United States
Environmental Protection
1=1 m m Agency
EPA/690/R-10/007F
Final
6-17-2010
Provisional Peer-Reviewed Toxicity Values for
Crotonaldehyde
(CASRN 123-73-9)
Superfund Health Risk Technical Support Center
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268

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Commonly Used Abbreviations
BMC
Benchmark Concentration
BMD
Benchmark Dose
BMCL
Benchmark Concentration Lower bound 95% confidence interval
BMDL
Benchmark Dose Lower bound 95% confidence interval
HEC
Human Equivalent Concentration
HED
Human Equivalent Dose
IRIS
Integrated Risk Information System
IUR
inhalation unit risk
LOAEL
lowest-observed-adverse-effect level
LOAELadj
LOAEL adjusted to continuous exposure duration
LOAELhec
LOAEL adjusted for dosimetric differences across species to a human
NOAEL
no-ob served-adverse-effect level
NOAELadj
NOAEL adjusted to continuous exposure duration
NOAELhec
NOAEL adjusted for dosimetric differences across species to a human
NOEL
no-ob served-effect level
OSF
oral slope factor
p-IUR
provisional inhalation unit risk
p-OSF
provisional oral slope factor
p-RfC
provisional reference concentration (inhalation)
p-RfD
provisional reference dose (oral)
POD
point of departure (oral)
RfC
reference concentration (inhalation)
RfD
reference dose
UF
uncertainty factor
UFa
animal to human uncertainty factor
UFC
composite uncertainty factor
UFd
incomplete to complete database uncertainty factor
UFh
interhuman uncertainty factor
UFl
LOAEL to NOAEL uncertainty factor
UFS
subchronic to chronic uncertainty factor
WOE
weight of evidence
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PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR
CROTONALDEHYDE (CASRN 123-73-9)
Background
On December 5, 2003, the U.S. Environmental Protection Agency's (EPA) Office of
Superfund Remediation and Technology Innovation (OSRTI) revised its hierarchy of human
health toxicity values for Superfund risk assessments, establishing the following three tiers as the
new hierarchy:
1)	EPA's Integrated Risk Information System (IRIS).
2)	Provisional Peer-Reviewed Toxicity Values (PPRTVs) used in EPA's Superfund
Program.
3)	Other (peer-reviewed) toxicity values, including
~	Minimal Risk Levels produced by the Agency for Toxic Substances and Disease
Registry (ATSDR),
~	California Environmental Protection Agency (CalEPA) values, and
~	EPA Health Effects Assessment Summary Table (HEAST) values.
A PPRTV is defined as a toxicity value derived for use in the Superfund Program when
such a value is not available in EPA's IRIS. PPRTVs are developed according to a Standard
Operating Procedure (SOP) and are derived after a review of the relevant scientific literature
using the same methods, sources of data, and Agency guidance for value derivation generally
used by the EPA IRIS Program. All provisional toxicity values receive internal review by two
EPA scientists and external peer review by three independently selected scientific experts.
PPRTVs differ from IRIS values in that PPRTVs do not receive the multiprogram consensus
review provided for IRIS values. This is because IRIS values are generally intended to be used
in all EPA programs, while PPRTVs are developed specifically for the Superfund Program.
Because new information becomes available and scientific methods improve over time,
PPRTVs are reviewed on a 5-year basis and updated into the active database. Once an IRIS
value for a specific chemical becomes available for Agency review, the analogous PPRTV for
that same chemical is retired. It should also be noted that some PPRTV documents conclude that
a PPRTV cannot be derived based on inadequate data.
Disclaimers
Users of this document should first check to see if any IRIS values exist for the chemical
of concern before proceeding to use a PPRTV. If no IRIS value is available, staff in the regional
Superfund and Resource Conservation and Recovery Act (RCRA) program offices are advised to
carefully review the information provided in this document to ensure that the PPRTVs used are
appropriate for the types of exposures and circumstances at the Superfund site or RCRA facility
in question. PPRTVs are periodically updated; therefore, users should ensure that the values
contained in the PPRTV are current at the time of use.
It is important to remember that a provisional value alone tells very little about the
adverse effects of a chemical or the quality of evidence on which the value is based. Therefore,
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users are strongly encouraged to read the entire PPRTV document and understand the strengths
and limitations of the derived provisional values. PPRTVs are developed by the EPA Office of
Research and Development's National Center for Environmental Assessment, Superfund Health
Risk Technical Support Center for OSRTI. Other EPA programs or external parties who may
choose of their own initiative to use these PPRTVs are advised that Superfund resources will not
generally be used to respond to challenges of PPRTVs used in a context outside of the Superfund
Program.
Questions Regarding PPRTVs
Questions regarding the contents of the PPRTVs and their appropriate use (e.g., on
chemicals not covered, or whether chemicals have pending IRIS toxicity values) may be directed
to the EPA Office of Research and Development's National Center for Environmental
Assessment, Superfund Health Risk Technical Support Center (513-569-7300), or OSRTI.
