible: 250 mg/m3
Based on TEELs
U.S. APHC (2013)
Cancer
IRIS
NV
NA
U.S. EPA (2024b)
HEAST
NV
NA
U.S. EPA (2011c)
DWSHA
NV
NA
U.S. EPA (2018a)
NTP
NV
NA
NTP (2021)
IARC
NV
NA
IARC (2021)
CalEPA
NV
NA
CalEPA (2022. 2020)
ACGIH
NV
NA
ACGIH (2021)
aSources: ACGIH = American Conference of Governmental Industrial Hygienists; ATSDR = Agency for Toxic
Substances and Disease Registry; CalEPA = California Environmental Protection Agency; DOE = U.S. Department
of Energy; DWSHA = Drinking Water Standards and Health Advisories; HEAST = Health Effects Assessment
Summary Tables; IARC = International Agency for Research on Cancer; IRIS = Integrated Risk Information
System; NIOSH = National Institute for Occupational Safety and Health; NTP = National Toxicology Program;
OSHA = Occupational Safety and Health Administration; TCEQ = Texas Commission of Enviromnental Quality;
USAPHC = U.S Army Public Health Command; WHO = World Health Organization.
Parameters: LClo = lowest reported lethal concentration; MEG = military exposure guideline; PAC = protective
action criteria; RfD = reference dose; TCLo = toxic concentration lowest; TEEL = temporary emergency exposure
limit.
°Reference date is the publication date for the database and not the date the source was accessed.
NA = not applicable; NV = not available.
Non-date-limited literature searches were conducted in June 2019 and updated most
recently in November 2023 for studies pertinent to understanding potential human health hazards
of/?-isopropyltoluene, CASRN 99-87-6. Searches were conducted using the U.S. EPA's Health
and Environmental Research Online (HERO) database of scientific literature. HERO searches
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the following databases: PubMed, TOXLINE1 (including TSCATS1), Scopus, and Web of
Science. The National Technical Reports Library (NTRL) was searched for government reports
from 2020 through December 20222. The following resources were searched outside of HERO
for health-related values: American Conference of Governmental Industrial Hygienists
(ACGIH), Agency for Toxic Substances and Disease Registry (ATSDR), California
Environmental Protection Agency (CalEPA), Defense Technical Information Center (DTIC),
European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC), European
Chemicals Agency (ECHA), the U.S. EPA Chemical Data Access Tool (CDAT), the U.S. EPA
ChemView, the U.S. EPA Integrated Risk Information System (IRIS), the U.S. EPA Health
Effects Assessment Summary Tables (HEAST), the U.S. EPA Office of Water (OW),
International Agency for Research on Cancer (IARC), the U.S. EPA TSCATS2/TSCATS8e, the
U.S. EPA High Production Volume (HPV), Chemicals via International Programme on Chemical
Safety (IPCS) INCHEM, Japan Existing Chemical Data Base (JECDB), Organisation for
Economic Co-operation and Development (OECD) Screening Information Data Sets (SIDS),
OECD International Uniform Chemical Information Database (IUCLID), OECD HPV, National
Institute for Occupational Safety and Health (NIOSH), National Toxicology Program (NTP),
Occupational Safety and Health Administration (OSHA), and World Health Organization
(WHO).
TOXLINE was retired in December 2019. Searches of this database were conducted through July 2019.
2NTRL was a subset of TOXLINE until December 2019 when TOXLINE was discontinued. Searches of NTRL
were conducted starting in 2020 to ensure that references were not missed due to delays in importing items into the
database.
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2. REVIEW OF POTENTIALLY RELEVANT DATA
(NONCANCER AND CANCER)
Tables 3A and 3B provide overviews of the relevant noncancer and cancer evidence
bases, respectively, for/>-isopropyltoluene and include all potentially relevant subchronic, and
chronic studies, as well as reproductive and developmental toxicity studies. These tables include
studies for which no-observed-adverse-effect levels (NOAELs)/lowest-observed-adverse-effect
levels (LOAELs) could be identified (the principal study is identified in bold). All
NOAELs/LOAELs were identified by the U.S. EPA unless noted otherwise. The phrase
"statistical significance" and term "significant," used throughout the document, indicate a
p-value of < 0.05 unless otherwise specified.
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Table 3A. Summary of Potentially Relevant Noncancer Data for />-Isopropyltoluene (CASRN 99-87-6)
Category3
Number of Male/Female, Strain
Species, Study Type, Reported
Doses, Study Duration
Dosimetryb
Critical Effects
NOAELb
LOAELb
Reference
(comments)
Notes0
Human
1. Oral (mg/kg-d)
ND
2. Inhalation (mg/m3)
ND
Animal:
1. Oral (mg/kg-d)
Short-term
ND
Subchronic
Chronic
10 M/10 F, Sprague Dawley
(Crl:CD) rat (gavage
administration)
F: 2 wk prior to mating,
continued through mating (up to
2 wk), gestation, and lactation
until sacrifice on PND 13 (~63 d)
M: 2 wk premating and
continued through mating (up to
2 wk) and postmating until
sacrifice (~35 d)
0,50,100,
200
Biologically significant (>10%) and
dose-related increases in liver weights
(absolute and relative) in Po female rats at
50 and 100 mg/kg-d.
Corroborative evidence of liver toxicity
included: biologically significant increases in
absolute and relative liver weights in
Po males at 200 mg/kg-d; dose-related
increases in ALP in Po rats (statistically
significant at 100 mg/kg-day in females and
200 mg/kg-day in males); increased
hepatocyte hypertrophy in Po males and
females mostly at >100 mg/kg-d.
Other effects occurring mostly at
>200 mg/kg-d included decreased hindlimb
grip strength and increased BUN and kidney
lesions in Po males.
NA
50
ECHA (2019b);
Svmrise (2018)
NPR,
PS
ND
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Table 3A. Summary of Potentially Relevant Noncancer Data for />-Isopropyltoluene (CASRN 99-87-6)
Category3
Number of Male/Female, Strain
Species, Study Type, Reported
Doses, Study Duration
Dosimetryb
Critical Effects
NOAELb
LOAELb
Reference
(comments)
Notes0
Reproductive/
Developmental
10 M/10 F, Sprague Dawley
(Crl:CD) rat (gavage
administration)
F: 2 wk prior to mating, continued
through mating (up to 2 wk),
gestation, and lactation until
sacrifice on PND 13 (~63 d)
M: 2 wk premating and continued
through mating (up to 2 wk) and
postulating until sacrifice (~35 d)
0, 50, 100,
200
Reproductive: decreased fertility index and
degenerative lesions in the testes and
epididymides of Po male rats at >100 mg/kg-d
(sperm retention, reduced luminal sperm, and/or
cribriform changes).
Alterations in estrous cyclicity, male
reproductive organ weight changes and
additional male reproductive histopathology in
Po rats at 200 mg/kg-d.
Developmental: marginally significant (>4.5%)
decreases in Fi female body weights on PND 1
at 50 and 100 mg/kg-d.
Decreased Fi offspring survival
(i.e., postimplantation survival index and live
birth index) and decreased Fi male body
weights on PND 1 at 100 mg/kg-d.
50
100
ECHA (2019b):
Svmrise (2018)
NPR, PS
NA
50d
ND
2. Inhalation (mg/m3)
"¦Duration categories are defined as follows: acute = exposure for <24 hours; short term = repeated exposure for 24 hours to <30 days; long-term (subclironic) = repeated
exposure for >30 days <10% life span for humans (>30 days up to approximately 90 days in typically used laboratory animal species); and chronic = repeated exposure
for >10% life span for humans (>~90 days to 2 years in typically used laboratory animal species) (U.S. EPA. 2002).
bDosimetry: Doses are presented as ADDs (mg/kg-day) for oral noncancer effects.
°Notes: NPR = not peer-reviewed; PS = principal study.
dTentative LOAEL based on marginally significant decreases in Fi female body weights on PND 1 (>5% is considered biologically significant for this health effect).
ADD = adjusted daily dose; ALP = alkaline phosphatase; BUN = blood urea nitrogen; F = female(s); HEC = human equivalent concentration;
LOAEL = lowest-observed-adverse-effect level; M = male(s); NA = not applicable; ND = no data; NOAEL = no-observed-adverse-effect level; PND = postnatal day.
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Table 3B. Summary of Potentially Relevant Cancer Data for />-Isopropyltoluene (CASRN 99-87-6)
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)
ND
2. Inhalation (mg/m3)
ND
ND = no data.
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2.1. HUMAN STUDIES
No studies investigating the effects of oral or inhalation exposure to />isopropyltoluene in
humans were identified.
2.2. ANIMAL STUDIES
2.2.1. Oral Exposures
The effects of oral exposure of animals to />isopropyltoluene were evaluated in a
combined repeated-dose systemic and reproductive/developmental toxicity study in rats exposed
for subchronic duration (ECHA 2019b; Symrise. 2018). Three short-term oral studies in rodents
exposed to p-isopropyltoluene were also identified that had significant reporting limitations (very
few details on methods and results; see Table 4B) lYLi et al.. 2020; DuPont 1992); Stel'makh et
al. (1983) as cited in ECHA (1986)1. As such, these studies were considered supplemental and
summarized in Section 2.3.
Subchronic Studies (Including Combined Reproductive and Developmental Screening)
Symrise (2018)
Symrise (2018) is non peer-reviewed, Good Laboratory Practice (GLP)-compliant OECD
guideline 422 (combined repeated-dose, systemic toxicity study with a reproductive/
developmental toxicity screening test) TSCA study in rats that was summarized in ChemView
and reported in ECHA (2019b). A combination of these documents was used to generate the
summary of this OECD 422 guideline study below. Details on methods of endpoint assessment
and results were limited in some cases and most data were provided qualitatively.
Commercially obtained Sprague Dawley (Crl:CD) rats (10 sex/group), 11-13 weeks old,
with initial body weights of 332-434 g (male) and 235-299 g (female) at study initiation, were
administered p-isopropyltoluene (purity not reported) in corn oil daily, via gavage, at doses of
0 (vehicle control), 50, 100, or 200 mg/kg-day. For parental (Po) males, dosing began 2 weeks
premating and continued through mating (up to 2 weeks) and postmating until sacrifice, for a
total of -35 days. Po females were dosed 2 weeks prior to mating, and dosing continued through
mating (up to 2 weeks), gestation, and lactation until sacrifice on postnatal day (PND) 13, for a
total of -63 days. Homogeneity, stability, and confirmation of doses of the test substance under
the storage conditions used in the study were analytically verified.
Rats were observed twice daily during the administration period for mortality and general
condition. Clinical signs of toxicity and body weights were evaluated in Po males once per week
until termination, in Po females once per week from initiation of exposure through mating, and in
mated females on gestation days (GDs) 0, 7, 14, and 20 and PNDs 1, 4, 7, and 14. Additionally,
daily observations of maternal behavior were recorded from GD 18 to PND 13. Food intake was
measured throughout the study. Functional observational battery (FOB) tests (sensory reactivity,
grip strength, and locomotor activity) were performed in five rats per sex per group during the
last week of administration in males and on PND 8 in females. Blood drawn at terminal necropsy
from five rats per sex per group was used for hematology evaluation (indices were not specified;
blood from an additional five rats per sex per group was used for coagulation evaluation) and
serum clinical chemistry evaluation (parameters were not specified). Additional blood was drawn
at study termination in up to 10 per sex per group for thyroid hormone analysis (serum thyroxine
[T4] and thyroid-stimulating hormone [TSH]). Postmortem macroscopic examinations conducted
on all Po animals included external and internal examinations focusing on reproductive system
organs. Organs weights were measured in up to five males per group and up to five lactating
females per group. Females that failed to deliver a litter were euthanized on GD 25 (organs were
not weighed in females that failed to deliver a litter). Microscopic pathology was conducted on
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all Po animals surviving until study termination. Livers were examined for all females, including
those from nonpregnant females that were euthanized during the gestation period. Very few
details were provided on the specific organs weighed and tissues that were examined
microscopically.
Assessment of reproductive endpoints included determinations of the mating and fertility
indices of males and females, sperm parameters, length of estrous cycle, number of days to
copulation, conception rate, numbers of pregnant dams, implantation index, gestation period,
gestation index, corpora lutea, implantations, pre- and postimplantation loss, birth indices, and
litter size. Reproductive parameters also included gross pathology of reproductive organs
(including organ weights) and histopathology of reproductive tissues. Developmental endpoints
included sex ratio, viability index, number of live pups on Days 1, 4, 7, 11, and 13,
postimplantation survival, pup body weight, and examination of pups at sacrifice on PND 13 for
gross abnormalities. Blood from Fi female pups (up to two per litter) was collected on PND 4
and blood from Fi males and females (up to two per litter) was collected on PND 13 for thyroid
hormone analysis (T4 and TSH). Thyroid glands from one randomly selected male and female
pup per litter were collected for histopathological examination.
No treatment-related mortalities were reported at doses up to 200 mg/kg-day for Po males
and 100 mg/kg-day for Po females; one (nonpregnant) female in the 200-mg/kg-day group was
euthanized on GD 24 in extremis. Microscopic examination of the euthanized female revealed
lesions in the liver and adrenal glands (micro/macro vesicular vacuolation), kidney (bilateral
tubular dilation and vacuolation in the cortex and bilateral tubular necrosis with degeneration/
regeneration in the papilla) and lymphoid tissues (decreased cellularity or necrosis/apoptosis in
the thymus, mesenteric lymph node, splenic white pulp, and Peyer's patch/gut-associated
lymphoid tissue, and atrophy of the splenic red pulp). Additionally, females that failed to become
pregnant were euthanized early on GD 25, including 1/10 in the control and 50-mg/kg-day
groups, 6/10 in the 100-mg/kg-day group, and 10/10 in the 200-mg/kg-day group. No other
general signs of toxicity were seen in males or females from any group during observations.
The FOB showed a dose-related, statistically significant decrease in hindlimb grip
strength (35%) at Week 5 in Po males in the 200-mg/kg-day group (see Table B-l). Decreases
(18-30%) in forelimb strength were also observed in treated Po males but the changes were not
dose-related or statistically significant. All other FOB observations of treated groups (including
females) were comparable to the control group (data were not provided). Body weights,
body-weight changes, and food consumption in Po males were similar to controls at all dose
levels (data were not provided). Body weights, body-weight changes, and food consumption in
Po females of all dose groups during premating and mating periods and during gestation and
lactation in dams treated with 50 and 100 mg/kg-day were similar to controls (data were not
provided). Females treated with 200 mg/kg-day could not be evaluated for body weight,
body-weight change, or food consumption during gestation and lactation due to lack of
pregnancy.
Statistically significant changes in hematology and clinical chemistry measures are
shown in Table B-2. Observed changes in reported hematological parameters for Po males at
>100 mg/kg-day (increased reticulocytes, red blood cell distribution width [RDW], and
prothrombin time [PT]) lacked a dose-response relationship and/or were of unknown
toxicological significance. No hematology results were provided for Po female rats. Clinical
chemistry measures in Po male rats showed statistically significant increases in alkaline
phosphatase (ALP) (45%) and blood urea nitrogen (BUN) (50%) at 200 mg/kg-day. Significant
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increases in ALP were also observed in Po females at 100 mg/kg-day (79%). The changes in
ALP and BUN levels in rats showed a dose-response relationship. Females in the 200-mg/kg-day
group were not evaluated for clinical chemistry since they were euthanized due to nonpregnancy.
All other observed changes in reported clinical chemistry parameters in Po males and females
were not dose-related and/or were of unknown toxicological significance (see Table B-2).
T4 values in Po males were decreased (44 and 63%) compared to controls at 100 and
200 mg/kg-day, respectively, at the end of the study (no other details were provided). There were
no corresponding thyroid weight changes or histopathological findings in the thyroid gland, and
TSH values were generally below the level of detection in Po males and females (data were not
provided); therefore, the toxicological significance of the T4 changes was unclear. Changes in
T4 levels in females were not noted.
The data for selected organ weights (percent difference to controls) are summarized in
Table B-3. Any discussion of relative organ weights is based on changes with respect to body
weight unless otherwise noted. Dose-related, biologically significant (>10%) increases in liver
weights were reported in both male and female rats. In Po males, absolute and relative liver
weights were biologically and statistically significantly increased in the 200-mg/kg-day group
(27 and 41% higher than controls, respectively). In Po females, absolute and relative liver
weights were biologically and/or statistically significantly increased at 50 and 100 mg/kg-day
(14-26%) higher than controls). Liver weight data for females at 200 mg/kg-day were
unavailable. Statistically significant decreases in absolute and/or relative reproductive organ
weights (testis, epididymis, and prostate) were reported in the Po males at 200 mg/kg-day (data
for other dose groups were not reported for the testis or the epididymides). Other organ weight
changes, including kidney weights in males, were not considered treatment-related; however,
quantitative data were not provided.
No gross changes were reported in Po females. Gross pathological findings were observed
in two Po males at 200 mg/kg-day: one with unilateral small testis and one with bilateral small
testes, both seen in conjunction with germ cell degeneration/depletion. A single male also
exhibited a small prostate and another male exhibited small levator ani/bulbocavernosus (LABC)
muscle complex.
Histopathological findings in the liver included minimal hepatocellular hypertrophy in
2/5 high-dose Po males and 1/6 and 1/10 Po females in the low- and high-dose groups,
respectively (see Table B-4). This liver lesion was not observed in the controls. Focal
hepatocellular necrosis and inflammatory cell infiltrates were reportedly sporadic in the dosed
Po animals or occurred at similar incidences in controls (data were not provided). These findings
were not considered treatment-related (but rather a background incidence). Kidney lesions
included increased incidence of minimal to slight hyaline droplet accumulation (three of
five rats) and minimal tubular epithelial vacuolation (two of five animals) in Po males in the
high-dose group compared to one of five and zero of five controls, respectively) (see Table B-4).
Minimal tubular basophilia was found only in treated Po males (one of five rats for all dose
groups). Testicular and epididymal lesions were reported in male Po rats mostly at
>100 mg/kg-day but not in the controls (see Table B-5). At 100 mg/kg-day, minimal spermatid
retention (7/10) was observed in the testes and minimal-to-slight reduced luminal sperm (2/10)
and slight cribriform changes (1/10) were observed in the epididymides. Male reproductive tract
effects observed at 200 mg/kg-day included minimal-to-slight spermatid retention (9/10) and
minimal-to-moderate germ cell degeneration/depletion (7/10) in the testis, and
slight-to-markedly reduced luminal sperm (10/10), sperm with minimal-to-slight cribriform
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changes (5/10), and minimal-to-moderate luminal cell debris (9/10) in the epididymides.
Additionally, unilateral or bilateral seminiferous tubular atrophy (with or without luminal cell
debris and reduced sperm) was observed in 1/10 males at 50 and 100 mg/kg-day (severity was
not reported; therefore, data were not summarized in Table B-5).
Changes in estrous cyclicity were observed in Po females during the premating period.
The number of females with regular cycles decreased (4/10 compared to 6/10 in controls) and the
number of females with at least one irregular cycle increased (6/10 compared to 4/10 in controls)
at the highest dose (200 mg/kg-day) (see Table B-6). The mean cycle duration and number of
cycles were reportedly comparable to controls in all treatment groups (data were not provided).
Select fertility and reproductive parameters are shown in Table B-7. There were effects on
fertility in Po females at doses >100 mg/kg-day, with no pregnant females in the 200-mg/kg-day
group. The numbers of pregnant animals were 9/10, 9/10, 4/10, and 0/10 in the control, 50-,
100-, and 200-mg/kg-day groups, respectively. There were no statistically significant differences
in the number of mated females, mean number of days to mating, gestation length, corpora lutea,
implantations, or preimplantation loss compared to controls.
Fi offspring survival was affectedly demonstrated by a reduction in postimplantation
survival index and live birth index in the 100-mg/kg-day group compared to controls
(see Table B-7); the effects on live birth index were statistically significant. Only one of
four litters had 100% viability in the 100 mg/kg-day group, compared to all the litters (nine of
nine) having 100% viability in the control group. Viability indices in litters in the 50- and
100-mg/kg-day group were comparable with controls on PNDs 4, 7, and 13. Dose-related
decreases in mean offspring body weights occurred in females at 50 and 100 mg/kg-day on
PND 1 compared to controls. The decrease at the lowest dose (50 mg/kg-day) was marginally
significant (>4.5%; >5% is considered biologically significant for this effect). Correspondingly,
decreased offspring body weight was observed in males (10%) on PND 1 but only at the highest
dose tested (100 mg/kg-day). Minimal changes in body weight (<5%) were observed on mean
offspring body weights on PND 4, 7, 11, or 13. No significant changes on sex ratio,
thyroid/parathyroid gland weights, or histopathological findings in offspring of the 50- or
100-mg/kg-day groups, or on T4 or TSH in Fi females on PND 4 or T4 levels in Fi males on
PND 13 were reported (data were not provided). The following clinical observations were made
in offspring: thin, cold to touch, partially absent appendages, little or no milk in stomach,
swollen, twisted, encrustation, ulceration, dark color, and pallor. The ECHA (2020b) report
stated that "gross pathological findings were not considered test material-related because they
occurred only in the control group, occurred in the control group in similar incidence, did not
occur in a treatment-related manner, were of short duration and/or were considered to be a
common finding of young pups in a laboratory situation." Due to the lack of quantitative data on
incidence and severity, the significance of these observations is unclear.
Despite reporting deficiencies (including lack of details on methods of endpoint
assessment and presentation of results) that to some extent limit interpretation of results, the
ECHA (2019b)/Svmrise (2018) study provided sufficient information to identify sensitive health
effects associated with repeated-dose oral exposure to p-isopropyltoluene. A systemic lowest-
observed-adverse-effect level (LOAEL) of 50 mg/kg-day is identified from this study based on
biologically significant increases (>10%) in absolute and relative liver weights in Po female rats.
A systemic no-observed-adverse-effect level (NOAEL) cannot be determined because the liver
weight changes occurred at the lowest dose. Increases (>10%) in liver weights were also
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observed in Po male rats and were accompanied by dose-related increases in serum ALP levels
and some evidence of hepatocyte hypertrophy in both sexes mostly at doses >100 mg/kg-day.
A reproductive NOAEL value of 50 mg/kg-day and LOAEL value of 100 mg/kg-day is
determined based on decreased fertility index and degenerative lesions in the testes and
epididymides of Po male rats (i.e., sperm retention, reduced luminal sperm, and cribriform
changes). At the highest dose (200 mg/kg-day), changes in estrous cyclicity and effects on male
reproductive organ weights and histopathology were found. For developmental effects, a
tentative LOAEL of 50 mg/kg-day (lowest dose tested) is identified based on a dose-related and
marginally significant decrease (>4.5%) in the body weights of Fi females on PND 1. A
developmental NOAEL cannot be identified. Significant reductions in Fi male body weights on
PND 1 (>5%) and offspring survival (decreased postimplantation survival index and live birth
index) also occurred at 100 mg/kg-day.
Other possible treatment-related effects include decreases in hindlimb grip strength,
kidney lesions (tubular epithelial vacuolation, hyaline droplet accumulation, and tubular
basophilia) and increased BUN levels reported in Po male rats mostly at doses >200 mg/kg-day.
As mentioned above, there is some uncertainty in the identified health effects and associated
effects levels due to the reporting deficiencies in the ECHA (2019b) study. The administered
doses of 0, 50, 100, and 200 mg/kg-day correspond to human equivalent doses (HEDs) of 0, 14,
28.1, and 56.2 mg/kg-day in Po males and 0, 13, 25.6, and 51.2 mg/kg-day in Po females3.
Chronic Studies
No chronic oral studies were identified forp-isopropyltoluene.
2.2.2. Inhalation Exposures
No inhalation studies suitable for use in risk assessment were located. One short-term
study in rats (Lam et al.. 1996) and two subchronic studies in dogs (DuPont 1992) were
identified for p-isopropyltoluene exposure via inhalation. The studies had outstanding design and
reporting limitations (e.g., small number of test animals, limited relevant endpoints evaluated,
and/or few details on methods and results; see Table 4B). As such, these studies were considered
supplemental and are summarized in Section 2.3.
2.3. OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS)
Other studies that examined p-isopropyltoluene but were not appropriate for the selection
of a point of departure (POD) are described here. These studies are not adequate for the
determination of provisional reference values but may provide supportive data supplementing a
weight-of-evidence (WOE) approach. These include genotoxicity, metabolism/toxicokinetic, and
acute-duration studies, studies using routes of exposure other than the oral or inhalation route,
and short-term and subchronic oral studies with significant design and reporting limitations that
Administered doses were converted to HEDs by multiplying by a dosimetric adjustment factor (DAF) of 0.281 for
males and 0.256 for females, calculated as follows: DAF = (BWaI/4 ^ BWh1'4), where BWa = animal body weight,
and BWh = human body weight. The study reported initial body weights of 0.332-0.434 and 0.235-0.299 kg for
Sprague Dawley male and female rats, respectively. In the absence of data for study-specific time-weighted average
(TWA) or final body weights in the animals, the upper end of the reported initial body weight for rats in this study
was used for males (0.434 kg) and females (0.299 kg) given that the initial body weights are higher than the
recommended default values for males and females Sprague Dawley rats in a subchronic study (0.267 and 0.204 kg,
respectively) (U.S. EPA. 1988). For humans, the reference value of 70 kg was used for body weight, as
recommended by U.S. EPA (1988).
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are considered inadequate for the derivation of toxicity values (see Section 2.3.2 for more
details).
2.3.1. Genotoxicity
Table 4A provides an overview of genotoxicity studies of />isopropyltoluene. The limited
data available from in vitro and in vivo animal model systems suggest thatp-isopropyltoluene is
not genotoxic. Genotoxicity data in human model systems are mostly lacking. The chemical was
negative for mutagenicity in both plate incorporation and preincubation tests in Salmonella
typhimnrium strains TA98, TA100, TA1535, and TA1537, Escherichia coli WPluvrA, and
Chinese hamster lung fibroblasts (CHL/V9) cells, both in the presence and absence of rat liver
S9 metabolic activation (ECHA. 2020a. 2018). Two other studies also reported that the chemical
was negative for mutagenicity in bacteria (S. typhimurium strains TA98 and TA100, and E. coli
Sd-4-73); both studies provided insufficient study details [Szybalski (1958) as cited in FFHPVC
(2005); (Rockwell and Raw. 1979)1. p-Isopropyltoluene did not induce chromosomal aberrations
(CAs) in human peripheral lymphocytes (ECHA. 2017) or Chinese hamster ovary (CHO) cells
(ECHA. 2019a) in the presence or absence of rat liver S9 metabolic activation.
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Table 4A. Summary of/>-Isopropyltoluene Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested3
Results
Without
Activationb
Results
With
Activationb
Comments
References
Genotoxicity studies in prokaryotic organisms
Mutation
Salmonella tvphimurium
TA98, TA100, TA1535,
TA1537 and
Escherichia coli WP2
uvrA, with and without
liver microsomal
metabolic activation
TA98: 0 or
3-5,000 ng/plate;
all other strains: 0 or
10-5,000 ng/plate
5,000 ng/plate
Plate incorporation and preincubation tests. No
evidence of mutagenicity in any of the strains of
S. tvphimurium or E. coli tested with or without
S9 activation.
Cytotoxicity at the highest concentration.
ECHA (2020a)
Mutation
S. tvphimurium TA98 and
TA100
ND
NDr
Plate incorporation assay. No evidence of
mutagenicity reported in any of the strains tested
with S9 activation (data not provided).
Limited details were provided. Doses were not
explicitly stated. Cytotoxicity was not evaluated
or not reported. Only two strains of
S. tvphimurium were tested.
The test substance in this study is identified as
"cymene;" it is not specified if it is /?-cymcnc.
Rockwell and Raw
(1979)
Mutation
S. tvphimurium TA98 and
TA100
0.05-100 |iL/platc
NDr
Plate incorporation assay. No evidence of
mutagenicity reported in any of the strains tested
with 24-h urinary extracts of Sprague Dawley
rats (;? = 2) that were administered 0.5 mL of
undiluted /?-isopropyltolucnc via gavage in the
presence of S9 activation.
The test substance in this study is identified as
"cymene;" it is not specified if it is /?-cymcnc.
Rockwell and Raw
(1979)
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Table 4A. Summary of/>-Isopropyltoluene Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested3
Results
Without
Activationb
Results
With
Activationb
Comments
References
Mutation
E. coli Sd-4-73,
0.01-0.025 inL or small
crystals of /j-cymcne
(applied to paper disk and
then placed on agar)
0.025 mL or small
crystals of
/j-cvmene
NDr
Paper-disk method. No evidence of
mutagenicity.
