AEPA
United States
Environmental Protection
Agency
EPA/690/R-21/001F | March 2021 | FINAL
Provisional Peer-Reviewed Toxicity Values for
frans-Crotonaldehyde
(CASRN 123-73-9)
U.S. EPA Office of Research and Development
Center for Public Health and Environmental Assessment
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Environmental Protection
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EPA/690/R-21/001F
March 2021
https://www.epa.gov/pprtv
Provisional Peer-Reviewed Toxicity Values for
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AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGER
Jeffry L. Dean II, 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
J. Andre Weaver, PhD
Center for Public Health and Environmental Assessment, Research Triangle Park, NC
J. Phillip Kaiser, PhD, DABT
Center for Public Health and Environmental Assessment, Cincinnati, OH
PRIMARY EXTERNAL REVIEWERS
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
J. Phillip Kaiser, PhD, DABT
Center for Public Health and Environmental Assessment, Cincinnati, OH
Questions regarding the content of this PPRTV assessment should 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
/ra/7.s-Crotonaldehyde
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TABLE OF CONTENTS
COMMONLY USED ABBREVIATIONS AND ACRONYMS v
BACKGROUND 1
QUALITY ASSURANCE 1
DISCLAIMERS 2
QUESTIONS REGARDING PPRTVs 2
INTRODUCTION 3
METHODS 7
Literature Search 7
Screening Process 7
LITERATURE SEARCH AND SCREENING RESULTS 8
REVIEW OF POTENTIALLY RELEVANT DATA (NONCANCER AND CANCER) 9
HUMAN STUDIES 13
ANIMAL STUDIES 13
Oral Exposures 13
Inhalation Exposures 18
OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS) 18
Genotoxicity Studies 18
Supporting Animal Toxicity Studies 25
Absorption, Distribution, Metabolism, and Excretion Studies 30
Mode-of-Action/Mechanistic Studies 31
DERIVATION 01 PROVISIONAL VALUES 32
DERIVATION OF PROVISIONAL ORAL REFERENCE DOSES 32
Derivation of a Subchronic Provisional Reference Dose 32
Derivation of a Chronic Provisional Reference Dose 35
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS 37
Summary ofNoncancer Provisional Reference Values 37
PROVISIONAL CARCINOGENICITY ASSESSMENT 38
APPENDIX A. LITERATURE SEARCH STRATEGY 39
APPENDIX B. DETAILED PECO CRITERIA 41
APPENDIX C. SCREENING PROVISIONAL VALUES 42
APPENDIX D. DATA TABLES 43
APPENDIX E. BENCHMARK DOSE MODELING RESULTS 49
APPENDIX F. REFERENCES 58
iv
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COMMONLY USED ABBREVIATIONS AND ACRONYMS
a2u-g
alpha 2u-globulin
LC50
median lethal concentration
ACGIH
American Conference of Governmental
LD50
median lethal dose
Industrial Hygienists
LOAEL
lowest-observed-adverse-effect level
AIC
Akaike's information criterion
MN
micronuclei
ALD
approximate lethal dosage
MNPCE
micronucleated polychromatic
ALT
alanine aminotransferase
erythrocyte
AR
androgen receptor
MOA
mode of action
AST
aspartate aminotransferase
MTD
maximum tolerated dose
atm
atmosphere
NAG
7V-acetyl-P-D-glucosaminidase
ATSDR
Agency for Toxic Substances and
NCI
National Cancer Institute
Disease Registry
NOAEL
no-observed-adverse-effect level
BMC
benchmark concentration
NTP
National Toxicology Program
BMCL
benchmark concentration lower
NZW
New Zealand White (rabbit breed)
confidence limit
OCT
ornithine carbamoyl transferase
BMD
benchmark dose
ORD
Office of Research and Development
BMDL
benchmark dose lower confidence limit
PBPK
physiologically based pharmacokinetic
BMDS
Benchmark Dose Software
PCNA
proliferating cell nuclear antigen
BMR
benchmark response
PND
postnatal day
BUN
blood urea nitrogen
POD
point of departure
BW
body weight
PODadj
duration-adjusted POD
CA
chromosomal aberration
QSAR
quantitative structure-activity
CAS
Chemical Abstracts Service
relationship
CASRN
Chemical Abstracts Service registry
RBC
red blood cell
number
RDS
replicative DNA synthesis
CBI
covalent binding index
RfC
inhalation reference concentration
CHO
Chinese hamster ovary (cell line cells)
RfD
oral reference dose
CL
confidence limit
RGDR
regional gas dose ratio
CNS
central nervous system
RNA
ribonucleic acid
CPHEA
Center for Public Health and
SAR
structure activity relationship
Environmental Assessment
SCE
sister chromatid exchange
CPN
chronic progressive nephropathy
SD
standard deviation
CYP450
cytochrome P450
SDH
sorbitol dehydrogenase
DAF
dosimetric adjustment factor
SE
standard error
DEN
diethylnitrosamine
SGOT
serum glutamic oxaloacetic
DMSO
dimethylsulfoxide
transaminase, also known as AST
DNA
deoxyribonucleic acid
SGPT
serum glutamic pyruvic transaminase,
EPA
Environmental Protection Agency
also known as ALT
ER
estrogen receptor
SSD
systemic scleroderma
FDA
Food and Drug Administration
TCA
trichloroacetic acid
FEVi
forced expiratory volume of 1 second
TCE
trichloroethylene
GD
gestation day
TWA
time-weighted average
GDH
glutamate dehydrogenase
UF
uncertainty factor
GGT
y-glutamyl transferase
UFa
interspecies uncertainty factor
GSH
glutathione
UFC
composite uncertainty factor
GST
glutathione-S-transferase
UFd
database uncertainty factor
Hb/g-A
animal blood-gas partition coefficient
UFh
intraspecies uncertainty factor
Hb/g-H
human blood-gas partition coefficient
UFl
LOAEL-to-NOAEL uncertainty factor
HEC
human equivalent concentration
UFS
subchronic-to-chronic uncertainty factor
HED
human equivalent dose
U.S.
United States of America
i.p.
intraperitoneal
WBC
white blood cell
IRIS
Integrated Risk Information System
IVF
in vitro fertilization
Abbreviations and acronyms not listed on this page are defined upon first use in the
PPRTV document.
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PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR
m4A«-CROTONALDEHYDE (CASRN 123-73-9)
BACKGROUND
A Provisional Peer-Reviewed Toxicity Value (PPRTV) is defined as a toxicity value
derived for use in the Superfund Program. PPRTVs are derived after a review of the relevant
scientific literature using established Agency guidance on human health toxicity value
derivations.
The purpose of this document is to provide support for the hazard and dose-response
assessment pertaining to chronic and subchronic exposures to substances of concern, to present
the major conclusions reached in the hazard identification and derivation of the PPRTVs, and to
characterize the overall confidence in these conclusions and toxicity values. It is not intended to
be a comprehensive treatise on the chemical or toxicological nature of this substance.
Currently available PPRTV assessments can be accessed on the U.S. Environmental
Protection Agency's (EPA's) PPRTV website at https://www.epa.gov/pprtv. PPRTV
assessments are eligible to be updated on a 5-year cycle and revised as appropriate to incorporate
new data or methodologies that might impact the toxicity values or affect the characterization of
the chemical's potential for causing adverse human-health effects. Questions regarding
nomination of chemicals for update can be sent to the appropriate U.S. EPA Superfund and
Technology Liaison (https://www.epa.gov/research/fact-sheets-regional-science).
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 was written
with guidance from the CPHEA Program Quality Assurance Project Plan (PQAPP), the QAPP
titled Program Quality Assurance Project Plan (PQAPP) for the Provisional Peer-Reviewed
Toxicity Values (PPRTVs) and Related Assessments/Documents (L-CPAD-0032718-QP), and the
PPRTV development contractor QAPP titled Quality Assurance Project Plan—Preparation of
Provisional Toxicity Value (PTV) Documents (L-CPAD-0031971-QP). 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 Center for Public
Health and Environmental Assessment (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 adverse effects
of the chemical and the evidence on which the value is based, including the strengths and
limitations of the data. All users are advised to review the information provided in this
document to ensure that the PPRTV used is appropriate for the types of exposures and
circumstances at the site in question and the risk management decision that would be supported
by the risk assessment.
Other U.S. EPA programs or external parties who may choose to use PPRTVs are
advised that Superfund resources will not generally be used to respond to challenges, if any, of
PPRTVs used in a context outside of the Superfund program.
This document has been reviewed in accordance with U.S. EPA policy and approved for
publication. Mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
QUESTIONS REGARDING PPRTVS
Questions regarding the content of this PPRTV assessment should be directed to the
U.S. EPA Office of Research and Development (ORD) CPHEA website at
https://ecomments.epa.gov/pprtv.
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INTRODUCTION
/ra/7.s-Crotonal dehyde, CASRN 123-73-9, is an a,P-unsaturated aldehyde with defined
stereochemistry (WHO, 2008). The commercial product crotonaldehyde is represented by
CASRN 4170-30-3 and consists of >95% trans-isomer (CASRN 123-73-9). c/s-Crotonaldehyde
is represented by CASRN 15798-64-8. ^ram,-Crotonaldehyde is primarily used in chemical
manufacturing as a precursor, intermediate, or solvent (WHO. 2008; HSDB. 2005). It is
synthesized by aldol condensation of acetaldehyde and a dehydration step (HSDB, 2005).
Commercial crotonaldehyde and /ra/7.s-crotonaldehyde are both listed on the U.S. EPA's Toxic
Substances Control Act (TSCA) public inventory (U.S. HP A. 2018b). and crotonaldehyde is
registered with Europe's Registration, Evaluation, Authorisation and Restriction of Chemicals
(REACH) program (ECHA. 2018).
The empirical formula for /ra//.s-crotonaldehyde is C4H6O (see Figure 1). Its
physicochemical properties are shown in Table 1. /ra/7.s-Crotonaldehyde is a reactive, clear
liquid, with high water solubility. In the air, it will exist in the vapor phase based on its vapor
pressure of 30.0 mm Hg. /ra//.s-Crotonaldehyde will be degraded in the atmosphere by reacting
with photochemically produced hydroxyl radicals. Direct photolysis of /ra//.s-crotonaldehyde in
the atmosphere does not occur [BUA (1993) as cited in WHO (2018)1. Volatilization of
/ra/7.s-crotonaldehyde from dry soil surfaces is expected based on the compound's vapor
pressure, and moderate volatilization from water or moist soil surfaces is expected based on its
estimated Henry's law constant of 1.45 x 10 5 atm-m3/mole. The estimated Koc for
/ra/7.s-crotonaldehyde indicates potential for mobility in soil and negligible potential to adsorb to
suspended solids and sediment in aquatic environments; however, /ra/7.s-crotonaldehyde
polymerizes readily and may react when released in the environment (HSDB, 2005). Hydrolysis
is not expected to be an important fate process under environmental conditions, although
/ra//.v-crotonaldehyde will undergo hydrolysis in low or high pH conditions (WHO, 2018). In
dilute acid aqueous solutions, /ra/7.s-crotonaldehyde reversibly hydrates to form the aldol,
3-hydroxybutanal (ECHA. 2018).
trans-Crotonaldehyde
c/.v-Crotonal dehyde
Figure 1. trans- (CASRN 123-73-9) and
cis-Crotonaldehyde (CASRN 15798-64-8) Structures
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Table 1. Physicochemical Properties of frans-Crotonaldehyde (CASRN 123-73-9) and
Commercial Crotonaldehyde (>95% trans-; CASRN 4170-30-3)
Property (unit)
trim \- C ro t o n a 1 d e hyd e Value3
Commercial Crotonaldehyde Value3
Physical state
Liquid
Liquid
Boiling point (°C)
103
103 (predicted average)
Melting point (°C)
-75.8
-76.0 (predicted average)
Density (g/cm3)
0.845 (predicted average)
0.820 (predicted average)
Vapor pressure (mm Hg)
30.0
30.9 (predicted average)
pH (unitless)
NA
NA
pKa (unitless)
NA
NA
Solubility in water (mol/L)
2.09
4.54 (predicted average)
Octanol-water partition constant
(log Kow)
0.573 (predicted average)
0.564 (predicted average)
Henry's law constant (atm-m3/mol)
1.45 x io~5 (predicted average)
1.48 x 10 " (predicted average)
Soil adsorption coefficient Koc (L/kg)
10.7 (predicted average)
10.7 (predicted average)
Atmospheric OH rate constant
(cm3/molecule-sec)
3.61 x 10-11
3.61 x io~n (predicted average)
Molecular weight (g/mol)
70.091
70.091
Flash point (°C)
12.1
4.58 (predicted average)
aData were extracted from the U.S. EPA CompTox Chemicals Dashboard (/raws-crotonaldehyde,
CASRN 123-73-9; https://comptox.epa.gOv/dashboard/dsstoxdb/results7searcliFDTXSID6020351#properties:
accessed February 4, 2021 and commercial crotonaldehyde, CASRN 15798-64-8; DTXSID 6020351). All values
are experimental averages unless otherwise specified.
NA = not applicable; SMILES = simplified molecular-input line-entry system.
A summary of available toxicity values for trans-crotonaldehyde from U.S. EPA and
other agencies/organizations is provided in Table 2. Toxicity values for commercial
crotonaldehyde (>95% trans-isomer) are also included in Table 2. A 2010 PPRTV assessment
from the U.S. EPA was previously available for "crotonaldehyde." The assessment herein
provides an updated evaluation of /ra//.s-crotonaldehyde based on recent scientific literature and
current PPRTV assessment practices. Information pertaining to commercial crotonaldehyde
mixtures (CASRN 15798-64-8) was also considered as this mixture is defined as containing at
least 95% /ra/zs-crotonal dehyde isomer. To promote evaluation of the trans-isomer of
crotonaldehyde in the context of human health, studies using unspecified isomers, or other
isomers of crotonaldehyde (i.e., cis-) are mentioned only for comparison, where necessary.
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Table 2. Summary of Available Toxicity Values for
fraws-Crotonaldehyde (CASRN 123-73-9) and
Commercial Crotonaldehyde (>95% trans-; CASRN 4170-30-3)
Source
(parameter)3'b
Value
(applicability)
Compound(s)
Notes
Reference0
Noncancer
IRIS
NV
NA
NA
U.S. EPA (2020)
HEAST
NV
NA
NA
U.S. EPA (2011a)
DWSHA
NV
NA
NA
U.S. EPA (2018a)
ATSDR
NV
NA
NA
ATSDR (2019)
IPCS
NV
NA
NA
IPCS (2020)
CalEPA
NV
NA
NA
CalEPA (2019)
OSHA (PEL)
2 ppm
123-73-9
4170-30-3
8-hr TWA for general industry,
construction, and shipyard
employment
OSHA (2018a):
OSHA (2018b):
OSHA (2020)
NIOSH (REL)
2 ppm
123-73-9
4170-30-3
TWA for up to a 10-hr workday
during a 40-hr workweek
NIOSH (2016):
NIOSH (1994)
NIOSH (IDLH)
50 ppm
123-73-9
Based on acute inhalation toxicity
data in humans and animals
NIOSH (1994)
ACGIH
(TLV-ceiling)
0.3 ppm
4170-30-3
Concentration that should not be
exceeded during any part of the
working exposure; based on eye
and upper respiratory tract
irritation; skin notation
ACGIH (2018)
AEGL (AEGL 1)
10 min: 0.19 ppm
30 min: 0.19 ppm
60 min: 0.19 ppm
4 hr: 0.19 ppm
8 hr: 0.19 ppm
123-73-9
4170-30-3
Based on mild eye irritation in
humans
U.S. EPA (2016):
NRC (2008)
AEGL (AEGL 2)
10 min: 27 ppm
30 min: 8.9 ppm
60 min: 4.4 ppm
4 hr: 1.1 ppm
8 hr: 0.56 ppm
123-73-9
4170-30-3
Based on impaired pulmonary
function in rats
U.S. EPA (2016):
NRC (2008)
AEGL (AEGL 3)
10 min: 44 ppm
30 min: 27 ppm
60 min: 14 ppm
4 hr: 2.6 ppm
8 hr: 1.5 ppm
123-73-9
4170-30-3
Based on lethality in rats
U.S. EPA (2016):
NRC (2008)
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Table 2. Summary of Available Toxicity Values for
fraws-Crotonaldehyde (CASRN 123-73-9) and
Commercial Crotonaldehyde (>95% trans-; CASRN 4170-30-3)
Source
(parameter)3'b
Value
(applicability)
Compound(s)
Notes
Reference0
Cancer
IRIS (WOE)
Classification C:
possibly carcinogenic
to humans
123-73-9
Based on an increased incidence of
hepatocellular carcinomas and
hepatic neoplastic nodules
(combined) in male rats in an oral
chronic study
U.S. EPA (2005)
HEAST (OSF)
1.9 (mg/kg-d) 1
123-73-9
Based on liver tumors in rats in an
oral chronic study
U.S. EPA (2011a)
DWSHA
NV
NA
NA
U.S. EPA (2018a)
NTP
NV
NA
NA
NTP (2016)
IARC
Group 3: not
classifiable as to its
carcinogenicity to
humans
4170-30-3
Based on inadequate evidence in
human and experimental animals
IARC (1995)
CalFPA
NV
NA
NA
CalEPA (2019)
ACGIH (WOE)
A3: confirmed animal
carcinogen with
unknown relevance to
humans
4170-30-3
Based on induction of
hepatocellular carcinomas and
neoplastic nodules in rats in an oral
chronic study
ACGIH (2018)
aSources: ACGIH = American Conference of Governmental Industrial Hygienists; ATSDR = Agency for Toxic
Substances and Disease Registry; CalEPA = California Environmental Protection Agency; DWSHA = Drinking
Water Standards and Health Advisories; HEAST = Health Effects Assessment Summary Tables;
IARC = International Agency for Research on Cancer; IPCS = International Programme on Chemical Safety;
IRIS = Integrated Risk Information System; NIOSH = National Institute for Occupational Safety and Health;
NTP = National Toxicology Program; OSHA = Occupational Safety and Health Administration.