INTRODUCTION
There is no RfD or RfC assessment for crotonaldehyde (2-butenal; see Figure 1 for
chemical structure) on IRIS (U.S. EPA, 2008), the Drinking Water Health Advisories list
(U.S. EPA, 2006), or the HEAST (U.S. EPA, 1997). The Chemical Assessments and Related
Activities (CARA) list (U.S. EPA, 1991, 1994) includes a Health and Environmental Effects
Document (HEED) for crotonaldehyde (U.S. EPA, 1989). The HEED (U.S. EPA, 1989)
concluded that a chronic or subchronic RfD could not be derived from the available animal data.
Crotonaldehyde has not been the subject of a toxicological profile by ATSDR (2008). A World
Health Organization (WHO, 2008) Concise International Chemical Assessment Document
(CICAD) declined to derive toxicity values due to the lack of suitable data. The American
Conference of Governmental Industrial Hygienists (ACGIH, 2008) has adopted a threshold limit
value-short-term exposure limit (TLV-STEL) (ceiling limit) of 0.3 ppm for crotonaldehyde based
on the potential for irritation to the eyes and upper respiratory tract (quantitation based on
analogy to formaldehyde). The time-weighted average-recommended exposure limit
(TWA-REL) established by the National Institute for Occupational Safety and Health (NIOSH,
2008), and the current enforceable Occupational Safety and Health Administration (OSHA,
2008) permissible exposure limit (PEL), is 2 ppm (6 mg/m3).
o	^ ch3
Figure 1. Chemical Structure of Crotonaldehyde
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IRIS (U.S. EPA, 2008) includes a Group C, Possible Human Carcinogen, cancer
weight-of-evidence designation for crotonaldehyde based on inadequate human evidence and
limited evidence for carcinogenicity in animals (increased incidence of hepatocellular neoplasms
in male rats exposed for up to 113 weeks to crotonaldehyde in drinking water by Chung et al.,
1986). The source document for this assessment, which was verified 03/07/91, was the HEED
(U.S. EPA, 1989). IRIS (U.S. EPA, 2008) does not include derivation of quantitative cancer risk
values for crotonaldehyde due to the limited nature of the positive cancer data (positive response
only in the low-dose group). The HEED (U.S. EPA, 1989) had derived an oral slope factor of
1.9 per mg/kg-day for this chemical by dropping the high-dose group, and this value is reported
in the HEAST (U.S. EPA, 1997). The International Agency for Research on Cancer (IARC,
1995) assigned crotonaldehyde to Group 3, Not Classifiable as to its Carcinogenicity to Humans,
based on inadequate data in humans and animals. ACGIH (2008) classified crotonaldehyde as a
Group A3 carcinogen {Confirmed Animal Carcinogen with Unknown Relevance to Humans)
based on the Chung et al. (1986) study.
Literature searches were conducted from the 1960s through February 2010 for studies
relevant to the derivation of provisional toxicity values for crotonaldehyde. Databases searched
included: MEDLINE, TOXLINE (and NTIS), CCRIS, DART, EMIC, HSDB, GENETOX,
RTECS, TSCATS, CANCERLIT, Chemical Abstracts, BIOSIS, and Current Contents (last
6 months).
REVIEW OF PERTINENT DATA
Human Studies
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 crotonaldehyde in hippocampi obtained at autopsy of Alzheimer's disease patients
and age-matched controls. Crotonaldehyde is formed endogenously during lipid peroxidation
and reacts with proteins to form stable adducts (WHO, 2008). In Alzheimer's disease patients,
statistically significant (p < 0.01) higher levels of protein-bound 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 Exposure
Subchronic Studies—Groups of 30 male and 30 female Sprague-Dawley rats were
treated with crotonaldehyde (purity not reported) in water by daily gavage for 91-93 days at dose
levels of 0, 5, 15, or 45 mg/kg-day (TRL, 1986). Groups of 10 rats per sex from each dose group
were sacrificed on Day 42 or 43, and remaining surviving rats were sacrificed on Days 91-93.