Use of positive controls is unclear. Limited
details are available in a secondary source.
Szybalski (1958) as
cited in FFHPVC
(2005)
Genotoxicity studies in mammalian cells—in vitro
Mutation
CHL/V79 (Cliinese
hamster lung fibroblasts)
tested for 4 h with (0, 5,
10, 20, 40, 50, 60, 70,
80 ng/mL) and without
S9 rat liver fraction (0,
1.25, 2.5, 5, 10, 20, 30,
40, 50 ng/mL)
50 iig/mL
No evidence of mutagenicity in any test
condition.
Precipitation was observed at 40 ng/mL and
precipitation and excessive toxicity were
observed at 50 ng/mL without metabolic
activation. Excessive cytotoxicity was observed
at 60 |ig/mL with metabolic activation. For the
experiment without metabolic activation, the
plates were discarded at 50 ng/mL due to
excessive toxicity and precipitate. The
experiments without metabolic activation were
not plated starting at 60 ng/mL after Day 0 due
to excessive toxicity.
ECHA (2018)
CA
Human peripheral
lymphocytes; cells tested
with or without activation
by S9 hepatic microsomal
fraction (0,
10-80 |ig/mL) for 4 h
and without metabolic
activation (0,
20-160 |ig/mL) for 24 h
160 |ig/mL
No increase in CAs in any test condition.
No cytotoxicity was seen. The main study used
doses determined to be noncytotoxic, based on a
preliminary test.
ECHA (2017)
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Table 4A. Summary of/>-Isopropyltoluene Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested3
Results
Without
Activationb
Results
With
Activationb
Comments
References
CA
CHO cells, with (0,
24-225 pg/mL) and
without (0,
7.0-200 pg/mL)
activation by S9 rat liver
fraction
225 pg/mL
No increase in CAs in any test condition. A
statistically significant increase in CAs was
observed at 156 pg/mL with metabolic activation
compared to vehicle control but was within the
historical control range. There was no
statistically significant increase compared to
untreated control cells.
Cytotoxicity at 200 pg/mL without metabolic
activation and at 225 pg/mL with metabolic
activation.
ECHA (2019a)
aLowest effective dose for positive results; highest dose tested for negative results.
b- = negative.
CA = chromosomal aberration; CHO = Chinese hamster ovary; ND = no data; NDr = not determined.
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In a study designed to investigate the mutagenicity of the urinary metabolites of a number
of food additives, two Sprague Dawley rats were given a single dose of 0.5 mL of
/?-isopropyltoluene by gavage, and their urine was collected for 24 hours. To assess the genotoxic
potential of urinary metabolites, the urine was assayed directly or extracted with ether after
dilution in a phosphate buffer and treatment with (3-glucuronidase to hydrolyze glucuronide
conjugates. Accordingly, three types of urine samples were tested in the Ames assay with
S. typhimurium strains TA98 and TA100 with metabolic activation: 24-hour urine samples
(500 |iL), ether extracts of the urine, and aqueous fractions of the extracts. No evidence of
mutagenicity was observed for p-isopropyltoluene in this study (Rockwell and Raw. 1979).
2.3.2. Supporting Animal Studies
Table 4B provides an overview of other supporting studies on p-isopropyltoluene. Studies
on acute lethality of p-isopropyltoluene indicate low acute toxicity in animals following oral,
inhalation, dermal, and intraperitoneal (i.p.) routes. The median lethal dose (LDso) in rats ranged
from 2,460 to 4,750 mg/kg (DuPont 1992; Jenner et al.. 1964; Mellon Institute. 1951). No effect
on motor activity was observed in mice following a single oral dose of 40 mg/kg (Siqueira
Quintans et al.. 2013). An analgesic effect (i.e., increased reaction time on the hot plate test) was
noted in mice exposed to 40 mg/kg (Siqueira Quintans et al.. 2013). Three short-term studies
available in rodents exposed via the oral route had outstanding reporting limitations (very few
details on methods and results; see Table 4B for more details). A Russian study noted reduced
mobility in mice at a reported dose of 1,650 mg/kg-day for 24-28 days [Stel'makh et al. (1983)
as cited in ECHA (1986)1. Another short-term oral study in rats reported no effects on mortality
or gross or microscopic pathology (unspecified) withp-isopropyltoluene exposures of
510 mg/kg-day, 5 days/week for 2 weeks (DuPont. 1992). The third short-term oral study
exposed male mice to low doses of /Msopropyltoluene (3 and 7 mg/kg-d) or 10-40 mg/kg-day of
the volatile oil of Chenopodium cimbrosioides L. (containing p-isopropyltoluene [24.25%]) for
27 days (Li et al.. 2020). The study reported significant increases in relative liver weights, serum
levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and
morphological changes in mouse liver, heart, and kidney with exposure to the volatile oil but no
significant effects were noted withp-isopropyltoluene exposure alone.
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Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Supporting studies in animals following oral exposure
Acute (oral)
Osborne-Mendel rats (10/sex/group)
were administered a single dose of
/?-isopropyltolucnc via gavage and
observed for 2 wk. The doses tested
were not provided.
Clinical signs included depression after
dosing, coma, bloody lacrimation, diarrhea,
irritation, and scrawny appearance.
Rat
LD50 (95% CI) = 4,750 (3,720-6,060) mg/kg.
Jenner et al.
(1964)
Acute (oral)
In an acute oral toxicity of
/?-isopropyltolucnc. white rats (strain,
sex, and number per group not
specified) were administered a single
dose ranging from 1,100 to 6,750 mg/kg
via gavage. Mortality, clinical signs,
gross necropsy, and histopathology
were evaluated.
Gross and microscopic evidence of gastritis
and liver damage (not further described)
were observed.
Rat LD50 = 3,000 mg/kg (approximate).
DuPont (1992)
Acute (oral)
Rats (five per group; strain and sex not
specified) were administered single oral
doses (1,880-3,230 mg/kg) of
/?-isopropyltolucnc.
Mortality occurred (incidence of deaths was
not reported).
Rat LD50 = 2,460 mg/kg.
Mellon Institute
(1951)
Acute (oral)
Male Swiss mice (eight per group) were
administered single doses of 0, 20, or
40 mg/kg /j-isopropyltolucne via
gavage. Motor activity was evaluated
after administration using a rotarod test.
Additional tests evaluated the protective
effect on pain.
No effect on motor performance in the
rotarod test was observed. Significantly
increased latency time (licking and jump
parameters) was reported in a hot plate test
at 40 mg/kg /j-isopropyltolucne.
An analgesic effect was noted at 40 mg/kg.
Motor performance was not affected.
Siaueira Ouintans
et al. ("20131
Short-term
(oral)
Groups of six rats (strain and sex not
specified) were administered
/?-isopropyltolucnc at a dose of
510 mg/kg-d via an unspecified oral
route for 5 d/wk for 2 wk. Rats were
sacrificed lid following the final dose.
No mortality occurred. No gross or
microscopic pathology was observed.
The study has data reporting limitations (lack
of details on experimental procedures and
results).
DuPont (1992)
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Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Short-term
(oral)
Mice (strain, sex, and number not
specified) were administered
/?-isopropyltolucnc (purity not reported)
at 0.22 mg/kg-d, via gavage, for 4 d;
doses were increased by a factor of
1.5 every 4 d for a total of 24-28 d.
Endpoints evaluated were not specified.
Reduced mobility was observed at a
reported dose of 1,650 mg/kg-d.
The study has data reporting limitations (lack
of details on experimental procedures and
results). Also, original study is in Russian.
Stel'makh et al.
(1983) as cited in
ECHA (1986)
Short-term
(oral)
Male Kunming mice (10/group) were
administered/?-isopropyltolucnc (purity
not reported) at 3 and 7 mg/kg-d (exact
method of administration not specified)
or Chenopodium ambrosioides L.
volatile oil (containing
/?-isopropyltolucnc [24.25%]) at 10, 25,
and 40 mg/kg-d for 27 d. Endpoints
evaluated included body weight, serum
enzymes levels (ALT and AST), and
liver, thymus, heart, and kidney weights
and histopathology.
Significant increases in relative liver
weights, serum ALT and ALP levels, and
morphological changes in liver, heart and
kidney were reported with exposure to the
volatile oil but no effects were noted with
/?-isopropyltolucnc exposure alone.
The study evaluated few relevant endpoints
and lacked details regarding compound
administration, methods and results for
statistical analysis, and presentation of
histopathological findings (quantitative data
were not provided and representative images
were difficult to interpret).
Li et al. (2020)
Supporting studies in animals following inhalation exposure
Acute
(inhalation)
Rats, guinea pigs, and mice were
exposed to /?-isopropyltolucnc at an
atmospheric concentration of 9.7 mg/L
(9,700 mg/m3) for 5 h and observed for
up to 1 d.
Mortality was observed in mice, but not in
rats or guinea pigs. Transient clonic
convulsions reported within 15 min in rats
and 90 min in guinea pigs. Necropsy in mice
revealed hyperemic lungs, mottled liver, and
pale kidneys.
Mouse LClo <9,700 mg/m3
Rat and guinea pig LCso >9,700 mg/m3
MacDonald
(1962a, b) as cited
in FFHPVC
(2005)
22
/Msopropyltoluene
-------
EPA 690 R-24 003F
Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Short-term
(inhalation)
Male Long-Evans rats (7-12/group)
were exposed whole body to
/?-isopropyltolucnc (purity >99%) as a
vapor via inhalation at concentrations of
0, 50, or 250 ppm (v/v) (equivalent to 0,
270, or 1,370 mg/m3, respectively) for
6 h/d, 5 d/wk, for 4 wk followed by an
8-wk untreated period. Endpoints
evaluated include mortality, clinical
signs, body weight, and brain weight
(dissected into regions—forebrain
[whole brain without cerebellum] and
cerebellum—and weighed separately).
Brain homogenates and synaptosomes
were prepared and analyzed for
neurotransmitters (NA, DA, and 5-HT),
and an aliquot of each was reserved for
determination of enzyme activities
(LDH, AcliE, and BuCliE) and protein
analysis. Neurotransmitters and
enzymes were compared either to
forebrain weight (total) or relative to
synaptosomal protein.
No effects on body weight or brain weight
(whole brain, cerebellum, or forebrain) were
observed. There were no effects on enzyme
activities, protein synthesis, or
neurotransmitter concentrations in the whole
brain, forebrain, or cerebellum. Statistically
significant decreases were observed in
relative (to forebrain weight) synaptosomal
protein yield and total amount of forebrain
synaptosomal protein at >270 mg/m3.
Statistically significant increases were
observed in the enzyme activity relative to
synaptosomal protein (defined as 1 |iIVI
min '/mg synaptosomal protein) of LDH,
AcliE, and BuCliE. No effect was observed
on total (forebrain) enzyme activity of LDH,
AcliE, or BuChE, or relative (to LDH)
synaptosomal concentrations of NA or DA.
Relative (to LDH) concentrations of 5-HT
were reduced (nonsignificantly) at both
concentrations. NA and DA concentrations
were significantly increased, relative (to
synaptosomal protein concentration),
whereas no effect was observed on total NA
or DA (forebrain) concentrations. 5-HT
concentrations (relative to synaptosomal
protein) were increased (nonsignificantly),
whereas total 5-HT concentrations were
decreased.
This study evaluated few relevant endpoints.
/?-Isopropyltolucnc exposure did not have
effects on mortality, clinical signs, body
weights, or brain weights. The synaptosomal
protein, enzyme activity, and
neurotransmitter data from this study suggest
that the density and total number of synapses
is reduced by /Msopropyltoluene.
Lam et al. (1996)
23
/Msopropyltoluene
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EPA 690 R-24 003F
Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Subchronic
(inlialation)
Dogs (four total; breed and sex not
specified) were exposed 6 h/d, 5 d/wk to
mean /j-isopropyltolucne vapor
concentrations of 75 ppm for 1 mo, then
to 100 ppm for 6 wk. Endpoints
evaluated included blood pressure,
respiration pulse rates, body
temperatures, mortality, body weights,
hematology, clinical chemistry, and
urinalysis. Measurements were
compared to baseline values established
prior to exposure. At sacrifice, animals
were subjected to gross and microscopic
examinations.
No mortality was observed, but one dog was
removed from the study on Day 12 due to an
illness unrelated to exposure (no other
details were provided). Pulse pressure was
consistently decreased in the three remaining
dogs. Changes in other blood pressure
endpoints were variable. Marked increases
in respiration rate were observed within the
first week of exposure in two dogs at
75 ppm, and in the third dog at 100 ppm.
Respiration changes were attributed to the
external temperature and humidity during
the summer months. Hemoglobin
concentrations were decreased in all three
animals in the absence of changes in RBC
counts. Animals did not become severely
anemic. No changes in the other endpoints
evaluated were reported.
The study included a small number of
animals and provided few details on
experimental procedures and results. There
was limited evidence of effects on blood
pressure and hematological changes.
DuPont (1992)
Subchronic
(inlialation)
Dogs (four total; breed and sex not
specified) were exposed 6 li/d, 5 d/wk to
mean /j-isopropyltolucne vapor
concentrations of 50 ppm for
40 exposures, 74 ppm for 24 exposures,
and 160 ppm for 28 exposures.
Endpoints evaluated included blood
pressure, respiration pulse rates, body
temperatures, mortality, body weights,
hematology, clinical chemistry, and
urinalysis. Measurements were
compared to baseline values established
prior to exposure. At sacrifice, animals
were subjected to gross and microscopic
examinations.
No mortalities occurred. Diastolic and pulse
pressure showed a downward trend in three
of four animals and systolic pressure was
reduced in four of four animals. Effects were
significant at 50 ppm in one dog and at
160 ppm in three dogs. No changes in
respiration were observed. Decreased
hemoglobin levels, increased percentages of
eosinophils, and elevated blood urea levels
were observed in one animal. A filarial
infestation was identified in this animal
during postmortem examinations. These
changes and the observed congestion of the
lungs and zones of fibrosis in the kidney
were attributed to the infestation.
The study included a small number of
animals and provided few details on
experimental procedures and results. There
was limited evidence of effects on blood
pressure and hematological changes.
DuPont (1992)
24
/Msopropyltoluene
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EPA 690 R-24 003F
Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Supporting studies in animals via other routes
Acute (i.p.)
Male albino Swiss mice (eight per
group) were administered a single dose
of /?-isopropyltolucnc via i.p. injection
at doses of 0, 25, 50, and 100 mg/kg.
Animals were observed for clinical
signs and behavioral screening 4 h after
the injection. /j-Isopropyltolucne was
also evaluated for effects preventing
pain and inflammation.
CNS depression, based on reduced
spontaneous activity, analgesia, and
sedation, was observed at 50 and 100 mg/kg.
Reduced urination and defecation were
noted at 100 mg/kg. Reaction time to the hot
plate test was significantly increased in all
dose groups for up to 1 h and lasting longer
at higher doses (up to 2 h at 100 mg/kg).
/?-Isopropyltolucnc induced clinical signs of
CNS depressive behaviors.
Boniardim et al.
(2012)
Acute (i.p.)
Male Swiss mice (six per group) were
administered/?-isopropyltolucnc as a
single i.p. injection of 0, 25, 50, or
100 mg/kg. Animals were sacrificed
after 1.5 h and whole brains were
collected and prepared for
immunofluorescence c-Fos staining.
The focus of the study was preventive
effects of /?-isopropyltolucnc on pain
and inflammation.
Histopathology immunofluorescence of the
periaqueductal grey brain region showed
significantly increased c-Fos-positive cells
at all doses, compared to vehicle control.
/?-Isopropyltolucnc increased c-Fos staining
in the periaqueductal grey brain region.
Santana et al.
(2015)
Acute (i.p.)
Male Swiss albino mice (five per group)
were administered single doses of
800 |iL/kg (equivalent to 680 mg/kg
using a density of 0.85 g/mL)
/?-isopropyltolucnc via i.p. injection. At
24 h after the dose, animals were
sacrificed. Endpoints evaluated include
clinical chemistry (serum ALT). No
other standard toxicological endpoints
were evaluated.
No effect on serum ALT was observed.
Limited data indicated no effects on ALT in
mice at a dose of 680 mg/kg.
Mansour et al.
(2001)
25
/Msopropyltoluene
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EPA 690 R-24 003F
Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Acute (i.p.)
Male albino Swiss mice (six to eight per
group) were administered a single dose
of 200 mg/kg /Msopropyltoluene via i.p.
injection. Motor activity was evaluated
using a rotarod test. Additional tests
evaluated the protective effect on pain
induction (acetic acid-induced writhing
and formalin-induced pain tests; 50,
100, and 200 mg/kg i.p.).
No effect on motor performance was
observed.
Treatment with /Msopropyltoluene produced
significant antinociceptive effects and
inhibition of licking response to injected
paw.
Significant antinociceptive effects in mice at
a dose of 200 mg/kg but no changes on motor
performance.
Ouintans-Junior et
al. ("20131
Acute (i.p.)
Male Swiss mice (eight per group) were
administered single doses of 0, 25, 50,
or 100 mg/kg /Msopropyltoluene via i.p.
injection. Motor activity was evaluated
at 0.5, 1, and 2 h after administration
using a rotarod test. Additional tests
evaluated the protective effect on
orofacial pain (formalin, capsaicin, and
glutamate tests; 0, 25, 50, or
100 mg/kg).
No effect on motor performance was
observed. Treatment with
/Msopropyltoluene produced significant
antinociceptive effects at all doses.
Significant antinociceptive effects in mice at
>25 mg/kg but not changes on motor
performance up to doses of 100 mg/kg.
Santana et al.
(2011)
Acute
(dermal)
In an unpublished acute toxicity study
summarized in a secondary source,
rabbits were dennally exposed to
/Msopropyltoluene.
Unpublished study where insufficient data
were reported in the secondary source.
Rabbit LD50 >5,000 mg/kg.
Moreno (1973) as
cited inFFHPVC
(2005)
MOA/Mechanistic Studies
Cytotoxicity
Human THP-1 monocytes were exposed
to /Msopropyltoluene concentrations of
1, 10, 100, and 1,000 ng/mL for 24 h.
Cell viability was determined using an
MTT assay.
Viability was reduced by 40% in the
1,000 ng/mL group. No effects on viability
were observed at concentrations
<100 ng/mL.
/>-Isopropyltoluene was cytotoxic in a human
monocyte cell line at high exposure levels
(1,000 ng/mL).
Kavoosi and
Teixeira da Silva
(2012)
26
/Msopropyltoluene
-------
EPA 690 R-24 003F
Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Cytotoxicity
Murine macrophage cells (RAW 264.7)
were exposed to /?-isopropyltolucnc
concentrations ranging from 0 to
428.65 |ig/mL for up to 18 h. Cell
viability was assessed using an MTT
assay.
No cytotoxicity was observed at
/?-isopropyltolucnc concentrations up to
428.65 ng/mL.
/?-Isopropyltolucnc was not cytotoxic to
RAW 264.7 cells at concentrations up to
428.65 ng/mL.
Zhone et al.
(2013)
Nerve
excitability
Sciatic nerves, isolated from Wistar rats
(both sexes), were mounted in moist
chambers and stimulated to record
baseline CAPs. When a stable CAP
peak-to-peak amplitude was achieved
for at least 30 min, nerves were exposed
to a solution containing
/?-isopropyltolucnc (analytical-grade) at
1.79 mg/mL for 180 min followed by a
washout recovery period of 180 min.
Throughout the experiment, rheobase,
chronaxie, peak-to-peak amplitude, and
conduction velocity of CAP
components were recorded.
Treatment with /Msopropyltoluene had no
significant effect on electrophysiological
parameters of sciatic nerve CAP.
/Msopropyltoluene did not affect nerve
excitability in an ex vivo nerve conduction
test in rat sciatic nerves.
Barbosa et al.
(2017)
Metabolism/Toxicokinetic Studies
Dermal
absorption
0.1 mL of |14 C | -/? - i so p ro py 11 o 1 lie nc was
applied to the shaved skin of male
albino mice (five to nine per group) for
15-60 min. Urine was collected for
72 h and radioactivity was measured.
The rate of skin absorption remained
constant throughout the exposure duration
(32 mnol/cm2/min).
/Msopropyltoluene was readily absorbed
through intact mouse skin.
Wemerre et al.
(1968)
27
/Msopropyltoluene
-------
EPA 690 R-24 003F
Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Metabolism/
toxicokinetics
Two male albino rats (strain not
reported) were administered a purified
diet containing 1% neomycin sulfate for
7 d and control urine was obtained for
analysis on Days 5-7. Rats were then
given 100 mg/kg /Msopropyltoluene as
a single gavage dose and urine was
collected for 48 h and analyzed for
phenolic metabolites. Samples were
incubated with p-glucuronidase
(type H-l containing sulfatase) for
hydrolysis prior to detection.
Phenolic metabolites, such as
5-isopropyl-2-methylphenol (carvacrol) and
2-isopropyl-5-methylphenol (thymol), were
not detected following administration of
/Msopropyltoluene. Administration of
purified diet containing neomycin sulfate
reduced the amount of phenolic compounds
typically found in rat urine.
The absence of detectable phenolic
metabolites indicates that ring hydroxylation
did not occur.
Bakke and
Scheline (1970)
Metabolism/
toxicokinetics
Three bushtail possums were
administered/Msopropyltoluene over a
period of 10 d (administered in bread at
varying volumes; dose not provided).
Urine and feces were collected for
analysis during dosing.
Urinary metabolites included
/Msopropylbenzoic acid and/?-crcsol. Trace
amounts of /Msopropyltoluene were
detected in feces. Data were not provided for
control animals; therefore, the source of
urinary /?-crcsol could not be conclusively
attributed to /Msopropyltoluene
administration.
Dealkylation of the isopropyl group to form
/?-crcsol is unlikely and this metabolite is not
reported in any other study.
Southwell et al.
(1980)
Metabolism/
toxicokinetics
A single male Japanese white rabbit was
administered/Msopropyltoluene as a
single gavage dose at 670 mg/kg. Urine
was collected daily for 3 d after
chemical administration and stored at
0-5°C until time of analysis. Samples
were incubated with p-glucuronidase-
arylsulfatase for hydrolysis prior to
detection.
Within 72 h after administration, 20% of the
administered dose was eliminated in the
urine as neutral or acidic metabolites. The
main metabolites identified in the urine were
2-/Molylpropan-l-ol and
2-/Molylpropan-2-ol (50 and 28%,
respectively, of the neutral metabolites). In
total, seven metabolites were identified.
Hydroxylation at the three possible aliphatic
sites of /Msopropyltoluene contributes to the
metabolite formation in this species.
However, the methyl group oxidation makes
a minor contribution compared with that
shown by hydroxylation of the isopropyl
group. No ring hydroxylation was observed.
Isliida et al. (1981)
28
/Msopropyltoluene
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EPA 690 R-24 003F
Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Metabolism/
toxicokinetics
Male Wistar rats and Dunkin Hartley
guinea pigs were administered
/?-isopropyltolucnc orally or by
inhalation at a dose of 100 mg/kg (two
to three per group). Urine was collected
for 48 h. Samples were incubated with a
glucuronidase and sulfatase preparation
for hydrolysis prior to detection.
Within 48 h after oral administration,
approximately 70-80% of the administered
dose (60-70% following inhalation) was
excreted in the form of extractable
metabolites in the urine. Similar urinary
metabolites were identified in both species
but in different quantities.
Oxidation of both the methyl and isopropyl
groups of /?-isopropyltolucnc contributed to
the metabolite formation in both species. No
ring-hydroxylation of /?-isopropyltolucnc
was detected in rats, but trace amounts of
the ring hydroxylation metabolite,
5-isopropyl-2-methylphenol (carvacrol), were
detected in the urine in guinea pigs and only
occurred ortho- to the methyl group.
Walde et al.
(1983)
Metabolism/
toxicokinetics
Four rabbits (two per sex) were given
/?-isopropyltolucnc orally at a dose of
1,000 mg/kg. Urine was collected 3 d
after dosing. The study was designed to
identify the stereochemistry of
/?-isopropyltolucnc metabolites.
Samples were incubated with a
glucuronidase and sulfatase preparation
for hydrolysis prior to detection.
Different hydroxylated and carboxylated
metabolites were recovered in the urine.
Four were optically active, and three were
optically inactive.
The enzymatic oxidation of
/?-isopropyltolucnc occurred stereoselective^.
Matsumoto et al.
(1992)
Metabolism/
toxicokinetics
A variety of species (rat, brushtail
possum, and greater glider [six per
group] and ringtail possum [three per
group]) were administered
/?-isopropyltolucnc orally at doses
equivalent to 50 and 200 mg/kg. Urine
and feces were collected for 48 h.
Control samples were taken prior to
dosing. Samples were incubated with
P-glucuronidase-arylsulfatase for
hydrolysis prior to detection.
The fraction of dose recovered within 48 h
ranged from 52 to 74%. Differences were
observed between species in the urinary
metabolic disposition of /?-isopropyltolucnc.
The rat excreted metabolites containing all
degrees of oxidation (one to four oxygen
atoms added) but predominantly a
monooxygenated metabolite. The brushtail
possum excreted metabolites with two to
four oxygens and the greater glider and
ringtail possum excreted metabolites
containing three or four oxygen atoms. A
conjugation reaction with glycine,
glucuronic acid, or glutathione was observed
in the rat and brushtail possum. No parent
compound or metabolites were detected in
feces.
All species exhibited a complex metabolic
pattern with extensive oxidation of the methyl
and isopropyl groups of /?-isopropyltolucnc.
Bovle et al. (1999)
29
/Msopropyltoluene
-------
EPA 690 R-24 003F
Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Metabolism/
toxicokinetics
In vitro metabolism of
/?-isopropyltolucnc was evaluated using
liver microsomes obtained from Wistar
rats, brushtail possums, and koalas.
Microsomes were also obtained from
possums exposed to mixed terpenes
prior to sacrifice.
The primary metabolite in each species was
/?-isopropylbcnzyl alcohol (84, 73, and 89%
of total metabolites using rat, possum, and
koala microsomes, respectively). Other
metabolites, each accounting for <10% of
the total, included 2-/?-tolylpropan-2-ol (or
/?,a,a-trimethylbenzylalcohol),
/?-isopropylbcnzoic acid, 2-p-(hydroxy-
methyl)phenyl-2-propan-2-ol,
2-/?-tolypropan-1,2-diol. and
2-/?-(hydroxymethyl)phenyl-propan-1 -ol. No
phenolic metabolites were detected. Kinetic
analysis revealed the following rank order
for intrinsic clearance (Clmt): terpene-treated
possum (128 |iL/mg protein-1 min ') >
control possum (107 |iL/mg protein-1 min-1)
> koala (69 ^L/mg protein-1 min-1) > rat
(38 |iL/mg protein 1 min-1).
In vitro metabolism occurred at the same sites
of oxidation as observed in the in vivo studies
in the same species (i.e., hydroxylation of the
methyl and isopropyl substituents without
ring hydroxylation). Metabolic capacity
(measured as Clint) was increased in possums
that were pretreated with terpenes, suggesting
induction of metabolic enzymes. Metabolism
was greater in possums and koalas, compared
to rats.
Pass et al. (2002)
30
/Msopropyltoluene
-------
EPA 690 R-24 003F
Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Metabolism/
toxicokinetics
In vitro incubation of
/?-isopropyltolucnc (purity 99%) with
human recombinant CYP enzymes
followed by GC-MS analysis.
A single volunteer swallowed 35 tablets
containing 1% tea tree oil and a
concentration of 0.5-12%
/?-isopropyltolucnc among other
monoterpenes (dose not further
described). Ingestion occurred over a
2-h period. Blood was collected 10 min
after the ingestion period, and urine
samples were taken at 60 min during the
ingestion period and 10 and 25 min
postingestion.
In the in vitro study,
2-isopropyl-5-methylphenol (thymol),
/?-isopropylbcnzyl alcohol, and
/9-isopropylbcnzaldchvdc were identified in
the extract. In the human study, thymol was
recovered from both blood and urine as
glucuronide or sulfate conjugate, while other
monoterpenes were detected in urine.
One predicted metabolite from an in vitro
study (thymol) was found in human blood
and urine following oral dosing with tea tree
oil containing /j-isopropyltolucne.
Meesters et al.