Parameters: AEGL = acute exposure guideline level; IDLH = immediately dangerous to life or health
concentrations; OSF = oral slope factor; PEL = permissible exposure limit; REL = recommended exposure limit;
TLV = threshold limit value; TWA = time-weighted average; WOE = weight of evidence.
°Reference date is the publication date for the database and not the date the source was accessed.
NA = not applicable; NV = not available.
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METHODS
Literature Search
Four online scientific databases (PubMed, Web of Science [WOS], TOXLINE, and Toxic
Substances Control Act Test Submissions [TSCATS] via TOXLINE) were searched by
U.S. EPA's Health and Environmental Research Online (HERO) staff and stored in the HERO
database.1 The literature search focused on chemical name and synonyms (identified as
"valid/validated" or "good" via the U.S. EPA's Chemistry Dashboard2 and ChemSpider3) with
no limitations on publication type, evidence stream (i.e., human, animal, in vitro, in silico), or
health outcomes. Full details of the search strategy for each database are presented in
Appendix A. The initial database searches were conducted in May 2019 and updated in
July 2020.
Screening Process
Two screeners independently conducted a title and abstract screen of the search results
using DistillerSR4 to identify study records that met the Population, Exposure, Comparator,
Outcome (PECO) eligibility criteria (see Appendix B for a more detailed summary):
• Population: Humans, laboratory mammals, and other animal models of established
relevance to human health (e.g., Xenopus embryos); mammalian organs, tissues, and
cell lines; and bacterial and eukaryote models of genetic toxicity.
• Exposure: In vivo (all routes), ex vivo, and in vitro exposure to the chemical of
interest, including mixtures to which the chemical of interest may contribute
significantly to exposure or observed effects.
• Comparison: Any comparison (across dose, duration, or route) or no comparison
(e.g., case reports without controls).
• Outcome: Any endpoint suggestive of a toxic effect on any bodily system or
mechanistic change associated with such effects. Any endpoint relating to disposition
of the chemical within the body.
Records that were not excluded based on title and abstract screening advanced to full-text
review using the same PECO eligibility criteria. Studies that have not undergone peer review
were included if the information could be made public and sufficient details of study methods
and findings were included in the report. Full-text copies of potentially relevant records
identified from title and abstract screening were retrieved, stored in the HERO database, and
independently assessed by two screeners using DistillerSR to confirm eligibility. At both
title/abstract and full-text review levels, screening conflicts were resolved by discussion between
the primary screeners with consultation by a third reviewer to resolve any remaining
disagreements. During title/abstract or full-text level screening, studies that were not directly
relevant to the PECO, but could provide supplemental information, were categorized (or
"tagged") relative to the type of supplemental information they provided (e.g., review,
commentary, or letter with no original data; conference abstract; toxicokinetics and mechanistic
1 U.S. EPA's HERO database provides access to the scientific literature supporting U.S. EPA science assessments.
The database includes more than 2,500,000 scientific references and data from the peer-reviewed literature used by
U.S. EPA to develop its regulations.
2U.S. EPA's Chemistry Dashboard: https://comptox.epa.gov/dashboard/dsstoxdb/results?search= 123-73-9.
'ChemSpider: http://www.chemspider. com/Chemical-Structure. 394562.html?rid=330d45ef-b664-44c4-b920-
67d738e55957.
4DistillerSR, a web-based systematic review software used to screen studies, is available at
https://www.evidencepartners.com/products/distillersr-svstematic-review-softwaiB.
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information aside from in vitro genotoxicity studies; studies on routes of exposure other than oral
and inhalation; acute exposure studies only; etc.). Conflict resolution was not required during
the screening process to identify supplemental information (i.e., tagging by a single screener was
sufficient to identify the study as potential supplemental information).
LITERATURE SEARCH AND SCREENING RESULTS
The database searches yielded 992 unique records. Of the 992 studies identified,
818 were excluded during title and abstract screening, 174 were reviewed at the full-text level,
and 49 were considered relevant to the PECO eligibility criteria (see Figure 2). This included
2 human health effect studies, 22 in vivo animal studies, and 12 in vitro genotoxicity studies.
Thirteen additional studies were tagged for inclusion as "supplemental/other." The detailed
search approach, including the query strings and PECO criteria, are provided in Appendix A and
Appendix B, respectively.
trans-Crotonaldehyde (CASRN123-73-9)
Literature Searches (through June 2020)
f
PubMed
(/? = 588)
t
TITLE AND ABSTRACT SCREENING
WOS
(n = 542)
TOXLINE
(n = 356)
Other
(n = 87)
Title & Abstract Screening
(992 records after duplicate removal)
Excluded (n= S18)
Not relevant to PECO (/i = 818)
FULL-TEXT SCREENING
Full-Text Screening
(n = 174)
J
Studies Considered Further (n = 36)
Human health effect studies (n = 2)
Animal health effect studies (n= 22)
Genotoxicity studies (n = 12)
Excluded (n - 125)
Not relevant to PECO (n = 125)
Tagged as Supplemental/Other (n- 13)
Other routes of exposure besides oral and
inhalation (n= 12), acute toxicity studies
(n = 0), mechanistic studies (in vitro or in
vivo; n = 0), ADME/PBPK studies (n = 0),
review articles in = 1)
Figure 2. Literature Search and Screening Flow Diagram for
frwf.v-Crotonaldehyde (CASRN 123-73-9)
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REVIEW OF POTENTIALLY RELEVANT DATA
(NONCANCER AND CANCER)
Tables 3A and 3B provide overviews of the relevant noncancer and cancer evidence
bases, respectively, for /ra//.s-crotonaldehyde and commercial crotonaldehyde, and include all
potentially relevant repeated short-term, subchronic, and chronic studies, as well as reproductive
and developmental toxicity studies. Principal studies are identified in bold. The phrase
"statistical significance" and the term "significant," used throughout the document, indicates a
p-walue of < 0.05 unless otherwise specified.
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Table 3A. Summary of Potentially Relevant Noncancer Data for frans-Crotonaldehyde (CASRN 123-73-9)
and Commercial Crotonaldehyde (>95% trans-; CASRN 4170-30-3)
Category"
Number of Male/Female, Strain,
Species, Study Type, Reported
Doses, Study Duration
Dosimetryb
Critical Effects
NOAELb
LOAELb
Reference
(comments)
Notes0
Human
ND
Animal
1. Oral (mg/kg-d)
Short term
5 M/5 F, S-D albino rat, diet, 14 d
Reported target doses: 0, 22, 44, 88,
or 175 mg commercial
crotonaldehyde/kg-d
M: 0, 19, 36, 73,
139
F: 0, 17, 36, 68,
136
No exposure-related changes in
clinical signs, body weight,
organ weight, or gross necropsy
139
NDr
Borriston (1980a)
NPR
Subchronic
10 M/10 F, F344 rat, gavage in corn
oil, 5 d/wk, 13 wk
Reported doses:
0, 2.5, 5,10, 20, or 40 mg
commercial crotonaldehyde/kg-d
0,1.8, 4, 7.1,14,
29
Epithelial hyperplasia of the
forestomach in male and
female rats. Thickened
forestomach in female rats.
Decreased absolute and
relative thymus weight in
female rats at 13 wk
7.1
14
Hazleton
NPR,
PS
Laboratories
(1986b): NTP-PWG
(1987)
Subchronic
10 M/10 F, B6C3F1 mice, gavage in
corn oil, 5 d/wk, 13 wk
Reported doses:
0,2.5, 5, 10, 20, or 40 mg
commercial crotonaldehyde/kg-d
0, 1.8, 4,7.1, 14,
29
Epithelial hyperplasia of the
forestomach (forestomach
thickening and nodules) in male
and female mice
NDr
NDr
Hazelton Laboratories
(1986a) as
summarized bv NTP-
PWG (1987)
NPR
10
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Table 3A. Summary of Potentially Relevant Noncancer Data for frans-Crotonaldehyde (CASRN 123-73-9)
and Commercial Crotonaldehyde (>95% trans-; CASRN 4170-30-3)
Category"
Number of Male/Female, Strain,
Species, Study Type, Reported
Doses, Study Duration
Dosimetryb
Critical Effects
NOAELb
LOAELb
Reference
(comments)
Notes0
Reproductive/
Developmental
20 M/20 F, F344 rat, gavage in corn
oil, 9 wk (males exposed 61 d prior to
mating and during 7-d mating period;
females exposed 30 d prior to mating,
through mating and gestation to
PND 5)
Reported doses:
0, 2.5, 5, or 10 mg commercial
crotonaldehyde/kg-d
0, 2.5, 5, 10
No adverse reproductive or
developmental effects
10
NDr
Hazleton Laboratories
(1987)
NPR
2. Inhalation (mg/m3)
ND
aDuration categories are defined as follows: acute = exposure for <24 hours; short term = repeated exposure for 24 hours to <30 days; long term (subchronic) = repeated
exposure for >30 days <10% lifespan for humans (>30 days up to approximately 90 days in typically used laboratory animal species); and chronic = repeated exposure
for >10% lifespan for humans (>~90 days to 2 years in typically used laboratory animal species) (U.S. EPA. 2002).
bDosimetry: Doses are presented as ADDs (mg/kg-day) for oral noncancer effects and as HECs (mg/m3) for inhalation noncancer effects. ADD (mg/kg-day) = reported
dose x (days treated per week/7 days per week).
°Notes: NPR = not peer reviewed; PS = principal study.
ADD = adjusted daily dose; F = female(s); HEC = human equivalent concentration; LOAEL = lowest-observed-adverse-effect level; M = male(s); ND = no data;
NDr = not determined; NOAEL = no-observed-adverse-effect level; PND = postnatal day; S-D = Sprague-Dawley.
11
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Table 3B. Summary of Potentially Relevant Cancer Data for frans-Crotonaldehyde (CASRN 123-73-9)
Number of Male/Female,
Strain, Species, Study Type,
Reported Doses, Study
Reference
Category
Duration
Dosimetry3
Critical Effects
(comments)
Notesb
Human
ND
Animal
1. Oral (mg/kg-d)
Carcinogenicity
23-27 M, F344 rat, drinking
0, 0.6, 4.6
Elevated incidence of liver neoplastic
Chung etal. (1986)
PR,
water, 113 wk
nodules at low dose, but not high dose
IRIS
Reported doses: 0, 0.6, or
6.0 mM trans-crotona ldehvde
2. Inhalation (mg/m3)
ND
aDosimetry: Oral exposures are expressed as HEDs (mg/kg-day); HEDs are calculated using DAFs. as recommended by U.S. EPA (2011b):
HED = ADD (mg/kg-day) x DAF. The DAF is calculated as follows: DAF = (BWa ^ BWh)1/4, where BWa = animal body weight and BWh = human body weight, using
study body-weight values for BWa and the reference value of 70 kg for BWh.
bNotes: IRIS = used by IRIS (U.S. EPA. 2005): PR = peer reviewed.
ADD = adjusted daily dose; BW = body weight; DAF = dosimetric adjustment factor; HED = human equivalent dose; IRIS = Integrated Risk Information System;
M = male(s); ND = no data.
12
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HUMAN STUDIES
Crotonaldehyde is irritating to the skin, eyes, and mucous membranes (ATSDR. 2014;
WHO. 2008; ATSDR, 2002; I ARC. 1995). Sensitization reactions have been reported in humans
following repeated skin exposure to commercial crotonaldehyde (Mellon Institute of Industrial
Research. 1942). Irritation of the eyes, mucous membranes, and respiratory tract were reported
in a small number of workers exposed to crotonaldehyde (isomer not specified [NS]), along with
several other chemicals, during the incineration of polypropylene syringes (Mehta and Liveright.
1986). Headache and irritation of the eyes, nose, and throat were also reported in the majority of
workers exposed to crotonaldehyde (NS) (along with several other chemicals) at a small printing
and finishing company; some workers also reported difficulty breathing, wheezing, and nausea
(Rosen steel and Tanaka. 1976). The measured levels of unspecified isomers of crotanaldehyde
ranged from 0.7 to 2.1 mg/m3. However, due to multiple chemical exposures, including other
known irritants such as formaldehyde, the potential irritative effects of //vmv-crotonal dehyde
cannot be adequately assessed in either of these health surveys.
Concern for increased cancer incidence in workers from an aldehyde factory was reported
in an English-language abstract of a German-language study by Bittersohl (1974). Evaluation of
this study by WHO (2008) indicated that no conclusions regarding the carcinogenicity of
crotonaldehyde (NS) could be made from this study because all workers were smokers and were
exposed to several different aldehydes. IARC (1995) also concluded that the data from this
study were too sparse to be conclusive.
In a study examining the role of oxidative stress in the etiology of Alzheimer's disease,
Kawaguchi-Niida et al. (2006) used immunohistochemical analysis to measure levels of
protein-bound /ra/7.s-crotonal dehyde in hippocampi obtained at autopsy of Alzheimer's disease
patients and age-matched controls. Intracellularly, crotonaldehyde (NS) is formed during lipid
peroxidation and reacts with proteins to form stable adducts with nucleic acids (WHO. 2008). In
Alzheimer's disease patients, statistically significant (p < 0.01) higher levels of protein-bound
/ra/7.s-crotonaldehyde were observed when compared with age-matched controls. In addition, the
protein-bound crotonaldehyde was localized in reactive astrocytes and microglia around senile
plaques in Alzheimer's patients.
ANIMAL STUDIES
Oral Exposures
Short-Term Studies
Borriston (1980a)
In a non-peer-reviewed study, groups of albino Sprague-Dawley (S-D) rats (5/sex/group)
were exposed to commercial crotonaldehyde (purity not reported) in the diet at target doses of 0,
22, 44, 88, or 175 mg/kg-day for 14 days (Borriston. 1980a). Daily observations and
measurements of food consumption were performed, and body weight was recorded weekly.
Based on food consumption and body-weight data, the study authors calculated doses of 19 ± 4,
36 ± 7, 73 ± 14, and 139 ± 21 mg/kg-day in males, and 17 ± 4, 36 ± 7, 68 ± 11, and
136 ± 27 mg/kg-day in females. At sacrifice, all animals were subjected to gross necropsy, and
the liver and kidney weights were recorded. No organs were examined microscopically.