Clinical signs and behavior were noted twice daily, and food consumption and body weight were
recorded weekly. Ophthalmoscopic examinations were performed before treatment and at
13 weeks. Blood and urine samples were collected before exposure (from a fifth group of
10 males and 10 females, and at the interim and final sacrifices. From these samples,
hematology, including hemoglobin [Hgb], hematocrit [Hct], erythrocyte count [RBC], mean cell
volume [MCV], mean cell hemoglobin [MCH], mean cell hemoglobin concentration [MCHC],
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total and differential white blood cell [WBC] count, and platelet count, was assessed. Serum
chemistry (alkaline phosphatase [ALP], blood urea nitrogen [BUN], aspartate aminotransferase
[AST], alanine aminotransferase [ALT], glucose, total protein, albumin, globulin, total bilirubin,
electrolytes, inorganic phosphate, carbon dioxide, cholesterol, and creatinine), and urine
chemistry (pH, specific gravity, glucose, protein, ketones, bilirubin, urobilinogen, and sediment
microscopy) were also assessed from these samples. Upon sacrifice, all animals were subjected
to gross necropsy, and the following organs were weighed: brain, heart, liver, spleen, kidneys,
adrenals, thyroid, parathyroid, testes with epididymides, and ovaries. Histopathology
(>25 tissues) was performed on all animals of the control and high-dose groups; the liver,
kidneys, heart, and forestomach only were examined histologically in all low- and mid-dose rats.
There were two mortalities unrelated to treatment; one low-dose female and one
high-dose female (TRL, 1986). Exposure-related clinical signs of toxicity were observed only in
the high-dose group. Immediately after dosing, salivation (lasting about 15 minutes) was
observed in about half of the high-dose rats; this effect was observed throughout the study.
Transient hypoactivity, lasting less than an hour immediately following dosing, was observed in
a maximum of 15% of high-dose rats. Labored respiration was also observed in 5-7% of
high-dose rats but only during Weeks 1 and 2. Scattered incidences of other clinical signs
including lacrimation, rales, prostration, tremors, retching, hypothermia, and/or ataxia occurred
in the high-dose animals; no further details were provided. Statistically significant (p < 0.05)
effects of treatment on food consumption, weight gain, and body weight were only apparent in
high-dose males; these changes were largely confined to Weeks 1 and 2 on study. During Weeks
10-12, food consumption in high-dose males was significantly (p < 0.05) lower than controls,
but there were no statistically significant effects on body weight or weight gain. By study
termination, the mean body weight of high-dose males was decreased by 6% compared with
controls; the difference was not statistically significant. Body weight, weight gain, and food
consumption were not significantly affected by treatment in high-dose females or at lower doses.
Ophthalmoscopy was not impacted by exposure to crotonaldehyde. Treatment-related serum
chemistry changes were confined to males, which exhibited statistically significantly (p < 0.05)
decreased ALP (22-42% lower than controls at all doses), AST (high-dose only), and ALT
(high-dose only) activities at the interim (but not final) sacrifice. The biological significance of
decreases in these serum biomarkers of liver toxicity is uncertain. Examination of the data
revealed no consistent exposure-related differences between the exposed and control groups in
hematology or urine chemistry variables, or in other serum chemistry parameters. Increases in
the relative, but not absolute, weights of brain (7.5% higher than controls, p < 0.05) and kidneys
(7.6%) higher than controls,/? < 0.05) were observed in high-dose males; these changes may have
been related to the lower body weight in this exposure group. Exposure-related necropsy and
histology findings were restricted to the forestomach of high-dose animals. At the interim
sacrifice, 8/10 high-dose males exhibited gross lesions described as white, raised, and/or pitted
areas of the forestomach; these lesions were also observed at the final sacrifice in 20/20 males
and 8/19 females in the high-dose group. Histology revealed hyperkeratosis in the forestomach
of all high-dose rats (except the female that died early) and parakeratosis in one high-dose
animal of each sex. No changes were observed in the glandular portion of the stomach. This
study identified a NOAEL of 15 mg/kg-day and a LOAEL of 45 mg/kg-day for hyperkeratosis of
the forestomach and clinical signs of toxicity (hypoactivity, labored respiration) in
Sprague-Dawley rats.
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In another subchronic gavage study, groups of 10 male and 10 female F344 rats were
administered crotonaldehyde (purity not reported) in corn oil by gavage at doses of 0 (vehicle
control), 2.5, 5, 10, 20, or 40 mg/kg-day, 5 days/week for up to 13 weeks (Hazleton Laboratories,
1986a). 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
and 16, and at the beginning of Week 13 for hematology (RBC, Hgb, Hct, MCV, MCH, MCHC,
total and differential WBC, reticulocyte count, platelet count, and RBC, WBC, and platelet
morphology) and serum chemistry (sorbitol dehydrogenase [SDH], gamma glutamyltransferase
[GGT], ALT, ALP, BUN, creatinine) analyses. The report suggested that urine samples were
also collected but did not discuss analysis of the samples. Assessment of sperm morphology and
vaginal cytology was performed at the end of the study on animals in the 0-, 2.5-, 5.0-, and
10-mg/kg-day groups. 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 where 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 convened a Pathology Working Group (NTP-PWG, 1987) to review
selected slides from this study and a companion study of B6C3F1 mice (Hazleton Laboratories,
1986b).