(2009)
aValues in the study report were given in ppm. Values in mg/m3 = exposure in ppm x MW of /?-isopropyltolucnc 24.45. The MW of/?-isopropyltoluene is
134.222 g/mol (U.S. EPA. 2022a).
AcliE = acetylcholinesterase; ALT = alanine aminotransferase; AST = aspartate aminotransferase; BuCliE = butyrylcholinesterase; CAP = compound action potential;
CI = confidence interval; CNS= central nervous system; CYP = cytochrome P450; DA = dopamine; 5-HT = 5-hydroxytrptamine; GC = gas chromatography;
i.p. = intraperitoneal; LCso = median lethal concentration; LClo = lowest reported lethal concentration; LD50 = median lethal dose; LDH = lactate dehydrogenase;
MOA = mode-of-action; MS = mass spectrometry; MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MW = molecular weight; NA = noradrenaline;
RBC = red blood cell.
31
/Msopropyltoluene
-------
EPA 690 R-24 003F
In an unpublished, acute inhalation study, summarized in secondary sources, mice, rats,
and guinea pigs were evaluated for acute lethality following a 5-hour exposure to a concentration
of 9,700 mg/m3 [MacDonald, (1962a, b) as cited in FFHPVC (2005)1. No mortality was
observed in rats or guinea pigs; however, transient clonic convulsions were reported within
15 minutes in rats and 90 minutes in guinea pigs. The lowest lethal concentration (LClo) in mice
was <9,700 mg/m3 as all three mice died.
A 4-week inhalation study in male rats exposed top-isopropyltoluene at concentrations
up to 1,370 mg/m3 showed no effects on mortality, clinical signs, body weight, or whole brain,
cerebellum, or forebrain weight (Lam et al.. 1996). Statistically significant decreases in relative
(to forebrain weight) synaptosomal protein yield and total amount of forebrain synaptosomal
protein were observed at >270 mg/m3. No effect was observed on total forebrain lactate
dehydrogenase (LDH), acetylcholinesterase (AchE), or butyrylcholinesterase (BuChE) activities
or noradrenaline (NA) or dopamine (DA) concentrations; however, enzyme activities and
concentrations of DA and NA relative to synaptosomal protein were significantly increased at
>270 mg/m3. In addition, total forebrain 5-hydroxytryptamine (5-HT) concentrations were
decreased at 1,370 mg/m3, and 5-HT concentrations relative to synaptosomal protein were
increased (nonsignificantly). These findings suggest that the density and total number of
synapses is reduced by p-isopropyltoluene, leading to changes in neurotransmitter concentrations
(Lam et al.. 1996). Subchronic inhalation studies in dogs suggest effects on blood pressure and
hematological changes; however, the evidence was limited (DuPont 1992).
Several acute-duration studies by other routes are also described in Table 4B. In an
unpublished study cited in a secondary source, the rabbit dermal LDso was >5,000 mg/kg
according to Moreno (1973) as cited in FFHPVC (2005) (no additional details regarding this
study were available). Several acute i.p. studies examined neurological changes in mice.
Reduced spontaneous activity, analgesia (i.e., increased reaction time on the hot plate test), and
reduced urination and defecation were observed at doses of 50-100 mg/kg (Boniardim et al..
2012). Increased c-Fos expression in the brain periaqueductal grey region (Santana et al.. 2015)
and antinociceptive effects (Quintans-Junior et al.. 2013; Santana et al.. 2011) were reported at
doses >25 mg/kg; however, no effects were observed on motor performance up to doses of
200 mg/kg (Quintans-Junior et al.. 2013; Santana et al.. 2011). No effects on serum ALT levels
were observed in mice administered a single i.p. dose of 680 mg/kg (Mansour et al.. 2001).
2.3.3. Mode-of-Action/Mechanistic Studies
Few noncancer mechanistic studies were identified. /Msopropyltoluene was not cytotoxic
to murine macrophage cells (RAW 264.7) (Zhong et al.. 2013). but was cytotoxic to human
THP-1 monocytes at high exposure concentrations (Kavoosi and Teixeira da Silva. 2012).
/Msopropyltoluene did not affect nerve excitability in sciatic nerves isolated from Wistar rats
(Barbosa et al.. 2017).
2.3.4. Metabolism/Toxicokinetic Studies
Studies in animals (rats, rabbits, guinea pigs, and marsupials [possum, greater glider, and
koala]) have shown that p-isopropyltoluene is well absorbed from the gastrointestinal tract,
widely distributed in the body, metabolized, and excreted mainly in the urine (Boyle et al.. 1999;
Matsumoto et al.. 1992; Walde et al.. 1983; Ishida et al.. 1981). Dermal absorption was also
demonstrated in mice (Wepierre et al.. 1968). No in vivo human or animal studies reporting
32
/Msopropyltoluene
-------
EPA 690 R-24 003F
distribution ofp-isopropyltoluene were identified; however, based on a log Kow (octanol-water
partition coefficient) value >4 (see Table 1), p-isopropyltoluene is expected to accumulate in
fatty tissues. The metabolism studies in laboratory animals summarized in Table 4B demonstrate
that /Msopropyltoluene undergoes extensive oxidation of the methyl substituent and isopropyl
side-chain to yield polar oxygenated metabolites (see Figure 2). The primary metabolites include
monohydric alcohols, diols, mono- and dicarboxylic acids, and hydroxy acids. These
metabolites are either excreted unchanged in the urine or undergo conjugation with glucuronic
acid and/or glycine, followed by excretion in the urine. Ring-hydroxylation was not observed in
in vivo animal studies (Boyle et al.. 1999; Matsumoto et al.. 1992; Walde et al.. 1983; Ishida et
al.. 1981; Bakke and Scheline. 1970). with the single exception of trace amounts of
5-isopropyl-2-methylphenol (carvacrol) found in the urine of guinea pigs (Walde et al.. 1983).
There are limited data in humans. An in vitro study using human recombinant cytochrome P450
(CYP) enzymes identified 2-isopropyl-5-methylphenol (thymol) as a metabolite in addition to
p-isopropylbenzyl alcohol, 2-/;-tolylpropan-2-ol (or /;,a,a-tri methyl benzyl alcohol), and
p-isopropylbenzyl aldehyde (quantitative data were not presented) (Meesters et al.. 2009). An in
vitro study using liver microsomes obtained from rats or marsupials (possum and koala) reported
the same sites of oxidation as observed in the in vivo studies discussed above (i.e., hydroxylation
of the methyl and isopropyl substituents without ring hydroxylation) and noted the absence of
phenolic metabolites (Pass et al.. 2002). Southwell et al. (1980) reported/?-cresol as a metabolite
ofp-isopropyltoluene in bushtail possums; however, this is a poorly reported study with
significant limitations. Dealkylation of the isopropyl group to form p-cresol is unlikely, and this
metabolite is not reported in any other study. In addition, endogenous/?-cresol is produced via
the digestion of tyrosine (from food proteins) in the intestine. Free /?-cresol formed in this way is
absorbed from the intestine and eliminated in the urine as conjugates (IPCS. 1995).
33
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p-isopropyltoluene
1
COOH
CH„
2-(p-tolyl)-1 -propanol
CH
/ \
H3C COOH
2-(p-tolyl)-
propionic acid
p-isopropylbenzoic acid
HOH2C
ch2oh h3c
H,C —C — CH,OH
3 I 2
2-(p-tolyl)- 2-p-(hydroxymethyl) 2-(p-tolyl)-
1,3-propanediol phenyl-1-propanol 1,2-propandiol
h3C-C—
OH
2-p-(hydroxymethyl)phenyl-2-propanol
COOH
COOH
COOH
CH
/ \
H3C COOH
2-p-(hydroxymethyl)
phenylpropionic acid
2-p-carboxyphenyl-
1-propanol
h3c-c—ch3
2-p-carboxyphenyl-
2-propanol
3 ^1 I2
p-isopropenylbenzoic
acid
COOH
CH
/ \
H3C COOH
2-p-carboxyphenylpropionic acid
Figure 2./>-Isopropyltoluene Metabolism in Laboratory Animals Based on Information
Presented in Walde et al. (1983)
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3. DERIVATION OF PROVISIONAL VALUES
3.1. DERIVATION OF ORAL REFERENCE DOSES
The database of repeated-dose oral exposure studies for p-isopropyltoluene adequate for
quantitative dose-response analysis consists of a non-peer-reviewed, repeated-dose systemic
toxicity study that included a reproductive/developmental toxicity screening test in rats (Symrise.
2018). This study was insufficient to support deriving a provisional toxicity value given its
non-peer-reviewed status, reporting limitations in assessment methods, and most data were
reported qualitatively from a secondary source . However, this study provides sufficient data to
develop screening subchronic and chronic provisional reference dose (p-RfD) values
(see Appendix A).
3.2. DERIVATION OF INHALATION REFERENCE CONCENTRATIONS
No adequate repeated-dose studies (including subchronic or chronic studies) were located
regarding toxicity of p-isopropyltoluene to humans or animals via inhalation exposure. Due to
the limited inhalation toxicity data for p-isopropyltoluene, subchronic and chronic provisional
reference concentrations (p-RfCs) were not derived using chemical-specific information. Instead,
screening subchronic and chronic p-RfCs are derived in Appendix A using an alternative
analogue approach.
3.3. SUMMARY OF NONCANCER PROVISIONAL REFERENCE VALUES
The noncancer screening provisional reference values forp-isopropyltoluene are
summarized in Table 5.
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Table 5. Summary of Noncancer Reference Values for
/7-Isopropyltoluene (CASRN 99-87-6)
Toxicity Type
(units)
Species/
Sex
Critical Effect
p-Reference
Value
POD
Method
POD
(HED/HEC)
UFc
Principal
Study
Screening
subchronic p-RfD
(mg/kg-d)
Rat/F
Increased ALP
4 x 1(T2
BMDL
3.6
100
Svmrise
(2018)
Screening
chronic p-RfD
(mg/kg-d)
Rat/F
Increased ALP
4 x 1(T3
BMDL
3.6
1,000
Svmrise
(2018)
Screening
subchronic p-RfC
(mg/m3)
Rat/M
Impaired motor
coordination
(decreased rotarod
performance)
1 x KT1
NOAEL
39 (based on
analogue
PODa)
300
Korsak et
al. (1994)
as cited in
U.S. EPA
(2009): and
U.S. EPA
(2003)
Screening
chronic p-RfC
(mg/m3)
Rat/M
Impaired motor
coordination
(decreased rotarod
performance
4 x 1(T2
NOAEL
39 (based on
analogue
PODa)
1,000
Korsak et
al. (1994)
as cited in
U.S. EPA
(2009): and
U.S. EPA
(2003)
aXylene (mixed isomers) was selected as a suitable source analogue for/j-isopropyltolucne as described in
Appendix A.
ALP = alkaline phosphatase; BMDL = benchmark dose lower confidence limit; F = female; HEC = human
equivalent concentration; HED = human equivalent dose; M = male; NOAEL = no-observed-adverse-effect level;
POD = point of departure p-RfC = provisional reference concentration; p-RfD = provisional reference dose;
UFC = composite uncertainty factor.
3.4. CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR
No oral or inhalation studies have been conducted to assess the carcinogenicity of
/?-isopropyltoluene. Limited data suggest that the chemical is not genotoxic (see Section 2.3).
Under the U.S. EPA Cancer Guidelines (U.S. EPA. 2005). there is "Inadequate Information to
Assess the Carcinogenic Potential" of p-isopropyltoluene by oral or inhalation exposure
(see Table 6).
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Table 6. Cancer WOE Descriptor for />-Isopropyltoluene (CASRN 99-87-6)
Possible WOE
Descriptor
Designation
Route of Entry (oral,
inhalation, or both)
Comments
"Carcinogenic to
Humans "
NA
NA
The available data do not support this
descriptor.
"Likely to be
Carcinogenic to
Humans "
NA
NA
The available data do not support this
descriptor.
"Suggestive Evidence of
Carcinogenic Potential"
NA
NA
The available data do not support this
descriptor.
"Inadequate
Information to Assess
Carcinogenic Potential"
Selected
Both
No adequate information is available to
assess the carcinogenic potential of
/7-isopropyltoluene by the inhalation or
oral routes of exposure.
"Not Likely to be
Carcinogenic to
Humans "
NA
NA
The available data do not support this
descriptor.
NA = not applicable; WOE = weight-of-evidence.
3.5. DERIVATION OF PROVISIONAL CANCER RISK ESTIMATES
The absence of data indicating a tumorigenic effect precludes development of cancer risk
estimates for />isopropyltoluene (see Table 7).
Table 7. Summary of Cancer Risk Estimates for
/7-Isopropyltoluene (CASRN 99-87-6)
Toxicity Type
Species/Sex
Tumor Type
Cancer Value
Principal Study
p-OSF (mg/kg-d) 1
NDr
p-IUR (mg/m3)
NDr
NDr = not determined; p-IUR = provisional inhalation unit risk; p-OSF = provisional oral slope factor.
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APPENDIX A. NONCANCER SCREENING PROVISIONAL VALUES
Due to the lack of evidence described in the main Provisional Peer-Reviewed Toxicity
Value (PPRTV) assessment, it is inappropriate to derive provisional toxicity values for
/?-isopropyltoluene because the limited database on the toxicity of/?-isopropyltoluene is
insufficient to support direct derivation. However, some information is available for this
chemical, which although insufficient to support deriving a provisional toxicity value under
current guidelines, may be of use to risk assessors. In such cases, the Center for Public Health
and Environmental Assessment (CPHEA) summarizes available information in an appendix and
develops a "screening value." Appendices receive the same level of internal and external
scientific peer review as the provisional reference values to ensure their appropriateness within
the limitations detailed in the document. Users of screening toxicity values in an appendix to a
PPRTV assessment should understand that there could be more uncertainty associated with the
derivation of an appendix screening toxicity value than for a value presented in the body of the
assessment. Questions or concerns about the appropriate use of screening values should be
directed to the CPHEA.
Screening subchronic and chronic provisional reference doses (p-RfDs) were derived for
p-isopropyl toluene as described below. For inhalation, an alternative analogue approach was
utilized to derive screening subchronic and chronic provisional reference concentration (p-RfC)
values.
ORAL NONCANCER TOXICITY VALUES
As discussed in the main body of the report, the database of repeated-dose oral studies for
p-isopropyl toluene adequate for quantitative dose-response analysis consists of a
non-peer-reviewed repeated-dose systemic toxicity study that included a reproductive/
developmental toxicity screening test in rats (Symrise. 2018). Other studies that examined
p-isopropyl toluene but are not appropriate for the selection of a point of departure (POD) are
described in Section 2.3. These include short-term oral studies with significant design and
reporting limitations, as well as genotoxicity, metabolism/toxicokinetic, and acute studies and
studies using routes of exposure other than the oral or inhalation route.
The ECHA (2019b)/Svmrise (2018) study is insufficient to derive p-RfD values because
of its non-peer-reviewed status and uncertainties surrounding reporting deficiencies (limited
details on methods and results for some endpoints and most data were reported qualitatively from
a secondary source). However, the study adhered to Good Laboratory Practice (GLP) and
Organisation for Economic Co-operation and Development (OECD) test guidelines, evaluated a
wide range of relevant toxicity endpoints (including body weight, food consumption, clinical
observations, functional observational battery [FOB], hematology, clinical chemistry, organ
weight, and histopathology) and provided sufficient information to identify toxicologically
relevant health effects and associated dose-response relationships for deriving screening-level
p-RfD values (see study summary in Section 2.2.1 for more details).
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The ECHA (2019b)/Svmrise (2018) study identified the liver as a sensitive target of
toxicity following subchronic oral exposure in rats. ECHA (2019b)/Svmrise (2018) reported
dose-related and biologically significant (>10%) increases in absolute and relative4 liver weight
at 50 and 100 mg/kg-day in Po female rats exposed for -63 days (50 mg/kg-day was the lowest
dose tested) that were the basis for the systemic lowest-observed-adverse-effect level (LOAEL)
value (LOAEL = 50 mg/kg-day; a no-observed-adverse-effect level [NOAEL] could not be
determined). These findings are supported by increases (>10%) in absolute and relative liver
weights in Po male rats at 200 mg/kg-day, statistically significant and dose-related increases in
serum alkaline phosphatase (ALP) levels (a biomarker of hepatobiliary injury) in Po males and
females at >100 mg/kg-day, and low incidence of hepatocyte hypertrophy (10-40%) in both
sexes at >50 mg/kg-day. The data across organ weights, serum chemistry, and histopathology
provide coherent evidence of liver toxicity following oral exposure to p-isopropyltoluene in rats.
Effects on the reproductive system and developing offspring were also reported in the
ECHA (2019b)/Svmrise (2018) study. A reproductive NOAEL of 50 mg/kg-day and a LOAEL
of 100 mg/kg-day were identified based on decreased fertility index (40 and 0% at 100 and
200 mg/kg-day, respectively, compared to 90% for controls) and degenerative male reproductive
lesions in the testes and epididymides of Po rats (i.e., increased minimal sperm retention and
minimal-to-slight reduced luminal sperm and sperm with slight cribriform changes,
respectively). These effects were accompanied by changes in estrous cyclicity (decreased
number of females with regular cycles and increased number of females with irregular cycles),
male reproductive organ weights (statistically significant decreases in absolute and/or relative
testes, epididymides, and prostate weights), and male histopathology (increased
minimal-to-slight spermatid retention and minimal-to-moderate germ cell degeneration/depletion
in the testis, and slight-to-markedly reduced luminal sperm, sperm with minimal-to-slight
cribriform changes, and minimal-to-moderate luminal cell debris in the epididymides) at the
highest dose (200 mg/kg-day). A tentative LOAEL of 50 mg/kg-day (lowest dose tested; a
NOAEL could not be determined) for developmental effects was based on marginally significant
(>4.5%; >5% is considered biologically significant) decreases in the body weights of female
offspring on postnatal day (PND) 1. The findings were dose-related, consistent with reductions
in male offspring body weights (-10%) on PND 1, and coherent with effects on offspring
survival (reductions in postimplantation survival index and live birth index) occurring at
100 mg/kg-day, which provide support for the biological significance of the reductions in female
Fi body weights. Furthermore, reduced birth weight has been associated with toxicologically
relevant effects such as neonatal and postnatal mortality, coronary heart disease, arterial
hypertension, chronic renal insufficiency, and diabetes mellitus in humans (Barken 2007; Reyes
and Manalich. 2005).
Kidney lesions occurred in Po male rats in the ECHA (2019b)/Svmrise (2018) study,
including increases in minimal tubular epithelium vacuolation (two of five rats at 200 mg/kg-day
compared to zero of five rats in the controls), minimal-to-slight hyaline droplets accumulation
(one of five and three of five rats at 50 and 200 mg/kg-day, respectively, compared to one of
five rats in the controls), and minimal tubular basophilia (one of five rats in all dose groups
compared to zero of five rats in the controls). Additionally, statistically significant increases in
blood urea nitrogen (BUN) levels occurred in males at the highest dose (200 mg/kg-day). In
4Any discussion of relative organ weights is based on changes with respect to body weight unless otherwise noted.
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general, the increases in the incidence and severity of the kidney lesions were relatively minor
and lacked dose-response correspondence. Therefore, the biological significance of these lesions
and accompanying clinical chemistry changes (increased BUN levels) in male rats occurring
mostly at the highest dose in the ECHA (2019b)/Svmrise (2018) study are unclear.
Statically significant decreases in hindlimb grip strength were observed in Po male rats at
the highest dose (200 mg/kg-day) (ECHA. 2019b; Symrise. 2018). but there was no
corroborative evidence of neurotoxicity from other FOB assays evaluating grip strength
(forelimb grip), sensory reactivity, and locomotor activity. Several acute/short-term supplemental
studies reported neurological effects associated withp-isopropyltoluene exposure that included
reduced mobility at 1,650 mg/kg-day for 24-28 days [Stel'makh et al. (1983) as cited in ECHA
(1986)1 and analgesic effects (i.e., increased reaction time on the hot plate test) after a dose of
40 mg/kg in mouse studies with oral exposure (Siqueira Quintans et al.. 2013); transient clonic
convulsions in rats and guinea pigs after a single inhalation exposure at 9,700 mg/m3
[MacDonald, (1962a, b) as cited in FFHPVC (2005)1; and reduced spontaneous activity,
analgesia, reduced urination and defecation, and antinociceptive effects in acute intraperitoneal
(i.p.) studies in mice at doses of 25-200 mg/kg (Quintans-Junior et al.. 2013; Boniardim et al..
2012; Santana et al.. 2011). The significance of these findings and association with potential
neurological effects (decreases in hindlimb grip strength) observed after repeated-dose exposure
to/Msopropyltoluene in the ECHA (2019b)/Svmrise (2018) study is unclear.
Overall, coherent evidence of liver toxicity based on organ weights, clinical chemistry,
and histopathology effects was identified in Po rats at doses >50 mg/kg-day after subchronic
exposure (ECHA. 2019b; Symrise. 2018). Similarly, developmental effects were observed at
>50 mg/kg-day in the ECHA (2019b)/Svmrise (2018) study that included marginally significant
reductions in body weight and survival of Fi rats. There is also evidence of reproductive effects
in Po rats, involving alterations in fertility index, estrous cyclicity, and male reproductive organ
weights and histopathology at >100 mg/kg-day. As such, liver, developmental, and reproductive
effects were considered further for the derivation of screening p-RfDs. Other treatment-related
effects that occurred only in Po male rats (i.e., kidney lesions, increased BUN, and decreased
hindlimb grip strength) in the ECHA (2019b)/Svmrise (2018) study were not considered for
dose-response analysis due to the limitations in the database for/?-isopropyltoluene, which
prevent further evaluation of the biological significance of these effects in animals.
Derivation of a Screening Subchronic Provisional Reference Dose
Data for liver, developmental, and reproductive effects in rats from the ECHA
(2019b)/Svmrise (2018) study were considered for modeling using the U.S. Environmental
Protection Agency (U.S. EPA) Benchmark Dose Software (BMDS, Version 3.3). As mentioned
previously, despite the non-peer-reviewed status and data reporting limitations, the ECHA
(2019b)/Svmrise (2018) study used an adequate design (repeated-dose systemic study in rats
with reproductive/developmental screening test according to GLP/OECD 422 guidelines),
included multiple doses and a comprehensive array of toxicity endpoints, and identified sensitive
health effects that are suitable for the derivation of the screening subchronic p-RfD. Increased
liver weights (>10%) in Po female rats observed at the lowest dose could not be modeled due to
the lack of quantitative data on this endpoint (only percent change relative to controls was
provided) but the LOAEL of 50 mg/kg-day was considered as a candidate POD. Dose-related
increases in ALP in both male and Po female rats were modeled as continuous data using a
40
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standard benchmark response (BMR) of 1 standard deviation (SD). Increased liver weights
(>10%) in Po male rats and increased hepatocyte hypertrophy in Po male and female rats were not
considered for dose-response analysis since these effects were mostly observed at the highest
dose (200 mg/kg-day) and there were more sensitive markers of liver toxicity available
(i.e., increased liver weights in females and increased ALP in males and females at
>50 mg/kg-day).
For developmental effects, dose-related decreases in body weights in Fi females on
PND 1 were modeled as continuous data using a BMR of 5% relative deviation (RD) because a
5% change in markers of growth/development in gestational studies (e.g., fetal weight) is
considered a minimally biologically significant response level (U.S. EPA. 2012b). Significant
decreases (>5%) in body weights of Fi males on PND 1 and in Fi offspring survival
(postimplantation survival index and live birth index) were only observed at 100 mg/kg-day;
therefore, these endpoints were not considered for dose-response analysis.
For reproductive effects, the changes in percent fertility index observed at
>100 mg/kg-day were considered for dose-response analysis by estimating the increased
incidence of nonpregnant Po female rats over the total mated Po females (1/10, 1/10, 6/10, and
9/9 at 0, 50, 100, and 200 mg/kg-day, respectively); however, the data were not ultimately
selected for BMD modeling because the response rate (60%) at the lowest dose group with
increased incidence over the control (100 mg/kg-day) is much higher than the standard BMR of
10% extra risk used for dichotomous data. Similarly, the increased incidence in testicular
spermatid retention in Po male rats at >100 mg/kg-day was not considered amenable for BMD
modeling because of the steep dose-repose curve (response rate at 100 mg/kg-day was 70%). As
such, the NOAEL of 50 mg/kg-day for these endpoints was considered for POD candidate
comparisons. For other dose-related male degenerative lesions (i.e., epididymal reduced luminal
sperm and sperm with cribriform changes in Po males at >100 mg/kg-day), the incidence data
were evaluated as dichotomous data using a standard BMR of 10% extra risk. Other reproductive
effects occurring only at the highest dose (200 mg/kg-day) were not amenable for BMDS
modeling (i.e., changes in estrous cyclicity, decreased male reproductive organ weights, and
increased incidence in testicular germ cell degeneration/depletion and epidydimal luminal cell
debris) and were not considered for POD candidate derivation due to the presence of more
sensitive endpoints.
Prior to modeling the selected liver, developmental and reproductive endpoints, exposure
doses used in ECHA (2019b)/Svmrise (2018) study were converted to human equivalent doses
(HEDs)5. In Recommended Use of Body Weight3 4 as the Defairft Method in Derivation of the
Oral Reference Dose (U.S. EPA. 201 le). the Agency endorses body-weight scaling to the
5Administered doses were converted to HEDs by multiplying by a dosimetric adjustment factor (DAF) of 0.281 for
males and 0.256 for females, calculated as follows: DAF = (BWaI/4 ^ BWh1'4), where BWa = animal body weight,
and BWh = human body weight. The study reported initial body weights of 0.332-0.434 and 0.235-0.299 kg for
Sprague Dawley male and female rats, respectively. In the absence of data for study-specific time-weighted average
(TWA) or final body weights in the animals, the upper end of the reported initial body weight for rats in this study
was used for males (0.434 kg) and females (0.299 kg) given that the initial body weights are higher than the
recommended default values for males and females Sprague Dawley rats in a subchronic study (0.267 and 0.204 kg,
respectively) (U.S. EPA. 1988). For humans, the reference value of 70 kg was used for body weight, as
recommended by U.S. EPA (1988).
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3/4 power (i.e., BW3 4) as a default to extrapolate toxicologically equivalent doses of orally
administered agents from all laboratory animals to humans for deriving an RfD from effects that
are not portal-of-entry effects or effects resulting from direct exposure of neonatal or juvenile
animals. In the ECHA (2019b)/Svmrise (2018) study, the observed decreases in body weight in
female offspring resulted from exposure of the Po female rats; there was no direct exposure of
neonates in this study. As such, HEDs for all the endpoints evaluated, including developmental
effects, were calculated using a dosimetric adjustment factor (DAF) of 0.256 for Po female rats.
Table A-l shows the data for liver, developmental, and reproductive endpoints that were
considered for dose-response assessment and Table A-2 summarizes the BMD modeling results
and provides candidate PODs for the derivation of the screening subchronic p-RfD. Details of
model fit for each data set are presented in Appendix C. Candidate PODs that could not be
evaluated via BMDS analysis (i.e., liver weights and fertility index in Po rats) are presented as
NOAEL/LOAEL values.
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Table A-l. Data for Sensitive Liver and Reproductive Effects in Po Rats and
Developmental Effects in Fi Offspring3
Po Females: exposed -63 Days [HED] (mg/kg-d)b
0 (control)
50 [13]
100 [25.6]
200 [51.2]c
Number of animals
5
5
4
0
ALP (U/L)d
151 ± 19.8°
210 ±70.9 (±39%)
270 ± 119.1* (±79%)
NA
Liver weight relative to body
weight (%)e
-
14
22*
NA
Absolute liver weight (%)e
-
16
26*
NA
Number of mated females
10
10
10
9
Increased incidence of
nonpregnant femalesf
1
1
6
9
Po Males: exposed -35 [HED] (mg/kg-d)b
0 (control)
50 [14]
100 [28.1]
200 [56.2]
Number of animals
5
5
5
5
ALP (U/L)d
160 ±23.5
166 ± 43.5 (±4%)
184 ± 22.8 (±15%)
232 ±61.4* (±45%)
Number of animals
10
10
10
10
Spermatid retention in the
testesf
0
0
7*
9*
Reduced luminal sperm in the
epididymidesf
0
0
2
10*
Sperm with cribriform changes
in the epididymidesf
0
0
1
5*
Fi Offspring: exposed during gestation and lactation until PND 13 [HED] (mg/kg-d)b
0 (control)
50 [13]
100 [25.6]
200 [51.2]c
Number ofpregnant females
9
9
4
0
Decreased Fi female body
weight on PND ld
6.6 ±0.66
6.3 ± 0.84 (-4.5%)
6.0 ±0.3 (-9.1%)
NA
aSvmrise (2018).
bADDs (mg/kg-day) were reported by the study authors; calculated HEDs appear in brackets.