As described by the study authors, all animals survived until scheduled sacrifice. No
clinical signs of toxicity were observed. Body weights, food consumption, and organ weights
were comparable among groups. No exposure-related changes were observed at gross necropsy.
13
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The study authors identified a no-observed-adverse-effect level (NOAEL) of
139 mg/kg-day based on a lack of exposure-related effects. A lowest-observed-adverse-effect
level (LOAEL) was not identified.
Subchronic Studies
Hazleton Laboratories (1986b); NTP-PWG (1987)
In a non-peer-reviewed study, groups of F344 rats (10/sex/group) were administered
commercial crotonaldehyde (CASRN 4170-30-3) at doses of 0, 2.5, 5, 10, 20, or 40 mg/kg-day
via gavage in corn oil, 5 days/week, for up to 13 weeks (Hazleton Laboratories. 1986b).
Adjusted daily doses (ADDs)5 calculated for this review were 0, 1.8, 4, 7.1, 14, or 29 mg/kg-day.
The animals were subjected to twice daily mortality/morbidity checks; food consumption and
body weight were recorded weekly. Blood samples were collected on Days 4, 16, and at the
beginning of Week 13 for hematology (hemoglobin [Hb], hematocrit [Hct], red blood cell [RBC]
count, mean cell volume [MCV], mean cell hemoglobin [MCH], mean cell hemoglobin
concentration [MCHC], total and differential white blood cell [WBC] count, platelet count, and
RBC, WBC, and platelet morphology) and serum chemistry (sorbitol dehydrogenase [SDH],
gamma glutamyl transferase [GGT], alanine aminotransferase [ALT], alkaline phosphatase
[ALP], blood urea nitrogen [BUN], and creatinine) analyses. The report suggested that urine
samples were also collected but analysis of the samples was not discussed. An assessment of
sperm morphology and vaginal cytology was performed at the end of the study on animals in the
three lower dose groups and control animals. At sacrifice, all animals were subjected to gross
necropsy, and the brain, heart, liver, right kidney, lung, and thymus were weighed. A complete
histopathological examination was performed on all gross lesions and tissue masses, all control
rats, all rats in the highest dose group with at least 60% survivors at time of sacrifice, and all rats
in the higher dose groups in which death occurred prior to study termination. Target organs
(nasal cavities and forestomach) from all rats in all groups were also examined microscopically.
The National Toxicology Program (NTP) convened a Pathology Working Group (PWG) (NTP-
PWG. 1987) to review selected data and slides from this study.
Early deaths (death prior to cessation of the study) occurred at the following incidences in
the control through highest dose groups: 0/10, 0/10, 0/10, 3/10, 3/10, and 5/10 for males, and
1/10, 0/10, 1/10, 1/10, 7/10, and 5/10 for females. The NTP-PWG (1987) concluded that nearly
all early deaths were associated with gavage trauma and/or oil in the lungs, and that the early
deaths should not be used as criteria for selecting doses for a chronic study. The study authors
reported that the highest dose males showed a decrease in body-weight gain from Week 11-13
(data not shown in the original report) and a statistically significant 9% decrease in terminal
body weight, compared to control. Terminal body weights were comparable to control in other
male groups and in females (see Table D-l and Table D-2). The study authors indicated that
statistically significant changes occasionally occurred in hematological and clinical chemistry
measures, but no dose- or time-related trends were observed (quantitative data not available).
Therefore, these changes were not considered toxicologically relevant by the study authors or the
NTP-PWG (1987). Sporadic (not dose dependent) statistically significant differences were also
observed in organ weights in some exposed groups (i.e., relative and absolute thymus weights,
relative liver weight, relative brain weight, and relative testicle weight as depicted in Table D-l
and Table D-2). Upon review, organ-weight changes were not considered toxicologically
significant by the NTP-PWG (1987). with no further explanation provided. Although the
sporadic nature of these changes was apparent for most of the above endpoints, it is unclear why
5ADD (mg/kg-day) = reported dose x (5 7).
14
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EPA/690/R-21/001F
the NTP determined that the decreased absolute and relative thymus-weight changes observed in
female rats were not toxicologically relevant. Statistically significant results were seen in these
animals only at the highest (29 mg/kg-day) dose. No exposure-related changes were observed in
male sperm morphology or female estrous cycle.
At gross necropsy, exposure-related lesions were observed only in the forestomach of rats
of both sexes in the two highest dose groups; these lesions included thickened forestomach
and/or forestomach hyperplasia (see Table D-3). Microscopic examination showed epithelial
hyperplasia in the forestomach at >7.1 mg/kg-day (adjusted daily dose [ADD]) in male rats
(see Table D-3) although the results were not statistically significant. The NTP-PWG (1987)
concluded that no-effect levels for forestomach lesions were 5 and 10 mg/kg-day (ADDs: 4 and
7.1 mg/kg-day) for males and females, respectively, noting that the lesion was equivocal in the
single affected female in the 7.1-mg/kg-day group. The study authors reported that statistical
significance was only reached at 14 mg/kg-day for females and 29 mg/kg-day for males.
Thickened forestomach was observed in male rats at 14 mg/kg-day and 29 mg/kg-day, but only
reached statistical significance in female rats at 29 mg/kg-day. The only other exposure-related
microscopic change reported was described as nasal inflammation in males at >7.1 mg/kg-day
and females at >14 mg/kg-day, but statistical significance was reached only at 29 mg/kg-day in
both sexes (see Table D-3). However, the NTP-PWG (1987) concluded that the nasal lesions
were serous exudation and not acute inflammation as reported by Hazleton Laboratories (1986b)
and that the effect was likely a localized effect from exhaled crotonaldehyde rather than an effect
from blood-circulated crotonaldehyde. Importantly, after histological review of lung tissues in
early-death rodents, it was determined that gavage error was the likely cause of early mortality in
these animals. NTP-PWG (1987) also observed that the serous exudation was usually present in
these early-death rats and may have been exacerbated by postmortem change.
In summary, a NOAEL of 7.1 mg/kg-day and a LOAEL of 14 mg/kg-day were identified
by the NTP-PWG based on a statistically significant increase in the incidence of forestomach
lesions in female rats.
Hazleton Laboratories (1986a) as summarized by NTP-PWG (1987); NTP (2018)
Hazleton Laboratories (1986a) is a companion study to the 13-week study in F344 rats by
Hazleton Laboratories (1986b) that is available only as a summary in a report by NTP-PWG
(1987). The Hazleton studies are non-peer-reviewed study reports but were reviewed by the
NTP-PWG. Selected data tables are also on the NTP Chemical Effects in Biological Systems
(CEBS) database (N I P. 2018). In this study, groups of B6C3F1 mice (10/sex/group) were
administered commercial crotonaldehyde (CASRN 4170-30-3) at doses of 0, 2.5, 5, 10, 20, or
40 mg/kg-day via gavage in corn oil, 5 days/week for up to 13 weeks. ADDs calculated for this
review were 0, 1.8, 4, 7.1, 14, or 29 mg/kg-day. The study followed the same protocol as the
companion study in F344 rats (Hazleton 1 .aboratories. 1986b) reported above, but it excluded the
hematology and serum chemistry analyses. The forestomach, designated as the target organ
based on lesions observed at initial histopathological examination, was examined
microscopically in all mice in the control and treated groups. The NTP-PWG (1987) reviewed
selected slides from this study.
All mice survived treatment to terminal sacrifice, no clinical signs were observed by the
study authors, and they reported no statistically significant differences between treated and
control groups for body-weight gain. The study authors reported statistically significant changes
in absolute organ weights and organ-/body-weight ratios between the treated and control groups
15
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EPA/690/R-21/001F
(quantitative data not available), but NTP-PWG (1987) did not consider these differences
toxicologically significant because of a lack of consistency or dose-related trend.
Treatment-related lesions were not observed at gross necropsy. Microscopic examination
showed hyperplasia of the forestomach mucosa in males and females from the highest dose
group only. Neither NTP-PWG (1987) nor NTP (2018) reported quantitative data for
forestomach lesions. No significant pathological findings were reported for lower dose groups.
The NTP-PWG (1987) identified the highest dose as a LOAEL (29 mg/kg-day) for epithelial
hyperplasia of the forestomach in both sexes of B6C3F1 mice exposed to commercial
crotonaldehyde. Due to the lack of available quantitative data, it is not possible for the purposes
of this PPRTV assessment to identify NOAELs and LOAELs for this study.
Reproductive/Developmental Studies
Hazleton Laboratories (1987)
In a non-peer-reviewed one-generation reproductive study, groups of F344 rats
(20/sex/group) were administered commercial crotonaldehyde (CASRN 4170-30-3) via daily
gavage in corn oil at doses of 0, 2.5, 5, or 10 mg/kg-day (Hazleton I .aboratories. 1987). Males
were dosed for 61 days prior to mating and during the 7-day cohabitation period and then
sacrificed. Females were dosed for 30 days prior to mating, during mating, and through
gestation to Postnatal Day (PND) 5, when they and their surviving pups were sacrificed.
Females that did not get pregnant were sacrificed 30 days after the cohabitation period.
Mortality checks were performed twice daily. Body weights were recorded weekly, and
pregnant females were weighed on Gestation Days (GDs) 0, 7, 14, and 20; at parturition; and
again, at sacrifice on PND 5. All females were subjected to a vaginal cytology evaluation prior
to mating and again just prior to termination for nonpregnant females. At study termination, all
males were subjected to a sperm morphology evaluation, and weights of the right testes and
epididymis were recorded. Blood was collected from all animals at termination for possible
hormone evaluations. Gross examination was conducted on all animals at necropsy, and
histopathology was performed on the reproductive tissues (testes, epididymis, vagina, uterus,
cervix, oviducts, and ovaries). Pups were weighed, counted, sexed, and examined at birth and on
PND 5.
All animals except for one mid-dose male survived to study termination; the cause of
death was reported as not treatment-related. As stated by the study authors, compound-related
clinical signs were not evident in any group at any phase of the study. No significant changes in
body weights, testis weights, or epididymis weights were observed among any treated male rats
compared with control (see Table D-4). In pregnant females, no significant differences from
controls in body weights were observed through gestation to PND 5 (see Table D-4). Among
female rats that did not get pregnant, there was a significant 20-21% decrease in body-weight
gain (and 10-14% decrease in absolute body weight) in the low- and high-dose groups relative to
controls during the postmating period (see Table D-4). However, the control group for this
analysis included only two animals, and the treated groups also included small numbers
(n = 3-5), which limits interpretation of these results and confounds interpretation of the
decreased body-weight effect described above. The study authors suggested that this effect was
probably not related to treatment due to "the limited number of non-pregnant females and lack of
significant findings in the pregnant females." The data for reproductive and litter parameters
showed no effect of treatment. No exposure-related histological lesions in reproductive tissues
were observed in males or females. In total, no treatment-related effects were observed for
survival or body weight data (parents and pups). Results of the sperm morphology and vaginal
cytology studies were not presented.
16
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EPA/690/R-21/001F
The high dose of 10 mg/kg-day is identified by the study authors as a NOAEL for
parental and reproductive toxicity in this study. No LOAEL is identified.
Chronic/Carcinogenicity Studies
Chuns et al. (1986)
In a peer-reviewed study, groups of male F344 rats (23-27/group) were exposed to
/ra/7.s-crotonaldehyde (>99% purity) in drinking water for 113 weeks at concentrations of 0, 0.6,
or 6.0 mM (0, 40, or 420 mg/L, based on molecular weight = 70.09 mg/mmol) beginning at
6 weeks of age (Chung et al .. 1986). Average daily doses of 0, 2, and 17 mg/kg-day (HEDs: 0,
0.6, and 4.6 mg/kg-day) were calculated for this review using reported body weights and water
consumption. Drinking water consumption was measured twice weekly, and body weight was
measured weekly for 40 weeks and then biweekly. Upon sacrifice at the end of exposure, gross
necropsy was performed on all animals, and histopathology was assessed on gross lesions and
major organs (not specified). Statistics were performed by the study authors for
histopathological findings only.
Survival percentages were >95% for the exposed and control groups through 70 weeks of
exposure but began to decline thereafter (see Table D-5). At 110 weeks, survival for the control,
low-, and high-dose groups was 70% (16/23), 63% (17/27), and 57% (13/23), respectively.
These differences in survival rate were not statistically significant. Body weight was decreased
in the high-dose group beginning at the 8th week of exposure and continuing throughout the
study. Based on estimates derived from graphically presented data, body weights in high-dose
animals were more than 10% lower than controls over the latter 6 months of the study,
suggesting that the high dose of 4.6 mg/kg-day is at or approaching the maximum tolerated dose
(MTD). Body weight in the low-dose group remained similar to controls throughout the study.
Incidence of neoplastic nodules in the liver was significantly elevated at the low dose
(9/27), but not the high dose (1/23) compared with controls (0/23) (see Table D-5). Two rats
(7%> of the 27 animals in the dose group) with neoplastic nodules in the low-dose group also
showed hepatocellular carcinomas. This finding was not significant as determined by the study
authors. Incidences of altered liver foci, considered to be a preneoplastic lesion by the study
authors, were significantly elevated at the low dose (23/27) and the high dose (13/23) compared
with controls (1/23) (see Table D-5). The number of altered liver foci per square centimeter was
also significantly increased at both doses, with greater increases at the low dose (see Table D-5).
Among high-dose rats, the 10/23 individuals that did not have preneoplastic (altered foci) or
neoplastic lesions in the liver instead showed moderate to severe degenerative liver damage
(fatty metamorphosis, focal liver necrosis, fibrosis, cholestasis, and mononuclear cell
infiltration). The report did not discuss whether these 10 rats were the ones that died prior to
study termination. The degenerative liver lesions described at the high dose were not reported in
the control or low-dose groups. Incidences of neoplastic lesions in tissues other than the liver
were not significantly affected by treatment, although it may be noteworthy that bladder tumors
were observed in two rats in the low-dose group but not in the control or high-dose rats. There
were no reports of non-neoplastic lesions in tissues other than the liver.
A NOAEL and LOAEL cannot be identified from this study due to the limited
assessment and reporting of noncancer endpoints and confounding due to the elevated incidence
of liver tumors at the low dose, but not at the high dose. Although the reduced body weight in
high-dose animals was considered as a potential LOAEL, no mention was made of degenerative
liver lesions (a potential precursor effect) in the control and low-dose groups, which were
17
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-------
EPA/690/R-21/001F
observed to accompany the decreased body weight in the high-dose animals. Due to this lack of
reporting on potential precursor effects, it is uncertain that attributing a LOAEL to these
non-neoplastic effects would be health protective. Therefore, this study was not included in the
"Summary of Potentially Relevant Noncancer Data" table (see Table 3A) above. The study is
also of limited value as a quantitative cancer bioassay due to the small group sizes, use of a
single sex and species, and the lack of the expected dose-response relationship in the observed
tumor data. The latter may reflect that the high dose in this study exceeded the MTD, which is
suggested by both the decrease in body weight and the occurrence of degenerative liver lesions
in the high-dose group.
Inhalation Exposures
No adequate inhalation studies have been identified.
OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS)
Genotoxicity Studies
The genotoxicity of /ra//.s-crotonaldehyde (CASRN 123-73-9) and commercial
crotonaldehyde (CASRN 4170-30-3) has been evaluated primarily in vitro, with a limited
number of in vivo studies. Available studies are summarized below (see Table 4A for more
details). Based on available data, /ra//.s-crotonaldehyde is clastogenic and forms
deoxyribonucleic acid (DNA) adducts, both in vitro and in vivo. It is also mutagenic under
certain conditions.
18
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EPA/690/R-21/001F
Table 4A. Summary of frans-Crotonaldehyde (CASRN 123-73-9) and Commercial Crotonaldehyde
(>95 trans-; CASRN 4170-30-3) Genotoxicity
Endpoint
Test System
Doses/
Concentrations Tested
Results
without
Activation3
Results
with
Activation3
Comments
References
Genotoxicity studies—prokaryotic organisms
Reverse
mutation
Salmonella typhimurium
TA98, TA1537, TA7001,
TA7002, TA7003, TA7004,
TA7005, TA7006, and a
mix of TA7000 series
50-1,000 |ig commercial
crotonaldehyde
+
(TA98,
TA7002,
TA7004,
TA7005,
TA7006, mix)
(TA1537,
TA7001,
TA7003)
NDr
Liquid preincubation study. The TA7000
series contains base-specific tester strains.