Early deaths occurred at the following incidences in the control through high-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 (Hazleton Laboratories, 1986a). The NTP-PWG (1987) concluded that nearly
all of the 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. High-dose
males showed decreases in body-weight gain at Weeks 11 and 13; mean terminal body weight
was about 10% lower than the control mean (p < 0.05). Review of the data showed that terminal
body weights in other exposed male groups and female groups were not different from controls.
Scattered statistically significant differences in organ weights and in hematological and clinical
chemistry variables occurred in some exposed groups, but none were considered biologically
significant by the NTP-PWG (1987). At gross necropsy, exposure-related lesions were only
observed in the forestomach (thickened forestomach and/or forestomach nodules) of rats of both
sexes in the two highest dose groups (20 and 40 mg/kg-day). Microscopic examination showed
epithelial hyperplasia in the forestomach beginning at the 10-mg/kg-day dose level. Incidences
of epithelial hyperplasia in the 10, 20, and 40 mg/kg-day groups were 3/10, 3/10, and 8/10 in
males and 1/10, 4/10, and 8/10 in females. None of the rats in the control or lower dose groups
showed epithelial hyperplasia of the forestomach. The NTP-PWG (1987) concluded that
no-effect levels for forestomach lesions were 5 and 10 mg/kg-day for males and females,
respectively, noting that the lesion was equivocal in the single affected female in the
10-mg/kg-day group. The corresponding LOAELs for forestomach lesions are 10 and
20 mg/kg-day for males and females, respectively. The only other exposure-related microscopic
change reported by Hazleton Laboratories (1986a) was described as nasal inflammation.
Incidences at control through 40 mg/kg-day were 0/10, 0/10, 0/10, 3/10, 3/10, and 7/10 in males;
and 1/10, 0/10, 1/10, 1/10, 4/10, and 6/10 in females. The NTP-PWG (1987) concluded that the
nasal lesions were serous exudation and not acute inflammation as reported by Hazleton
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Laboratories (1986a), and that the effect was likely a localized effect from exhaled
crotonaldehyde rather than an effect from blood-circulated crotonaldehyde. For the purpose of
this review, a LOAEL of 10 mg/kg-day is identified based on forestomach lesions in males; the
NOAEL is 5 mg/kg-day.
Hazleton Laboratories (1986b) also conducted a study of crotonaldehyde exposure in
mice. Groups of 10 male and 10 female B6C3F1 mice were administered crotonaldehyde in corn
oil by gavage at doses of 0 (vehicle control), 2.5, 5, 10, 20, or 40 mg/kg-day, 5 days/week for up
to 13 weeks. The original report for this study was not available, and the NTP-PWG (1987)
review of the data did not report results. According to NTP-PWG (1987), the study followed the
same protocol as for the companion study in F344 rats (Hazleton Laboratories, 1986a), excluding
the clinical laboratory studies. Histopathological evaluation and selection of tissues examined
microscopically from the control and treated-mouse groups followed the criteria used in the
F344 rat study (Hazleton Laboratories, 1986a). The forestomach was designated as the target
organ due to lesions observed at initial histopathological examination. According to study
protocol, tissue sections of the forestomach from all mice in the control and treated groups were
examined microscopically. The NTP-PWG (1987) reviewed selected slides from this study.
All mice survived treatment to terminal sacrifice, and there were no statistically
significant differences between treated and control groups for body-weight gain (Hazleton
Laboratories, 1986b; NTP-PWG, 1987). Although statistically significant changes in absolute
organ weights and organ/body-weight ratios were seen between treated and control groups, these
differences were not considered toxicologically significant. Treatment related lesions were not
observed at gross necropsy. Microscopic lesions were observed in the forestomach mucosa of
males and females from the high-dose group (40 mg/kg) only. These lesions consisted of
focal-to-diffuse thickening of the epithelium, with an irregular basal layer of the epithelial lining
of the forestomach (hyperplasia). The NTP-PWG (1987) report did not give incidences of these
effects, and the original report was not available. In addition to the epithelial hyperplasia, two
males in the high-dose (40 mg/kg-day) group had moderate chronic inflammatory lesions into
the forestomach submucosa. Inflammation of the forestomach was not seen in the controls,
high-dose female mice, or any of the lower dose groups. No significant pathological findings
were reported for lower dose groups. The NTP-PWG (1987) identified a NOAEL of
20 mg/kg-day and a LOAEL of 40 mg/kg-day for epithelial hyperplasia of the forestomach in
both sexes of B6C3F1 mice exposed to crotonaldehyde; these effect levels were adopted for this
review.
Chronic Studies—In the only chronic study available, male F344 rats (23-27 per group)
were exposed to crotonaldehyde (>99% pure) in drinking water for 113 weeks at concentrations
of 0, 0.6, or 6.0 mM (0, 42, or 421 mg/L) beginning at 6 weeks of age (Chung et al., 1986).