Data for females exposed to 200 mg/kg-day were not collected due to failure to become pregnant.
dData represent mean ± SD. Value in parentheses is % change relative to control = ([treatment mean - control
mean] control mean) x 100.
eData represent percent difference relative to controls [actual measurement data for control and treatment groups
were not available in the study report (ECHA. 2019b)l.
fData represent number of animals showing changes.
* Significantly different from control (p < 0.05) as reported by the study authors.
- = reported in ECHA as not test item-related; ALP = alkaline phosphatase; ECHA = European Chemicals Agency;
HED = human equivalent dose; NA = not available; SD = standard deviation.
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Table A-2. Comparison of Candidate POD Values in Sprague Dawley Rats
Exposed to /7-Isopropyltoluene via Subchronic Gavage Exposure
(-35-63 Days) or During Gestation and Lactation until Postnatal Day 13a
Endpoint
Best-Fitting Model
BMR
BMDL (HED)
(mg/kg-d)
POD type
POD (HED)
(mg/kg-d)
Increased absolute and
relative liver weight in
Po female rats
Data not amenable for BMD modeling13
LOAEL
13
Increased ALP in
Po female rats
Exponential
(degree 3)
1 SD from control
(1SD)
3.6
BMDL
3.6
Increased ALP in
Po male rats
Linear
1 SD from control
(1SD)
18
BMDL
18
Increased incidence of
nonpregnant Po female
rats
Data not amenable for BMD modeling0
NOAEL
13
Increased incidence of
spermatid retention in
the testes of P0 male
rats
Data not amenable for BMD modeling0
NOAEL
14
Increased incidence of
reduced luminal sperm
in the epididymides of
Po male rats
Log-logistic
10% extra risk
20
BMDL
20
Increased incidence of
sperm with cribriform
changes in the
epididymides of
P0 male rats
Multistage (degree 3)
10% extra risk
14
BMDL
14
Decreased Fi female
body weight on PND 1
Linear
5% RD from
control
(0.05 RD)
7.1
BMDL
7.1
aSvmrise (2018).
bData were not considered amenable for BMD modeling due to the lack of quantitative data on mean liver weights
and variance.
Data were not considered amenable for BMD modeling since the response rates (60-70%) at the lowest dose
group (100 mg/kg-day) with increased incidence over the controls is much higher than the standard BMR used for
dichotomous data (10% extra risk).
BMD = benchmark dose; BMDL = benchmark dose lower confidence limit; BMR = benchmark response;
HED = human equivalent dose; LOAEL = lowest-adverse-effect level; PND = postnatal day 1; POD = point of
departure; RD = relative deviation; SD = standard deviation.
The benchmark dose lower confidence limit with one standard deviation (BMDLisd)
(HED) of 3.6 mg/kg-day for increased ALP in Po female rats exposed -63 days (ECHA. 2019b;
Symrise. 2018) provides the lowest candidate POD for liver effects. A freestanding LOAEL
(HED) of 13 mg/kg-day for increased liver weights in Po female rats is also available but the
changes (16 and 14% for absolute and relative liver weights, respectively) at the identified
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LOAEL (HED) were close to the minimally biologically significant response level (10%).
Therefore, it is likely that the POD for increased ALP, which is 70% lower, is protective of the
POD for increased liver weights. Further, these endpoints are coherent and reliable markers of
liver toxicity occurring in the same species and sex. In particular, increased ALP is associated
with obstruction of hepatic bile flow and damage to the biliary epithelial cells (EMEA. 2008;
Boone et al.. 2005). Additionally, there is higher confidence in the POD estimate for increased
ALP based on the availability of quantitative data (mean ± SD) for this endpoint for BMD
modeling. As mentioned above, data for liver weights in Po rats from the Symrise (2018) study
were only available as percent change relative to controls from the ECHA (2019b) report.
Finally, the POD for increased ALP in Po female rats (BMDLisd [HED] of 3.6 mg/kg-day) is
protective of the lowest PODs derived for developmental effects (a benchmark dose lower
confidence limit with 5% relative deviation [BMDLo.osrd] [HED] of 7.1 mg/kg-day for decreased
Fi female body weights on PND 1) and reproductive effects (aNOAEL [HED] of 13 mg/kg-day
for increased incidence of nonpregnant Po females). Altogether, the evidence suggests that the
liver is a primary target for />-isopropyltoluene via oral exposure and the BMDLisd (HED)
of 3.6 mg/kg-day for increased ALP in Po female rats exposed ~63 days (ECHA, 2019b;
Symrise, 2018) is selected as the most sensitive POD for the derivation of the screening
subchronic p-RfD.
The screening subchronic p-RfD forp-isopropyltoluene is derived by applying a
composite uncertainty factor (UFc) of 100 (reflecting an interspecies uncertainty factor [UFa] of
3, a database uncertainty factor [UFd] of 3, and an intraspecies uncertainty factor [UFh] of 10) to
the selected POD of 3.6 mg/kg-day.
Screening Subchronic p-RfD = POD (HED) UFc
= 3.6 mg/kg-day -M00
= 4 x 10"2 mg/kg-day
Table A-3 summarizes the uncertainty factors for the screening subchronic p-RfD for
p-i sopropy ltoluene.
45
/Msopropyltoluene
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EPA 690 R-24 003F
Table A-3. Uncertainty Factors for the Screening Subchronic p-RfD for
/7-Isopropyltoluene (CASRN 99-87-6)
UF
Value
Justification
UFa
3
A UFa of 3 (10"5) is applied to account for uncertainty associated with extrapolating from animals to
humans when cross-species dosimetric adjustment (HED calculation) is performed.
UFd
3
A UFd of 3 is applied to account for deficiencies and uncertainties in the database. The oral database
of relevant studies for/j-isopropyltolucne includes a non-peer-reviewed, repeated-dose systemic
toxicity studv with a reoroductive/develomnental toxicity screening test (ECHA. 2019b: Svmrise.
2018). The studv had data reoortine limitations but adhered to GLP and OECD test guidelines,
evaluated a wide range of relevant toxicity endpoints (including body weight, food consumption,
clinical observations, FOB, hematology, serum chemistry, organ weights, histopathology, and
reproductive and developmental endpoints [mating and fertility indices, reproductive parameters,
sperm evaluations, estrous cycle, histopathology of reproductive organs, and offspring viability, body
weight, gross abnormalities, and thyroid hormone levels]) and provided sufficient information to
identify toxicologically relevant health effects and associated dose-response relationships. The lack of
teratogenic studies examining potential effects in utero and multigenerational reproductive studies
represent a significant limitation in the database for/?-isopropyltoluene.
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 /?-isopropyltolucnc in humans.
UFl
1
A UFl is applied because the POD is a BMDL.
UFS
1
A UFS of 1 is applied because the POD was derived from a study of suitable duration (35-63 days)
for a subchronic value.
UFC
100
Composite UF = UFA x UFD x UFH x UFL x UFS.
BMDL = benchmark dose lower confidence limit; FOB = functional observational battery; GLP = Good
Laboratory Practice; HED = human equivalent dose; LOAEL = lowest-observed-adverse-effect level;
NOAEL = no-observed-adverse-effect level; OECD = Organisation of Economic Co-operation and Development;
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.
Derivation of Screening Chronic Provisional Reference Dose
No chronic studies are available for p-isopropyltoluene via any exposure route.
Therefore, the POD (BMDLisd [HED] of 3.6 mg/kg-day) used for the derivation of the screening
subchronic p-RfD based on increased ALP in Po female rats exposed for -63 days (ECHA.
2019b; Symrise, 2018) is also selected for the derivation of the chronic p-RfD. The rationale for
the selection of this POD is provided in the section above (Derivation of a Screening Subchronic
Provisional Reference Dose). The screening chronic p-RfD is derived by applying a UFc of
1,000 to the selected POD of 3.6 mg/kg-day. The UFc of 1,000 was derived by applying a UFa
of 3, a UFd of 3, a UFh of 10, and a subchronic-to-chronic uncertainty factor (UFs) of 10.
Screening Chronic p-RfD = POD (HED) UFc
= 3.6 mg/kg-day ^ 1,000
= 4 x 10"3 mg/kg-day
Table A-4 summarizes the uncertainty factors for the screening subchronic p-RfD for
p-i sopropy ltoluene.
46
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Table A-4. Uncertainty Factors for the Screening Chronic p-RfD for
/7-Isopropyltoluene (CASRN 99-87-6)
UF
Value
Justification
UFa
3
A UFa of 3 (10"5) is applied to account for uncertainty associated with extrapolating from animals to
humans when cross-species dosimetric adjustment (HED calculation) is performed.
UFd
3
A UFd of 3 is applied to account for deficiencies and uncertainties in the database. The oral database
of relevant studies for/j-isopropyltolucne includes a non-peer-reviewed, repeated-dose systemic
toxicity studv with a reoroductive/develomnental toxicity screening test (ECHA. 2019b: Svmrise.
2018). The studv had data reoortine limitations but adhered to GLP and OECD test guidelines,
evaluated a wide range of relevant toxicity endpoints (including body weight, food consumption,
clinical observations, FOB, hematology, serum chemistry, organ weights, histopathology, and
reproductive and developmental endpoints [mating and fertility indices, reproductive parameters,
sperm evaluations, estrous cycle, histopathology of reproductive organs, and offspring viability, body
weight, gross abnormalities and thyroid hormone levels]) and provided sufficient information to
identify toxicologically relevant health effects and associated dose-response relationships. The lack of
teratogenic studies examining potential effects in utero and multigenerational reproductive studies
represent a significant limitation in the database for/j-isopropyltolucne. Additionally, the lack of
chronic studies, which is accounted by applying a UFS of 10, represents a limitation in the database
for /j-isopropyltolucne.
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 /?-isopropyltolucnc in humans.
UFl
1
A UFl is applied because the POD is a BMDL.
UFS
10
A UFS of 10 is applied because the POD was derived from a study of subchronic duration
(35-63 days) for a chronic value.
UFC
1,000
Composite UF = UFA x UFD x UFH x UFL x UFS.
BMDL = benchmark dose lower confidence limit; FOB = functional observational battery; GLP = Good
Laboratory Practice; HED = human equivalent dose; LOAEL = lowest-observed-adverse-effect level;
NOAEL = no-observed-adverse-effect level; OECD = Organisation of Economic Co-operation and Development;
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.
INHALATION NONCANCER TOXICITY VALUES
As discussed in the main body of the report, no repeated-dose inhalation studies adequate
for quantitative dose-response analysis are available forp-isopropyltoluene. Instead, an
alternative analogue approach was taken to support the derivation of inhalation noncancer
toxicity values.
APPLICATION OF AN ALTERNATIVE ANALOGUE APPROACH (METHODS)
The analogue approach allows for the use of data from related compounds to calculate
screening values when data for the target chemical are limited or unavailable. Details regarding
searches and methods for analogue analysis are adapted from Wang et al. (2012) and Lizarraga et
al. (2023) and chemical-specific parameters of read-across tools can be found in Appendix D.
Candidate analogues are identified on the basis of three similarity categories (structure,
toxicokinetics [metabolism], and toxicodynamics [toxicity and mode of action; MO A]) to
facilitate the final source analogue selection. The analogue approach may or may not be route-
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specific or applicable to multiple routes of exposure. All information is considered together as
part of the final weight-of-evidence (WOE) approach to select the most suitable source analogue.
In this assessment, an expanded analogue identification approach was utilized to collect
an augmented set of candidate analogues for the target chemical. As described below, this
approach applies a variety of tools and methods for identifying candidate analogues that are
similar to the target chemical based on structural features; metabolic relationships; or related
toxic effects and mechanisms of action. The application of a variety of different tools and
methods to identify candidate analogues minimizes the impact of limitations of any individual
tool or method on the pool of chemicals included, chemical fragments considered, and methods
for assessing similarity. Further, the inclusion of techniques to identify analogues based on
metabolism and toxicity or bioactivity expands the pool of candidates beyond those based
exclusively on structural similarity. The specific tools described below used for the expanded
analogue searches were selected because they are publicly available, supported by U.S. and
OECD agencies, updated regularly, and widely used.
To identify structurally-related compounds, an initial pool of analogues is identified using
automated tools, including ChemlDplus6 (NLM. 2022a). the CompTox Chemicals Dashboard7
(U.S. EPA 2022a). and the OECD Quantitative Structure-Activity Relationship (QSAR)
Toolbox8 (NLM. 2022a). Additional analogues identified as ChemlDplus-related substances,
mixtures, and CompTox "related substances"9 are also considered. CompTox General
Read-Across (GenRA)10 analogues are collected using the methods deployed on the publicly
available GenRA Beta version, which may include Morgan fingerprints, Torsion fingerprints,
ToxPrints and the use of ToxCast, Tox21, and ToxRef data (Patlewicz and Shah. 2023). For
compounds that have very few analogues identified by structural similarity using a similarity
threshold of 0.8 or 80%, substructure searches may be performed in the QSAR Toolbox, or
similarity searches may be rerun using a reduced similarity threshold (e.g., <80%). Structural
6ChemIDplus is a free, web search system that provides access to the structure and nomenclature authority files used
for the identification of chemical substances cited in National Library of Medicine (NLM) databases, including the
TOXNET system. The database contains over 350,000 chemical records, of which over 80,000 include chemical
structures and allows users to draw a chemical structure to search for similar substances using PubChem
Substructure fingerprints (NLM. 2009: Liwanag et al.. 2000). NLM retired ChemlDplus in December 2022.
7The U.S. EPA's CompTox Chemicals Dashboard provides publicly accessible chemistry, toxicity, and exposure
information for over one million chemicals (Williams et al„ 2017). Using EPAM's Bingo fingerprints, the "Similar
Compounds" tab provides a list of chemicals that are similar in structure to the selected chemical, based on the
Tanimoto similarity search metric with a minimum similarity factor threshold of 0.8 (EPAM. 2024).
8The OECD QSAR Toolbox is a software application intended to be used by government, industry and other
stakeholders to fill gaps in data needed for assessing the hazards of chemicals. The application allows users to search
for analogues based on structure similarity criteria and input similarity thresholds (OECD. 2017). It also contains
metabolism simulators which are simplified versions of the simulators in CATALOGIC and TIMES and consist of
hierarchically ordered molecular transformations (Yordanova et al.. 2019).
9The CompTox Chemicals Dashboard "Related Substances" tab provides a chemical list of all chemicals related to
the queried chemical through mapped relationships underlying the database. Relationships include searched
chemical (self-relationship), salt form, monomer, polymer, predecessor component, component, Markush parent,
Markush child, transformation parent, and transformation product (Williams et al.. 2021).
luOperationalized within the CompTox Chemicals Dashboard, GenRA is an algorithmic approach that makes read-
across predictions on the basis of a similarity weighted activity of source analogues (nearest neighbors). GenRA
gives users the ability to identify candidate analogues based on structural and bioactivity information (U.S. EPA.
2022c).
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analogues are clustered using the Chemical Assessment Clustering Engine (ChemACE)11 (U.S.
EPA 2011b) based on chemical fragments to support expert-driven refinement of the candidate
pool. The ChemACE output is reviewed by an experienced chemist, who narrows the list of
structural analogues based on expert judgment of multiple lines of evidence including known or
expected structure-activity relationships, reactivity, and known or expected metabolic pathways.
Initially, candidate analogues are screened for structural and chemical similarity to confirm that
the analogues have the same reactive functional groups and similar overall size and structural
features as the target chemical. Chemicals lacking key functionality or bearing additional
functionality relative to the target are less desirable as analogues and are not selected as
structural analogues. The selection may be expanded to include chemicals expected to be part of
a metabolic series (either as metabolic precursors or as metabolites) of the target chemical.
Chemicals that produce metabolites in common with the target may also be selected if the
metabolite is known or suspected to be part of the mechanism of action. All candidate analogues
are then screened for structural features that can influence their activity relative to the target.
Examples of such features include steric influences of bulky substituent groups, branching,
rigidity, presence of blocking groups on a functional group, and differing substitution patterns on
aromatic rings. Finally, key physical and chemical properties of the candidate analogues are
compared with the target to confirm that they can be expected to have similar bioavailability,
similar transport, and similar abiotic transformation properties.
Toxicokinetic studies tagged as potentially relevant supplemental material during
screening are used to identify metabolic analogues (metabolites and metabolic precursors).
Metabolites are also identified from two OECD QSAR Toolbox metabolism simulators (in vivo
rat metabolism simulator and rat liver S9 metabolism simulator). Targeted PubMed searches are
conducted to identify metabolic precursors and other compounds that share any of the observed
or predicted metabolites identified for the target chemical.
In vivo toxicity data for the target chemical (if available) are evaluated to determine
whether characteristic effects associated with a particular mechanism of toxicity are observed
(e.g., cholinesterase inhibition, inhibition of oxidative phosphorylation). In addition, in vitro
mechanistic data tagged as potentially relevant supplemental material during screening or
obtained from tools including GenRA, ToxCast/Tox2112, and Comparative Toxicogenomics
Database (CTD)13 (CTD. 2022) are also evaluated for this purpose. ToxCast/Tox21 data
available from the CompTox Chemicals Dashboard are collected for the target chemical to
determine bioactivity in in vitro assays that may indicate potential mechanism(s) of action. The
GenRA tool is used to search for analogues using Morgan, Torsion and ToxPrints fingerprint
"ChemACE clusters chemicals into groups based on structural features and a reasonable presumption that toxicity
may be influenced by such structural characteristics (e.g., structural alerts, toxicophores). ChemACE identifies
structural diversity in a large chemical inventory and highlights analogous clusters for potential read across. In the
expanded analogue approach, clustering with ChemACE supports expert refinement of the candidate analogue pool.
The ChemACE methodology is based on logic implemented in the Analog Identification Methodology (AIM) tool
(http://aim.epa. gov) that identifies analogues based on the presence of common fragments using a tiered approach
(U.S. EPA 2011a).
12ToxCast and Tox21 are publicly available databases containing high-throughput assay endpoints covering a range
of high-level cell responses (Thomas et al„ 2018: U.S. EPA. 2018b).
13The CTD is a publicly available database that provides manually curated information about chemical-gene/protein
interactions, chemical-disease and gene-disease relationships. The CTD allows users to identify chemicals that
induce gene interactions similar to those induced by the target chemical (Davis et al„ 2021).
49
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similarities and activity in ToxCast/Tox21 in vitro assays or ToxRef data (10 analogues collected
from each neighbors data set). Using the ToxCast/Tox21 bioactivity data, nearest neighbors
identified may be considered potential candidate analogues. The CTD is searched to identify
compounds with gene interactions similar to those induced by the target chemical; compounds
with gene interactions similar to the target chemical (similarity index >0.5) may be considered
potential candidate analogues.
Candidate analogues identified on the basis of the structural, metabolic, and
toxicodynamic similarity contexts are interrogated through the CompTox Chemicals Dashboard,
where QSAR-ready simplified molecular-input line-entry system (SMILES) are collected and
toxicity value availability is determined (e.g., from the Agency for Toxic Substances and Disease
Registry [ATSDR], California Environmental Protection Agency [CalEPA] Office of
Environmental Health Hazard Assessment [OEHHA), the U.S. EPA's Integrated Risk
Information System [IRIS], PPRTVs). Analogues that have subchronic or chronic toxicity data
or toxicity values available from other public health agencies are flagged for potential
consideration as supportive evidence.
Analogue Search Results for />-Isopropyltoluene (Inhalation Exposure)
As mentioned above, candidate analogues for p-isopropyltoluene for inhalation exposure
were identified based on structural, metabolic, and toxicity/mechanisms/MOA relationships. For
candidates identified through these approaches, the U.S. EPA (IRIS and PPRTV), ATSDR, and
CalEPA sources were searched for subchronic, intermediate, and chronic inhalation toxicity
values. Details are provided below.
Identification of Structural Analogues with Established Toxicity Values
/Msopropyltoluene is not a member of an existing OECD or New Chemical category.
Candidate structural analogues for p-isopropyltoluene were identified using similarity searches in
the OECD Toolbox (OECD. 2022). the U.S. EPA CompTox Chemicals Dashboard (U.S. EPA.
2022a). and ChemlDplus tools (NLM. 2022a). A total of 547 unique structural analogues were
identified for/?-isopropyltoluene in the Dashboard, OECD QSAR Toolbox, and ChemlDplus
(80% similarity threshold).
The list of potential analogues was reviewed by a chemist with expertise in read-across.
Based on the structural features expected to influence toxicokinetics and/or toxicity, the criteria
for including candidate analogues were as follows:
1. One aromatic ring with two or fewer substituents.
2. Preferred substituents on the ring are one methyl group and one isopropyl group;
however, .sec-butyl groups are also acceptable since they have active benzylic positions
analogous to isopropyl groups.
a. The carbon atoms at the benzylic positions are either primary (methyl) or tertiary
(isopropyl or .sec-butyl) because radicals can form at these positions. Analogues that
can form similar radicals at a benzylic position were included, since radical formation
may be part of the biological activity of p-isopropyltoluene.
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b. The benzylic positions are sites for metabolic transformations, and the substitution
patterns at the benzylic position can influence the metabolites formed.
3. In order to select analogues with similar bioavailability, preferred candidate chemicals
will have a log Kow (octanol-water partition coefficient) within 1 log unit of the target
chemical.
4. Toluene was excluded due to expected differences in its metabolic pathways relative to
p-i sopropy ltoluene.
a. p-Isopropyltoluene is primarily metabolized via side-chain oxidation pathways in
laboratory animals. Ring-oxidation products are not formed in large quantities (Boyle
et al.. 1999; Matsumoto et al.. 1992; Walde et al.. 1983; Ishida et al.. 1981; Bakke and
Scheline. 1970).
b. Toluene is metabolized via ring oxidation pathways in addition to oxidation of the
methyl group. Its metabolites include more epoxides and phenols than have been
reported for p-i sopropy ltoluene (ATSDR. 2017).
5. Ethylbenzene and ethyl-substituted benzenes were excluded due to expected differences
in their metabolic pathways relative top-isopropyltoluene.
a. p-Isopropyltoluene is primarily metabolized via side-chain oxidation pathways.
Ring-oxidation products are not formed in large quantities (Boyle et al.. 1999;
Matsumoto et al.. 1992; Walde et al.. 1983; Ishida et al.. 1981; Bakke and Scheline.
1970).
b. Ethylbenzene is metabolized via ring oxidation pathways in addition to oxidation of
the ethyl group. Its metabolites include more epoxides and phenols than have been
reported forp-isopropyltoluene (ATSDR. 2010).
c. Oxidation of the ethyl group occurs at the benzylic position first, yielding
acetophenone (ATSDR. 2010). This transformation is not possible for
p-isopropyltoluene.
6. Xylene isomers were not reported in the similarity tool outputs (OECD QSAR Toolbox,
Dashboard, or ChemlDplus) but were included based on shared structural features,
including a single aromatic ring with up to two methyl or isopropyl substituents.
a. Metabolism of xylenes occurs primarily through oxidation of the methyl
substituents (ATSDR. 2007). which is similar to p-i sopropy ltoluene.
Using these criteria, a total of 10 candidate structural analogues for p-i sopropy ltoluene
were identified, as shown in Table A-5. Two of the identified structural analogues,
isopropylbenzene and xylenes (mixed isomers), had available inhalation toxicity values from the
searched databases.
51
p-Isopropyltoluene
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EPA 690 R-24 003F
Table A-5. Candidate Structural Analogues Identified for
/7-Isopropyltoluene Based on Tools and Expert Judgment
H3C / V
j-Q-~
Tool (method)
Analogue (CASRNs) Selected
for Toxicity Value Searches
Structure
Dashboard (Tanimoto), OECD
Toolbox (method not described)3
w-Isopropyltoluene (CASRN 535-77-3)
a.
Dashboard (Tanimoto)3
o-Isopropyltoluene (CASRN 527-84-4)
a.
Benzene, l,2-bis(l-methylethyl)- (CASRN 577-55-9)
H3C\ /CH3
ch3
h3c
l-(Butan-2-yl)-3-methylbenzene
(CASRN 1772-10-7) '
CH3
h3c
1 -(Butan-2-yl)-4-methylbenzene
(CASRN 1595-16-0) '
CH,
9
chj
sec-Butylbenzene (CASRN 135-98-8)
»3cr
Isopropylbenzene (cumene) (CASRN 98-82-8)
nsc /=\
r\)
h3c \ '
Expert judgment13
Xylene, mixed isomers (CASRN 1330-20-7)
ch3 ch3
u ex.
V
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EPA 690 R-24 003F
Identification of Toxicokinetic Precursors or Metabolites with Established Toxicity
Values
/>-Isopropyltoluene metabolism has been studied in rats, rabbits, guinea pigs, and
marsupials [possum and greater glider]) (Boyle et al.. 1999; Matsumoto et al.. 1992; Walde et al..
1983; Ishida et al.. 1981; Bakke and Scheline. 1970). These studies demonstrated that
p-isopropyl toluene undergoes extensive oxidation of the methyl substituent and isopropyl
side-chain to yield polar oxygenated metabolites (see Section 2.3.4 for more details). The
primary metabolites include monohydric alcohols, diols, mono- and dicarboxylic acids, and
hydroxy acids. These metabolites are either excreted unchanged in the urine or undergo
conjugation with glucuronic acid and/or glycine, followed by excretion in the urine.
Ring-hydroxylation was not observed in in vivo animal studies (Boyle et al.. 1999; Matsumoto et
al.. 1992; Walde et al.. 1983; Ishida et al.. 1981; Bakke and Scheline. 1970). with the single
exception of trace amounts of 5-isopropyl-2-methylphenol (carvacrol) found in the urine of
guinea pigs (Walde et al.. 1983). An in vitro study using human recombinant cytochrome P450
(CYP) enzymes identified 2-isopropyl-5-methylphenol (thymol) as a major metabolite (Meesters
et al.. 2009). Phenolic metabolites were not observed in an in vitro study using liver microsomes
obtained from rats or marsupials (possum and koala) (Pass et al.. 2002). Southwell et al. (1980)
reportedp-cresol as a metabolite ofp-isopropyltoluene in bushtail possums; however, this is a
poorly reported study with significant limitations. Dealkylation of the isopropyl group to form
p-cresol is unlikely, and this metabolite is not reported in any other study. In addition,
endogenous /?-cresol is produced via digestion of tyrosine (from food proteins) in the intestine.
Free/?-cresol formed in this way is absorbed from the intestine and eliminated in the urine as
conjugates (IPCS. 1995). Data were not provided for control animals; therefore, the source of
urinary p-cresol could not be conclusively attributed top-isopropyltoluene administration.
/?-Cresol was not included as a candidate metabolic analogue of /?-isopropyltoluene.
Predicted metabolites were collected from the OECD QSAR Toolbox (OECD. 2022).
Predicted metabolites of/Msopropyltoluene collected from the OECD QSAR Toolbox also
consist of monohydric alcohols, diols, mono- and dicarboxylic acids, and hydroxy acid
metabolites. All of the predicted metabolites were also identified as observed metabolites
except for/?-isopropylbenzaldehyde (CASRN 122-03-2); 2-p-tolylpropionaldehyde
(CASRN 99-72-9); 4-(l -hydroxy-l-methyl-ethyl)benzaldehyde (CASRN 81036-81-9);
2-[4-(hydroxymethyl)phenyl]propanal, 4-(l-hydroxy-2-propanyl)benzaldehyde
(CASRN 1512868-96-0); and 2-[4-(hydroxymethyl)phenyl]-l,2-propanediol
(CASRN 1822818-61-0).
PubMed searches (searching "/^-isopropyl toluene" or "99-87-6" and "metabolite") were
conducted to identify metabolic precursors top-isopropyltoluene. No metabolic precursors were
identified. PubMed was also searched to identify other compounds that are metabolized to any of
the observed or predicted metabolites of p-isopropyltoluene (searching the metabolite name or
[CASRN if available] and "metabolite"). No compounds that share at least one metabolite with
/?-isopropyltoluene were identified in these searches.