Compound tested up to a toxic dose.
Geeetal. (1998)
Reverse
mutation
S. typhimurium TA98,
TA100, TA1535, TA1537,
TA1538
0.005-10 commercial
crotonaldehyde/plate
Plate incorporation assay. Cytotoxicity
observed at >1 |iL/platc.
Litton Bionetics
(1979)
DNA damage
Escherichia coli PQ37
100 mM commercial
crotonaldehyde
—
—
SOS chromotest.
von der Hude et al.
(1988)
Genotoxicity studies—mammalian cells in vitro
Mutation
L5178Y/7/i'+/- mouse
lymphoma cells
0, 1, 10, 25, 50 nM
commercial crotonaldehyde
+
NDr
Thymidine-kinase mutation assay.
Detnir et al. (2011)
CA
Human lymphoblastoid
cells (Namalwa cell line)
0, 5, 10, 20, 40, 50, 100,
150, 200, 250 nM
/raws-crotonaldehyde
+
NDr
The total number of structural aberrations
was increased at >100 |iM
Dittbemer et al.
(1995)
CA
Primary human blood
lymphocytes
0, 5, 10, 20, 40, 50, 100,
150, 200, 250 nM
/raws-crotonaldehyde
+
NDr
The total number of structural aberrations
was increased at >10 |iM
Dittbemer et al.
(1995)
CA
CHO cells
0.5-16 |ig commercial
crotonaldehyde/mL
+
+
The total number of structural aberrations
was increased at 1.6 ng/mL (-S9) and
16 ng/mL (+S9).
Gallowav et al.
(1987)
19
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Table 4A. Summary of frans-Crotonaldehyde (CASRN 123-73-9) and Commercial Crotonaldehyde
(>95 trans-; CASRN 4170-30-3) Genotoxicity
Endpoint
Test System
Doses/
Concentrations Tested
Results
without
Activation3
Results
with
Activation3
Comments
References
SCE
Human lymphoblastoid
cells (Namalwa cell line)
0, 5, 10, 20, 40, 50, 100,
150, 200, 250 nM
/raws-crotonaldehyde
+
NDr
SCE induced at >20 |iM
Dittbemer et al.
(1995)
SCE
Primary human blood
lymphocytes
0, 5, 10, 20, 40, 50, 100,
150, 200, 250 nM
/raws-crotonaldehyde
+
NDr
SCE induced at >10 |iM
Dittbemer et al.
(1995)
SCE
CHO cells
1.6-160 ng commercial
crotonaldehyde/mL
+
+
The total number of SCE was increased at
>0.5 ng/mL (-S9) and >1.6 ng/mL (+S9).
Gallowav et al.
(1987)
MN
Human lymphoblastoid
cells (Namalwa cell line)
0, 5, 10, 20, 40, 50, 100,
150, 200, 250 nM
/raws-crotonaldehyde
+
NDr
MN induced at >40 |iM
Centromere-positive MN were not
significantly increased with exposure.
Dittbemer et al.
(1995)
MN
Primary human blood
lymphocytes
0, 5, 10, 20, 40, 50, 100,
150, 200, 250 nM
/raws-crotonaldehyde
+
NDr
MN induced at >40 |iM
Centromere-positive MN were not
significantly increased with exposure.
Dittbemer et al.
(1995)
Aneuploidy
Primary human blood
lymphocytes
0, 5, 10, 20, 40, 50, 100,
150, 200, 250 nM
/raws-crotonaldehyde
NDr
The number of aneuploid metaphases was
not increased at any concentration.
Dittbemer et al.
(1995)
Genotoxicity studies—mammalian species in vivo
Dominant
lethal mutation
Swiss albino mice (20 M)
were exposed via i.p.
injection in olive oil for 5 d.
After final treatment, males
were mated with unexposed
females for 5 wk. Dams
were sacrificed on
GDs 14-16 and examined
for live and dead implants.
0, 8, 16, 32 nL
/ra«.Y-crotonaldchvdc/kg
Positive control: 40 mg
cyclophosphamide
+
NA
Significant increase in the number of
dominant lethal mutations in all treated
groups. Maximum lethality was observed
in the group exposed to 32 |iL/kg and
mated 15-21 d post-treatment.
J ha et al. (2007)
20
/ra/7.s-Crotonaldehyde
-------
EPA/690/R-21/001F
Table 4A. Summary of frans-Crotonaldehyde (CASRN 123-73-9) and Commercial Crotonaldehyde
(>95 trans-; CASRN 4170-30-3) Genotoxicity
Endpoint
Test System
Doses/
Concentrations Tested
Results
without
Activation3
Results
with
Activation3
Comments
References
CA
Swiss albino mice (3 M,
2 F/group) were exposed
once via i.p. injection in
olive oil. Mice were
sacrificed 6, 13, and 24 hr
after treatment. Bone
marrow was evaluated for
CAs.
0, 8, 16, 32 nL
/ra«.Y-crotonaldchvdc/kg
Positive control: 1.5 mg/kg
mitomycin C
+
NA
Significant increase in CAs/cell and
percent abnormal cells in all treated
groups at all time points.
J ha et al. (2007)
CA
Swiss albino mice
(5 M/group) were exposed
once via i.p. injection in
olive oil. Mice were
sacrificed 24 hr after
treatment. Spermatocytes
were evaluated for CAs.
0, 8, 16, 32 nL
/ra«.Y-crotonaldchvdc/kg
Positive control: 25 mg
cyclophosphamide
+
NA
Significant increase in percent abnormal
cells at >16 |iL/kg.
J ha et al. (2007)
MN
B6C3F1 mice
(10/sex/group) were
exposed via gavage in corn
oil for 90 d. Erythrocytes
were evaluated for MN.
0, 2.5, 5, 10, 20, 40 mg
commercial
crotonaldehyde/kg-d
NA
NA
Witt et al. (2000)
DNA adducts
F344 rats (4 F/group) were
exposed once via gavage in
corn oil. Rats were
sacrificed 12 or 20 hr after
exposure. Major organs
were evaluated for adducts.
0, 200, 300 mg
/ra«.v-crotonaldchvdc/kg
+
NA
l. Y2-Propanodco\yguanosinc adducts
were identified in treated animals at both
dose levels. The highest levels were
detected in liver, followed by lung,
kidney, and large intestine. Adduct levels
at 200 and 300 mg/kg were 2.9 and
3.4 adducts per 108 nucleotides,
respectively. No adducts were detected in
controls (detection limit 3 adducts per
109 nucleotides).
Budiawati and
Eder (2000);
Budiawati et al.
(2000)
21
/ra/7.s-Crotonaldehyde
-------
EPA/690/R-21/001F
Table 4A. Summary of frans-Crotonaldehyde (CASRN 123-73-9) and Commercial Crotonaldehyde
(>95 trans-; CASRN 4170-30-3) Genotoxicity
Endpoint
Test System
Doses/
Concentrations Tested
Results
without
Activation3
Results
with
Activation3
Comments
References
DNA adducts
F344 rats (4 F/group) were
exposed for 6 wk (5 d/wk)
via gavage in corn oil. Rats
were sacrificed 20 hr after
final exposure.
1, 10 mg
/ra«.v-crotonaldchvdc/kg-d
+
NA
l. Y2-Propanodco\yguanosinc adducts
were identified in treated animals at both
dose levels. Adduct levels at 1 and
10 mg/kg were 2.0 and 6.2 adducts per
108 nucleotides, respectively. No adducts
were detected in controls (detection limit
3 adducts per 109 nucleotides).
Eder and
Budiawan (2001);
Budiawan and
Eder (2000)
DNA adducts
F344 rats (F, NS) were
exposed for 4 wk (5 d/wk)
via gavage in corn oil. Rats
were sacrificed 24 hr, 1 wk,
or 2 wk after final
exposure.
10 mg
/ra«.v-crotonaldchvdc/kg-d
+
NA
l. Y2-Propanodco\yguanosinc adducts
were identified in treated animals at all
time points. The numbers of adducts at
1 and 2 wk postexposure were 69 and
18%, respectively, of the number of
adducts 24 hr after final exposure. No
adducts were detected in controls
(detection limit 3 adducts per
109 nucleotides).
Eder and
Budiawan (2001);
Budiawan and
Eder (2000)
Genotoxicity studies—invertebrates in vivo
Sex-linked
recessive lethal
Drosophila melanogaster;
males were exposed for 3 d
via feeding prior to mating
to unexposed females.
4,000 ppm commercial
crotonaldehyde
NA
NA
Woodruff et al.
(1985)
Sex-linked
recessive lethal
D. melanogaster; males
were injected with
0.2-0.3 24-48 hr prior
to mating to unexposed
females.
3,500 ppm commercial
crotonaldehyde
+
NA
3,500 ppm dose promoted 15% mortality
and 4% increase in male sterility.
Woodruff et al.
(1985)
22
/ra/7.s-Crotonaldehyde
-------
EPA/690/R-21/001F
Table 4A. Summary of frans-Crotonaldehyde (CASRN 123-73-9) and Commercial Crotonaldehyde
(>95 trans-; CASRN 4170-30-3) Genotoxicity
Endpoint
Test System
Doses/
Concentrations Tested
Results
without
Activation3
Results
with
Activation3
Comments
References
Reciprocal
translocation
D. melanogaster; males
were injected with
0.2-0.3 24-48 hr prior
to mating to unexposed
females.
3,500 ppm commercial
crotonaldehyde
+
NA
Increased reciprocal translocations
observed in sperm after 3 d of storage.
Woodruff et al.
(1985)
Mitotic
recombination
(white/white+
eye mosaic
bioassay)
D. melanogaster larvae
were exposed via feeding
for 48 hr.
0, 10, 25, 50 mM
commercial crotonaldehyde
+
NA
Mitotic recombination observed at
>25 mM.
Detnir et al. (2013)
Genotoxicity studies—subcellular systems
DNA adduct
Calf thymus DNA; 8-48-hr
incubation.
0, 90 nL
/raws-crotonaldehyde
+
NDr
Formed cyclic l. Y2-propanodco\y-
guanosine adducts. More adducts formed
with longer incubation.
Budiawan and
Eder (2000);
Budiawan et al.
(2000)
a+ = positive; ± = weakly positive; - = negative.
CA = chromosomal aberration; CHO = Chinese hamster ovary; DNA = deoxyribonucleic acid; F = female(s); GD = gestation day; i.p. = intraperitoneal; M = male(s);
MN = micronuclei; NA = not applicable; NDr = not determined; SCE = sister chromatid exchange.
23
/ra/7.s-Crotonaldehyde
-------
EPA/690/R-21/001F
Mutagenicity
Results of Salmonella typhimurium mutation assays for commercial crotonaldehyde in
nonmammalian species are mixed (no study specifically indicated that it was testing
/ra/M-crotonaldehyde). A study by Gee et al. (1998) using the liquid suspension method reported
that commercial crotonaldehyde was mutagenic without metabolic activation in S. typhimurium,
and was not tested under conditions of external metabolic activation. A single study using the
plate incorporation method reported that commercial crotonaldehyde was not mutagenic, both
with and without metabolic activation (I.itton Bionctics. 1979). Research has suggested that
contradictory results in bacterial assays may be associated with the high cellular toxicity of
crotonaldehyde, particularly in standard plate assays using lower bacterial cell densities (Demir
et al.. 2011).
In mammals, increased dominant lethal mutations were observed in mice following male
exposure to /ra//.s-crotonaldehyde via intraperitoneal (i.p.) injection for 5 days prior to mating
(Jha et al.. 2007). In an in vitro mutation study in mammalian cells, commercial crotonaldehyde
induced mutations in mouse lymphoma cells without metabolic activation (Demir et al.. 2011).
In Drosophila melanogaster, sex-linked recessive mutations were induced following exposure to
commercial crotonaldehyde via injection, but not oral exposure (Woodruff et al.. 1985).
Clastogenicity
Chromosomal aberrations (CAs) were induced in both bone marrow cells and
spermatocytes in mice exposed once to /ram-crotonal dehyde via i.p. injection (Jha et al.. 2007).
Micronuclei (MN) were not induced in erythrocytes of mice following exposure to commercial
crotonaldehyde via gavage for 90 days (Witt et al.. 2000). CAs, sister chromatid exchanges
(SCEs), and MN were all induced by /ra//.s-crotonaldehyde in primary human blood lymphocytes
and cultured human lymphoblastoid cells (Dittberner et al.. 1995). However,
/ra/7.s-crotonaldehyde did not induce aneuploidy or centromere-positive MN, indicating that
observed effects were clastogenic in nature, rather than aneugenic (Dittberner et al.. 1995). CAs
and SCEs were also induced in Chinese hamster ovary (CHO) cells following in vitro exposure
to commercial crotonaldehyde (Galloway et al.. 1987). A significant increase in reciprocal
translocations and mitotic recombinations were also observed in D. melanogaster following
exposure to commercial crotonaldehyde (Demir et al .. 2013; Woodruff et al .. 1985).
DNA Ad ducts, Damage, and Repair
Numerous studies report that /ra//.s-crotonaldehyde can directly bind to DNA, forming
DNA adducts. 1 ,A'2-Propanodeoxyguanosine DNA adducts were observed in multiple tissues
(the highest levels of adducts were detected in the liver, lung, kidney, and large intestine) of
F344 rats exposed to /ra//.s-crotonaldehyde via a single gavage exposure to doses >200 mg/kg or
repeated gavage exposures to doses >1 mg/kg-day (Eder and Budiawan. 2001; Budiawan and
Eder. 2000; Budiawan et al.. 2000). DNA adducts persisted several weeks after exposure.
1 ,A'2-Propanodeoxyguanosine DNA adducts were also observed in CHO cells, human fibroblast
cells, and isolated calf DNA exposed to /ram-crotonaldehyde in vitro (Budiawan and Eder.
2000; Budiawan et al.. 2000).
DNA damage was not induced in Escherichia coli exposed to commercial
crotonaldehyde (with and without activation) in the SOS chromotest (von der Hude et al.. 1988).
24
/ra/7.s-Crotonal dehyde
-------
EPA/690/R-21/001F
Supporting Animal Toxicity Studies
Numerous acute oral and inhalation studies, studies available only from secondary
sources or as abstracts, and studies via other routes (e.g., dermal, injection) were identified. The
relevant studies are summarized below (see Table 4B for additional details).
25
/ra/7.s-Crotonaldehyde
-------
EPA/690/R-21/001F
Table 4B. Other frans-Crotonaldehyde (CASRN 123-73-9) and
Commercial Crotonaldehyde (>95% trans-; CASRN 4170-30-3) Studies
Test3
Materials and Methods
Results
Conclusions
References
Supporting evidence—noncancer effects in animals following oral exposure
Acute (oral)
In a range-finding study, S-D rats
(2/sex/group) were exposed to 50, 160,
500, 1,600, or 5,000 mg commercial
crotonaldehyde/kg via gavage. Rats
were observed for 3 d after dosing.
Convulsions observed in all rats exposed to
>500 mg/kg immediately after dosing, and all rats in
these dose groups died within 0.5 hr of dosing. One
female died at 160 mg/kg within 1 hr of dosing. No
rats died at 50 mg/kg.
FEL: 160 mg/kg (death)
Borristoti
(1980b):
Borristoti (1980c)
Acute (oral)
In an LD5o study, S-D rats (5/sex/group)
were exposed once to 64.5, 107.5, 180,
300, or 500 mg commercial
crotonaldehyde/kg via gavage. At the
end of the 14-d observation period, all
surviving rats were sacrificed.
Endpoints evaluated included mortality,
clinical signs, body weight, and gross
necropsy.
All rats exposed to >300 mg/kg died, and 4/5 males
and 3/5 females at 180 mg/kg died. All rats
survived at <107.5 mg/kg. All observed deaths
occurred within 24 hr of dosing. Clinical signs of
toxicity (salivation, lacrimation, ataxia, lethargy,
and convulsions) and decreased body weight were
observed prior to death. Gross lesions were
observed in lungs (discoloration, mottling, and
congestion) and stomach and intestines (distended
with gas or fluid) of dead animals. No
exposure-related changes were observed in
surviving animals.