Total doses were reported to be 0, 9.5, or 70 mmol/rat based on average water consumption data
for an exposure period of 113 weeks. Corresponding daily doses of 0, 2, and 17 mg/kg-day were
calculated using approximate average body weights of 425 g for the control and 0.6-mM groups,
and 375 g for the 6.0-mM group (from Figure 2 in Chung et al., 1986). 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).
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Survival percentages were greater than 95% for exposed and control groups through
70 weeks of exposure but began to decline thereafter (Chung et al., 1986). At 110 weeks,
respective survival percentages for the control, low-, and high-dose groups were 70% (16/23),
63%) (17/27), and 56% (13/23). Differences from controls were not statistically significant.
Body weight was decreased in the high-dose group beginning at the eighth week of exposure and
continuing throughout the study (approximately 10%> lower than controls at study termination
based on visual inspection of data presented graphically). Water consumption was reduced in
high-dose animals (15 mL/day) compared with low-dose animals (20 mL/day), but water
consumption in controls was not reported.
Liver tumors, described as neoplastic nodules or hepatocellular carcinomas, were found
at statistically significant (p < 0.001) elevated incidences in the low-dose, but not high-dose,
group compared with controls (Chung et al., 1986). Incidences of neoplastic nodules in the
control through high-dose groups were 0/23, 9/27, and 1/23. Two rats in the low-dose group
showed hepatocellular carcinomas in addition to the neoplastic nodules. Incidences of altered
liver foci, considered to be a preneoplastic lesion, were elevated in both exposed groups
compared with controls (1/23, 23/27, and 13/23 in the control, low-, and high-dose groups,
respectively; p < 0.001). The number of altered liver foci per square centimeter was also
increased at both doses (0.1 ± 0.4, 12.4 ± 7.4, and 3.5 ± 3.8 in control through high-dose groups;
p < 0.001). Moderate-to-severe liver damage (fatty metamorphosis, focal liver necrosis, fibrosis,
cholestasis, and mononuclear cell infiltration) occurred in the 10/23 high-dose rats that did not
have preneoplastic or neoplastic lesions in the liver. No mention was made of degenerative liver
lesions in the control or low-dose groups. Inspection of the tumor data showed that incidences of
neoplastic lesions in tissues other than the liver were not affected by treatment. However, two
rats at the low-dose level had bladder tumors as well. No mention was made of nonneoplastic
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.
Reproductive/Developmental Studies—Hazleton Laboratories (1987) also conducted a
reproduction study in rats. Groups of 20 male and 20 female F344 rats were administered
crotonaldehyde in corn oil by daily gavage at doses of 0 (vehicle control), 2.5, 5, or
10 mg/kg-day. 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 Postpartum Day 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 0, 7, 14, and 20; at parturition; and again at
termination. 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 were taken of the right testes and
epididymis. 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 again 5 days later.
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All animals except for one mid-dose male survived to study termination; the cause of
death was reported not to be treatment-related (Hazleton Laboratories, 1987). Clinical signs
were few and not related to treatment. The data showed no significant changes in body weights,
testes weights, or epididymis weights among any treated male rats. The data also showed no
significant differences from controls in body weights among pregnant females through gestation
to Lactation Day 5. Among female rats that did not get pregnant, there appeared to be a decrease
in body weight in the treated groups relative to controls during the postmating period. 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. The researchers
suggested that this effect was probably not related to treatment. No significant histological
lesions were observed in males or females. The data for reproductive and litter parameters
showed no effect of treatment. Results of the sperm morphology and vaginal cytology studies
were not presented. The high dose of 10 mg/kg-day was a NOAEL for parental and reproductive
toxicity in this study.
Inhalation Exposure
WHO (2008) briefly summarized a subchronic inhalation study of crotonaldehyde
published in Russian (Voronin et al., 1982). Efforts to obtain the original study were not
successful. WHO (2008) reported that rats and mice were exposed continuously to
crotonaldehyde for 3 months (exposure concentrations not reported). According to WHO
"3
(2008), exposure to 1.2 mg/m crotonaldehyde was associated with alterations of motor activity
and blood levels of hemoglobin. It was not clear from the summary whether both species were
affected, or the direction or magnitude of the effects; no other information was available.
In a study published only as an abstract, Hilkevich (2002) tested the acute and chronic
effects of inhaled crotonaldehyde in rats. According to the abstract, concentrations of 1, 5, 87,
"3
and 130 mg/m were tested in the acute experiments, while concentrations of 0.14, 0.56, and
5.18 mg/m were tested for chronic toxicity; exposure durations were not reported. The authors
"3
reported that acute exposure to concentrations of 87-130 mg/m resulted in "oppression" of the
blood circulation and toxicity to WBCs but no changes in peripheral nerve, muscle, or liver
"3
function. Chronic exposure to concentrations of 0.56 and 5.18 mg/m reportedly resulted in
stimulation of the central nervous system, "oppression" of respiratory receptor function,
pulmonary hypoventilation, and cytotoxicity but no effects on liver function (no other details
provided). The authors stated that there were no effects at 0.14 mg/m3. No other information
was available in the abstract.