Table A-6 summarizes the 22 candidate metabolic analogues for p-isopropyltoluene
(17 observed metabolites and 5 additional predicted metabolites). Searches for relevant toxicity
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values for the observed or predicted candidate metabolic analogues of p-isopropyltoluene did not
identify candidate analogues with inhalation toxicity values.
Table A-6. Candidate Metabolic Analogues of/>-Isopropyltoluene
Relationship to
/j-Isopropyltoluene
Compound (CASRN)
Structure
Metabolic precursor
None identified
Not applicable
Metabolite
5-Isopropyl-2-methylphenol (carvacrol)
(CASRN 499-75-2)a'b
HO
N ' CH$
Hydroxycarvacrol (location of second hydroxyl group
undefined)abc
Not provided
2-(/?-Tolyl)propanoic acid (CASRN 938-94-3 J"1,
a
t
ch3
2-Isopropyl-5-methylphenol (thymol)
(CASRN 89-83-8)a'b
CHj
2-(p-1 oly 1)-1 -propanol (CASRN 4371-50-0)"1,
a i.
2-/j-Carbo.\vphcn\i-1 -propanol (CASRN 88416-61-9) '1,
OH
2-p-Carboxyphenylpropionic acid
(CASRN 67381-50-4)ab
H3C / \ 0
0
/?-Isopropylbenzoic acid (cumic acid)
(CASRN 536-66-3)ab
H^C CM,
T
H0*^^*0
2-/?-tolylpropan-2-ol (CASRN 1197-01-9)"h
/ ^
OH
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Table A-6. Candidate Metabolic Analogues of/>-Isopropyltoluene
Relationship to
/j-Isopropyltoluene
Compound (CASRN)
Structure
2-/?-(Hydroxymethyl)phenyl-2-propanol
(CASRN 88416-58-4)ab
HO /
-\ CHi
/—f'Hj
J (X
/?-Isopropylbenzyl alcohol (CASRN 536-60-7)"h
II,C ,—
\_J
H,C
—V Oil
2-/j-(Hvdro.\Ymcth\i)phcn\ipropanoic acid
(CASRN 88416-60-8; 1630333-50-4, 1630342-91-4)ab
HO
L/..
CH3
2-(/?-Tolyl)-2-propanol (CASRN 3609-50-5)"h
H,C
ks
HO
H
CH,
J)
^*o
N-(4-Isopropylbenzoyl)glycine (CASRN 88416-62-0)"h
.....
Glucuronides of 2-(/?-tolyl)-l-propanol;
4-(l-carboxyethyl)benzoic acid; /?-isopropylbenzoic
acid, 2-p-carboxyphenylpropionic acid; and
2-(/Mohl)-2-propanol'll,c
Not Provided
2-/?-(Hydroxymethyl)phenyl-2-propanol
(CASRN 88416-59-5)ab
r
2-(/?-Tolyl)-l,2-propanediol (CASRN 88416-64-2) '1'
c
?
/Msopropylbcnzaldchvdc (CASRN 122-03-2)b
HjC
J
2-/?-Tolylpropionaldehyde (CASRN 99-72-9)b
X
jX,-
4-(l-Hydroxy-l-methyl-ethyl)benzaldehyde
(CASRN 81036-81-9)b
0
ll H3°
OH
55
/Msopropyltoluene
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Table A-6. Candidate Metabolic Analogues of/>-Isopropyltoluene
Relationship to
/j-Isopropyltoluene
Compound (CASRN)
Structure
4-(l-hydroxy-2-propanyl)benzaldehyde
(CASRN 1512868-96-0)b
CH3
fO^"
2-[4-(Hydroxymethyl)phenyl] -1,2-propanediol
(CASRN 1822818-6 l-0)b
sC"
OH
Share common
metabolite(s)
None identified
Not applicable
"¦Observed metabolites reported in the scientific literature.
Predicted metabolites from OECD QSAR Toolbox metabolism simulators (OECD. 2022).
°CASRN not available for this metabolite; consequently, the chemical structure is not provided.
OECD = Organisation for Economic Co-operation and Development; QSAR = quantitative structure-activity
relationship.
Identification of Analogues on the Basis of Toxicity/Mechanistic/MOA Information
and Established Toxicity Values
The mechanistic and supplemental data forp-isopropyltoluene (summarized in
Section 2.3), described in the main document above, do not suggest any characteristic effects
associated with a particular MOA (e.g., cholinesterase inhibition, inhibition of oxidative
phosphorylation) that could be used to identify candidate analogues.
/?-Isopropyltoluene was only active in 9 out of 845 ToxCast assays reported in the
Dashboard (invitro version 3.3) (U.S. EPA. 2020a). There were no PubChem assays in which
/?-isopropyltoluene was active (0 out of 479 assays) (U.S. EPA 2022a. 2020b). The GenRA
option within the Dashboard enables a search for analogues based on similarities in activity in
ToxCast in vitro assays. Using the ToxCast bioactivity data, none of the nearest neighbors
identified by GenRA had a similarity index >0.5. No candidate analogues were identified from
bioactivity data on the basis of toxicodynamic similarity (U.S. EPA. 2022b).
The CTD (2022) identified several compounds with gene interactions similar to
interactions induced by /?-isopropyltoluene. In the CTD, similarity is measured by the Jaccard
index, calculated as the size of the intersection of interacting genes for chemical A and
chemical B divided by the size of the union of those genes (range 0 [no similarity] to 1 [complete
similarity]). Among the compounds with gene interactions similar top-isopropyltoluene,
similarity indices ranged from 0.4 to 0.5. There were several compounds with a similarity index
of 0.5 (2-(4'-chlorophenyl)benzothiazole, 2-mercaptomethylbenzimidazole, 2-methylanthracene,
4'-chloroflavone, 4'-iodoflavone, acetyleugenol, AG 494, dibenzo(q/)anthracene, M50354, and
mineral waters). Although the similarity indices for these compounds were 0.5, the similarities
were based on only two gene interactions, so these compounds were not considered candidate
56
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analogues. No candidate mechanistic analogues for/>isopropyltoluene were identified using the
methods outlined above.
Candidate Analogues Moving Forwardfor Evaluation
Searches for metabolic, structural, and toxicity/mechanistic analogues for
/>isopropyltoluene yielded a total of 32 unique candidate analogues: 10 structural analogues and
22 metabolites. Of the candidates, two structural analogues have inhalation toxicity values
(isopropylbenzene and xylene, mixed isomers); these analogues were further evaluated on basis
of structural, physicochemical properties, toxicokinetic, and toxicodynamic similarity
comparisons.
Structural Analogues
Table A-7 summarizes available structural and physicochemical properties data for
/>isopropyltoluene and the structurally similar compounds (isopropylbenzene and xylene, mixed
isomers) identified as candidate analogues. The target compound and candidate analogues share
a similar general structure that consists of a single benzene ring containing one or two methyl
and/or isopropyl substituents. Specifically, />-isopropyltoluene contains methyl and isopropyl
groups in para- positions on the benzene ring. Isopropylbenzene contains a single isopropyl
substituent, while xylene (mixed isomers) contains two methyl substituents in the ortho-, meta-,
and/or para- positions. All compounds are liquids, with melting points <25°C. Measured vapor
pressures indicate that these chemicals will exist mostly in the vapor (gas) phase in the
atmosphere. />-Isopropyltoluene and the candidate analogues are expected to volatilize from
water to air and soil to air, based on their Henry's law constants and vapor pressures,
respectively. />-Isopropyltoluene, isopropylbenzene, and xylene are slightly to moderately soluble
in water and have similar measured log Kow values. The target compound and candidate
analogues are expected to be bioavailable by the oral and inhalation routes (based on vapor
pressure, water solubility, and log Kow values). Based on the available data, both candidate
analogues appear to be suitable analogues for />isopropyltoluene on the basis of structural and
physicochemical properties.
57
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Table A-7. Physicochemical Properties of />-Isopropyltoluene
(CASRN 99-87-6) and its Candidate Structural Analogues3
Property
Target Chemical
/j-Isopropyltoluene"
Candidate Analogues
Isopropylbenzeneb
Xylene (mixed isomers)
Structure
h,c
H,c
H,C
H,C
CASRN
99-87-6
98-82-8
Mixture: 1330-20-7°
w;-Xylene: 108-38-3d
o-Xylene: 95-47-6e
^-Xylene: 106-42-3f
Molecular weight
(g/mol)
134.222
120.195
106.168
Melting point (°C)
-68.2
-96.0
Mixture: no data
«/-Xylene: -47.5
o-Xylene: -28.3
/^-Xylene: 12.8
Boiling point (°C)
177
152
Mixture: 137-1408
m -Xylene: 139
o-Xylene: 143
/^-Xylene: 138
Vapor pressure (mm
Hg at 25°C)
0.772
4.50
Mixture: 6-16g
w;-Xylene: 8.29
o-Xylene: 6.61
/^-Xylene: 8.84
Henry's law constant
(atm-m3/mole at 25°C)
7.94 x 10 3 (predicted)
1.15 x 10~2
Mixture: no data
«/-Xylene: 7.18 x 10 3
o-Xylene: 5.18 x 10~3
/^-Xylene: 6.90 x 10~3
Solubility in water
(mg/L at 25°C)
23.2 (reported as
1.73 x 10~4 mol/L)
63.0 (reported as
5.24 x 10-4 mol/L)
Mixture: 160 (reported as
1.51 x 10"3 mol/L)
«/-Xylene: 160 (reported as
1.51 x 10"3 mol/L)
o-Xylene: 175 (reported as
1.65 x 10 3 mol/L)
/j-Xvlcne: 171 (reported as
1.61 x 10 3 mol/L)
58
/Msopropyltoluene
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Table A-7. Physicochemical Properties of />-Isopropyltoluene
(CASRN 99-87-6) and its Candidate Structural Analogues3
Property
Target Chemical
Candidate Analogues
/j-Isopropyltoluene"
Isopropylbenzeneb
Xylene (mixed isomers)
Octanol-water partition
coefficient (log Kow)
4.10
3.66
Mixture: 3.12-3.20g
«/-Xylene: 3.20
o-Xylene: 3.12
/^-Xylene: 3.15
aUnless otherwise noted, average values for/j-isopropyltolucne were extracted from the U.S. EPA CompTox
Chemicals Dashboard (https://comptox.epa.gov/dashboard/chemical/details/DTXSID3026645. Accessed May 21,
2024); U.S. EPA (2024a). Values are experimental unless otherwise specified.
bUnless otherwise noted, average values for isopropylbenzene were extracted from the U.S. EPA CompTox
Chemicals Dashboard (https://comptox.epa.gov/dashboard/chemical/details/DTXSID1021827. Accessed May 29,
2024); U.S. EPA (2024a). Values are experimental unless otherwise specified.
°Unless otherwise noted, average values for mixed xylenes were extracted from the U.S. EPA CompTox Chemicals
Dashboard (https://comptox.epa.gov/dashboard/chemical/details/DTXSID2021446. Accessed May 29, 2024); U.S.
EPA (2024a). Values are experimental unless otherwise specified.
dUnless otherwise noted, average values for w;-xylene were extracted from the U.S. EPA CompTox Chemicals
Dashboard (https://comptox.epa.gov/dashboard/chemical/properties/DTXSID6026298. Accessed May 29, 2024);
U.S. EPA (2024a). Values are experimental unless otherwise specified.
eUnless otherwise noted, average values for o-xylene were extracted from the U.S. EPA CompTox Chemicals
Dashboard (https://comptox.epa.gov/dashboard/chemical/properties/DTXSID3021807. Accessed May 29, 2024);
U.S. EPA (2024a). Values are experimental unless otherwise specified.
fUnless otherwise noted, average values for/?-xylene were extracted from the U.S. EPA CompTox Chemicals
Dashboard (https://comptox.epa.gov/dashboard/chemical/properties/DTXSID2021868 Accessed May 29, 2024);
U.S. EPA (2024a). Values are experimental unless otherwise specified.
gU.S. EPA (2003). Values are experimental unless otherwise specified.
Relevant structural alerts and toxicity predictions for noncancer health effects were
identified using computational tools from the OECD (2022) QSAR Toolbox profilers, ToxAlerts
OCHEM (2022) ToxAlerts, and IDEAconsult (2018) Toxtree. The model results for
/?-isopropyltoluene and its analogue compounds (isopropylbenzene and xylene, mixed isomers)
are shown in Figure A-l. Concerns for protein binding, developmental/reproductive toxicity, and
metabolism/reactivity were indicated for p-isopropyltoluene and its structural analogues.
59
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Structural Category
Compounds
Target
Chemical
Candidate
Analogues
Source
99-87-6
/7-Isopropyltoluene
98-82-8
Isopropylbenzene
1330-20-7
Xylene (mixed isomers)
Protein Binding
Protein binding (based on a Michael acceptor alert)
Toxtree
Protein binding (based on SN2-nucleophilic aliphatic substitution alert)
Toxtree
Hepatotoxicity and Renal Toxicity
Hepatotoxicity (based on mefenamic acid and acetaminophen alerts);
HESS model
OECD QSAR
Toolbox
Renal toxicity (based on styrene and toluene alerts); HESS model
OECD QSAR
Toolbox
Renal toxicity (based on acetaminophen alert); HESS model
OECD QSAR
Toolbox
Renal toxicity (based on propanolol alert); HESS model
OECD QSAR
Toolbox
Developmental/Reproductive Toxicity
Known precedent of reproductive and developmental toxic potential
(based on toluene and small alkyl benzene or toluene derivatives);
DART scheme
OECD QSAR
Toolbox
Metabolism/Reactivity
Cytochrome P450-mediated drug metabolism predicted (based on sp3
and sp2 hybridized carbon atoms)
ToxAlerts
~ Model results or structural alerts indicating concern for noncancer toxicity/endpoint of interest.
~ Model results or structural alert indicating no concern for noncancer toxicity/endpoint of interest.
aModels with results are presented in the heat map (models without results indicate that the queried chemical fell
outside of the applicability domain and are omitted).
DART = developmental and reproductive toxicity; HESS = Hazard Evaluation Support System;
OECD = Organisation for Economic Co-operation and Development; QSAR = quantitative structure-activity
relationship
Figure A-l. Structural Alerts for />-Isopropyltoluene and its Candidate Analogues
Toxtree indicated potential for protein binding based on being Michael acceptors for
p-isopropyl toluene and the candidate analogues. A potential for protein binding was also
indicated for p-isopropyltoluene and isopropylbenzene based on SN2-nucleophilic aliphatic
substitution; this was not indicated for xylene (mixed isomers).
60
/Msopropyltoluene
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The OECD QSAR Toolbox Hazard Evaluation Support System (HESS) model showed a
concern for hepatotoxicity for both analogues based on structural similarity to mefenamic acid
(allergic, acute, hepatocyte damage) and acetaminophen (oxidative stress, hepatocellular injury);
the HESS model did not show a hepatoxicity concern for the target chemical, /?-i sopropy ltoluene.
The OECD QSAR Toolbox HESS model showed a concern for renal toxicity for both
analogues based on structural similarity to styrene (epithelial necrosis of renal tubules) and
toluene (renal tubular acidosis, proteinurea). The HESS model also showed a concern for renal
toxicity for xylene based on structural similarity to acetaminophen (proximal tubular toxicity,
interstitial nephritis) and for isopropylbenzene based on structural similarity to propanolol
(nephrotoxicity not specified). No renal toxicity concerns were predicted for the target chemical,
p-i sopropy ltoluene.
The OECD QSAR Toolbox developmental and reproductive toxicity (DART) scheme
indicated a potential for developmental and/or reproductive toxicity for p-i sopropy ltoluene and
both analogues based on the benzene or toluene structure with an alkyl chain substituent of fewer
than five carbon atoms. The alert for reproductive and/or developmental toxicity for toluene and
small alkyl benzene or toluene derivatives is based on the training set of chemicals that includes
o-, m-, and ^-xylene, butyltoluene, and 4-/m-butyl toluene.
The ToxAlerts tool showed potential for CYP-mediated drug metabolism for
p-i sopropy ltoluene and both analogues based on the presence of sp3 and sp2 hybridized carbon
atoms.
In summary, /?-isopropyltoluene and its candidate analogues showed structural alert data
for protein binding, reproductive and developmental toxicity, and metabolism by CYP.
Isopropylbenzene and xylene also showed positive alerts for liver and kidney toxicity. While
these alerts were negative for p-i sopropy ltoluene, available toxicity data for p-i sopropy ltoluene
showed liver and kidney effects in experimental animals treated by oral exposure (Symrise.
2018) (see Section 2.2.1).
Metabolic Analogues
Table A-8 summarizes available toxicokinetic data for p-i sopropy ltoluene and the
structurally similar compounds identified as candidate analogues (isopropylbenzene and xylene,
mixed isomers).
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Table A-8. Comparison of ADME Data for />-Isopropyltoluene (CASRN 99-87-6) and its Candidate Analogues
Type of Data
/7-Isopropyltoluene
Isopropylbenzene
Xylene (mixed isomers)
Structure
H3C , V
H C N '
H,v^
h3c x '
CH, CH,
(J cX
CASRN
99-87-6
98-82-8
1330-20-7
Absorption
Rate and extent of
absorption
Laboratory animals (all routes):
• Excretion data suggest absorption in rats,
rabbits, guinea pigs, and marsupials by oral
route (Bovle et al.. 1999; Matsumoto et al..
1992: Walde et al.. 1983: Isliida et al.. 1981):
in rats and guinea pigs by inhalation route
(Walde et al.. 1983); and in mice bv dermal
route (Wemerre et al.. 1968).
• Urinary excretion data in rats and guinea pigs
suggest that absorption is rapid and extensive
by the oral and inhalation routes (60-80% of
administered or inhaled dose excreted within
48 h) (Walde et al.. 1983).
Humans (inhalation):
• Absorption was demonstrated in volunteer
studies; respiratory tract absorption ranged from
45 to 64% [Senczuk and Litewka (1976) as cited
in WHO (1999); and U.S. EPA (1997)1.
Laboratory animals (all routes):
• Rapid absorption by inhalation; blood
concentrations detectable within 5 min [Research
Trianele Institute (1989) as cited in WHO (1999);
and U.S. EPA (1997)1.
• Readily absorbed following gavage
administration, with maximum blood
concentrations at 4 h (earliest time point
measured) for the lower dose (33 mg/kg-d) and
8-16 h for the higher dose (1,350 mg/kg-d)
[Research Triangle Institute (1989) as cited in
WHO (1999); and U.S. EPA (1997)1.
• Dermal absorption was demonstrated in rats and
rabbits [Monsanto Co. (1984) as cited in WHO
(1999)1.
Humans (inhalation):
• Absorption was demonstrated in volunteer
studies and was similar for the m-. o-, and
p- isomers [Wallen et al. (1985); Riihimaki
and Savolainen (1980); David et al. (1979);
Astrad et al. (1978); Riihimaki and Pfaffli
(1978); Sedivec andFlek (1976a); Ogata et
al. (1970) as cited in ATSDR (2007); and
U.S. EPA (2003)1.
- Respiratory tract absorption ranged from
50 to 73%'
• Blood-air partition coefficients for the
three isomers ranged from 26.4 to 39,
suggesting that xylene (m-. o-, and
p- isomers) is readily transferred to blood
[Thrall et al. (2002); Pierce et al. (1996);
Sato and Nakajima (1979) as cited in
ATSDR (2007)1.
• Dermal absorption was demonstrated in
humans (w;-xylene); however, the extent of
absorption is lower than that resulting from
inhalation [Riihimaki (1979b); Riihimaki
and Pfaffli (1978); Engstrom et al. (1977) as
cited in ATSDR (2007)1.
Laboratory animals (all routes):
• Oral absorption is extensive: 87-92%
absorption of xylene (/?/-, o-, and p- isomers)
62
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Table A-8. Comparison of ADME Data for />-Isopropyltoluene (CASRN 99-87-6) and its Candidate Analogues
Type of Data
/7-Isopropyltoluene
Isopropylbenzene
Xylene (mixed isomers)
in rats via gavage [Bray et al. (1949) as cited
in ATSDR (2007)1.
• Oral absorption is rapid. Blood levels of
m-xylene peaked within 20 min after gavage
and the half-life absorption was faster in
female rats (tic = 0.31 h) compared to male
rats (ti/2 = 0.64 h) [Turkall et al. (1992) as
cited in ATSDR (2007); and U.S. EPA
(2003)1.
• Absorption via inhalation has not been
quantified but it can be inferred by recovery
of urine metabolites after inhalation of
xylene [Elovaara et al. (1987); Elovaara
(1982); Carlsson (1981); David et al. (1979);
Patel et al. (1978) as cited in ATSDR
(2007)1.
• Blood-air partition coefficients in rats for the
three isomers ranged from 37 to 46 [Thrall et
al. (2002); Kumarathasan et al. (1998);
Kaneko et al. (1991a); Gargas et al. (1989)
as cited in ATSDR (2007)1.
• Dermal absorption of w;-xylene was
demonstrated in rats [Morgan et al. (1991);
McDougal et al. (1990); Skowronski et al.
(1990) as cited in ATSDR (2007)1.
Distribution
Extent of
distribution
Laboratory animals (all routes):
• No data are available on blood or tissue
concentrations following exposure by any
route.
• Based on a log Kow value >4,
/?-isopropyltolucnc is hydrophobic and is
likely to partition to fat compartments.
Laboratory animals (all routes):
• Isopropylbenzene is widely distributed
throughout the body; found in endocrine organs
(not specified), the central nervous system (not
further specified), bone marrow, spleen and liver
TFabre et al. (1955) as cited in WHO (1999)1.
• Elevated tissue/blood ratios were detected in
adipose tissue, liver, and kidney in rats via oral.
Humans (inhalation):
• Accumulation of xylene isomers in human
adipose tissue ranged from 4 to 10% of the
absorbed dose [Astrand (1982); Riihimaki et
al. (1979b); Engstrom and Bjurstrom (1978)
as cited in ATSDR (2007); and U.S. EPA
(2003)1.
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Table A-8. Comparison of ADME Data for />-Isopropyltoluene (CASRN 99-87-6) and its Candidate Analogues
Type of Data
/j-Isopropyltoluene
Isopropylbenzene
Xylene (mixed isomers)
inhalation, and intravenous exposure routes
[Research Triangle Institute (1989) as cited in
WHO (1999); and U.S. EPA (1997)1.
• Following inlialation exposure in rats, the t'A for
elimination from blood was 3.9-6.6 h [Research
Trianele Institute (1989) as cited in WHO (1999);
and U.S. EPA (1997)1.
• Following gavage exposure in rats, the t'A for
elimination from blood was 9-16 h [Research
Trianele Institute (1989) as cited in WHO (1999);
and U.S. EPA (1997)1.
• Fat-air partition coefficients for xylene (in-.
o-, and p- isomers) ranged from 1,919 to
2,460 [Pierce et al. (1996) as cited in
ATSDR (2007)1.
• The milk-air partition coefficient (/?/-, o-, and
/^-xylenes) was 134 [Fisher et al. (1997) as
cited in ATSDR (2007)1.
• Elimination from the blood was biphasic,
with t!/2 values of 0.5-1 and 20-30 h
[Riihimaki and Savolainen (1980) as cited in
U.S. EPA (2003)1.
Laboratory animals (oral and inhalation):
• Inlialation studies in rats and mice showed
wide distribution of m- or ^-xylenes with
accumulation occurring primarily in adipose
tissue [Ito et al. (2002); Ghantous and
Danielsson (1986); Bergman (1983);
Carlsson (1981) as cited in ATSDR (2007);
and U.S. EPA (2003)1.
• Accumulation in fat tissues lias also been
demonstrated in oral rat studies with
«/-xylene [Turkall et al. (1992) as cited in
ATSDR (2007)1.
• Xylenes (p- and o- isomers) readily cross the
placenta and have been detected in amniotic
fluid and fetal tissue [Ghantous and
Danielsson 1986; Ungvary et al. 1980b as
cited in ATSDR (2007)1."
• Oil-blood partition coefficients for the
three isomers ranged from 98 to 146,
suggesting transfer to lipid-rich tissues [Sato
and Nakaiima (1979) as cited in U.S. EPA
(2003)1.
• Fat-air partition coefficient for xylenes (/?/-,
o-, and p- isomers) ranged from 1,748 to
64
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Table A-8. Comparison of ADME Data for />-Isopropyltoluene (CASRN 99-87-6) and its Candidate Analogues
Type of Data
/7-Isopropyltoluene
Isopropylbenzene
Xylene (mixed isomers)
2,930 [Kumarathasan et al. (1998); Pierce et
al. (1996); Kaneko et al. (1991a); Gargas et
al. (1989) as cited in ATSDR (2007)1.
• Tissue-blood partition coefficients were
1.5-3.7 for brain, muscle and kidney,
3.2-5.7 for liver, and 37-67 for adipose
tissue [Kumarathasan et al. (1998) as cited in
U.S. EPA (2003)1.
Metabolism
Rate;
primary
metabolites
Humans (in vitro):
• Human recombinant CYP enzymes identified
one phenolic metabolite
(2-isopropyl-5-methylphenol) in addition to
metabolites of side-chain oxidation
(/?-isopropyl benzylalcohol,
2-p-tolylpropan-2-ol, and /?-isopropylbcnzyl
aldehvde) (Meesters et al.. 2009).
Laboratory animals (all routes):
• Extensive oxidation of the methyl substituent
and isopropyl side chain in rats, rabbits,
euinea Diss, and marsupials (Bovlc et al..
1999; Matsumoto et al.. 1992; Walde et al..
1983; Isliida et al.. 1981; Bakke and Scheline.
1970).
Primary metabolites include monohydric
alcohols, diols, mono- and dicarboxylic
acids, and hydroxyacids.
Oxidative metabolites were conjugated
with glucuronic acid and/or glycine.
Ring-hydroxylation was not observed in
in vivo studies in rats, rabbits, or
marsupials.
Humans (inhalation):
• 2-Phenyl-2-propanol was measured in urine
(isopropyl side-chain oxidation) [Senczuk and
Litewka (1976) as cited in WHO (1999); and
U.S. EPA (1997)1.
Laboratory animals (all routes):
• Oxidized via CYP in liver and lung, with the
primary metabolite identified as
2-phenyl-2-propanol [Sato and Nakajima (1987)
as cited in WHO (1999); and U.S. EPA (1997)1.
• 2-Phenyl-l,2-propanediol and an unknown
metabolite were also detected in rats and rabbits;
dicarboxylic acid formation was suggested,
although not confirmed [MAK (1996); Isliida and
Matsumoto (1992); Research Triangle Institute
(1989) as cited in WHO (1999)1.
No phenolic metabolites were detected.
Oxidative metabolites were conjugated with
glucuronic acid and sulfate esters.
Humans (inhalation):
• The primary metabolic pathway of xylenes
(in-, o-, and p- isomers) involves methyl
group oxidation. Methylbenzyl alcohols are
converted to methylbenzoic acids, followed
by glycine conjugation to form
methylhippuric acids [Norstrom et al. (1989);
Ogata et al. (1979); Riihimaki et al. (1979a);
Astrand et al. (1978); Senczuk and Orlowski
(1978); Sedivec and Flek (1976b); Ogata et
al. (1970) as cited in ATSDR (2007); and
U.S. EPA (2003)1.
- Metabolism to phenols accounts for <2%
of total metabolites formed.
Laboratory animals (all routes)
• Similar metabolism as in humans, with
methylhippuric acid as the primary
metabolite [van Doom et al. (1980); Ogata et
al. (1979); Sugihara and Ogata (1978);
Bakke and Scheline (1970); Bray et al.
(1949) as cited in ATSDR (2007); and U.S.
EPA (2003)1.
- In a minor pathway, methylbenzoic acids
are conjugated with glucuronide or
sulfate.
65
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Table A-8. Comparison of ADME Data for />-Isopropyltoluene (CASRN 99-87-6) and its Candidate Analogues
Type of Data
/7-Isopropyltoluene
Isopropylbenzene
Xylene (mixed isomers)
• Trace amounts of
5-isopropyl-2-methylphenol (carvacrol) were
found in the urine of euinea tries (Walde et
al.. 1983).
Laboratory animals (in vitro):
• Eight metabolites were detected from
microsomes obtained from Wistar rats,
brushtail possums, and koalas; same sites of
oxidation as observed in in vivo studies in the
same species (Pass et al.. 2002).
- Ring oxidation to form phenols is
negligible (<1%).
Elimination
Elimination
half-time; route of
excretion
Laboratory animals (oral, inhalation):
• Metabolites are either excreted unchanged or
as glucuronide or glycine conjugates in the
urine of rats, rabbits, guinea pigs, and
marsupials (Bovlc et al.. 1999; Matsumoto et
al.. 1992; Walde et al.. 1983; Isliida et al..