RatLDso (95% CI)
Male: 165 (107-254) mg/kg
Female: 175 (105-292) mg/kg
Combined: 174 (131-231) mg/kg
FEL: 180 mg/kg (death)
Borristoti
(1980b):
Borristoti (1980c)
Acute (oral)
The LD50 for commercial
crotonaldehyde was determined in
groups of rats. No further details were
provided.
The reported LD50 (95% CI) was 0.22
(0.20-0.25) g/kg.
RatLDso (95% CI) =
220 (200-250) mg/kg
Mellon Institute
of Industrial
Research (1948)
Acute (oral)
Albino rats (6 M/group) were exposed
once to 0.1 or 1 g commercial
crotonaldehyde/kg via gavage in water
plus 1% Tergitol. Animals were
observed for mortality and clinical
signs. Gross necropsy was conducted.
At 1 g/kg, death occurred within 10 min. Prior to
death, animals exposed to 1 g/kg showed clinical
signs of toxicity (pain, jumping around). Gross
necropsy showed pale kidney, excessive peritoneal
fluid, and congestion in the liver, stomach, and
intestine. The LD50 was reported to be
approximately 0.3 g/kg.
Rat LD50 = 300 mg/kg
Mellon Institute
of Industrial
Research (1942)
26
/ra/7.s-Crotonaldehyde
-------
EPA/690/R-21/001F
Table 4B. Other frans-Crotonaldehyde (CASRN 123-73-9) and
Commercial Crotonaldehyde (>95% trans-; CASRN 4170-30-3) Studies
Test3
Materials and Methods
Results
Conclusions
References
Acute (oral)
The LD50 for commercial
crotonaldehyde was determined in
groups of rats. No further details were
provided.
The study authors classified crotonaldehyde as
having moderate toxicity via the oral route.
Rat LD50 = 300 mg/kg
Kennedy and
Graeoel (1991)
Supporting evidence—noncancer effects in animals following inhalation exposure
Acute
(inhalation)
The LC50 for commercial
crotonaldehyde was determined in
groups of rats. No further details were
provided.
The 4-hr rat LC50 was 100 ppm. The study authors
classified crotonaldehyde as having moderate
toxicity via the inhalation route.
4-hr rat LC50 = 286 mg/m3 b
Kennedy and
(inaenel (1991)
Acute
(inhalation)
In an LC50 study, rats (3/group; sex and
strain not reported) were exposed to
0.099, 0.16-0.28, 0.38, 0.48, 1.03, 2.6,
3.6, 6, 37, or 46.5 mg/L of commercial
crotonaldehyde for up to 6 hr.
Mortality, clinical signs, and body
weight were recorded over 2-wk
observation period.
Reported LC50 was <0.48 mg/L but
>0.16-0.28 mg/L. All rats exposed to >1.03 mg/L
died. At >2.6 mg/L, rats died during the first
43-120 min of exposure; at 1.03 mg/L, death
occurred 3.5-24 hr after exposure. At 0.48 mg/L,
2/3 rats died within 1 d; surviving rats lost weight.
At 0.38 mg/L, all rats died within 48 hr. No deaths
were observed in groups exposed to 0.099 or
0.16-0.28 mg/L. Clinical signs were observed in all
exposure groups (gasping, nasal irritation, pink
extremities, and labored breathing), with serious
signs (tremors, prostration, and convulsions) at
lethal doses.
6-hr rat LC50 (range) =
280-480 mg/m3 0
FEL: 380 mg/m3 (death)
Eastman Kodak
(1961)
Acute
(inhalation)
Rats (4/group; strain and sex not
specified) were exposed to air saturated
with commercial crotonaldehyde for 1
or 10 min. Rats were held for 7-d
observation period.
All rats exposed for 10 min died on the same day of
exposure; all rats exposed for 1 min survived. The
LT50 was determined to be 3 min.
Exposure to air saturated with
commercial crotonaldehyde will
kill 50% of animals after
approximately 3 min.
Mellon Institute
of Industrial
Research (1942)
Acute
(inhalation)
Groups of guinea pigs were exposed to
1,000 or 2,000 ppm commercial
crotonaldehyde for up to 30 min. No
further details were provided.
Mortality at 1,000 ppm was 0% at 5 min, 20% at
10 min, and 50% at 30 min. Mortality at 2,000 ppm
was 50% at 15 min and 100% at 30 min.
FEL: 2,870 mg/m3 (death)b
Mellon Institute
of Industrial
Research (1942)
27
/ra/7.s-Crotonaldehyde
-------
EPA/690/R-21/001F
Table 4B. Other frans-Crotonaldehyde (CASRN 123-73-9) and
Commercial Crotonaldehyde (>95% trans-; CASRN 4170-30-3) Studies
Test3
Materials and Methods
Results
Conclusions
References
Supporting evidence—noncancer effects in animals following dermal or ocular exposure
Acute (dermal
lethality)
The LD50 for commercial
crotonaldehyde was determined in
groups of rabbits. No further details
were provided.
Rabbit LD50 (95% CI) = 0.38 (0.27-0.52) mL/kg
Rabbit LD50 (95%CI) =
330 (230-450) mg/kgd
Mellon Institute
of Industrial
Research (1948)
Acute (dermal
lethality)
Male and female guinea pigs
(6-12/group; strain and sex not
specified) were exposed to 0.1 or 1 g/kg
commercial crotonaldehyde under
occluded conditions for 2 or 24 hr.
Mortality was 6/6 at 1 g/kg and 0/6 at 0.1 g/kg for
both durations. The LD50 was reported to be
approximately 0.3 g/kg. Skin was tanned brown.
Guinea pig LD50 values:
2-hr LD50 = 300 mg/kg
24-hr LD50 = 300 mg/kg
Mellon Institute
of Industrial
Research (1942)
Short term
(dermal lethality)
Male and female guinea pigs
(6-12/group; strain and sex not
specified) were exposed to 0.01, 0.1, or
1 g/kg commercial crotonaldehyde
under occluded conditions for 4 d.
Mortality was 1/12 at 0.01 g/kg and 6/6 at >0.1 g/kg.
The LD5o was reported to be approximately
0.03 g/kg. Skin was tanned a dark brown with slight
necrosis.
4-d guinea pig LD50 = 30 mg/kg
Mellon Institute
of Industrial
Research (1942)
Acute (ocular
irritation)
An eye irritation study with commercial
crotonaldehyde was conducted in
rabbits. No further details were
provided.
Very severe necrosis was observed at 0.001 mL.
Crotonaldehyde is a severe eye
irritant.
Mellon Institute
of Industrial
Research (1942)
Supporting evidence—noncancer effects in animals following exposure via other routes
Acute (i.p.)
Swiss albino mice (5 M/group) were
exposed to /raws-crotonaldehyde once
via i.p. injections of 0, 8, 16, or
32 |iL/kg (0, 7, 14, or 27 mg/kg). The
frequency of abnormal sperm heads was
determined at 1, 3, or 5 wk in
spermatozoa, spermatid, and
preleptotene spermatogonia,
respectively.
Sperm head abnormalities were observed in
spermatozoa and spermatid at >14 mg/kg and in
preleptotene spermatogonia at 27 mg/kg. Common
types observed included short hooked, without hook,
giant amorphous, and banana.
NA
J ha and Kumar
(2006)
28
/ra/7.s-Crotonaldehyde
-------
EPA/690/R-21/001F
Table 4B. Other frans-Crotonaldehyde (CASRN 123-73-9) and
Commercial Crotonaldehyde (>95% trans-; CASRN 4170-30-3) Studies
Test3
Materials and Methods
Results
Conclusions
References
Short term (i.p.)
Swiss albino mice (5 M/group) were
exposed to /raws-crotonaldehyde via
daily i.p. injections of 0, 8, 16, or
32 |iL/kg-d (0, 7, 14, or 28 mg/kg-d) for
5 d. Males were then mated with
groups of 40 unexposed females for
5 wk.
A significant decrease in fertility index was
observed at >14 mg/kg-d during the 2nd and 3rd wk
of mating and at 28 mg/kg-d during the 4th wk of
mating.
NA
J ha et al. (2007)
aAcute = exposure for <24 hours; short term = repeated exposure for >24 hours, <30 days; subchronic = repeated exposure for >30 days, <10% lifespan (>30 days up to
approximately 90 days in typically used laboratory animal species); chronic = repeated exposure for >10% lifespan for humans (more than approximately 90 days to
2 years in typically used laboratory animal species) (U.S. EPA. 2002).
bConcentration in mg/m3 = concentration in ppm x molecular weight (70.09 g/mol) 24.45.
Concentration in mg/m3 = concentration in mg/L x 1,000 L/m3.
dDose in mg/kg = dose in mL/kg x density (0.869 g/mL) x 1,000 mg/g.
eDose inmg = dose innmol x molecular weight (70.09 ng/nmol) x 1 ^g/1,000 ngx 1 mg/1,000 |ig.
CI = confidence interval; FEL = frank effect level; i.p. = intraperitoneal; LC50 = median lethal concentration; LD5o = median lethal dose; LT5o = medial lethal time;
M = male(s); NDr = not determined; S-D = Sprague-Dawley.
29
/ra/7.s-Crotonaldehyde
-------
EPA/690/R-21/001F
Supporting Studies for Noncarcinogenic Effects in Animals
Acute Oral Toxicity
Acute oral lethality studies with commercial crotonaldehyde reported median lethal dose
(LDso) values in rats ranging from 165 to 300 mg/kg (Kennedy and Graepei. 1991; B orris ton.
1980b. c; Mellon Institute of Industrial Research. 1948. 1942). Mortality was observed at acute
oral doses as low as 160 mg/kg, with no mortality observed at doses <107.5 mg/kg (Borriston.
1980b. c). Clinical signs observed at lethal doses included salivation, lacrimation, ataxia,
excitability followed by lethargy, and convulsions. In the animals that died, gross lesions were
observed in the lungs (discoloration, mottling, congestion), stomachs, and intestines (distended
with gas or fluid).
Acute Inhalation Toxicity
/ra/7.s-Crotonaldehyde is a potent respiratory irritant. Acute inhalation lethality studies
with commercial crotonaldehyde reported a 6-hour median lethal concentration (LC50) in rats of
280-480 mg/m3 (Eastman Kodak. 1961) and a 4-hour LC50 in rats of 286 mg/m3 (Kennedy and
Graepei. 1991). Exposure to air saturated with commercial crotonaldehyde resulted in the death
of 50% of exposed rats within 3 minutes; all rats died within 10 minutes (Mellon Institute of
Industrial Research. 1942). Thirty-minute exposure to 2,870 or 5,740 mg/m3 killed 50% and
100%) of exposed guinea pigs, respectively (Mellon Institute of Industrial Research. 1942).
Clinical signs observed at lethal concentrations in these studies included excitation, tremors,
convulsions, marked respiratory distress (labored breathing, gasping), lacrimation, nasal
irritation, pink extremities, and weight loss. Mild clinical signs of respiratory distress, nasal
irritation, and lacrimation were also observed at nonlethal concentrations as low as 99 mg/m3.
Hemorrhage and hyperemia were observed in the lungs, heart, liver, and kidneys of some
animals that died.
Ocular and Dermal Toxicity
Commercial crotonaldehyde is a severe eye irritant in rabbits and is classified as a
corrosive substance (Mellon Institute of Industrial Research, 1942). In dermal lethality studies,
the LD50 values in rabbits or guinea pigs following exposure up to 24 hours were
300-330 mg/kg; the 4-day LD50 value in guinea pigs was 30 mg/kg (Mellon Institute of
Industrial Research. 1948. 1942).
Other Route Toxicity
Available injection studies include acute and short-term studies primarily focused on
acute lethality or toxicity to the reproductive, hematological, or immune systems.
Decreased male fertility was observed when male mice were given i.p. injections
>14 mg/kg-day for 5 days prior to mating with unexposed females; fertility was comparable to
control at 7 mg/kg-day (Jha et al.. 2007). Damage to male germ cells at all stages of
spermatogenesis were also observed in mice following single i.p. injections to
/ra/7.s-crotonaldehyde at a dose of 14 mg/kg, but sperm abnormalities were not observed at
7 mg/kg (Jha and Kumar. 2006).
Absorption, Distribution, Metabolism, and Excretion Studies
No studies evaluating absorption, distribution, or excretion of /ra//.s-crotonaldehyde and
commercial crotonaldehyde have been identified.
30
/ra/7.s-Crotonal dehyde
-------
EPA/690/R-21/001F
Available studies indicate that trans-crotonaldehyde can be metabolized via oxidation
and conjugation with thiols. Specifically, /ra//.s-crotonaldehyde has been shown to conjugate
with thiols in vitro, including glutathione (GSH) and A-acetyl cysteine, and is a substrate for
glutathione S-transferase (GST) (van lersel et al.. 1996; Wang et al.. 1992). The reaction rates of
thiols with crotonaldehyde were 2-mercaptoethanesulfonate > GSH > iV-acetylcysteine (Wang et
al.. 1992).
Mode-of-Action/Mechanistic Studies
A target of non-neoplastic toxicity in rodents following gavage exposure is the
forestomach [Hazleton Laboratories (1986a) as cited in NTP-PWG (1987); Hazleton
Laboratories (1986b)1. Observed lesions at this portal of entry are likely due to the irritative and
corrosive nature of crotonaldehyde (TEL. 1986). This mechanism is also relevant to observed
nasal lesions in the sub chronic rat study by Hazleton Laboratories (1986b) because nasal effects
were considered to be a localized effect from exhaled crotonaldehyde rather than an effect from
blood-circulated crotonaldehyde (NTP-PWG. 1987).
Several in vitro studies in cells from the human or mammalian respiratory tract report
alterations following exposure to /ra//.s-crotonal dehyde, including cytotoxicity, apoptosis,
alterations in immune parameters (e.g., increased cytokine secretion, decreased phagocytic
activity of alveolar macrophages), and induction of genes associated with inflammation and
oxidative stress (Yang et al.. 2013b; Yang et al.. 2013a).
As discussed in the "Genotoxicity Studies" section and Table 4A, /ra//.s-crotonaldehyde is
mutagenic under certain conditions. It has been proposed that cyclic propano DNA adducts
associated with crotonaldehyde exposure are highly stable promutagenic lesions and may
underlie mutagenicity and subsequent tumor formation following exposure to
/ra//.v-crotonaldehyde (Voulgaridou et al.. 2011). In support, DNA-containing
/ra/7.s-crotonaldehyde-induced adducts was transfected into mammalian COS-7 cells, and
replication in the presence of these adducts resulted in the induction of mutations (Fernandes et
al .. 2005). Known endogenous production of aldehyde cyclic adducts makes the potential role of
/ra/7.s-crotonaldehyde-mediated adduct formation in carcinogenicity unclear. To this point, the
liver, which is the site of tumor formation following /ram-crotonal dehyde exposure (Chung et
al.. 1986). shows the highest number of DNA adducts in rats following oral exposure (Budiawan
and Eder. 2000; Budiawan et al.. 2000).
31
/ra/7.s-Crotonal dehyde
-------
EPA/690/R-21/001F
DERIVATION OF PROVISIONAL VALUES
DERIVATION OF PROVISIONAL ORAL REFERENCE DOSES
The database of potentially relevant studies for deriving oral reference values for
/ra/M-crotonaldehyde includes one subchronic gavage study in rats (Hazleton Laboratories.
1986b). a subchronic gavage study in mice [Hazleton Laboratories (1986a) as cited in NTP-PWG
(1987)1. a reproductive study in rats (Hazleton Laboratories. 1987). and a chronic drinking water
study in rats (Chung et at.. 1986). Of the available studies, only Chung et al. (1986) is peer
reviewed; however, data reporting for non-neoplastic endpoints in this study is inadequate for
evaluation. The two subchronic gavage studies in mice and rats, while not peer reviewed, were
performed by a contract research laboratory with extensive documentation of the experimental
conditions and methodologies employed. A comprehensive array of endpoints was evaluated in
each study, and a review of the pathology results was conducted and confirmed by the
NTP-PWG. As part of this review, the NTP-PWG examined the pathology results reported by
two independent pathologists, the pathology quality assessment report, and other toxicological
data and determined each to be of sufficient scientific quality. Therefore, although there is no
explicit indication that these studies were performed under Good Laboratory Practice (GLP)
guidelines, review of the study design and data report by the NTP-PWG sufficiently increases
confidence in the ability to use these data to derive provisional reference dose (p-RfD) values.