Other Studies
Acute or Short-term Studies
Borriston Research Laboratories (1980) evaluated the toxicity of crotonaldehyde (purity
not reported) in Sprague-Dawley rats (groups of 5/sex/dose) exposed via the diet to target doses
of 0, 22, 44, 88, or 175 mg/kg-day for 14 days. Daily observations and measurements of food
consumption were performed, and body weight was recorded weekly. At sacrifice at the end of
exposure, all animals received gross necropsy, and liver and kidney weights were measured.
Based on food consumption and body-weight data, the authors calculated doses of 19, 36, 73,
and 139 mg/kg-day in males, and 17, 36, 68, and 136 mg/kg-day in females. Inspection of the
data indicated that there were no treatment-related effects on clinical signs, body weight, food
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consumption, organ weights, or gross necropsy findings. No organs were examined
microscopically.
DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC ORAL RfD
VALUES FOR CROTONALDEHYDE
Oral studies of crotonaldehyde include two subchronic gavage studies (TRL, 1986;
Hazleton Laboratories, 1986a; NTP-PWG, 1987) and a reproduction study (Hazleton
Laboratories, 1987) in rats, one subchronic gavage study in mice (Hazleton Laboratories, 1986b;
NTP-PWG, 1987), and a chronic drinking water study conducted primarily as a cancer bioassay
in rats (Chung et al., 1986). Table 1 summarizes these data. The three 13-week subchronic
gavage studies all showed effects in the forestomach of both rats and mice. The chronic drinking
water study did not report forestomach lesions but observed altered foci, tumors, and
degenerative changes in the liver. No significant effects on reproduction were observed in rats
treated by gavage with up to 10 mg/kg-day of crotonaldehyde.
Subchronic p-RfD
Hazleton Laboratories, 1986a and NTP-PWG, 1987 observed forestomach lesions at
doses >10 mg/kg-day in rats and at 40 mg/kg-day in mice. The absence of forestomach lesions
in the chronic drinking water study suggests that the observed lesions may be due in part to bolus
dosing in the subchronic studies. However, crotonaldehyde is one of the class of a,P-unsaturated
aldehydes that has been noted for its ability to bind covalently to protein and DNA. It is possible
that this binding may be a mechanism of forestomach lesion development and would be relevant
in humans. In the absence of more definitive information, the forestomach lesions are
considered a valid endpoint for human health risk assessment.
The lowest LOAEL and NOAEL were observed in the Hazleton Laboratories (1986a;
NTP-PWG, 1987) study of rats; this study was chosen for dose-response modeling (see Table 2).
Data for forestomach lesion incidence in both male and female rats were modeled. Appendix A
contains details of the modeling and plots of the best fitting models. The BMDio for male rats
was 7.9 mg/kg-day, and the BMDLio was 4.8 mg/kg-day. The BMDio for female rats was
10.0 mg/kg-day, and the BMDLio was 5.5 mg/kg-day. The lower BMDLio of 4.8 mg/kg-day
from the male rats was selected as the point-of-departure (POD) for derivation of the subchronic
p-RfD. The BMDLio (4.8 mg/kg-day) based on gavage dosing 5 days/week was converted to an
equivalent continuous (7 days/week) dose of 3.4 mg/kg-day.