1981; Bakke and Scheline. 1970).
No metabolites were identified in the
feces.
• In rats and guinea pigs, 60-80% of the
administered dose was excreted as
metabolites in the urine within 48 h of oral or
inhalation dosins (Walde et al.. 1983).
Humans (inhalation):
• 2-Phenyl-2-propanol excretion was maximal at
6-8 h after exposure and near complete at 40 h
after exposure [Senczuk and Litewka (1976) as
cited in (WHO. 1999; and U.S. EPA. 1997)1.
- Urinary t'A values show a rapid early phase
(t'A = 2 h) and a slower late phase (VA = 40 h).
Laboratory animals (all routes):
• Urine is the primary excretion route in rats after
oral, inhalation, and intravenous exposure (>70%
of administered dose was excreted in urine)
[Research Triangle Institute (1989) as cited in
(WHO. 1999; and U.S. EPA. 1997)1.
- Total body clearance in rats was rapid, with
<1% of the absorbed fraction remaining after
72 h.
- Metabolites are either excreted unchanged or
as glucuronide or sulfate conjugates in urine.
Humans (inhalation):
• 95% of absorbed xylene (mixed isomers) is
excreted in urine; 5% is excreted unchanged
in exhaled air [Pellizzari et al. (1992); Ogata
et al. (1979); Riihimaki et al. (1979b);
Astrand et al. (1978); Senczuk and Orlowski
(1978); Sedivec and Flek (1976b) as cited in
ATSDR (2007); and U.S. EPA (2003)1.
- Excretion is rapid with metabolites
detected within 2 h of exposure; 50-60%
excreted by 18 h after exposure.
• Urinary VA values show a rapid early phase
([A = 1 h) and a slower late phase
([A = 20 h) [Riihimaki and Savolainen
(1980) as cited in as cited in U.S. EPA
(2003)1.
Laboratory animals (oral, inhalation):
• Excreted primarily in the urine (-74-96% of
administered dose) over 48 h after oral
dosing with the remainder excreted
unchanged in expired air [Turkall et al.
(1992) as cited in ATSDR (2007)1.
66
/Msopropyltoluene
-------
EPA 690 R-24 003F
Table A-8. Comparison of ADME Data for />-Isopropyltoluene (CASRN 99-87-6) and its Candidate Analogues
Type of Data
/j-Isopropyltoluene
Isopropylbenzene
Xylene (mixed isomers)
- Cumulative amounts of methylhippuric
acid (primary excretion product) in urine
increased through 12 h and then reached a
plateau.
- Elimination in urine after oral dosing is
rapid, with 52-59% of the administered
dose excreted within the first 12 h.
• Following dermal administration, elimination
within 48 h occurred in expired air (-60%)
and urine (-40%) [Skowronski et al. (1990)
as cited in ATSDR (2007)1.
References:
Meesters et al. (2009); Pass et al. (2002); Bovle
et al. (1999); Matsumoto et al. (1992); Walde et
al. (1983); Isliida et al. (1981); Bakke and
Scheline (1970)
WHO (1999); U.S. EPA (1997)
ATSDR (2007); U.S. EPA (2003); David et al.
(1979)
ADME = absorption, distribution, metabolism, and excretion; CYP = cytochrome P450; Kow = octanol-water partition coefficient; t'A = half-life.
67
/Msopropyltoluene
-------
EPA 690 R-24 003F
Animal studies demonstrate absorption ofp-isopropyltoluene via oral, inhalation, and
dermal routes (see Table A-8). Absorption of p-isopropyltoluene in rats and guinea pigs was
rapid and extensive (60-80% of administered or inhaled dose within 48 hours) (Walde et al..
1983) following oral and inhalation exposure. Absorption data in humans and animals were
similar for the candidate analogues, showing that isopropylbenzene and xylene (m-, o-, and
p- isomers) are well absorbed by multiple routes (oral, inhalation, and dermal routes). For
example, respiratory tract absorption ranges were 45-64 and 50-73% for isopropylene and
xylene, respectively, in volunteer studies (see Table A-8).
No in vivo human or animal studies reporting the distribution ofp-isopropyltoluene were
identified; however, based on a log Kow value >4, it is expected to accumulate in fatty tissues.
For the candidate analogues, initial distribution is rapid and widespread throughout the body
(see Table A-8). Tissue-blood ratios were elevated in liver, kidney, and adipose tissue in rats
after oral, inhalation, and intravenous exposure to isopropylbenzene. For xylene, accumulation
occurs primarily in fat tissue in both humans and rats, and it has also been detected in amniotic
fluid and fetal tissues.
The primary metabolic pathway for p-isopropyltoluene in laboratory animals involves
extensive oxidation of the methyl substituent and isopropyl side chain (see Table A-8 and
Figure 2 in Section 2.3.4 for more details). Metabolites include monohydric alcohols, diols,
mono- and dicarboxylic acids, and hydroxy acids. Ring-hydroxylation was not observed in in
vivo studies of rats, rabbits, or marsupials. Trace amounts of one phenolic metabolite
(5-isopropyl-2-methylphenol) of p-isopropyltoluene were reported in guinea pigs. Oxidative
metabolites were conjugated with glucuronic acid and/or glycine. Similar to the target
compound, the candidate analogues are primarily metabolized by side-chain oxidation pathways
in animals (see Table A-8). The primary metabolite for isopropylbenzene was identified as
2-phenyl-2-propanol. No phenolic metabolites arising from ring oxidation were seen. Oxidative
metabolites were conjugated with glucuronic acid and sulfate esters. Xylene undergoes methyl
group oxidation. Methylbenzyl alcohols are converted to methylbenzoic acids, followed by
glycine conjugation to form methylhippuric acids. In a minor pathway, methylbenzoic acids are
conjugated with glucuronide or sulfate. Ring oxidation to form phenols is negligible (<1%).
Metabolism data in humans for/Msopropyltoluene is limited. A phenolic product
(2-isopropyl-5-methylphenol) was identified as a metabolite ofp-isopropyltoluene in in vitro
human recombinant CYP assays (see Table A-8); however, the evidence is insufficient to
determine whether ring hydroxylation constitutes an important metabolic pathway in humans.
For the analogues, isopropylbenzene and xylene, the available evidence in humans suggests that
ring hydroxylation is not a major pathway (see Table A-8).
/Msopropyltoluene metabolites are excreted unchanged or as glucuronide or glycine
conjugates in the urine of experimental animals (60-80% of the administered dose in rats and
guinea pigs within 48 hours; see Table A-8). No metabolites were identified in the feces. Urine is
also the primary excretion route for isopropylbenzene. Oxidative metabolites are excreted
unchanged or as glucuronide or sulfate conjugates in urine (>70% of administered oral,
inhalation, and intravenous dose within 72 hours; see Table A-8). Xylene are excreted primarily
as methylhippuric acid in the urine (-74-96% of administered oral dose within 48 hours), with
the remainder excreted unchanged in expired air (see Table A-8).
68
/Msopropyltoluene
-------
EPA/690/R-24/003F
In summary, toxicokinetic data suggest that the target compound and candidate analogues
are well absorbed via multiple routes (including inhalation exposure). No distribution data are
available for />isopropyltoluene; however, the log Kow value indicates hydrophobicity and
suggests that/>isopropyltoluene will partition to fat compartments. Both candidate analogues
were shown to be distributed widely throughout the body and to accumulate in adipose tissue.
Metabolic pathways are similar for the target compound and both candidate analogues in
laboratory animals, involving oxidation of side-chain substituents (i.e., methyl and/or isopropyl
substituents). Ring oxidation to form phenolic metabolites was negligible for all three
compounds. Oxidative metabolites were conjugated with glycine, glucuronic acid, and/or sulfate
and excreted in the urine. Fecal excretion was not reported for the target compound or the
candidate analogues. A small fraction of unchanged xylene was excreted in expired air following
inhalation. Based on the available toxicokinetic data, both candidate analogues appear to be
suitable analogues for /;-isopropyltoluene.
Toxicodynamic Analogues
As mentioned in Section 2.2, no adequate subchronic or chronic inhalation toxicity
studies are available for the target chemical, /?-isopropyltoluene. Inhalation toxicity values for the
candidate analogues are presented in Table A-9. The critical effects for the two candidate
analogue compounds include respiratory irritation (POD human equivalent concentration [HEC]
of 61 mg/m3) and neurological effects (impaired motor coordination, decreased pain sensitivity,
and floating sensation; POD (HEC) range of 39-217 mg/m3) for xylene (mixed isomers) and
increased kidney and adrenal weights (POD (HEC) of 435 mg/m3) for isopropylbenzene. A
discussion of available toxicity data for the target compound and analogues for these (and other)
relevant endpoints is provided following Table A-9.
69
/>Isopropyltoluene
-------
EPA 690 R-24 003F
Table A-9. Comparison of Available Inhalation Toxicity Values for />-Isopropyltoluene (CASRN 99-87-6) and its
Candidate Analogues
Chemical
/j-Isopropyltolucnc
Isopropylbenzene
Xylene (Mixed Isomers)
Structure
H3C / V
,K>"'
h3c n '
VQ
h3c N '
CHj CH3
cr ex -o-
CASRN
99-87-6
98-82-8
1330-20-7
Subchronic inhalation toxicity values
Source
U.S. EPA (2009)
ATSDR (2007)
POD
ND
ND
39 mg/m3 (9.0 ppm)a
50 ppm (217 mg/m3)
POD type
ND
ND
NOAELhec
LOAELhec
Subchronic UFC
ND
ND
100 (UFa, UFd, UFh)
90 (UFa, UFh, UFl)
Subchronic p-RfC (mg/m3)
ND
ND
0.4 mg/m3
0.6 ppm (2.6 mg/m3)
Critical effects
ND
ND
Neurological effects
(impaired motor coordination)
Decreased mean latency of
the paw-lick response
Species
ND
ND
Rat
Duration
ND
ND
3 mo (6 h/d, 5 d/wk)
Route (method)
ND
ND
Inhalation
Source
ND
ND
Korsak et al. (1994) as cited in U.S. EPA (2009); and
ATSDR (2007)
70
/Msopropyltoluene
-------
EPA 690 R-24 003F
Table A-9. Comparison of Available Inhalation Toxicity Values for />-Isopropyltoluene (CASRN 99-87-6) and its
Candidate Analogues
Chemical
/j-Isopropyltoluene
Isopropylbenzene
Xylene (Mixed Isomers)
Chronic inhalation toxicity values
POD
ND
435 mg/m3 (88.5 ppm)b
39 mg/m3 (9.0 ppm)a
14 ppm (61 mg/m3)
POD type
ND
NOAELhec
NOAELhec
LOAEL
Chronic UFC
ND
1,000 (UFa, UFd, UFh, UFs)
300 (UFa, UFd, UFh, UFs)
300 (UFh, UFd, UFl)
Chronic p-RfC/MRL
ND
0.4 mg/m3
0.1 mg/m3
0.05 ppm (0.2 mg/m3)
Critical effects
ND
Increased kidney weights and
adrenal weights
Neurological effects
(impaired motor coordination)
Respiratory and neurological
effects (nose and throat
irritation; floating sensation)
Species
ND
Rat
Rat
Human
Duration
ND
13 wk
3 mo (6 h/d, 5 d/wk)
7 yr
Route (method)
ND
Inhalation
Inhalation
Inhalation (occupational)
Source
ND
Cushman et al. (1995) as cited in
U.S. EPA (1997)
Korsak et al. (1994) as cited
in U.S. EPA (2003)
Ucliida et al. (1993) as cited
in ATSDR (2007)
Acute inhalation lethality data
Inhalation LCso (mg/m3)
19,500-24,000 (mouse, 2-4 h)
10,000-15,300 (mouse, 2-7 h);
35,000-39,000 (rat, 4 h)
17,000-23,000 (mouse, 6 h)
21,000-29,000 (rat, 4 h)
Toxicity at LCso
ND
Effects on liver, kidney (changes in
tubules and glomeruli), ureter,
bladder, and spleen
ND
Source
NLM (2022b)
NLM (2022c)
ATSDR (2007)
TOD has been adjusted for duration to continuous exposure. Unadjusted values reported as 50 ppm (217 mg/m3) according to U.S. EPA (2003).
bPOD has been adjusted for duration to continuous exposure. Unadjusted values reported as 496 ppm (2,438 mg/m3) according to U.S. EPA (1997).
HEC = human equivalent concentration; LCso = median lethal concentration; LOAEL = lowest-observed-adverse-effect level; MRL = minimal risk level; ND = no data;
NOAEL = no-observed-adverse-effect level; POD = point of departure; p-RfC = provisional reference concentration; 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.
71
/Msopropyltoluene
-------
EPA/690/R-24/003F
Figure A-2 compares hepatic, renal, and adrenal effects for the candidate analogues (no
data available for /;-isopropyl toluene) from repeated-dose inhalation toxicity studies in rats.
Liver effects (hepatocyte hypertrophy and/or increased liver weights) occurred at similar
concentrations for isopropylbenzene (214.6 ppm or 1,055 mg/m3) and xylene (164 ppm or
710 mg/m3). Kidney and adrenal effects (increased kidney and adrenal weights) were observed
for isopropylbenzene only (>108 ppm or 530 mg/m3 for kidney weight and 214.6 ppm or
1,055 mg/m3 for adrenal weight). Liver effects (increased liver weights, serum ALP levels, and
hepatocyte hypertrophy in male and female rats) were reported for/>isopropyltoluene after
subchronic oral exposure at >50 mg/kg-day (see Figure A-3) and were used for the derivation of
screening subchronic and chronic p-RfDs (see ORAL NONCANCER TOXICITY VALUES
above). Increased liver weights and ALT levels in rats were also reported for xylene at
>571 mg/kg-day following oral exposure, while no effects on liver histopathology were reported
after isopropylbenzene oral exposure up to 551 mg/kg-day (see Figure A-3). Possible renal
effects occurred in male rats exposed to/>isopropyltoluene (hyaline droplet accumulation,
tubular epithelial vacuolation and basophilia, and increased BUN at 200 mg/kg-day) following
oral dosing (see Figure A-4). Renal effects in female rats were found after oral exposure to
isopropylbenzene at >331 mg/kg-day (increased kidney weight) and xylene at >750 mg/kg-day
(increased kidney weight and mild nephropathy) (see Figure A-4).
72
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73
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74
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Figure A-4. Renal Effects Following Oral Exposure to p-Isopropyltoluene and its Candidate Analogues
75
p-Isopropyltoluene
-------
EPA 690 R-24 003F
Neurological effects were observed following inhalation exposure to the candidate
analogue, xylene (see Figure A-5). Altered rotarod performance, latency in paw-lick response,
motor activity, passive avoidance test, and auditory effects were noted in rats exposed to xylene
at >18 ppm (78 mg/m3) across several repeated-dose studies. In the case of isopropylbenzene, no
reproducible effects in FOB tests, motor activity tests, or neurohistopathology were observed in a
13-week inhalation rat study up to 214.6 ppm (1,055 mg/m3) (see Figure A-5). No subchronic or
chronic inhalation data are available for the target compound. There is limited evidence of
neurological effects following repeated-dose oral exposure top-isopropyltoluene and xylene
(see Figure A-6), although the measured endpoints reported for these compounds were different
(reduced grip strength in male rats exposed to 200 mg/kg-day ofp-isopropyltoluene and
aggression and hyperactivity in rodents exposed to >714 mg/kg-day of xylene). Oral
neurotoxicity data were not available for isopropylbenzene. Neurological effects were reported
after acute/short-term exposure to the target chemical via multiple exposure routes (reduced
mobility in mice orally exposed to 1,650 mg/kg-day for 24-28 days; analgesic effects in mice
after an oral dose of 40 mg/kg; transient clonic convulsions in rats and guinea pigs after a single
inhalation exposure at 9,700 mg/m3; reduced spontaneous activity, analgesia, reduced urination
and defecation, and antinociceptive effects in mice exposed to 25-200 mg/kg via acute i.p.
injection) (see Section 2.3.2 for more details). This is consistent with evidence for the candidate
analogues and other solvents that produce acute neurological effects in humans and/or animals
indicative of central nervous system depressant activity mostly at high exposure levels
(>500 ppm or 2,460 mg/m3 for isopropylbenzene and >100 ppm or 434 mg/mg3 for xylene after
inhalation exposure) (U.S. EPA 2003. 1997).
76
/Msopropyltoluene
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Figure A-5. Neurological Effects Following Inhalation Exposure to /;-Isopropyltoluene Candidate Analogues
77
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/Msopropyltoluene
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Developmental and reproductive toxicity have not been examined following inhalation
exposure to />isopropyltoluene. For isopropylbenzene, limited evidence of developmental
toxicity (increased nonviable implants and early resorptions and decreased percentage of live
fetuses; effects were not statistically significant but showed a coherent pattern) in the presence of
decreased maternal body weight gain and food consumption was found at 574.3 ppm
(2,823 mg/m3) in an inhalation rabbit study with gestational exposure (gestation days
[GDs] 6-18) (see Figure A-7). For xylene, reversible neurodevelopmental effects (impaired
Morris water maze performance in offspring) were reported in rats exposed in utero on
GDs 7-20 at 125 ppm (543 mg/m3), while effects on offspring survival and development
(decreased fetal body weight) occurred at higher concentrations (>345.5 ppm or >1,500 mg/m3)
in animals exposed during gestation (see Figure A-7); maternal toxicity (decreased maternal
weight and failure to deliver live fetuses) was observed at >230 ppm (or >1,000 mg/m3) in the
gestational exposure studies (see Figure A-7). No effects on reproductive parameters were
detected in a one-generation rat study at 89.4 ppm (388 mg/m3) or a subchronic study examining
male fertility and testicular toxicity in rats at 749.9 ppm (3,256 mg/m3) after inhalation exposure
to xylene (see Figure A-7). Effects on the reproductive system (decreased fertility, altered
estrous cyclicity, and male reproductive organ weights and histopathology at >100 mg/kg-day)
and developing offspring (decreases in survival and body weights at >50 mg/kg-day) were
observed for />isopropyltoluene after subchronic oral exposure in rats (see Figure A-8). No data
on potential reproductive/developmental effects are available for isopropylbenzene via the oral
route. Data on xylene include a gestational gavage study in mice that reported effects on fetal
development (decreased fetal body weight and increased fetal malformations [i.e., cleft palate])
at >2,060 mg/kg-day accompanied by maternal toxicity (decreased uterine weight) and
subchronic oral studies in rats and mice that reported no effects on reproductive organ weights or
histopathology up to 1,429 mg/kg-day.
79
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Analogues
80
p-Isopropyltoluene
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81
p-Isopropyltoluene
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EPA 690 R-24 003F
Figure A-9 illustrates that respiratory irritation occurs following acute inhalation
exposure top-isopropyltoluene and isopropylbenzene in animals and acute/short-term inhalation
exposure to xylene in humans. Correspondingly, sensory irritation RD50 values (i.e., the
concentration that elicits a respiratory rate decrease of 50%) in mice were similar for
isopropylbenzene (2,058 ppm or 10,117 mg/m3) and xylene (1,300-2,440.2 ppm or
5,645-10,595 mg/m3) (see Figure A-10). No RDso data are available for the target compound.
The inhalation LCso values in rodents (see Table A-9) were similar for the target compound and
both candidate analogues, suggesting low acute inhalation toxicity following exposure to
these compounds (19,500-24,000 mg/m3 for^-isopropyltoluene; 10,000-39,000 mg/m3 for
isopropyltoluene; and 17,000-29,000 mg/m3 for xylene).
82
^-Isopropyltoluene
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Figure A-9. Irritation Effects Following Inhalation Exposure tojP-Isopropyltoluene and Candidate Analogues
83
p-Isopropyltoluene
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84
/Msopropyltoluene
-------
EPA 690 R-24 003F
Although there is limited toxicity data onp-isopropyltoluene to clearly identify or rule
out suitable analogues based on toxicity comparisons, the available evidence suggests some
commonalities between the target compound and candidate analogues. For example, all three
compounds target the liver, kidney, and developmental systems to some extent after
repeated-dose inhalation and/or oral exposures, albeit at different potencies. Further,
p-i sopropyl toluene and its candidate analogues induce acute/short-term neurological effects,
respiratory irritation, and low acute lethality following inhalation exposure.
Weight-of-Evidence Approach
A WOE approach is used to evaluate information available for candidate analogues as
described by Wang et al. (2012) and Lizarraga et al. (2023). Similarities between candidate
analogues and the target chemical are identified across three major categories of evidence:
structural/physicochemical properties; toxicokinetics (absorption, distribution, metabolism,
excretion; ADME) and toxicodynamics (toxicity or MO A). Evidence of toxicological and/or
toxicokinetic similarity is prioritized over evidence of similarity in structural/physicochemical
properties. Candidate analogues are excluded if they demonstrate substantial differences from the
pool of candidate analogues as a whole and/or the target chemical in any of the three categories
of evidence. From the remaining pool of candidate analogues, the most suitable analogue
(i.e., the analogue that displays the closest biological or toxicological similarity to the target
chemical) with the greatest structural similarity and/or most health-protective point-of-departure
is selected. Additional considerations include preference for evidence from existing U.S. EPA
assessments and suitability of study duration (i.e., chronic studies are preferred over subchronic
studies when selecting an analogue for the derivation of a chronic value).
/Msopropyltoluene and both candidate analogues (isopropylbenzene and xylene [mixed
isomers]) share important structural features expected to influence toxicokinetics and/or toxicity
(i.e., a single benzene ring containing one or two methyl and/or isopropyl substituents).
Similarities in physicochemical properties (i.e., vapor pressure, water solubility, K0c, and log
Kow) suggest that the target compound and candidate analogues are expected to be bioavailable
by the oral and inhalation routes. p-Isopropyltoluene and candidate analogues showed similar
structural alerts for protein binding, reproductive and developmental toxicity, and metabolism by
CYP. In summary, similarities in structural features, physicochemical properties, and structural
alerts suggest that both isopropylbenzene and xylene are suitable structural analogues for
p-i sopropy ltoluene.
The target compound and candidate analogues share similarities in absorption,
metabolism, and distribution based on available in vivo data from experimental animals. Indeed,
all three compounds exhibit extensive absorption by multiple exposure routes (including by
inhalation) and are primarily metabolized through the oxidation of side-chain substituents in
laboratory animals (i.e., methyl and/or isopropyl substituents) and excreted via the urine as
glycine-, glucuronide-, and sulfate-conjugated metabolites. No in vivo distribution data are
available for p-i sopropy ltoluene; however, based on the log Kow value >4, it is expected to
accumulate in fatty tissues, similar to the candidate analogue compounds. Taken together, these
data suggest that both isopropylbenzene and xylene are suitable analogues for p-i sopropy ltoluene
based on toxicokinetic properties.
There are limited toxicity data, particularly by the inhalation route, to draw comparisons
between p-i sopropy ltoluene and the candidate analogues and to clearly identify or rule out
suitable analogues based on toxicodynamic properties. Additionally, comparison of toxicities by
85
p-Isopropyltoluene
-------
EPA 690 R-24 003F
the oral route may or may not inform toxicities by the inhalation route. However, the available
evidence suggests that these compounds may share some common toxicity targets (i.e., liver,
kidney, and developmental system) after repeated-dose exposure via inhalation and/or oral routes
and induce neurological effects, respiratory irritation, and low acute lethality potency after
acute/short-term inhalation exposure.
In summary, both isopropylbenzene and xylene (mixed isomers) are considered suitable
analogues for p-isopropyltoluene on the basis of structural and toxicokinetic properties and
limited toxicity data. Xylene provides the only subchronic inhalation toxicity value (U.S. EPA.
2009) and the more health-protective chronic inhalation toxicity value (U.S. EPA. 2003).
ATSDR Minimal Risk Levels (MRLs) for xylene are available for both intermediate and chronic
durations (ATSDR. 2007); however, the U.S. EPA (2009. 2003) values for this analogue are
more health-protective than the ATSDR values. Therefore, the subchronic and chronic p-RfC
values for xylene (mixed isomers) (U.S. EPA. 2009. 2003) will be used to derive the screening
p-RfC values for p-isopropyltoluene.
Derivation of a Screening Subchronic Provisional Reference Concentration
Based on the overall analogue approach presented in this PPRTV assessment, xylene
(mixed isomers) is selected as the analogue for p-isopropyltoluene for derivation of screening
subchronic and chronic p-RfCs. The principal study used for the screening subchronic and
chronic p-RfC values for p-isopropyltoluene is a 3-month inhalation study of w-xylene in rats
[Korsak et al. (1994) as cited in (U.S. EPA. 2009. 2003YI14. The PPRTV assessment for xylenes
(CASRN 1330-20-7) provided the following summary:
Korsak et al. (1994) exposed groups of 12 male Wistar rats by inhalation
to 0, 50 or 100 ppm m-xylene or n-butyl alcohol or a 1:1 mixture (parity of
chemicals not provided) for 6 hoars per day, 5 days per week for 3 months and
evaluated similar endpoints as in the earlier study (Korsak et al, 1992). Rotarod
performance and spontaneous motor activity were assayed. The report does not
specify the timing of the neurologic examinations; however, given that the 1994
study was conducted by the same group of investigators as a 1992 study (Korsak
et al, 1992) and that one of the tests (rotarod performance) was the same in both
studies, it appears reasonable to assume that the tests were administered 24 hoars
after termination of exposure. The rotarod test was used as a measure of motor
coordination disturbances fi'om exposure to m-xylene. The rotarod test involves
placing the subject animals on a rotating rod and evaluating their ability to
remain on the rod for a period of 2 minutes. The animals were trained to perform
the task, exposed to chemical or control gas and evaluated at defined intervals.
By the time interval after exposure, considerable proportions of absorbed xylenes
are expected to have been eliminatedfrom the body (see Toxicological Review,
U.S. EPA, 2003). Body weights and weights of seven organs were measured.
Bloodfor clinical biochemistry (e.g., alanine aminotransferase, aspartate
aminotransferase, sorbitol dehydrogenase, alkaline phosphatase and total
protein) and hematologic analysis (erythrocyte counts, hemoglobin concentration,
hematocrit, leukocyte count and differential leukocyte counts) was collected
24 hours after termination of exposure. Statistical evaluations (using a
14Korsak, Z; Wisniewska-Knypl, J; Swiercz, R. 1994. Toxic effects of subchronic combined exposure to n-butyl
alcohol and m-xylene in rats. Int J Occup Med Environ Health 7:155-166. [as cited in U.S. EPA (2009. 2003)1.
86
/Msopropyltoluene
-------
EPA 690 R-24 003F
p = 0.05 level of significance) of the collected data included analysis of variance,
Dunne It's test and Fisher's exact test.
No statistically significant exposure-related changes were noted in
body-weight gain, absolute or relative organ weights, hepatic activities of
microsomal monooxygenases, lipid peroxidation or levels of triglycerides in the
liver (Korsak et al., 1994). Statistically significant decreases in erythrocyte
number were seen in animals exposed to 50 ppm (93% of controls) or 100 ppm
(80.5% of controls) ofm-xylene alone. Similarly, decreased levels of hemoglobin
were reported in both groups (92% of controls for both groups). At 100 ppm, a
statistically significant increase in leukocyte number (35% increase over
controls) was reported. Exposure to 50 or 100 ppm m-xylene alone also resulted
in decreased rotarod performance starting at 1 month of exposure, which
remained at the same level until the end of the 3-month exposure. Decreases were
statistically significant in the 100 ppm group when compared with the controls.
The results were presented in graphical form; the actual numerical data are not
provided. The decreases in performance were roughly 8% and 33% for the 50 and
100 ppm groups, respectively, versus 0% for the controls.
Sensitivity to pain was assessed using the hot plate behavior test, in which
the animals are placed on a hot (54°C) surface and the time interval between
being placed on the plate and licking of the paws is measured (Korsak et al,
1994). Rats exposed to 50 or 100 ppm m-xylene alone had statistically
significantly increased sensitivity to pain at the end of the 3-month exposure
(latency of the paw-lick response was 8.7 and 8.6 seconds, respectively, us .
12.2 seconds for the controls). The LOAEL is 100 ppm, based on decreased
rotarod performance and decreased latency in the paw-lick response in the
hot-plate test and the NOAEL is 50 ppm.