Derivation of a Subchronic Provisional Reference Dose
Available unpublished repeated-dose subchronic gavage studies in rats and mice identify
the forestomach as the most sensitive target of toxicity [Hazelton Laboratories (1986a) as cited in
NTP-PWG (1987); Hazleton Laboratories (1986b )1. The lowest identified LOAEL for
forestomach lesions is 14 mg/kg-day in female rats. As described above, although not peer
reviewed, the Hazleton Laboratories [(1986a) as cited in NTP-PWG (1987); (1986b)1 studies
were performed by a contract laboratory with extensive documentation of experimental and
laboratory conditions. Furthermore, the accompanying data and histological results were
evaluated by the NTP-PWG for accuracy. Because forestomach lesions were observed in both
sexes of mice and rats and the data were reviewed by an independent party (NTP-PWG), the
database supporting the identification of forestomach lesions as a viable critical effect is of
sufficient confidence to warrant the development of p-RfDs within the main body of this
assessment.
In addition to the significantly elevated rates of forestomach lesions, several other
pathologies were observed to be significantly altered relative to control-treated animals. An
increased rate of nasal inflammation was reported in male and female rats (Hazleton
Laboratories. 1986b). Upon review of the reported data, the NTP-PWG concluded that the
observed nasal changes should be considered serous exudation (clear, thin, and watery exudate
associated with tissue repair and inflammation), and not acute inflammation as concluded in the
laboratory report. The exudates were thought to be reactions to the highly volatile
/ra/7.s-crotonaldehyde. As such, exudates were usually present in the early-death rats, which
were also observed to have significant levels of oil droplets within the lungs, indicating that these
early deaths were likely the result of gavage errors (accidental administration into the lung).
Taken together, these data suggest that early-death animals likely had /ra//.s-crotonaldehyde
deposited into the lungs, where it could readily volatilize into nasal passages. The NTP-PWG
suggested that the apparent serous exudations may have been exacerbated by postmortem change
in these animals. Combined, these results suggest that the observed nasal effects may be the
32
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result of gavage error and may have increased in severity after death of the rats, precluding the
use of nasal lesions/serous nasal exudations as a potential critical effect.
Hazleton Laboratories (1986b) also identified significant absolute and relative thymus
weight decreases in female rats at the highest dose examined. This was the only evidence to
suggest effects in the thymus. Because these effects were observed only in female rats (and not
male rats or male or female mice) and because there was 50% mortality in this dose group (only
five rats alive at the end of the study), evidence of effects within the thymus are not sufficiently
supported and were, therefore, not considered as a potential POD.
Data for forestomach lesions in male and female F344 rats (see Appendix D) reported by
Hazleton Laboratories (1986b) were modeled using all available dichotomous models, as
appropriate, in the U.S. EPA's Benchmark Dose Software (BMDS, Version 2.6)
(see Appendix E). Data for forestomach lesions in mice were not modeled because incidence
data are not available [Hazelton Laboratories (1986a) as cited in NTP-PWG (1987)1. The
modeled data for the F344 rat study are shown in Table D-3. ADDs were used for modeling. In
Recommended Use of Body Weight3/4 as the Default Method in Derivation of the Oral Reference
Dose (U.S. EPA. 2011b). the Agency endorses body-weight scaling to the 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 (calculation of HED) for the purpose of deriving an RfD from
effects that are not portal-of-entry effects or effects resulting from direct exposure of neonatal or
juvenile animals. Because forestomach lesions following gavage exposure may be
portal-of-entry effects, doses were not converted into HEDs prior to modeling. The standard
reporting value benchmark response (BMR) for dichotomous data of 10% extra risk was used.
The benchmark dose lower confidence limit (BMDL) (ADD) of 3 mg/kg-day, based on
incidence of forestomach hyperplasia in male rats exposed to crotonaldehyde via gavage
5 days/week for 13 weeks (Hazleton Laboratories. 1986b). provides the lowest candidate point of
departure (POD value), and was selected as the POD for deriving the subchronic p-RfD.
Forestomach lesions are generally believed to be a manifestation of chronic irritation at the portal
of entry associated with oral exposure to corrosive chemicals. The forestomach thickening,
nodule formation, and hyperplasia reported in male and female rats [Hazelton Laboratories
(1986a) as cited in NTP-PWG (1987); Hazleton Laboratories (1986b )1 and forestomach
hyperplasia reported in male and female mice [Hazelton Laboratories (1986a) as cited in NTP-
PWG (1987)1 are consistent with the potential consequence of portal-of-entry exposure to an
agent promoting chronic irritation.
The subchronic p-RfD is derived by applying a composite uncertainty factor (UFc) of
300 (reflecting an interspecies uncertainty factor [UFa] of 10, an intraspecies uncertainty factor
[UFh] of 10, and a database uncertainty factor [UFd] of 3) to the selected POD of 3 mg/kg-day.
Subchronic p-RfD = POD (ADD) UFc
= 3 mg/kg-day -^300
= 1 x 10"2 mg/kg-day
Table 5 summarizes the uncertainty factors for the subchronic p-RfD for
^ram'-crotonal dehy de.
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Table 5. Uncertainty Factors for the Subchronic p-RfD for
fraws-Crotonaldehyde (CASRN 123-73-9)
UF
Value
Justification
UFa
10
A UFa of 10 is applied to account for uncertainty associated with extrapolating from animals to
humans. Cross-species dosimetric adjustment (HED calculation) was not performed because the
critical endpoint may be a portal-of-entry effect.
UFd
3
A UFd of 3 (10°5) is applied to account for deficiencies and uncertainties in the database. The
database for oral exposure to irans-crotona 1 dc h v dc consists of subchronic toxicity studies in
two species, a single chronic carcinogenicity study in male rats, and a 1-generation reproduction study
in rats. There are no developmental toxicity studies available following exposure via any route.
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 /raws-crotonaldehyde in humans.
UFl
1
A UFl of 1 is applied because the POD is a BMDL.
UFS
1
A UFS of 1 is applied because the subchronic POD was derived from a 13-wk study.
UFC
300
Composite UF = UFA x UFD x UFH x UFL x UFS.
BMDL = benchmark dose lower confidence limit; HED = human equivalent dose;
LOAEL = lowest-observed-adverse-effect level; NOAEL = no-observed-adverse-effect level; POD = point of
departure; p-RfD = provisional reference dose; UF = uncertainty factor; UFa = interspecies uncertainty factor;
UFC = composite uncertainty factor; UFD = database uncertainty factor; UFH = intraspecies uncertainty factor;
UFl = LOAEL-to-NOAEL uncertainty factor; UFS = subchronic-to-chronic uncertainty factor.
Confidence in the subchronic p-RfD for /ra//.s-crotonaldehyde is medium, as described in
Table 6.
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Table 6. Confidence Descriptors for the Subchronic p-RfD for
fraws-Crotonaldehyde (CASRN 123-73-9)
Confidence Categories
Designation
Discussion
Confidence in study
M
Confidence in the orincioal studv (Hazleton Laboratories. 1986b) is
medium. This study is not peer reviewed; however, it used an adequate
number of animals (10 rats/sex) at 6 dose levels (including controls).
Furthermore, despite a lack of access to the complete data set, a
comprehensive suite of endpoints was examined (clinical symptoms, body
and organ weights, hematological and clinical chemistry, and pathology),
and the data was subseauentlv reviewed bv the NTP-PWG (NTP-PWG.
1987).
Confidence in database
M
Confidence in the database for /raws-crotonaldehyde is medium. The
relevant database consists of one short-term dietary study in rats
(Borristott. 1980a). as well as subchronic savase studies in rats (Hazleton
Laboratories. 1986b) and mice IHa/elton Laboratories (1986a). as cited in
NTP-PWG (1987)1. one sinele-eeneration reproduction studv (Hazleton
Laboratories. 1987). and one chronic carcinogenicity studv (Chung et al..
1986). None of the subchronic studies are peer reviewed: however, the
NTP-PWG reviewed and confirmed the data and pathology in the
subchronic eavaee studies (NTP-PWG. 1987). Generally, data from the
mouse study corroborated the effects observed in the similarly designed rat
gavage study identified as the principal study. Neurotoxicity and
teratogenicity were not evaluated.
Confidence in
subchronic p-RfDa
M
The overall confidence in the subchronic p-RfD is medium.
aThe overall confidence cannot be greater than the lowest entry in the table (medium).
M = medium; NTP-PWG = National Toxicology Program's Pathology Working Group; p-RfD = provisional
reference dose.
Derivation of a Chronic Provisional Reference Dose
No adequate chronic oral non-neoplastic data are available. Data from the Chung et al.
(1986) study were not considered as the basis for deriving a chronic p-RfD due to incomplete
reporting of relevant data as well as inconsistencies in the dose-response of observed liver
tumors and inconsistent reporting of degenerative liver lesions accompanying body-weight
changes observed in the high-dose male rats. This lack of reporting precludes an analysis of
potential precursor effects at the low dose that could underly the chronic toxicity manifested
through reduced body weight at the high dose (Chung et al.. 1986). Therefore, the
BMDLio (ADD) of 3 mg/kg-day for increased incidence of forestomach hyperplasia in male rats
exposed to crotonaldehyde via gavage 5 days/week for 13 weeks (Hazleton 1 .aboratories. 1986b)
was also selected as the POD for derivation of the chronic p-RfD.
The chronic p-RfD is derived by applying a UFc of 3,000 (reflecting a UFa of 10, a UFh
of 10, a UFd of 3, and a subchronic-to-chronic extrapolation uncertainty factor [UFs] of 10) to
the selected POD of 3 mg/kg-day.
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Chronic p-RfD = POD (ADD) UFc
= 3 mg/kg-day ^ 3,000
= 1 x 10"3 mg/kg-day
Table 7 summarizes the uncertainty factors for the chronic p-RfD for
^ram'-crotonal dehy de.
Table 7. Uncertainty Factors for the Chronic p-RfD for
fraws-Crotonaldehyde (CASRN 123-73-9)
UF
Value
Justification
UFa
10
A UFa of 10 is applied to account for uncertainty associated with extrapolating from animals to
humans. Cross-species dosimetric adjustment (HED calculation) was not performed because the
critical endpoint may be a portal-of-entry effect.
UFd
3
A UFd of 3 (100 5) is applied to account for deficiencies and uncertainties in the database. The
database for oral exposure to /raws-crotonaldehyde consists of subchronic toxicity studies in
two species, one chronic carcinogenicity study in rats, and a one-generation reproduction study in rats.
There are no developmental toxicity studies available following exposure via any route.
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 /raws-crotonaldehyde in humans.
UFl
1
A UFl of 1 is applied because the POD is a BMDL.
UFS
10
A UFS of 10 is applied because the chronic POD was derived from a 13-wk study.
UFC
3,000
Composite UF = UFA x UFD x UFH x UFL x UFS.
BMDL = benchmark dose lower confidence limit; HED = human equivalent dose;
LOAEL = lowest-observed-adverse-effect level; NOAEL = no-observed-adverse-effect level; POD = point of
departure; p-RfD = provisional reference dose; UF = uncertainty factor; UFA = interspecies uncertainty factor;
UFc = composite uncertainty factor; UFD = database uncertainty factor; UFH = intraspecies uncertainty factor;
UFl = LOAEL-to-NOAEL uncertainty factor; UFS = subchronic-to-chronic uncertainty factor.
Confidence in the chronic p-RfD for /ra/7.s-crotonaldehyde is low, as described in
Table 8.
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Table 8. Confidence Descriptors for the Chronic p-RfD for
fraws-Crotonaldehyde (CASRN 123-73-9)
Confidence Categories
Designation
Discussion
Confidence in study
M
Confidence in the orincroal studv (Hazleton Laboratories. 1986b) is
medium. This study is not peer reviewed; however, it used a standard
number of animals (10 rats/sex) at 6 dose levels (including controls).
Furthermore, despite a lack of access to the complete data set, a
comprehensive suite of endpoints was examined (clinical symptoms, body
and organ weights, hematological and clinical chemistry, and pathology),
and the data was subseauentlv reviewed bv the NTP-PWG (NTP-PWG.
1987).
Confidence in database
L
There were no chronic studies identified that investigated non-neoplastic
endpoints, so confidence in the chronic database for /raws-crotonaldehyde
is low. The relevant database consists of one short-term dietary study in
rats (Borristoti. 1980a). as well as subchronic savase studies in rats
(Hazleton Laboratories. 1986b) and mice IHazelton Laboratories (1986a)
as cited in NTP-PWG (1987)1 and one sinsle-seneration reproduction
studv (Hazleton Laboratories. 1987) which found no effects. None of
these studies are peer reviewed; however, the NTP-PWG reviewed and
confirmed the data and patholoev in the subchronic eavaee studies (NTP-
PWG. 1987). Generally. data from this mouse studv corroborated the
effects observed in the similarly designed rat gavage study identified as the
principal study. Neurotoxicity and teratogenicity were not evaluated.
Confidence in chronic
p-RfDa
L
The overall confidence in the chronic p-RfD is low.
aThe overall confidence cannot be greater than the lowest entry in the table (low).
L = low; M = medium; NTP-PWG = National Toxicology Program's Pathology Working Group;
p-RfD = provisional reference dose.
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS
No studies have been identified that were adequate for deriving inhalation toxicity values.
SUMMARY OF NONCANCER PROVISIONAL REFERENCE VALUES
A summary of the noncancer provisional reference values is shown in Table 9.
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Table 9. Summary of Noncancer Reference Values for
fraws-Crotonaldehyde (CASRN 123-73-9)
Toxicity Type
(units)
Species/
Sex
Critical
Effect
p-Reference
Value
POD
Method
POD
(ADD)
UFc
Principal
Study
Subchronic p-RfD
(mg/kg-d)
Rat/M
Forestomach
hyperplasia
1 X 10-2
BMDLio
3
300
Hazleton
Laboratories
(1986b)
Chronic p-RfD
(mg/kg-d)
Rat/M
Forestomach
hyperplasia
1 x 1(T3
BMDLio
3
3,000
Hazleton
Laboratories
(1986b)
Subchronic p-RfC
(mg/m3)
NDr
Chronic p-RfC
(mg/m3)
NDr
ADD = adjusted daily dose; BMDL = benchmark dose lower confidence limit (subscripts denote benchmark
response: i.e., 10 = dose associated with a 10% extra risk in parameter); M = male(s); NDr = not derived;
POD = point of departure; p-RfC = provisional reference concentration; p-RfD = provisional reference dose;
UFC = composite uncertainty factor.
PROVISIONAL CARCINOGENICITY ASSESSMENT
A provisional cancer assessment was not prepared for /ra/7.s-crotonaldehyde. Although
IRIS (U.S. EPA. 2005) conducted a cancer assessment for this compound (weight of evidence
[WOE] = "C; possible human carcinogen"), the data were not adequate for deriving quantitative
estimates of carcinogenic risk by oral or inhalation exposure. Additionally, liver tumor
incidence data reported in Chung et al. (1986) could not be modeled due to a lack of
dose-response as indicated by a lack of tumorigenesis in the high-dose group (see Table 10).
Table 10. Summary of Cancer Risk Estimates for
fraws-Crotonaldehyde (CASRN 123-73-9)
Toxicity Type
Species/
Cancer Risk
(units)
Sex
Tumor Type
Estimate
Principal Study
p-OSF (mg/kg-d) 1
NDr
p-IUR (mg/m3)-1
NDr
NDr = not derived; p-IUR = provisional inhalation unit risk; p-OSF = provisional oral slope factor.