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Table 1. Summary of Oral Noncancer Dose-response Information
Species and
Study Type
(«/sex/group)
Exposure
NOAEL
(mg/kg-day)
LOAEL
(mg/kg-day)
Duration-
adjusted"
NOAEL
(mg/kg-day)
Duration-
adjusted"
LOAEL
(mg/kg-day)
Responses at the
LOAEL
Comments
Reference
Sprague-Dawley
Rat
Subchronic
Gavage
30/sex/dose
0, 5, 15, and 45
mg/kg-day daily for up
to 93 days
15
45
15
45
Hyperkeratosis of
the forestomach
and clinical signs

TRL, 1986
F344 Rat
Subchronic
Gavage
10/sex/dose
0, 2.5, 5, 10, 20, and
40 mg/kg-day, 5
days/week for 13
weeks
5
10
3.6
7.1
Forestomach
lesions

Hazleton
Laboratories,
1986a; NTP-PWG,
1987
B6C3F1 Mouse
Subchronic
Gavage
10/sex/dose
0, 2.5, 5, 10, 20, and
40 mg/kg-day, 5
days/week for 13
weeks
20
40
14
29
Forestomach
lesions

Hazleton
Laboratories,
1986b; NTP-PWG,
1987
F344 Rat
Chronic
Drinking water
23-27 M/dose
Approximately 0, 2,
and 17 mg/kg-day
daily in drinking water
for 113 weeks
NA
NA
NA
NA
NA
Altered hepatic foci
and liver tumors at
low dose; also
degenerative liver
lesions at high dose
Chung etal., 1986
F344 Rat
Reproduction
Gavage
20/sex/dose
0, 2.5, 5, and 10
mg/kg-day daily prior
to and during mating
and through gestation
and lactation
10
NA
10
NA
NA

Hazleton
Laboratories, 1987
aAdjusted for continuous exposure
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Table 2. Dose-response Data for Forestomach Hyperplasia in F344 Rats"
Dose (mg/kg-day)
Incidence of Forestomach Hyperplasia
Males
Females
0
0/10
0/10
2.5
0/10
0/10
5
0/10
0/10
10
3/10
1/10
20
3/10
4/10
40
8/10
8/10
aHazleton Laboratories, 1986a; NTP-PWG, 1987
A subchronic p-RfD was derived by dividing the duration-adjusted BMDLio of
3.4 mg/kg-day by a UF of 300, as shown below:
Subchronic p-RfD = BMDLio UF
= 3.4 mg/kg-day300
= 0.01 mg/kg-day or 1 x 10"2 mg/kg-day
The composite UF of 300 was composed of the following UFs:
•	UFh: A factor of 10 is applied for extrapolation to a potentially susceptible human
subpopulation because data for evaluating susceptible human response are
insufficient.
•	UFa: A factor of 10 is applied for animal-to-human extrapolation because data for
evaluating relative interspecies sensitivity are insufficient.
•	UFd: The database for oral exposure to crotonaldehyde consists of subchronic
toxicity studies in two species and a single-generation reproduction study in rats.
A factor of 3 (10°5) is applied for database inadequacies because data for
evaluating developmental toxicity are inadequate.
Confidence in the principal study (Hazleton Laboratories, 1986a; NTP-PWG, 1987) is
high because the study tested 10 rats per sex at six dose levels (including controls), a broad array
of endpoints were evaluated, and a review of the pathology results was conducted and confirmed
by NTP-PWG. Confidence in the database is medium. Additional subchronic gavage studies in
rats and mice provided support for the findings of the principal study, and a single generation
reproduction study found no effects. However, it is unclear from the existing database the extent
to which the findings of the gavage studies reflect the bolus-dosing protocol. In addition,
teratogenicity has not been evaluated. Reflecting high confidence in the principal study and
medium confidence in the database, confidence in the subchronic p-RfD is medium.
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Chronic p-RfD
The chronic drinking water study in rats observed altered foci, tumors, and degenerative
changes in the liver (Chung et al., 1986). However, this study cannot be used as the basis for a
chronic p-RfD due to the limited assessment and reporting of noncancer endpoints and
confounding due to the elevated incidence of liver tumors at the low-dose.
In the absence of adequate chronic data, the POD used to derive the subchronic p-RfD
(adjusted BMDLio of 3.4 mg/kg-day for forestomach lesions in male rats) was also used to
derive the chronic p-RfD. The chronic p-RfD for crotonaldehyde was derived as follows:
Chronic p-RfD = BMDLio UF
= 3.4 mg/kg-day3000
= 0.001 mg/kg-day or 1 x 10 3
The composite UF of 3000 was composed of the following UFs:
•	UFh: A factor of 10 is applied for extrapolation to a potentially susceptible human
subpopulation because data for evaluating susceptible human response are
insufficient.
•	UFa: A factor of 10 is applied for animal-to-human extrapolation because data for
evaluating relative interspecies sensitivity are insufficient.
•	UFd: The database for oral exposure to crotonaldehyde consists of subchronic
toxicity studies in two species and a single-generation reproduction study in rats.
A factor of 3 (10°5) is applied for database inadequacies because data for
evaluating developmental toxicity are inadequate.
•	UFS: A factor of 10 is applied for using data from a subchronic study to assess
potential effects from chronic exposure because data for evaluating response after
chronic exposure are inadequate.
As discussed for the subchronic p-RfD, confidence in the principal study is high.
However, confidence in the database is reduced to low for the chronic p-RfD due to the absence
of an adequate chronic study and uncertainty whether the value based on forestomach lesions
would be protective for degenerative liver lesions, which were seen in the chronic study but not
in the subchronic studies. Overall, confidence in the chronic p-RfD is low.
FEASIBILITY OF DERIVING PROVISIONAL SUBCHRONIC AND CHRONIC
INHALATION RfC VALUES FOR CROTONALDEHYDE
The database for inhalation toxicity of crotonaldehyde is limited to a subchronic study
(Voronin et al., 1982) reported only in summary form by WHO (2008), and a chronic study
reported only as an abstract (Hilkevich, 2002). These studies are inadequate for derivation of
inhalation toxicity values for crotonaldehyde.