The NOAELhec based on impaired motor coordination in rats (decreased rotarod
performance) used to derive the subchronic p-RfC value for xylene (mixed isomers), is described
by U.S. EPA (2009. 2003) as follows:
The NOAEL of 50 ppm (217 mg/m3) was duration adjusted as follows:
NOAELadj = NOAEL x (5 days/7 days) x (6 hrs day / 24 hrs day)
= 217 mg/m3 x 5/7x6/24
= 39 mg/m3
The NOAEL[ADjjwas used to derive a human equivalent concentration
(HEC), as described in U.S. EPA (1994b). Xylene is considered a category 3 gas
because of its low water solubility and its potential for accumulation in blood
during exposure and because its most sensitive effect is an extrarespiratory effect.
The NOAELpEcjwas calculated using the equation
(II((H= 46.0/26.4 = 1.7
NOAELhec = NOAELadj x (H^a
= 39 mg/m3 x 1
= 39 mg/m3
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where Hb/g = blood gas partition coefficient for the species in question,
animal (A) or human (H)
Tardif et al. (1995) reported an (Hb/^Hof 26.4 for m-xylene, and an earlier
study fi'om the same group (Tardif et al, 1993a) reported an (Hb/g)A of 46.0 for
m-xylene in the rat.
However, when (Hb/g)A > (Hb/g)H, a value of 1 is usedfor the ratio
(U.S. EPA, 1994b).
To derive the screening subchronic p-RfC for p-isopropyltoluene, the same POD
(NOAELhec) of 39 mg/m3 based on impaired motor coordination (decreased rotarod
performance) in rats exposed to m-xylene is adopted. Persistent neurological effects have been
associated with exposure to individual xylene isomers and mixed xylenes as described by U.S.
EPA (2009. 2003). A UFc of 300 is applied to the POD to account for residual interspecies
differences after default NOAELhec dosimetric adjustments (UFa of 3), database uncertainties
due to the absence of adequate repeated-dose inhalation toxicity data for p-isopropyltoluene
(UFd of 10), and human variability and sensitive populations (UFh of 10).
Screening Subchronic p-RfC = Analogue POD (HEC) ^ UFc
= 39 mg/m3 ^ 300
= 1 x 10"1 mg/m3
Table A-10 summarizes the uncertainty factors for the screening subchronic p-RfC for
p-i sopropy ltoluene.
Table A-10. Uncertainty Factors for the Screening Subchronic p-RfC for
/7-Isopropyltoluene (CASRN 99-87-6)
UF
Value
Justification
UFa
3
A UFa of 3 (10"5) is applied to account for uncertainty associated with extrapolating from animals
to humans when cross-species dosimetric adjustment (HEC calculation) is performed.
UFd
10
A UFd of 10 is applied to reflect database limitations for the target compound, /?-isopropyltolucnc.
For/j-isopropyltolucne. there were no adequate subchronic or chronic inhalation toxicity studies or
any repeated-dose studies evaluating developmental/reproductive toxicity.
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 /?-isopropyltolucnc in humans.
UFl
1
A UFl of 1 is applied for LOAEL-to-NOAEL extrapolation because the POD is a NOAEL.
UFS
1
A UFS of 1 is applied because the POD was derived from a subchronic 3-month study.
UFC
300
Composite UF = UFA x UFD x UFH x UFL x UFS.
HEC = human equivalent concentration; LOAEL = lowest-observed-adverse-effect level; NOAEL = no-observed-
adverse-effect level; POD = point of departure; p-RfC = provisional 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.
Derivation of a Screening Chronic Provisional Reference Concentration
The NOAELhec 39 mg/m3 based on impaired motor coordination in rats used in the
derivation of the chronic p-RfC for xylene (mixed isomers) [Korsak et al. (1992) as cited in U.S.
88
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EPA (2009. 2003)1 is also selected as the POD for the derivation of the screening chronic p-RfC
forp-isopropyltoluene. The screening chronic p-RfC forp-isopropyltoluene is derived by
applying a UFc of 1,000 to the POD (HEC) of 39 mg/m3. The UFc of 1,000 was derived by
applying aUFA of 3, a UFd of 10, a UFh of 10, and a UFs of 3. A factor of 10 is not used for
duration extrapolation from subchronic to chronic because the changes in rotarod performance
associated with xylene inhalation exposure did not increase with time from 1 to 3 months, and
they were similar to those described in a separate study of 6 months in duration [Korsak et al.
(1992) as cited in U.S. EPA (2009. 2003)1. The same rationale and UFs of 3 was used in the U.S.
EPA (2003) assessment to derive the chronic RfC for xylene.
Screening Chronic p-RfC = Analogue POD (HEC) UFc
= 39 mg/m3 ^ 1,000
= 4 x 10"2 mg/m3
Table A-l 1 summarizes the uncertainty factors for the screening chronic p-RfC for
p-i sopropy ltoluene.
Table A-ll. Uncertainty Factors for the Screening Chronic p-RfC for
/7-Isopropyltoluene (CASRN 99-87-6)
UF
Value
Justification
UFa
3
A UFa of 3 (10"5) is applied to account for uncertainty associated with extrapolating from animals
to humans when cross-species dosimetric adjustment (HEC calculation) is performed.
UFd
10
A UFd of 10 is applied to reflect database limitations for the target compound, /?-isopropyltolucnc.
For/j-isopropyltolucne. there were no adequate subchronic or chronic inhalation toxicity studies or
any repeated-dose studies evaluating developmental/reproductive toxicity.
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 /?-isopropyltolucnc in humans.
UFl
1
A UFl of 1 is applied for LOAEL-to-NOAEL extrapolation because the POD is a NOAEL.
UFs
3
A UFS of 3 is applied because the POD was derived from a subchronic 3-mo study. A factor of
10 was not used because the changes in rotarod performance did not increase with time from 1 to
3 mo, and they were similar to those described in a separate study of a 6-mo duration
IKorsak et al (1992) as cited in U.S. EPA (2009. 2003)1.
UFC
1,000
Composite UF = UFA x UFD x UFH x UFL x UFS.
HEC = human equivalent concentration; 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.
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APPENDIX B. DATA TABLES
Table B-l. Select Functional Observational Battery Findings in Male
Sprague Dawley Rats Exposed to />-Isopropyltoluene via Gavage for up to
~35 Days3
Males: -35 Days
[HED] (mg/kg-d)b
0 (control)
50 [14]
100 [28.1]
200 [56.2]
Number of animals
5
5
5
5
Forelimb grip strength (g)
1,401.7 ± 346.3C
1,109.3 ±413.1
(-21%)d
1,143.7 ±261.7
(-18%)
984.4 ±231.9
(-30%)
Hindlimb grip strength (g)
837.7 ±284.5
680.5 ± 54.2
(-19%)
628.2 ± 152.0
(-25%)
541.6 ±227.7*
(-35%)
aSvmrise (2018).
bADDs (mg/kg-day) were reported by the study authors; calculated HEDs appear in brackets.
Data are mean ± SD.
dValue in parentheses is % change relative to control = ([treatment mean - control mean] control mean) x 100.
* Significantly different from control (p < 0.05) as reported by the study authors.
ADD = adjusted daily dose; HED = human equivalent dose; SD = standard deviation.
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Table B-2. Select Hematology, Coagulation, and Serum Chemistry Findings
in Sprague Dawley Rats Exposed to />-Isopropyltoluene via Gavage for up to
~35 Days (Males) or ~63 Days (Females)3
Endpoint
Males: -35 Days [HED] (mg/kg-d)b
0 (control)
50 [14]
100 [28.1]
200 [56.2]
Hematology and coagulation
Number of animals
4
5
5
5
Retic (xl09/L)
163.4 ±9.51c
166.3 ± 20.02 (±2%)d
151.8 ± 15.96 (-7%)
211.7 ±36.29** (±30%)
RDW (%)
12.5 ±0.19
12.7 ± 0.49 (±2%)
12.3 ± 0.36 (-2%)
13.4 ±0.8* (±7%)
PT (sec)
18 ±0.76
18.7 ± 0.54 (±4%)
19.5 ± 0.77** (±8%)
19.6 ± 0.4** (±9%)
Serum chemistry
Number of animals
5
5
5
5
ALP (U/L)
160 ±23.5
166 ± 43.5 (±4%)
184 ± 22.8 (±15%)
232 ±61.4* (±45%)
BUN (mg/dL)
12 ± 0e
13 ± 0.9 (±8%)
14 ± 1.3 (±17%)
18 ± 1.8** (±50%)
Triglyceride (mg/dL)
86 ± 14.4
69 ±38.1 (-20%)
45 ±9.1* (-48%)
43 ± 18.1** (-50%)
Na+ (mEq/L)
143 ± 1.4
143 ± 0.8 (0%)
143 ± 0.5 (0%)
141 ±0.5* (-1%)
CI (mEq/L)
103 ± 1.1
102 ± 1.2* (-1%)
102 ± 0.9* (-1%)
102 ± 0.8* (-1%)
PHOS (mg/dL)
8.5 ±0.41
7.9 ± 0.5 (-7%)
7.8 ±0.11* (-8%)
8.5 ± 0.43 (0%)
Endpoint
Mated Females: -63 Days [HED] (mg/kg-d)
0 (control)
50 [13]
100 [25.6]
200 [51.2]
Serum chemistry
Number of animals
5
5
4
0
ALT (U/L)
183 ±39
124 ± 25.9** (-32%)
93 ± 14** (-49%)
NA
ALP (U/L)
151 ± 19.8
210 ±70.9 (±39%)
270 ± 119.1* (±79%)
NA
Cholesterol (mg/dL)
120 ±30.8
95 ± 12 (-21%)
85 ± 5.4* (-29%)
NA
Albumin (g/dL)
3.5 ±0.29
3.5 ±0.15(0%)*
3.2 ±0.21* (-9%)
NA
aSvmrise (2018).
bADDs (mg/kg-day) were reported by the study authors; calculated HEDs appear in brackets.
Data are mean ± SD.
dValue in parentheses is % change relative to control = ([treatment mean - control mean] control mean) x 100.
* Significantly different from control (p < 0.05) as reported by the study authors.
**Significantly different from control (p < 0.01) as reported by the study authors.
ADD = adjusted daily dose; ALP = alkaline phosphatase; ALT = alanine aminotransferase; BUN = blood urea
nitrogen; CI = chloride; HED = human equivalent dose; Na+ = sodium; NA = not available; PHOS = inorganic
phosphate; PT = prothrombin time; RDW = red blood cell distribution width; RETIC = reticulocyte count;
SD = standard deviation.
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Table B-3. Select Organ Weights (Percent Difference Relative to Controls)
in Sprague Dawley Rats Exposed to />-Isopropyltoluene via Gavage for
~35 Days (Males) or ~63 Days (Females)3
Endpoint'
Males: ADD [HED] (mg/kg-d)b
50 [14]
100 [28.1]
200 [56.2]
Testes weight
Absolute (%)
-
-
-14*
Relative to body weight (%)
-
-
-8
Relative to brain weight (%)
-
-
-12
Epididymides
Absolute (%)
-
-
-14*
Relative to body weight (%)
-
-
-8
Relative to brain weight (%)
-
-
-14*
Levator ani/bulbocavernosus muscle
Absolute (%)
-
-
-14
Relative to body weight (%)
-
-
-9
Relative to brain weight (%)
-
-
-15
Seminal vesicles/coagulating glands
Absolute (%)
-19
-23
-22
Relative to body weight (%)
-20
-22
-14
Relative to brain weight (%)
-16
-23
-18
Prostate
Absolute (%)
-26
2
-24*
Relative to body weight (%)
-27*
5
-16
Relative to brain weight (%)
-23
3
-20
Liver
Absolute (%)
8
6
27*
Relative to body weight (%)
6
8*
41*
Relative to brain weight (%)
13
6
35*
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Table B-3. Select Organ Weights (Percent Difference Relative to Controls)
in Sprague Dawley Rats Exposed to />-Isopropyltoluene via Gavage for
~35 Days (Males) or ~63 Days (Females)3
Females: ADD [HED] (mg/kg-d)b
Endpoint'
50 [13]
100 [25.6]
200 [51.2]
Liver weight
Absolute (%)
16
26*
NA
Relative to body weight (%)
14
22*
NA
Relative to brain weight (%)
14
22
NA
aSvmrise (2018).
bADDs (mg/kg-day) were reported by the study author; calculated HEDs appear in brackets.
Data are percent difference relative to controls [actual measurement data for control and treatment groups were not
available in the study report (ECHA. 2019b) I: number of animals = 5; organs were not weighed in females that
failed to deliver a litter).
* Significantly different (p < 0.05) between mean values for treated and control groups, as reported by the study
authors.
- = reported in ECHA as not test item-related; ADD = adjusted daily dose; ECHA = European Chemicals Agency;
HED = human equivalent dose; NA = not available; no organ weights were taken for females that failed to deliver a
litter.
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Table B-4. Histopathology Findings in Liver and Kidney of
Adult Po Sprague Dawley Rats Exposed to />-Isopropyltoluene
via Gavage for ~35 Days (Males) or ~63 Days (Females)3
Lesions0
Dose Group, mg/kg-d [HED]b
Po Males
Po Females
0
50
[14]
100
[28.1]
200
[56.2]
0
50
[13]
100
[25.6]
200
[51.2]
Liver
Hepatocellular
hypertrophy
0/5
(0%)
0/5
(0%)
0/5
(0%)
2/5+
(40%)
0/6
(0%)
1/6+
(17%)
0/10
(0%)
1/10+
(10%)d
Kidney
Tubular dilation
l/5++
(20%)
0/5
(0%)
0/5
(0%)
0/5
(0%)
0/5
(0%)
1/5-"
(20%)
0/4
(0%)
NA
Tubular epithelium
vacuolation
0/5
(0%)
0/5
(0%)
0/5
(0%)
2/5+
(40%)
0/5
(0%)
0/5
(0%)
0/4
(0%)
NA
Hyaline droplets
accumulation
l/5+
(20%)
l/5+
(20%)
0/5
(0%)
3/5++
(60 %r
0/5
(0%)
0/5
(0%)
0/4
(0%)
NA
Tubular basophilia
0/5
(0%)
l/5+
(20%)
l/5+
(20%)
1/5+
(20%)
0/5
(0%)
0/5
(0%)
0/4
(0%)
NA
aSvmrise (2018).
bADDs (mg/kg-day) were reported by the study author; calculated HEDs appear in brackets.
°Values denote number of animals showing changes / total number of animals examined; severity of lesions
indicated by + (minimal) and ++ (slight).
dThe number of livers examined includes those from nonpregnant females that were euthanized during the gestation
period.
e2/5 had minimal severity and 1/5 had slight severity.
HED = human equivalent dose; NA = not available; kidneys from females in the 200-mg/kg-day group were not
examined.
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Table B-5. Select Histological Findings in Reproductive Organs of Male
Sprague Dawley Rats Administered />-Isopropyltoluene via Gavage for
~35 Days3
Lesions0
Dose Group, mg/kg-d [HED]b
0 (control)
50 [14]
100 [28.1]
200 [56.2]
Testis
Degeneration/depletion, germ cell
Minimal
0/10 (0%)
0/10 (0%)
0/10 (0%)
1/10 (10%)
Slight
0/10 (0%)
0/10 (0%)
0/10 (0%)
5/10 (50%)*
Moderate
0/10 (0%)
0/10 (0%)
0/10 (0%)
1/10 (10%)
Total
0/10 (0%)
0/10 (0%)
0/10 (0%)
7/10 (70%)*
Depletion, germ cell
Minimal
0/10 (0%)
0/10 (0%)
0/10 (0%)
2/10 (20%)
Total
0/10 (0%)
0/10 (0%)
0/10 (0%)
2/10 (20%)
Retention, spermatid
Minimal
0/10 (0%)
0/10 (0%)
7/10 (70%)*
2/10 (20%)
Slight
0/10 (0%)
0/10 (0%)
0/10 (0%)
7/10 (70%)*
Total
0/10 (0%)
0/10 (0%)
7/10 (70%)*
9/10 (90%)*
Epididymides
Sperm, reduced, luminal
Minimal
0/10 (0%)
0/10 (0%)
1/10 (10%)
0/10 (0%)
Slight
0/10 (0%)
0/10 (0%)
1/10 (10%)
4/10 (40%)*
Moderate
0/10 (0%)
0/10 (0%)
0/10 (0%)
5/10 (50%)*
Marked
0/10 (0%)
0/10 (0%)
0/10 (0%)
1/10 (10%)
Total
0/10 (0%)
0/10 (0%)
2/10 (20%)
10/10 (100%)*
Cribriform change
Minimal
0/10 (0%)
0/10 (0%)
0/10 (0%)
2/10 (20%)
Slight
0/10 (0%)
0/10 (0%)
1/10 (10%)
3/10 (30%)
Total
0/10 (0%)
0/10 (0%)
1/10 (10%)
5/10 (50%)*
95
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Table B-5. Select Histological Findings in Reproductive Organs of Male
Sprague Dawley Rats Administered />-Isopropyltoluene via Gavage for
~35 Days3
Lesions0
Dose Group, mg/kg-d [HED]b
0 (control)
50 [14]
100 [28.1]
200 [56.2]
Cell debris, luminal
Minimal
0/10 (0%)
0/10 (0%)
0/10 (0%)
2/10 (20%)
Slight
0/10 (0%)
0/10 (0%)
0/10 (0%)
3/10 (30%)
Moderate
0/10 (0%)
0/10 (0%)
0/10 (0%)
4/10 (40%)*
Total
0/10 (0%)
0/10 (0%)
0/10 (0%)
9/10 (90%)*
aSvmrise (2018).
bADDs (mg/kg-day) were reported by the study author; calculated HEDs appear in brackets.
°Values denote number of animals showing changes / total number of animals examined (% incidence); all changes
were bilateral.
* Statistically significant from control (p < 0.05) based on one-tailed Fisher's exact test performed for this review.
HED = human equivalent dose.
Table B-6. Select Estrous Cycle Evaluations in Female Sprague Dawley Rats
Exposed to /7-Isopropyltoluene via Gavage for ~63 Days3
Endpoints0
Dose Group, mg/kg-d [HED]b
0
50
[13]
100
[25.6]
200
[51.2]
Females with regular cycles'1
6/10 (60%)
7/10 (70%)
7/10 (70%)
4/10 (40%)
Females with irregular cycles6
4/10 (40%)
3/10 (30%)
3/10 (30%)
6/10 (60%)
Females with extended estrusf
0/10 (0%)
0/10 (0%)
0/10 (0%)
0/10 (0%)
Acyclic females8
0/10 (0%)
0/10 (0%)
0/10 (0%)
0/10 (0%)
Total number of pregnant females
10
10
10
10
aSvmrise (2018).
bADDs (mg/kg-day) were reported by the study author; calculated HEDs appear in brackets.
°Values denote number of animals with estrus cycle observations / total number of animals examined
(% incidence).
dAll regular cycles (4, 4/5, and 5 days).
eAt least one cycle of <4 or >5 days.
fAt least 4 consecutive days of estrus.
gAt least 10 days without estrus.
HED = human equivalent dose.
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Table B-7. Select Fertility and Offspring Survival Parameters in Sprague
Dawley Rats Exposed to />-Isopropyltoluene via Gavage for Gavage for
~35 Days (Males) or ~63 Days (Females)3
Endpoints
Dose Group, mg/kg-d [HED]
0
50
[13]
100
[25.6]
200
[51.2]
Mating and fertility
Number of females paired with males
10
10
10
10
Number mated
10
10
10
9
Mating index (%)b
100
100
100
90
Total number pregnant females
9
9
4
0
Fertility index (%)°
90
90
40
0
Reproductive and offspring survival
indices
Number
9
9
4
Gestation length (days)
21.8 ±0.44
21.7 ±0.50
21.3 ± 0.58d
-
Number of corpora lutea
16.3 ±2.40
14.9 ±3.06
17.3 ± 1.89
-
Number of implantations
16.0 ±2.06
14.0 ±3.08
16.0 ± 1.41
-
Preimplantation loss (%)e
1.8 ±3.64
5.9 ±6.81
7.1 ± 1.94
-
Postimplantation survival index (%)f
95 ±6.55
97.7 ±4.65
87.3 ± 14.50
-
Live birth index (%)g
100 ±0
97.7 ± 4.77
94.3 ±4.17*
-
Viability index, Day 4 (%)h
100 ±0
100 ±0
98.3 ±3.33
-
Viability index, Day 7 (%)h
86.7 ± 1.24
84.3 ±4.28
82.5 ±4.33
-
Viability index, Day 13 (%)h
84.5 ±3.27
84.3 ±4.28
82.5 ±4.33
-
97
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Table B-7. Select Fertility and Offspring Survival Parameters in Sprague
Dawley Rats Exposed to />-Isopropyltoluene via Gavage for Gavage for
~35 Days (Males) or ~63 Days (Females)3
Endpoints
Dose Group, mg/kg-d [HED]
0
50
[13]
100
[25.6]
200
[51.2]
Mean body weight for offspring (g)
Number
9
9
4
Males, Day 1
6.8 ±0.62
6.8 ±0.91 (0%)
6.1 ±0.44 (-10.3%)
NA
Males, Day 4
8.9 ±0.89
9.8 ± 1.61 (10.1%)
8.7 ± 0.50 (-2.2%)
NA
Males, Day 7
13.1 ± 1.31
14.8 ±2.68 (13.0%)
13.4 ±0.78 (2.3%)
NA
Males, Day 11
20.4 ±2.08
23.0 ±4.77 (12.7%)
21.2 ± 1.58(3.9%)
NA
Males, Day 13
24.5 ± 2.70
26.8 ± 5.22 (9.4%)
25.1 ± 1.72 (2.4%)
NA
Females, Day 1
6.6 ±0.66
6.3 ± 0.84 (-4.5%)
6.0 ±0.3 (-9.1%)
NA
Females, Day 4
8.4 ±0.90
9.0 ± 1.75 (7.1%)
8.6 ± 0.53 (2.4%)
NA
Females, Day 7
12.7 ± 1.51
14.0 ±2.8 (10.2%)
12.8 ±0.81d (0.8%)
NA
Females, Day 11
20.0 ±2.74
22.2 ±4.85 (11.0%)
19.9 ± 1.04d (-0.5%)
NA
Females, Day 13
23.7 ±3.25
26.0 ± 5.53 (9.7%)
23.7 ± 1.16d (0%)
NA
aSvmrise (2018).
bMating index = (number of females with confirmed mating + number of pregnant females without evidence of
mating) / (number of females placed with males) x 100.
"Fertility index = ([number pregnant] / [number copulated]) x 100.
dw = 3 for Days 7-13 in the 100-mg/kg-day group.
^reimplantation loss (%) = (number of corpora lutea - number of implantation sites) / (number of corpora
lutea) x 100.
fPostimplantation survival index = ([number of implantation sites - total number of live pups on Day 1] / [number
of implantation sites]) x 100.
gLive birth index = ([total number of live pups on Day 1] / [total number of pups born]) x 100.
''Viability index = ([number of pups alive on the specified day] / [total number of live pups on Day 1]) x 100.
* Significantly different from controls, as reported by study authors (p-level was not specified).
HED = human equivalent dose; NA = not available.
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APPENDIX C. BENCHMARK DOSE MODELING RESULTS
MODELING PROCEDURE FOR NONCANCER EFFECTS
Continuous Data
The benchmark dose (BMD) modeling of continuous data was conducted with the
U.S. Environmental Protection Agency (U.S. EPA) Benchmark Dose Software (BMDS)
(version 3.3). For these data, the Exponential, Linear, Polynomial, Hill, and Power continuous
models available within the software were used. The continuous models available within the
software were fit using a benchmark response (BMR) of 1 standard deviation (SD) or alternative
BMRs may be used where appropriate as outlined in the Benchmark Dose Technical Guidance
(U.S. EPA 2012a). A standard BMR of 1 SD was used for increased alkaline phosphatase (ALP)
in Po male and female rats. For developmental effects (i.e., decreased female offspring body
weight), a BMR of 5% relative deviation (RD) is considered a minimally biologically significant
response during growth/development in gestational studies (e.g., fetal weight) and was applied in
this assessment for BMD modeling purposes. An adequate fit was judged based on the
X2 goodness-of-fit /rvalue (p > 0.1), magnitude of the scaled residuals in the vicinity of the
BMR, and visual inspection of the model fit. In addition to these three criteria forjudging
adequacy of model fit, a determination was made as to whether the variance across dose groups
was constant. If a constant variance model was deemed appropriate (i.e., Test 2 in BMDS;
/> value > 0.05), the final BMD results were estimated from a constant variance model. If the test
for homogeneity of variance was rejected (/> value < 0.05), the model was run again while
modeling the variance as a power function of the mean to account for this nonconstant variance.
If this nonconstant variance model did not adequately fit the data (i.e., Test 3 in BMDS;
/rvalue < 0.05), the data set was considered unsuitable for BMD modeling. Among all models
providing adequate fit, the lowest lower confidence limit on the benchmark dose (BMDL) is
selected if the BMDLs estimated from different models varied less than threefold; otherwise, the
BMDL from the model with the lowest Akaike's information criterion (AIC) is selected as a
potential point of departure (POD) from which to derive the screening provisional reference
values.
Dichotomous Data
The BMD modeling of dichotomous data was conducted with the U.S. EPA's BMDS
(version 3.3). The Gamma, Logistic, Log-Logistic, Probit, Log-Probit, Hill, Multistage, and
Weibull dichotomous models available within the software were fit using a standard BMR of
10% extra risk (ER). In general, the BMR should be near the low end of the observable range of
increased risk in the study. BMRs that are too low can result in widely disparate BMDL
estimates from different models (i.e., model dependence is high). Adequacy of model fit was
judged on the basis of the x2 goodness-of-fit /rvalue (p > 0.1), magnitude of scaled residuals
(absolute value <2.0), and visual inspection of the model fit. Among all models providing
adequate fit, the BMDL from the model with the lowest AIC is selected as a potential POD, if
the BMDLs are sufficiently close (less than approximately threefold); if the BMDLs are not
sufficiently close (greater than approximately threefold), model dependence is indicated, and the
model with the lowest reliable BMDL is selected.
99
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EPA 690 R-24 003F
BMD MODELING TO IDENTIFY POTENTIAL PODS FOR DERIVATION OF
SCREENING SUBCHRONIC AND CHRONIC PROVISIONAL REFERENCE DOSES
Increased ALP in Po Male Sprague Dawley Rats After Oral Exposure to />-Isopropyltoluene
for -35 Days (ECHA. 2019b: Svmrise. 2018)
The procedure outlined above for continuous data was applied to the data for increased
ALP in Po male Sprague Dawley rats orally exposed top-isopropyltoluene for -35 days (ECHA.
2019b; Svmrise. 2018). The constant variance model provided an adequate fit to the variance
data, and the Exponential (degree 3), Polynomial (degree 2), Power, and Linear models provided
adequate fit to the means. Visual inspection of the dose-response curves suggested adequate fit,
BMDLs were not 10 times lower than the lowest nonzero dose, and scaled residuals did not
exceed ±2 units at the data point closest to the predefined BMR. BMDLs for models providing
adequate fit were sufficiently close (differed by less than threefold), so the model with the lowest
AIC was selected (Linear). The estimated human equivalent BMDisd and BMDLisd values of
28 and 18 mg/kg-day, respectively, were selected from this model. The results of the BMD
modeling are summarized in Table C-l. Figure C-l shows the fit of the Linear model to the data.
Table C-l. BMD Modeling Results (Constant Variance) for Increased ALP
in Po Male Sprague Dawley Rats Orally Exposed to />-Isopropyltoluene for
~35 Days3
Model
Variance
/>-Valucb
Means
/>-Valuec
Scaled Residual
at Dose Nearest
BMD
AIC
BMDisd
(HED,
(mg/kg-d)
BMDLisd
(HED,
(mg/kg-d)
Exponential (model 3)d
0.07737514
0.9003972
0.065709363
208.883831
37.08159
21.43453
Exponential (model 5)d
0.07737514
NA
7.5654E-08
210.8681661
35.42935
30.92168
Hill
0.07737514
NA
-8.85231E-07
210.8681661
35.53358
14.59477
Polynomial (3-degree)6
0.07737514
NA
-0.002490259
211.208119
45.13184
18.04798
Polynomial (2-degree)e
0.07737514
0.8023287
-0.0530054
208.9308334
34.25201
18.41255
Power6
0.07737514
0.9244079
0.048369367
208.8771688
36.6691
31.59494
Lineardf
0.07737514
0.7738805
-0.371230114
207.3808417
27.85133
17.83316
aSvmrise (2018).
bValues <0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.1 fail to meet conventional goodness-of-fit criteria.
dPower restricted to be >1.
eCoefficients restricted to be positive.