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APPENDIX A. LITERATURE SEARCH STRATEGY
Non-date-limited literature searches were conducted in May 2019 and updated in
July 2020 for studies relevant to the derivation of provisional toxicity values for
/ra/7.s-crotonaldehyde (CASRN 123-73-9) and the commercial crotonaldehyde mixture
(CASRN 4170-30-3; >95% /ra/7.s-isorner). Searches were conducted using U.S. EPA's Health
and Environmental Research Online (HERO) database of scientific literature. HERO searches
the following databases: PubMed, TOXLINE (including TSCATS1), and Web of Science. The
following databases were searched outside of HERO for health-related values: American
Conference of Governmental Industrial Hygienists (ACGIH), 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), U.S. EPA Chemical Data Access
Tool (CDAT), U.S. EPA ChemView, U.S. EPA Integrated Risk Information System (IRIS),
U.S. EPA Health Effects Assessment Summary Tables (HEAST), U.S. EPA Office of Water
(OW), International Agency for Research on Cancer (IARC), U.S. EPA TSCATS2/TSCATS8e,
U.S. EPA High Production Volume (HPV), Chemicals via IPCS INCHEM, European Centre for
Ecotoxicology and Toxicology of Chemicals (ECETOC), Japan Existing Chemical Data Base
(JECDB), European Chemicals Agency (ECHA), Organisation for Economic Cooperation 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).
LITERATURE SEARCH STRINGS
Pubmed
"123-73-9" OR "4170-30-3" OR "2 butenal"[tw] OR "2 E butenal"[tw] OR "2E but 2
enal"[tw] OR "b methylacrolein"[tw] OR "but 2 enal, E"[tw] OR "crotonal"[tw] OR
"crotonaldehyde"[tw] OR "crotonaldehydes"[tw] OR "croton aldehyde"[tw] OR "crotonic
aldehyde"[tw] OR "crotylaldehyde"[tw] OR "E but 2 en 1 al"[tw] OR "E but 2 enal"[tw] OR "E
crotonaldehyd"[tw] OR "trans 2 buten 1 al"[tw] OR "trans but 2 enal"[tw] OR "topanel CA"[tw]
OR "UN 1143"[tw]
WOS
TS="123 73 9" OR TS="4170 30 3" OR TS="2 butenal" OR TS="2 E butenal" OR
TS="2E but 2 enal" OR TS="b methylacrolein" OR TS="but 2 enal E" OR TS="crotonal" OR
TS="crotonaldehyde" OR TS="crotonaldehydes" OR TS="croton aldehyde" OR TS="crotonic
aldehyde" OR TS="crotylaldehyde" OR TS="E but 2 en 1 al" OR TS="E but 2 enal" OR TS="E
crotonaldehyd" OR TS="trans 2 buten 1 al" OR TS="trans but 2 enal" OR TS="topanel CA" OR
TS="UN 1143") AND ((WC=("Toxicology" OR "Endocrinology & Metabolism" OR
"Gastroenterology & Hepatology" OR "Gastroenterology & Hepatology" OR "Hematology" OR
"Neurosciences" OR "Obstetrics & Gynecology" OR "Pharmacology & Pharmacy" OR
"Physiology" OR "Respiratory System" OR "Urology & Nephrology" OR "Anatomy &
Morphology" OR "Andrology" OR "Pathology" OR "Otorhinolaryngology" OR
"Ophthalmology" OR "Pediatrics" OR "Oncology" OR "Reproductive Biology" OR
"Developmental Biology" OR "Biology" OR "Dermatology" OR "Allergy" OR "Public,
Environmental & Occupational Health") OR SU=("Anatomy & Morphology" OR
"Cardiovascular System & Cardiology" OR "Developmental Biology" OR "Endocrinology &
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Metabolism" OR "Gastroenterology & Hepatology" OR "Hematology" OR "Immunology" OR
"Neurosciences & Neurology" OR "Obstetrics & Gynecology" OR "Oncology" OR
"Ophthalmology" OR "Pathology" OR "Pediatrics" OR "Pharmacology & Pharmacy" OR
"Physiology" OR "Public, Environmental & Occupational Health" OR "Respiratory System" OR
"Toxicology" OR "Urology & Nephrology" OR "Reproductive Biology" OR "Dermatology" OR
"Allergy")) OR (TS="rat" OR TS="rats" OR TS="mouse" OR TS="murine" OR TS="mice"
OR TS="guinea" OR TS="muridae" OR TS=rabbit* OR TS=lagomorph* OR TS=hamster* OR
TS=ferret* OR TS=gerbil* OR TS=rodent* OR TS="dog" OR TS="dogs" OR TS=beagle* OR
TS="canine" OR TS="cats" OR TS="feline" OR TS="pig" OR TS="pigs" OR TS="swine" OR
TS="porcine" OR TS=monkey* OR TS=macaque* OR TS=baboon* OR TS=marmoset* OR
TS="child" OR TS="children" OR TS=adolescen* OR TS=infant* OR TS="worker" OR
TS="workers" OR TS="human" OR TS=patient* OR TS="mother" OR TS="fetal" OR
TS="fetus" OR TS="citizens" OR TS="milk" OR TS="formula" OR TS=epidemio* OR
TS=population* OR TS=exposure* OR TS="questionnaire" OR SO=epidemio*) OR TI=toxic*
TOXLINE
@SYNO+@OR+("2+butenal"+"2+E+butenal"+"2E+but+2+enal"+"b+methylacrolein"+"
but+2+enal+E"+crotonal+crotonaldehyde+crotonaldehydes+"croton+aldehyde"+"crotonic+alde
hyde"+crotylaldehyde+"E+but+2+en+l+al"+"E+but+2+enal"+"E+crotonaldehyd"+"trans+2+bu
ten+1+al"+"trans+but+2+enal"+"topanel+C A"+"UN+1143 "+@TERM+@rn+123-73 -9+@TER
M+@rn+4170-30-3)+@NOT+@org+pubmed+pubdart+nih
TSCATS
@TERM+@rn+" 123-73 -9"+@org+T SCATS
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APPENDIX B. DETAILED PECO CRITERIA
Table B-l. Population, Exposure, Comparison, and Outcome (PECO) Criteria
PECO Element
Evidence
Population
Humans, laboratory mammals, and other animal models of established relevance to human
health (e.g.. Xenopus embryos); mammalian organs, tissues, and cell lines; and bacterial and
eukaryote models of genetic toxicity.
Exposure
In vivo (all routes), ex vivo, and in vitro exposure to the chemical of interest, including
mixtures to which the chemical of interest may contribute significantly to exposure or
observed effects.
Comparison
Any comparison (across dose, duration, or route) or no comparison (e.g., case reports without
controls).
Outcome
Any endpoint suggestive of a toxic effect on any bodily system, or mechanistic change
associated with such effects. Any endpoint relating to disposition of the chemical within the
body.
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APPENDIX C. SCREENING PROVISIONAL VALUES
No screening provisional values are derived for /ra/7.s-crotonaldehyde.
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APPENDIX D. DATA TABLES
Table D-l. Terminal Body Weight and Select Organ Weights in Male F344 Rats Exposed to Commercial Crotonaldehyde
(CASRN 4170-30-3) via Gavage in Corn Oil for 13 Weeks (5 Days/Week)3
Dose Group, mg/kg-d (ADD)
Endpoint
0
2.5 (1.8)
5(4)
10 (7.1)
20 (14)
40 (29)
Mortality13
0/10 (0%)
0/10 (0%)
0/10 (0%)
3/10 (30%)
3/10 (30%)
5/10 (50%)
Terminal body
weight (g)c d
320.8 ± 18.7
323.3 ± 13.6 (+1%)
336.5 ± 20.9 (+5%)
333.7 ±7.2 (+4%)
318.4 ± 17.8 (-0.7%)
290.4 ± 15.5* (-9%)
Liver weight0, d
Absolute (g)
13.35 ± 1.00
12.98 ± 0.53 (-3%)
13.03 ± 1.70 (-2%)
14.03 ± 1.03 (+5%)
13.21 ±1.19 (-1%)
12.46 ±1.01 (-7%)
Relative (%)
4.163 ±0.217
4.023 ± 0.259 (-3%)
3.859 ±0.284 (-7%)
4.205 ±0.291 (+1%)
4.147 ±0.259 (-0.4%)
4.294 ±0.314 (+3%)
Kidney weight0, d
Absolute (g)
1.14 ±0.07
1.11 ±0.09 (-3%)
1.18 ±0.13 (+4%)
1.13 ±0.11 (-0.9%)
1.15 ±0.12 (+0.9%)
1.09 ±0.11 (-4%)
Relative (%)
0.355 ±0.016
0.344 ± 0.020 (-3%)
0.349 ±0.019 (-2%)
0.340 ± 0.036 (-4%)
0.360 ± 0.027 (+1%)
0.378 ± 0.049 (+6%)
Thymus weight0, d
Absolute (g)
0.308 ±0.023
0.303 ± 0.039 (-2%)
0.299 ± 0.046 (-3%)
0.314 ±0.030 (+2%)
0.287 ± 0.068 (-7%)
0.272 ± 0.049 (-12%)
Relative (%)
0.0966 ±0.0102
0.0939 ±0.0118 (-3%)
0.0888 ±0.0140 (-8%)
0.0941 ±0.0102 (-3%)
0.0895 ±0.0168 (-7%)
0.0933 ±0.0130 (-3%)
Brain weight0, d
Absolute (g)
2.15 ±0.09
2.10 ±0.06 (-2%)
2.13 ±0.09 (-0.9%)
2.15 ±0.03 (0%)
2.09 ±0.11 (-3%)
2.08 ± 0.09 (-3%)
Relative (%)
0.671 ±0.033
0.649 ±0.015 (-3%)
0.633 ± 0.026* (-6%)
0.645 ± 0.009 (-4%)
0.658 ± 0.042 (-2%)
0.718 ±0.045 (+7%)
43
/ra/7.s-Crotonaldehyde
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EPA/690/R-21/001F
Table D-l. Terminal Body Weight and Select Organ Weights in Male F344 Rats Exposed to Commercial Crotonaldehyde
(CASRN 4170-30-3) via Gavage in Corn Oil for 13 Weeks (5 Days/Week)3
Endpoint
Dose Group, mg/kg-d (ADD)
0
2.5 (1.8)
5(4)
10 (7.1)
20 (14)
40 (29)
Testicle weight0, d
Absolute (g)
Relative (%)
1.532 ±0.095
0.4784 ± 0.0288
1.476 ±0.114 (-4%)
0.4560 ± 0.0245 (-5%)
1.509 ±0.128 (-2%)
0.4490 ±0.0211 (-6%)
1.450 ±0.080 (-5%)
0.4347 ± 0.0257* (-9%)
1.469 ±0.131 (-4%)
0.4618 ±0.0376 (-3%)
1.452 ±0.106 (-5%)
0.5001 ±0.0214 (+5%)
aHazleton Laboratories (1986b).
bValues denote number of animals showing changes total number of animals examined (% incidence).
Data are mean ± SD; n = 5-10/group.
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; SD = standard deviation.
44
/ra/7.s-Crotonaldehyde
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EPA/690/R-21/001F
Table D-2. Terminal Body Weight and Select Organ Weights in Female F344 Rats Exposed to Commercial Crotonaldehyde
(CASRN 4170-30-3) via Gavage in Corn Oil for 13 Weeks (5 Days/Week)3
Dose Group, mg/kg-d (ADD)
Endpoint
0
2.5 (1.8)
5(4)
10 (7.1)
20 (14)
40 (29)
Mortality13
1/10 (10%)
0/10 (0%)
1/10 (10%)
1/10 (10%)
7/10 (70%)
5/10 (50%)
Terminal body
weight (g)° d
198.9 ±7.7
200.1 ±6.6 (+0.6%)
202.8 ± 3.3 (+2%)
200.8+ 13.1 (+1%)
201.9 + 4.4 (+2%)
194.3 + 8.2 (-2%)
Liver weight0, d
Absolute (g)
6.80 ±0.35
7.05 ± 0.48 (+4%)
7.20 ± 0.47 (+6%)
6.48 + 0.63 (-5%)
7.24 + 0.12 (+6%)
7.13 + 1.11 (+5%)
Relative (%)
3.419 ±0.142
3.525 ±0.198 (+3%)
3.556 ±0.264 (+4%)
3.226 + 0.186 (-6%)
3.587 + 0.039* (+5%)
3.666 + 0.542 (+7%)
Kidney weight0, d
Absolute (g)
0.74 ± 0.06
0.73 ± 0.05 (-1%)
0.76 ± 0.05 (+3%)
0.75 + 0.05 (+1%)
0.77 + 0.03 (+4%)
0.77 + 0.04 (+4%)
Relative (%)
0.371 ±0.029
0.366 ± 0.024 (-1%)
0.373 ± 0.026 (+0.5%)
0.376 +0.018 (+1%)
0.381 + 0.011 (+3%)
0.396 + 0.240 (+7%)
Thymus weight0, d
Absolute (g)
0.261 ±0.029
0.259 ± 0.024 (-0.8%)
0.251 ±0.026 (-4%)
0.245 + 0.022 (-6%)
0.260 + 0.062 (-0.4%)
0.206 + 0.023* (-21%)
Relative (%)
0.1310 ± 0.0131
0.1295 ±0.0098 (-1%)
0.1239 + 0.0131 (-5%)
0.1219 + 0.0087 (-7%)
0.1283 + 0.0293 (-2%)
0.1059 + 0.0102* (-19%)
Brain weight0, d
Absolute (g)
2.00 ±0.10
1.98 ±0.05 (-1%)
2.01 ± 0.06 (+0.2%)
2.01 + 0.08 (+0.5%)
2.04 + 0.07 (+2%)
1.99 + 0.07 (-0.5%)
Relative (%)
1.004 ±0.055
0.992 ± 0.035 (-1%)
0.990 + 0.038 (-1%)
1.005+ 0.057 (+0.1%)
1.011+ 0.046 (+0.7%)
1.026 + 0.054 (+2%)
aHazleton Laboratories (1986b).
bValues denote number of animals showing changes + total number of animals examined (% incidence).
Data are mean ± SD; n = 3-10/group.
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; SD = standard deviation.
45
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EPA/690/R-21/001F
Table D-3. Forestomach and Nasal Lesions in F344 Rats Exposed to Commercial
Crotonaldehyde (CASRN 4170-30-3) via Gavage in Corn Oil for 13 Weeks (5 DaysAVeek)3
Endpointb
Dose Group, mg/kg-d (ADD)
0
2.5 (1.8)
5(4)
10 (7.1)
20 (14)
40 (29)
Thickened forestomach
Males
Females
0/10 (0%)
0/10 (0%)
0/10 (0%)
0/10 (0%)
0/10 (0%)
0/10 (0%)
0/10 (0%)
0/10 (0%)
2/10 (20%)
0/10 (0%)
1/10 (10%)
5/10* (50%)
Forestomach nodule
Males
Females
0/10 (0%)
0/10 (0%)
0/10 (0%)
0/10 (0%)
0/10 (0%)
0/10 (0%)
0/10 (0%)
0/10 (0%)
0/10 (0%)
0/10 (0%)
3/10 (30%)
2/10 (20%)
Forestomach hyperplasia
Males
Females
0/10 (0%)
0/10 (0%)
0/10 (0%)
0/10 (0%)
0/10 (0%)
0/10 (0%)
3/10 (30%)
1/10° (10%)
3/10 (30%)
4/10* (40%)
8/10* (80%)
8/10* (80%)
Nasal inflammation"1
Males
Females
0/10 (0%)
1/10 (10%)
0/10 (0%)
0/10 (0%)
0/10 (0%)
1/10 (10%)
3/10 (30%)
1/10 (10%)
3/10 (30%)
4/10 (40%)
7/10* (70%)
6/10* (60%)
aHazleton Laboratories (1986b): NTP-PWG (1987).
bValues denote number of animals showing changes total number of animals examined (% incidence).
"Lesion reported as "equivocal" by NTP-PWG (1987).
'Reported as nasal inflammation by Hazleton Laboratories (1986b). reclassified as serous exudation by NTP-PWG
(1987).
* Significantly different from control by Fisher's exact probability test (p < 0.05), conducted for this review.
ADD = adjusted daily dose.
46
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EPA/690/R-21/001F
Table D-4. Body-Weight Data in F344 Rats Exposed to Commercial Crotonaldehyde
(CASRN 4170-30-3) via Gavage in Corn Oil for 9 Weeks (Premating through PND 5)a
Endpointb'c
Dose Group, mg/kg-d
0
2.5
5
10
Males
Body weight (g)
Initial
Wk 9
115.7 ± 8.17
305.8 ±20.24
115.8 ±7.38 (+0.1%)
301.1 ± 18.64 (-2%)
119.9 ±6.22 (+4%)
307.3 ±21.78 (+0.5%)
115.6 + 8.99 (-0.1%)
305.6+ 16.83 (-0.1%)
Body-weight gain (g)
Wk 0-9
190.1 ± 16.08
185.4 ± 16.50 (-2%)
187.5 + 17.00 (-1%)
190.0+ 14.71 (0%)
Females
Body weight (g)
Initial (all)
GD 0 (pregnant)
GD 20 (pregnant)
PND 0 (pregnant)
PND 5 (pregnant)
Wk 9 (nonpregnant)
106.3 ±5.74
158 ±6.0
240 ± 16.9
195.1 ±9.73
196.2 ± 11.00
202.9 ±0.21
108.0 ± 4.69 (+2%)
159 ± 7.9 (+0.6%)
244 ± 14.2 (+2%)
196.9 ± 10.78 (+0.9%)
198.2 ± 9.77 (+1%)
183.6 ±8.35 (-10%)
107.8 + 7.39 (+1%)
161 + 8.9 (+2%)
250 + 16.4 (+4%)
197.6+ 13.57 (+1%)
201.0+ 12.08 (+2%)
188.9 + 10.72 (-7%)
106.6 + 4.49 (+0.3%)
162 + 4.4 (+3%)
249 + 14.0 (+4%)
196.6 + 9.64 (+0.8%)
199.7+ 11.06 (+2%)
175.2 + 7.43 (-14%)
Body-weight gain (g)
GDs 0-20 (pregnant)
Wk 0-9 (nonpregnant)
82.0 ± 14.48
90.6 ±3.32
85.4 ± 9.08 (+4%)
72.6 ± 6.05* (-20%)
88.6 + 12.24 (+8%)
82.1 + 4.46 (-9%)
87.8+ 13.58 (+7%)
72.0 + 2.91* (-21%)
aHazleton Laboratories (1987).
bData are mean ± SD; n =19-20 males/group, 20 total females/group (14-16 pregnant females/group,
2-5 nonpregnant females/group).
0Value 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.
GD = gestation day; PND = postnatal day; SD = standard deviation.
47
/ra/7.s-Crotonaldehyde
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EPA/690/R-21/001F
Table D-5. Survival and Preneoplastic and Neoplastic Lesions in Male F344 Rats Exposed
to fraws-Crotonaldehyde (CASRN 123-73-9) in Drinking Water for 113 Weeks3
Dose Group, mg/kg-d (HED)
Endpoint
0
2 (0.6)
17 (4.6)
Survival13
70 wk
23/23 (100%)
27/27 (100%)
22/23 (96%)
90 wk
21/23 (91%)
25/27 (93%)
18/23 (78%)
110 wk
16/23 (70%)
17/27 (63%)
13/23 (57%)
Liver tumorsb
Neoplastic nodule
0/23 (0%)
9/27* (33%)
1/23 (4%)
Hepatocellular carcinoma
0/23 (0%)
2/27 (7%)
0/23 (0%)
Neoplastic nodule or
hepatocellular carcinoma
0/23 (0%)
9/27* (33%)
1/23 (4%)
Preneoplastic lesions
Altered liver focib
1/23 (4%)
23/27* (85%)
13/23* (57%)
Number of altered foci/cm2 0
0.1 ±0.4
12.4 ± 7.4* (+124-fold)
3.5 ±3.8* (+35-fold)
aChung et al. (1986).
bValues denote number of animals showing changes ^ total number of animals examined (% incidence). The
differences in terminal survival across groups were not significant based on Fisher's exact tests conducted for this
review.
Data are mean ± SD for 23-27 rats; value in parentheses is fold-change relative to control = treatment
mean control mean.
* Significantly different from control (p < 0.001), as reported by the study authors.
HED = human equivalent dose; SD = standard deviation.
48
/ra/7.s-Crotonaldehyde
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EPA/690/R-21/001F
APPENDIX E. BENCHMARK DOSE MODELING RESULTS
MODELING PROCEDURE
Dichotomous Noncancer Data
The benchmark dose (BMD) modeling of dichotomous data is conducted with the
U.S. EPA's Benchmark Dose Software (BMDS; Version 2.6 was used for this document). For
these data, the Gamma, Logistic, Log-Logistic, Log-Probit, Multistage, Probit, and Weibull
dichotomous models available within the software are fit using a benchmark response (BMR) of
10% extra risk. Alternative BMRs may also be used where appropriate, as outlined in the
Benchmark Dose Technical Guidance (U.S. EPA, 2012). 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 benchmark dose lower confidence limit (BMDL) estimates from
different models (high model-dependence). Adequacy of model fit is judged based on the
X2 goodness-of-fitp-wdXut (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 Akaike's information criterion (AIC) is selected as a potential point of
departure (POD), if the BMDLs are sufficiently close (threefold), model-dependence is indicated, and the model with the lowest
reliable BMDL is selected.
BMD MODELING TO IDENTIFY POTENTIAL POINTS OF DEPARTURE FOR
DERIVATION OF SCREENING PROVISIONAL REFERENCE DOSES
The data sets for forestomach lesions in male and female rats exposed to crotonaldehyde
for 13 weeks via gavage (Hazleton Laboratories. 1986b) were modeled to determine potential
PODs for the screening subchronic and chronic provisional reference dose (p-RfD), using BMD
analysis. Table D-3 shows the data that were modeled. Summaries of modeling approaches and
results (see Tables E-l and E-2 and Figures E-l and E-2) for each data set follow.
Increased Incidence of Forestomach Hyperplasia in Male F344 Rats Exposed to
Commercial Crotonaldehyde via Gavage for 13 Weeks (Hazleton Laboratories, 1986b)
The procedure outlined above for dichotomous data was applied to the data for
forestomach hyperplasia in male F344 rats exposed to commercial crotonaldehyde
(>95% ^ram'-isomer) via gavage in corn oil 5 days/week for 13 weeks (see Table D-3).
Table E-l summarizes the BMD modeling results. All models provided an adequate fit to the
data. BMDL values were sufficiently close (differed by
-------
EPA/690/R-21/001F
Table E-l. BMD Modeling Results for Increased Incidence of Forestomach Hyperplasia in
Male F344 Rats Exposed to Commercial Crotonaldehyde (CASRN 4170-30-3) via Gavage
for 13 Weeks (5 DaysAVeek)3
Model
DF
x2
x2
Goodness-of-Fit
/>-Valueb
Scaled Residual
at Dose Nearest
BMD
AIC
BMDio
(ADD)
(mg/kg-d)
BMDLio
(ADD)
(mg/kg-d)
Gamma0
4
2.99
0.5587
1.39
41.7056
5.7
2.7
Logistic
4
5.22
0.2656
1.854
44.3109
8.2
5.7
LogLogisticd
4
3.08
0.5439
1.347
41.8037
5.7
2.9
Log-Probitd' *
4
2.83
0.5865
1.259
41.4867
5.71482
3.46158
Multistage (2-degree)6
4
3.28
0.5121
1.498
42.0748
5.7
2.5
Multistage (3-degree)6
4
3.28
0.5121
1.498
42.0748
5.7
2.5
Probit
4
4.95
0.2922
1.86
43.7931
7.7
5.4
Weibull0
4
3.03
0.5532
1.405
41.8359
5.6
2.6
aHazleton Laboratories (1986b).
bValues <0.10 fail to meet conventional goodness-of-fit criteria.
Tower restricted to >1.
dSlope restricted to >1.
"Betas restricted to >0.
* Selected model. All models provided adequate fit. BMDLs were sufficiently close (differed by
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EPA/690/R-21/00 IF
LogProbit Model, with BMR of 10% Extra Riskfor the BMD and 0.95 Lower Confidence Limit for the BMDL
dose
15:14 11/20 2019
Figure E-l. Fit of Log-Probit Model to Data for Increased Incidence of Forestomach
Hyperplasia in Male F344 Rats Exposed to Commercial Crotonaldehyde
(CASRN 4170-30-3) via Gavage for 13 Weeks (Hazleton Laboratories, 1986b)
BMD Model Output for Figure E-l:
Probit Model. (Version: 3.4; Date: 5/21/2017)
Input Data File: C:/Users/jdean04/BMDS2704/Data/lnp_Crotonaldehyde-hazleton
male forestomach hyperplasia_Lnp-BMR10-Restrict.(d)
Gnuplot Plotting File:
C:/Users/jdean04/BMDS2704/Data/lnp_Crotonaldehyde-hazleton male forestomach
hyperplasia_Lnp-BMR10-Restrict.pit
Wed Nov 20 15:14:54 2019
BMDS Model Run
The form of the probability function is:
P[response] = Background
+ (1-Background) * CumNorm(Intercept+Slope*Log(Dose)) ,
where CumNormf .) is the cumulative normal distribution function
Dependent variable = Effect
Independent variable = Dose
Slope parameter is restricted as slope >= 1
51
/ra/7.s-Crotonaldehyde
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EPA/690/R-21/001F
Total number of observations = 6
Total number of records with missing values = 0
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
User has chosen the log transformed model
Default Initial (and Specified) Parameter Values
background = 0
intercept = -2.76936
slope = 1
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -background
have been estimated at a boundary point, or have been specified by
the user,
and do not appear in the correlation matrix )
intercept slope
intercept 1 -0.96
slope -0.96 1
Interval
Variable
Limit
background
intercept
0.889948
slope
1.91102
Estimate
0
-3.44396
-5.18822
1.24058
Parameter Estimates
Std. Err.
NA
-1.69969
0.34207
NA - Indicates that this parameter has hit a bound
implied by some ineguality constraint and thus
has no standard error.
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
0.570132
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-17.2213
-18.7434
-32.5964
# Param's
6
2
1
Deviance Test d.f.
3.04412
30.7501
P-value
0.5505
<.0001
AIC:
41.4867
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size Residual
0.0000 0.0000 0.000 0.000 10.000 0.000
1.8000 0.0033 0.033 0.000 10.000 -0.182
52
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EPA/690/R-21/001F
4.0000 0.0423 0.423 0.000 10.000 -0.665
7.1000 0.1557 1.557 3.000 10.000 1.259
14.0000 0.4325 4.325 3.000 10.000 -0.846
29.0000 0.7684 7.684 8.000 10.000 0.237
Chi^2 = 2.83 d.f. = 4 P-value = 0.5865
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 5.71482
BMDL = 3.4 6158
BMDU = 8.4939
53
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EPA/690/R-21/001F
Increased Incidence of Forestomach Hyperplasia in Female F344 Rats Exposed to
Commercial Crotonaldehyde via Gavage for 13 Weeks (Hazleton Laboratories, 1986b)
The procedure outlined above for dichotomous data was applied to the data for
forestomach hyperplasia in female F344 rats exposed to crotonaldehyde via gavage in corn oil
5 days/week for 13 weeks (see Table D-3). Table E-2 summarizes the BMD modeling results.
All models provided adequate statistical fit to the data; however, based on visual inspection,
scaled residuals, relatively poor statistical fit, and an outlier result relative to the other models,
the Multistage 1-degree model was not considered an adequate fit to the data. Remaining
BMDLs differed by -Valucb
Scaled Residual
at Dose Nearest
BMD
AIC
BMDio
(ADD)
(mg/kg-d)
BMDLio
(ADD)
(mg/kg-d)
Gamma0
4
0.39
0.983
0.161
34.5698
7.6
4.3
Logistic
4
2.55
0.635
0.219
37.3479
9.3
6.4
LogLogisticd
4
0.29
0.990
0.192
34.4614
7.6
4.5
Log-Probitd
4
0.16
0.997
0.179
34.2354
7.5
4.6
Multistage (2-degree)6' *
5
0.65
0.986
0.018
32.9994
7.166
3.8462
Multistage (3-degree)6
5
0.65
0.986
0.018
32.9994
7.2
3.9
Probit
4
2.08
0.721
0.272
36.6479
8.9
6.1
Weibull0
4
0.68
0.954
0.076
34.9882
7.4
4.0
aHazleton Laboratories (1986b).
bValues <0.10 fail to meet conventional goodness-of-fit criteria.
Tower restricted to >1.
dSlope restricted to >1.
"Betas restricted to >0.
* Selected model. All models provided adequate statistical fit to the data; however, based on visual inspection,
scaled residuals, relatively poor statistical fit, and an outlier result relative to the other models, the Multistage
1-degree model was not considered an adequate fit to the data. Remaining BMDLs differed by
-------
EPA/690/R-21/00 IF
Multistage Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL
dose
15:45 11/20 2019
Figure E-2. Fit of Multistage 2-Degree Model to Data for Increased Incidence of
Forestomach Hyperplasia in Female F344 Rats Exposed to Commercial Crotonaldehyde
(CASRN 4170-30-3) via Gavage for 13 Weeks (Hazleton Laboratories, 1986b)
BMD Model Output for Figure E-2:
Multistage Model. (Version: 3.4; Date: 05/02/2014)
Input Data File: C:/Users/jdean04/BMDS2704/Data/mst_Crotonaldehyde-hazleton
female forestomach hyperplasia_Mst3-BMR10-Restrict.(d)
Gnuplot Plotting File:
C:/Users/jdean04/BMDS2704/Data/mst_Crotonaldehyde-hazleton female forestomach
hyperplasia_Mst3-BMR10-Restrict.pit
Wed Nov 20 15:45:42 2019
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose/sl-beta2*dose/s2-beta3* doseA3)]
The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Total number of observations = 6
55
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EPA/690/R-21/001F
Total number of records with missing values = 0
Total number of parameters in model = 4
Total number of specified parameters = 0
Degree of polynomial = 3
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0
Beta(1) = 0.015664
Beta(2) = 0.0014296
Beta(3) = 0
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background -Beta(l) -Beta (3)
have been estimated at a boundary point, or have been specified by
the user,
and do not appear in the correlation matrix )
Beta(2)
Beta (2) 1
Parameter Estimates
Interval
Variable
Limit
Background
Beta(1)
Beta(2)
0.00325204
Beta(3)
Estimate
0
0
0.00205175
0
Std. Err.
NA
NA
0.000612406
NA
NA - Indicates that this parameter has hit a bound
implied by some ineguality constraint and thus
has no standard error.
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
0.000851457
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-14.985
-15.4997
-31.3594
# Param's Deviance Test d.f. P-value
6
1 1.02949 5 0.9602
1 32.7488 5 <.0001
AIC:
32.9994
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size Residual
0.0000 0.0000 0.000 0.000 10.000 0.000
1.8000 0.0066 0.066 0.000 10.000 -0.258
56
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EPA/690/R-21/001F
4.0000
7.1000
14.0000
29.0000
Chi^2 = 0.65
0.0323
0.0983
0.3311
0.8219
d.f. = 5
0.323 0.000 10.000
0.983 1.000 10.000
3.311 4.000 10.000
8.219 8.000 10.000
P-value = 0.9857
-0.578
0. 018
0. 463
-0.181
Benchmark Dose Computation
Specified effect
Risk Type
Confidence level
BMD
BMDL
BMDU
0.1
Extra risk
0. 95
7.166
3.8462
11.1204
Taken together, (3.8462 , 11.1204) is a 90
interval for the BMD
two-sided confidence
Table E-3 summarizes the BMD best-fit modeling results for the modeled endpoints.
Table E-3. BMD and BMDL Values from Best-Fitting Models for Forestomach
Hyperplasia in F344 Rats Exposed to frans-Crotonaldehyde (CASRN 123-73-9) and
Commercial Crotonaldehyde (>95% trans-; CASRN 4170-30-3)
via Gavage for 13 Weeks (5 DaysAVeek)3
BMD (ADD)
BMDL (ADD)
Sex
Best Fitting Model
BMR
(mg/kg-d)
(mg/kg-d)
Male
Log-Probit
10% extra risk
6
3
Female
Multistage 2-degree
10% extra risk
7
4
aHazleton Laboratories (1986b).
ADD = adjusted daily dose; BMD = benchmark dose; BMDL = benchmark dose lower confidence limit;
BMR = benchmark response.
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