'Selected model.
AIC = Akaike's information criterion; ALP = alkaline phosphatase; BMD = maximum likelihood estimate of the
dose associated with the selected BMR; BMDL = 95% lower confidence limit on the BMD (subscripts denote
BMR: i.e., 1SD = dose associated with 1 standard deviation from the control); BMR = benchmark response;
NA = test for fit is not valid; HED = human equivalent dose.
100
/Msopropyltoluene
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EPA 690 R-24 003F
Frequentist Linear Model with BMR of 1 Standard Deviation for
the BMD and 0.95 Lower Confidence Limit for the BMDL
Figure C-l. Fit of Linear Model to Data for Increased Alkaline Phosphatase in Po Male
Sprague Dawley Rats After Oral Exposure to />-Isopropyltoluene for ~35 Days (ECHA,
2019b; Symrise, 2018)
BMD Model Output for Linear Model to Data for Increased ALP in Po Male
Sprague Dawley Rats After Oral Exposure to />-Isopropyltoluene for ~35 Days (ECHA,
2019b; Symrise, 2018)
Data
Increased ALP in P0 males
Dose
N
Mean
Std. Dev.
HED (mg/kg-day)
[Custom]
[Custom]
[Custom]
0
5
160
23.5
14
5
166
43.5
28
5
184
22.8
56
5
232
61.4
Model Results
Benchmark Dose
BMD
27.85132587
BMDL
17.83316026
BMDU
62.79187172
AIC
207.3808417
Test 4 p-Value
0.773880476
D.O.F.
2
101
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EPA 690 R-24 003F
Model Parameters
# of Parameters
3
Variable
Estimate
Std Error
Lower Conf
Upper Conf
g
152.8000008
12.87668371
127.562164
178.037837
beta
1.334693861
0.401408791
0.54794708
2.12144064
alpha
1381.831454
603824.1589
-1182091.8
1184855.45
Goodness of Fit
Dose
Size
Estimated
Median
Calc'd
Median
Observed
Mean
Estimated
SD
Calc'd SD
Observed
SD
Scaled Residual
0
5
152.8000008
160
160
37.1729936
23.5
23.5
0.433101723
14
5
171.4857149
166
166
37.1729936
43.5
43.5
-0.329982339
28
5
190.1714289
184
184
37.1729936
22.8
22.8
-0.371230114
56
5
227.5428571
232
232
37.1729936
61.4
61.4
0.268110626
Likelihoods of Interest
Model
Log Likelihood*
# of Parameters
AIC
A1
-100.434083
5
210.868166
A2
-97.0169938
8
210.033988
A3
-100.434083
5
210.868166
fitted
-100.6904209
3
207.380842
R
-105.0905624
2
214.181125
*Includes additive constant of -18.37877. This constant was not included in the LL derivation prior to BMDS 3.0.
Tests of Interest
Test
-2*Log(Likelihood Ratio)
Test df
p-Value
1
16.14713719
6
0.01298564
2
6.834178449
3
0.07737514
3
6.834178449
3
0.07737514
4
0.512675682
2
0.77388048
Increased ALP in Po Female Sprague Dawley Rats After Oral Exposure to
/7-Isopropyltoluene for ~63 Days (ECHA, 2019b; Symrise, 2018)
The procedure outlined above for continuous data was applied to the data for increased
ALP in Po female Sprague Dawley rats orally exposed top-isopropyltoluene for -63 days
(ECHA 2019b; Symrise. 2018). The constant variance model did not provide an adequate fit to
the variance data (p < 0.05; see Table C-2). Data were modeled using a nonconstant variance
model, which provided an adequate fit to the variance data (see Table C-3). The Exponential
(model 3), and Power models provided adequate fit to the means. Visual inspection of the dose-
response curves suggested adequate fit, BMDLs were not 10 times lower than the lowest nonzero
dose, and scaled residuals did not exceed ±2 units at the data point closest to the predefined
102
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EPA 690 R-24 003F
BMR. BMDLs for models providing adequate fit were sufficiently close (differed by less than
threefold), so the model with the lowest AIC was selected (Exponential degree 3). The estimated
human equivalent BMDisd and BMDLisd values of 5.6 and 3.6 mg/kg-day, respectively, were
selected from this model. The results of the BMD modeling are summarized in Table C-3.
Figure C-2 shows the fit of the Exponential (degree 3) model to the data.
Table C-2. BMD Modeling Results (Constant Variance) for Increased ALP
in Po Female Sprague Dawley Rats Orally Exposed to />-Isopropyltoluene for
~63 Days3
Model
Variance
/>-Valucb
Means
/>-Valuec
Scaled
Residual at
Dose Nearest
BMD
AIC
BMDisd
(HED,
(mg/kg-d)
BMDLisd
(HED,
(mg/kg-d)
Exponential (model 3)d
0.00472624
0.8340455
0.164569433
163.8104955
16.67688
11.32402
Exponential (model 5)d
0.00472624
NA
-9.855E-09
167.7665986
14.20689
0.208399
Hill
0.00472624
NA
3.89928E-08
167.7665986
14.2768
0
Polynomial (2-degree)e
0.00472624
NA
-0.008198238
165.7667504
14.78772
8.693893
Power6
0.00472624
NA
0.000329145
165.7665988
14.90258
8.692696
Linear"1
0.00472624
0.9894642
-0.010564001
163.766773
14.80401
8.693664
aSvmrise (2018).
bValues <0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.1 fail to meet conventional goodness-of-fit criteria.
dPower restricted to be >1.
eCoefficients restricted to be positive.
AIC = Akaike's information criterion; ALP = alkaline phosphatase; BMD = maximum likelihood estimate of the
dose associated with the selected BMR; BMDL = 95% lower confidence limit on the BMD (subscripts denote
BMR: i.e., 1SD = dose associated with 1 standard deviation from the control); BMR = benchmark response;
NA = test for fit is not valid; HED = human equivalent dose.
103
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EPA 690 R-24 003F
Table C-3. BMD Modeling Results (Nonconstant Variance) for Increased
ALP in Po Female Sprague Dawley Rats Orally Exposed to
/7-Isopropyltoluene for ~63 Days3
Model
Variance
/>-Valucb
Means
/>-Valuce
Scaled
Residual at
Dose Nearest
BMD
AIC
BMDisd
(HED,
(mg/kg-d)
BMDLisd
(HED,
(mg/kg-d)
Exponential (model 3)d e
0.65440701
0.5701245
0.107050508
154.3815366
5.615738
3.608899
Exponential (model 5)e
0.65440701
NA
0.324414859
160.1941198
0.089199
0.032063
Hill
0.65440701
NA
0.278150212
159.8411931
5.403369
0.405075
Polynomial (2-degree)f
-
-
-
-
-
-
Power'
0.65440701
0.4169571
0.236217929
155.9166117
4.331736
2.568022
Linear"1
-
-
-
-
-
-
aSvmrise (2018).
bValues <0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.1 fail to meet conventional goodness-of-fit criteria.
Coefficients restricted to be positive.
Selected model.
fPower restricted to be >1.
AIC = Akaike's information criterion; ALP = alkaline phosphatase; BMD = maximum likelihood estimate of the
dose associated with the selected BMR; BMDL = 95% lower confidence limit on the BMD (subscripts denote
BMR: i.e., 1SD = dose associated with 1 standard deviation from the control); BMR = benchmark response;
NA = test for fit is not valid; HED = human equivalent dose.
Frequentist Exponential Degree 3 Model with BMR of 1
Standard Deviation for the BMD and 0.95 Lower Confidence
Limit for the BMDL
512
412
£
O 3n
+-»
to
!=L 212 ^
119 J-
±±Z.
12
ii
HED
16
21
26
Estimated Probability
^^Response at BMD
O Data
BMD
BMDL
Figure C-2. Fit of Exponential (Degree 3) Model to Data for Increased Alkaline
Phosphatase in Po Female Sprague Dawley Rats After Oral Exposure to
/7-Isopropyltoluene for ~63 Days (ECHA, 2019b; Symrise, 2018)
104 />-Isopropyltoluene
-------
EPA 690 R-24 003F
BMD Model Output for Exponential (Degree 3) Model to Data for Increased ALP in
Po Female Sprague Dawley Rats After Oral Exposure to />-Isopropyltoluene for ~63 Days
(ECHA. 2019b: Svmrise. 2018)
Data
Increased ALP in Po females
Dose
N
Mean
Std. Dev.
HED
[Custom]
[Custom]
[Custom]
0
5
151
19.8
13
5
210
70.9
26
4
270
119.1
Model Results
Benchmark Dose
BMD
5.615737565
BMDL
3.608899049
BMDU
12.47634342
AIC
154.3815366
Test 4 p-Value
0.570124523
D.O.F.
2
Model Parameters
# of Parameters
5
Variable
Estimate
Std Error
Lower Conf
Upper Conf
a
149.8959152
9.323998674
131.621213
168.170617
b
0.025483328
5.72E-03
0.01426484
0.03670182
d
Bounded
NA
NA
NA
rho
4.845641848
9.54E-02
4.65873323
5.03255047
log-alpha
Bounded
NA
NA
NA
Goodness of Fit
Dose
Size
Estimated
Median
Calc'd
Median
Observed
Mean
Estimated
SD
Calc'd SD
Observed SD
Scaled Residual
0
5
149.8959152
151
151
23.0620926
19.8
19.8
0.107050508
13
5
208.768173
210
210
51.4613106
70.9
70.9
0.053524656
26
4
290.7627603
270
270
114.832012
119.1
119.1
-0.361619725
105
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EPA 690 R-24 003F
Likelihoods of Interest
Model
Log Likelihood*
# of Parameters
AIC
A1
-78.88329932
4
165.766599
A2
-73.52867333
6
159.057347
A3
-73.62886784
5
157.257736
fitted
-74.19076832
3
154.381537
R
-81.68323444
2
167.366469
*Includes additive constant of -12.86514. This constant was not included in the LL derivation prior to BMDS 3.0.
Tests of Interest
Test
-2*Log(Likelihood Ratio)
Test df
p-Value
1
16.30912223
4
0.00263122
2
10.70925199
2
0.00472624
3
0.200389036
1
0.65440701
4
1.123800961
2
0.57012452
Increased Incidence of Reduced Epididymal Luminal Sperm in Po Male Sprague Dawley
Rats After Oral Exposure to />-Isopropyltoluene for ~35 Days (ECHA, 2019b; Symrise,
2018).
The procedure outlined above for dichotomous data was applied to the data for reduced
epididymal luminal sperm in Po male Sprague Dawley rats orally exposed top-isopropyltoluene
for -35 days (ECHA. 2019b; Symrise. 2018). The BMD modeling results are summarized in
Table C-4 and Figure C-3. The Dichotomous Hill, Gamma, Log-Logistic, Multistage (degree 3
and 2), Weibull, Logistic, Log-Probit and Probit models provided adequate fit to the means
(/> value > 0.1). The BMDLs for the models providing adequate fit were sufficiently close
(differed by less than threefold), so the model with the lowest AIC (Log-Logistic) was selected.
For reduced epididymal luminal sperm, the BMDLioer of 20 mg/kg-day from this model was
selected.
106
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EPA 690 R-24 003F
Table C-4. BMD Modeling Results for Increased Incidence of Reduced
Epididymal Luminal Sperm in Po Male Sprague Dawley Rats Orally
Exposed to /7-Isopropyltoluene for ~35 Days3
Model
/>-Valueb
Scaled Residual
at Dose Nearest
BMD
AIC
BMDioer
(HED,
(mg/kg-d)
BMDLioer
(HED,
(mg/kg-d)
Dichotomous Hill
0.9999994
-0.000113059
12.00837362
26.76641
20.14922
Gamma
0.9876456
-0.148364038
12.2508008
24.56389
18.72623
Log-Logisticc
0.9999994
-0.000113087
12.00837331
26.76641
20.14922
Multistage 3
0.6691959
-0.680591644
14.60019152
18.5518
12.49361
Multistage 2
0.2254552
-1.117668833
19.1047269
13.24523
8.821077
Multistage 1
0.0170617
-0.000390256
27.83415841
5.442348
3.421249
Weibull
0.9999067
0.007002091
12.01795831
25.72946
0
Logistic
0.9999959
0.000240027
12.00928765
26.6336
18.55289
Log-Probit
0.9999998
3.73132E-09
14.00804908
26.91278
19.8688
Probit
0.9999998
1.14334E-06
14.00804939
26.63133
17.75566
Quantal Linear
0.0170617
-0.000390256
27.83415841
5.442347
3.421303
aSvmrise (2018).
bValues <0.10 fail to meet conventional goodness-of-fit criteria.
°Selected model (bold). Lowest AIC among models with adequate fit was selected (Hill).
AIC = Akaike's information criterion; BMD = maximum likelihood estimate of the dose associated with the
selected BMR; BMDL = 95% lower confidence limit on the BMD (subscripts denote BMR: i.e., 10ER = dose
associated with 10% extra risk from the control); BMR = benchmark response.
Frequentist Log-Logistic Model with BMR of 10% Extra Risk
for the BMD and 0.95 Lower Confidence Limit for the BMDL
i
0.8
E 0.6
o
+-»
to
3 0.4
0.2
0(£
-4
Figure C-3. Fit of Log-Logistic Model to Data for Reduced Epididymal Luminal Sperm
in Po Male Sprague Dawley Rats After Oral Exposure to />-Isopropyltoluene for
-35 Days (ECHA. 2019b: Svmrise. 2018)
107
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EPA 690 R-24 003F
BMD Model Output for Log-Logistic Model to Data for Reduced Epididymal Luminal
Sperm in Po Male Sprague Dawley Rats After Oral Exposure to />-Isopropyltoluene for
-35 Days (ECHA. 2019b: Svmrise. 2018)
Data
Reduced luminal sperm
Dose
N
Incidence
[Custom]
[Custom]
[Custom]
0
10
0
14
10
0
28
10
2
56
10
10
Model Results
Benchmark Dose
BMD
26.76641336
BMDL
20.14922027
BMDU
29.75486686
AIC
12.00837331
p-Value
0.999999449
D.O.F.
3
Chi2
0.000162429
Model Parameters
# of Parameters
3
Variable
Estimate
Std Error
Lower Conf
Upper Conf
g
Bounded
NA
NA
NA
a
-61.36588622
0.790507797
-62.915253
-59.816519
b
Bounded
NA
NA
NA
Goodness of Fit
Dose
Estimated Probability
Expected
Observed
Size
Scaled
Residual
0
1.52301E-08
1.52301E-07
0
10
-0.0003903
14
9.68989E-07
9.68989E-06
0
10
-0.0031129
28
0.200014305
2.000143049
2
10
-0.0001131
56
0.999984743
9.999847428
10
10
0.0123521
108
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EPA 690 R-24 003F
Analysis of Deviance
Model
Log Likelihood
# of Parameters
Deviance
Test df
p-Value
Full Model
-5.004024235
4
-
-
NA
Fitted Model
-5.004186657
1
0.00032484
3
0.9999984
Reduced Model
-24.43457208
1
38.8610957
3
<0.0001
Increased Incidence of Epididymal Sperm with Cribriform Changes in Po Male
Sprague Dawley Rats After Oral Exposure to />-Isopropyltoluene for ~35 Days (ECHA,
2019b; Symrise, 2018)
The procedure outlined above for dichotomous data was applied to the data for
epididymal sperm with cribriform changes in Po male Sprague Dawley rats orally exposed to
p-isopropyl toluene for -35 days (ECHA. 2019b; Symrise. 2018). The BMD modeling results are
summarized in Table C-5 and Figure C-4. All models provided adequate fit to the means
(/;-value > 0.1). The BMDLs for the models providing adequate fit were sufficiently close
(differed by less than threefold), so the model with the lowest AIC (Multistage Degree 3) was
selected. For epididymal sperm with cribriform changes, the BMDLioer of 14 mg/kg-day from
this model was selected.
Table C-5. BMD Modeling Results for Increased Incidence of Epididymal
Sperm with Cribriform Changes in Po Male Sprague Dawley Rats Orally
Exposed to /7-Isopropyltoluene for ~35 Days3
Model
/>-Valucb
Scaled Residual
at Dose Nearest
BMD
AIC
BMDioer
(HED,
(mg/kg-d)
BMDLioer
(HED,
(mg/kg-d)
Dichotomous Hill
0.9999975
5.60817E-06
24.36461322
28.00001
16.06515
Gamma
0.9545159
0.174487442
24.5156259
29.46519
15.36218
Log-Logistic
0.9429148
0.183751807
24.56091147
29.59281
15.43774
Multistage 3C
0.9860244
0.182964274
22.61622846
29.76698
14.03198
Multistage 2
0.8929701
-0.332772397
23.34733897
23.76345
12.22134
Multistage 1
0.431135
-1.041320469
26.10691469
14.32795
7.791204
Weibull
0.9265372
0.2231386
24.61371866
30.11822
14.91087
Logistic
0.8045946
0.45253127
24.9917569
33.31747
21.77432
Log-Probit
0.9755454
0.115685467
24.44800513
28.89839
16.02347
Probit
0.866618
0.363213801
24.78200744
31.66984
20.41978
Quantal Linear
0.431135
-1.041320478
26.10691469
14.32795
7.791332
aSvmrise (2018).
bValues <0.10 fail to meet conventional goodness-of-fit criteria.
°Selected model (bold). Lowest AIC among models with adequate fit was selected (Hill).
AIC = Akaike's information criterion; BMD = maximum likelihood estimate of the dose associated with the
selected BMR; BMDL = 95% lower confidence limit on the BMD (subscripts denote BMR: i.e., 10ER = dose
associated with 10% extra risk from the control); BMR = benchmark response.
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Frequentist Multistage Degree 3 Model with BMR of 10% Extra
Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL
i
0.9
0.8
0.7
£ 0.6
tt 0.5
3 0.4
0.3
0.2
0.1 -
0&
-4
Figure C-4. Fit of Multistage (Degree 3) Model to Data for Epididymal Sperm with
Cribriform Changes in Po Male Sprague Dawley Rats After Oral Exposure to
/7-Isopropyltoluene for ~35 Days (ECHA, 2019b; Symrise, 2018)
BMD Model Output for Multistage (Degree 3) Model to Data for Epididymal Sperm with
Cribriform Changes in Po Male Sprague Dawley Rats After Oral Exposure to
/7-Isopropyltoluene for ~35 Days (ECHA, 2019b; Symrise, 2018)
Data
Sperm with cribriform changes
Dose
N
Incidence
[Custom]
[Custom]
[Custom]
0
10
0
14
10
0
28
10
1
56
10
5
Estimated Probability
Response at BMD
— — Linear Extrapolation
O Data
BMD
BMDL
110
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Model Results
Benchmark Dose
BMD
29.7669847
BMDL
14.03198061
BMDU
38.44259094
AIC
22.61622846
p-Value
0.986024401
D.O.F.
3
Chi2
0.14438555
Slope Factor
0.007126578
Model Parameters
# of Parameters
4
Variable
Estimate
Std Error
Lower Conf
Upper Conf
g
Bounded
NA
NA
NA
bl
Bounded
NA
NA
NA
b2
Bounded
NA
NA
NA
b3
3.9946E-06
0.291299158
-0.57093187
0.57093986
Goodness of Fit
Dose
Estimated
Probability
Expected
Observed
Size
Scaled
Residual
0
1.523E-08
1.523E-07
0
10
-0.0003903
14
0.010901344
0.109013436
0
10
-0.3319863
28
0.083954719
0.839547191
1
10
0.1829643
56
0.504166826
5.04166826
5
10
-0.0263542
Analysis of Deviance
Model
Log Likelihood
# of Parameters
Deviance
Test df
p-Value
Full Model
-10.18230154
4
-
-
NA
Fitted Model
-10.30811423
1
0.25162538
3
0.9688539
Reduced Model
-16.90836351
1
13.4521239
3
0.0037542
Decreased Body Weight in Fi Female Sprague Dawley Rats on Postnatal Day (PND) 1
After Exposure to />-Isopropyltoluene During Gestation and Lactation until PND 13
(ECHA. 2019b: Svmrise. 2018)
The procedure outlined above for continuous data was applied to the data for decreased
body weight in Fi female Sprague Dawley rats on postnatal day (PND) 1 after exposure to
/Msopropyltoluene during gestation and lactation until PND 13 (ECHA. 2019b; Svmrise. 2018).
The constant variance model provided an adequate fit to the variance data. The Linear model
111
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provided adequate fit to the means. Visual inspection of the dose-response curve suggested
adequate fit, the BMDL was not 10 times lower than the lowest nonzero dose, and scaled
residual did not exceed ±2 units at the data point closest to the predefined BMR. Therefore, the
BMDLo 05rd value of 7.1 mg/kg-day was selected from this model. The results of the BMD
modeling are summarized in Table C-6. Figure C-5 shows the fit of the Linear model to the data.
Table C-6. BMD Modeling Results (Constant Variance) for Decreased Body
Weight in Fi Female Sprague Dawley Rats on PND 1 Exposed to
/7-Isopropyltoluene During Gestation and Lactation until PND 13a
Model
Variance
/>-Valucb
Means
/>-Valuec
Scaled
Residual at
Dose Nearest
BMD
AIC
BMDo.osrd
(HED,
(mg/kg-d)
BMDLo.osrd
(HED,
(mg/kg-d)
Exponential (model 3)d
0.09017585
NA
4.23952E-06
51.72503846
14.28686
6.722934
Exponential (model 5)d
0.09017585
NA
-3.56272E-07
53.72503846
14.27673
0.470149
Hill
0.09017585
NA
-3.96764E-07
53.72503846
14.256
0.0096
Polynomial (2-degree)e
0.09017585
NA
-0.034757938
51.72713056
14.79179
7.146885
Power6
0.09017585
NA
-0.000293854
51.72503862
14.30375
7.147386
Lineardf
0.09017585
1
-5.60327E-09
49.72503846
14.3
7.147205
aSvmrise (2018).
bValues <0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.1 fail to meet conventional goodness-of-fit criteria.
dPower restricted to be >1.
eCoefficients restricted to be positive.
'Selected model.
AIC = Akaike's information criterion; BMD = maximum likelihood estimate of the dose associated with the
selected BMR; BMDL = 95% lower confidence limit on the BMD (subscripts denote BMR: i.e., 0.05 RD = dose
associated with 5% relative deviation from the control); BMR = benchmark response; NA = test for fit is not valid;
HED = human equivalent dose; PND = postnatal day.
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Figure C-5. Fit of Linear Model to Data for Decreased Body Weight in Fi Female
Sprague Dawley Rats on Postnatal Day (PND 1) After Exposure to />-Isopropyltoluene
During Gestation and Lactation until PND 13 (ECHA, 2019b; Symrise, 2018)
BMD Model Output for Linear Model to Data for Decreased Body Weight in Fi Female
Sprague Dawley Rats on Postnatal Day (PND 1) After Exposure to />-Isopropyltoluene
During Gestation and Lactation until PND 13 (ECHA, 2019b; Symrise, 2018)
Data
Decreased body weight in Fi females on PND 1
Dose
N
Mean
Std. Dev.
HED
[Custom]
[Custom]
[Custom]
0
9
6.6
0.66
13
9
6.3
0.84
26
4
6
0.3
Model Results
Benchmark Dose
BMD
14.29999876
BMDL
7.147205466
BMDU
Infinity
AIC
49.72503846
Test 4 p-value
1
D.O.F.
1
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Model Parameters
#of
Parameters
3
Variable
Estimate
Std Error
Lower Conf
Upper Conf
g
6.599999989
0.202291962
6.20351503
6.99648495
beta
-0.023076922
1.46E-02
-0.05168652
0.00553268
alpha
0.427254548
5.50E-02
0.31938096
0.53512813
Goodness of Fit
Dose
Size
Estimated
Median
Calc'd
Median
Observed
Mean
Estimated
SD
Calc'd SD
Observed
SD
Scaled Residual
0
9
6.599999989
6.6
6.6
0.65364711
0.66
0.66
4.85877E-08
13
9
6.300000001
6.3
6.3
0.65364711
0.84
0.84
-5.60327E-09
26
4
6.000000013
6
6
0.65364711
0.3
0.3
-3.98628E-08
Likelihoods of Interest
Model
Log Likelihood*
# of Parameters
AIC
A1
-21.86251923
4
51.7250385
A2
-19.45652557
6
50.9130511
A3
-21.86251923
4
51.7250385
fitted
-21.86251923
3
49.7250385
R
-23.04602664
2
50.0920533
*Includes additive constant of -20.21665. This constant was not included in the LL derivation prior to BMDS 3.0.
Tests of Interest
Test
-2*Log(Likelihood Ratio)
Test df
p-Value
1
7.179002137
4
0.12672578
2
4.811987326
2
0.09017585
3
4.811987326
2
0.09017585
4
0
1
1
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APPENDIX D. PARAMETERS OF TOOLS USED FOR READ-ACROSS
Table D-l. Parameters of Tools Used for Read-Across Evaluation of p-Isopropyltoluene
Similarity Context
[569]a
Tool Name
[4]
Settings/Parameters
Searched by (date)
Structural [547]
U.S. EPA CompTox Chemicals
Dashboard [489]
Tanimoto similarity threshold of 0.8 and related substances
CASRN
(December 2021-
ChemlDplus [2]
ChemlDplus similarity search (default method) with >80% threshold and related
substances, parent (or exact structure match), salts, and mixturesb
February 2, 2022)
GenRA Beta version (in the U.S. EPA
CompTox Chemicals Dashboard) [56]
Collect 10 nearest neighbors by each similarity setting and combination
available:
• Morgan Fingerprints
• Torsion Fingerprints
• ToxPrints
• Morg2TorlBiol
• CTl:Bio3
Using each of the following data sources: ToxCast, Tox21, and ToxRef
OECD QSAR Toolbox [10]
Similarity search with >80% similarity threshold using default settings:
• Dice similarity
• Atom centered fragments
• Hologram calculation
• All features combined
• Atom characteristics: atom type, count H attached, and hybridization
QSAR Toolbox Profilers0
No settings or parameters; results obtained from:
• DART scheme
• DNA binding by OECD
SMILESd
(December 2021)
ToxAlerts0
No settings or parameters; structural alerts obtained from:
• Cytochrome P450-mediated drug metabolism alert
• Idiosyncratic toxicity (Arenes alert)
SMILESd
(December 2021)
Toxtree0
No settings or parameters; results obtained from:
• Protein binding
• DNA binding
SMILESd
(December 2021)
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Table D-l. Parameters of Tools Used for Read-Across Evaluation of p-Isopropyltoluene
Similarity Context
[569]a
Tool Name
[4]
Settings/Parameters
Searched by (date)
OECD QSAR Toolbox Metabolism
Simulators [22]
No settings or parameters; results obtained from:
• Rat liver S9 metabolism simulator version 3.7
• in vivo rat metabolism simulator version 3.5
SMILES6
(January 2022)
T oxicity/mechanistic
[0]
GenRA beta version (in the U.S. EPA
CompTox Chemicals Dashboard) [0]
Collected 10 nearest neighbors using the ToxCast similarity settings.
• Nearest neighbors with a similarity index >0.5 considered for use as analogue
CASRN
(February 2022)
Comparative Toxicogenomics Database
(CTD) [0]
Compounds identified with gene interactions similar to those induced by
p-isopropy ltoluene:
• Used the interacting genes comparison search
• A similarity index of >0.5 is considered for use as a mechanistic analogue
• The number of gene interactions is also considered for use as a mechanistic
analogue if similarity index is >0.5
aUnique analogues identified using analogue identification search tools.
bFor more information, see https://www.nlm.nih.gov/pubs/techbull/ma06/ma06 technote.html.
Tool used for candidate analogue evaluation.
dSMILES collected from the U.S. EPA CompTox Chemicals Dashboard batch search of the structural analogues CASRN.
ep-Isopropyltoluene SMILES: CC(C)C1=CC=C(C)C=C1 (CASRN: 99-87-6).
DART = developmental and reproductive toxicity; DNA = deoxyribonucleic acid; GenRA = General Read-Across; OECD = Organisation for Economic Co-operation
and Development; QSAR = quantitative structure-activity relationship; SMILES = simplified molecular input line entry system; U.S. EPA = U.S. Enviromnental
Protection Agency.
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