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PROVISIONAL CARCINOGENICITY ASSESSMENT
FOR CROTONALDEHYDE
A provisional carcinogenicity assessment was not prepared for crotonaldehyde because
IRIS (U.S. EPA, 2008) includes a cancer assessment for this compound.
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REFERENCES
ACGIH (American Conference of Governmental Industrial Hygienists). 2008. 2008 Threshold
Limit Values for Chemical Substances and Physical Agents and Biological Exposure Indices.
ACGIH, Cincinnati, OH.
ATSDR (Agency for Toxic Substances and Disease Registry). 2008. Toxicological Profile
Information Sheet. U.S. Department of Health and Human Services, Public Health Service.
Online, http://www.atsdr.cdc.gov/toxpro2.html.
Borriston Laboratories. 1980. 14-Day subchronic toxicity study in rats with 2-Butenal. Study
no. 80-022. Final Report.
Chung, F.-L., T. Tanaka and S. Hecht. 1986. Induction of liver tumors in F344 rats by
crotonaldehyde. Cane. Res. 46:1285-1289.
Hazleton Laboratories. 1986a. Thirteen week subchronic study in F344 rats. Crotonaldehyde.
Final Report. Submitted to U.S. National Toxicology Program by Hazleton Laboratories
America, Inc. Rockville, MD.
Hazleton Laboratories. 1986b. Thirteen week subchronic study in B6C3F1 mice.
Crotonaldehyde. Final Report. Submitted to U.S. National Toxicology Program by Hazleton
Laboratories America, Inc. Rockville, MD.
Hazleton Laboratories. 1987. Amendment I to final report: Subchronic reproductive toxicity
study in F344 rats. Submitted to U.S. National Toxicology Program by Hazleton Laboratories
America, Inc. Rockville, MD.
Hilkevich, T.V. 2002. Effect of acute and chronic crotonic aldehyde inhalation on functional
state of the organs of rats. Toxicol. Lett. 135:S144.
IARC (International Agency for Research on Cancer). 1995. IARC Monographs on the
Evaluation of Carcinogenic Risks to Humans. Vol. 63. Dry cleaning, some chlorinated solvents
and other industrial chemicals. Lyon, France, p. 373.
Kawaguchi-Niida, M., N. Shibata, S. Morikawa et al. 2006. Crotonaldehyde accumulates in
glial cells of Alzheimer's disease brain. Acta Neuropathol. Ill :422-429.
NIOSH (National Institute for Occupational Safety and Health). 2008. NIOSH Pocket Guide to
Chemical Hazards. Index by CASRN. Online, http://www2.ede.gov/nioshtic-2/nioshtic2.htm.
NTP-PWG (National Toxicology Program - Pathology Working Group). 1987. 13-Week
subchronic toxicity test with crotonaldehyde (C56279B) in Fischer 344 rats and B6C3F1 mice.
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OSHA (Occupational Safety and Health Administration). 2008. OSHA Standard 1910.1000
Table Z-l. Part Z, Toxic and Hazardous Substances. Online, http://www.osha-slc.gov/
OshStd data/1910 1000 TABLE Z-l.html.
TRL (Toxicity Research Laboratories). 1986. Rat oral subchronic toxicity study of
crotonaldehyde. TRL Study #032-008. Report submitted to Research Triangle Institute,
Research Triangle Park, NC by Toxicity Research Laboratories, LTD, Muskegon, MI.
U.S. EPA. 1989. Health and Environmental Effects Document for Crotonaldehyde. Prepared
by the Office of Health and Environmental Assessment, Environmental Criteria and Assessment
Office, Cincinnati, OH for the Office of Solid Waste and Emergency Response, Washington,
DC.
U.S. EPA. 1991. Chemical Assessments and Related Activities (CARA). Office of Health and
Environmental Assessment, Washington, DC. April.
U.S. EPA. 1994. Chemical Assessments and Related Activities (CARA). Office of Health and
Environmental Assessment, Washington, DC. December.
U.S. EPA. 1997. Health Effects Assessment Summary Tables. FY-1997 Update. Prepared by
the Office of Research and Development, National Center for Environmental Assessment,
Cincinnati OH for the Office of Emergency and Remedial Response, Washington, DC. July.
EPA/540/R-97/036. NTIS PB97-921199.
U.S. EPA. 2000. Benchmark Dose Technical Guidance Document. External Review Draft.
Risk Assessment Forum. EPA/630/R-00/001. October.
U.S. EPA. 2006. 2006 Edition of the Drinking Water Standards and Health Advisories. Office
of Water, Washington, DC. EPA 822-R-06-013. Washington, DC. Online.
http://www.epa.gov/waterscience/drinking/standards/dwstandards.pdf.
U.S. EPA. 2008. Integrated Risk Information System (IRIS). Online. Office of Research and
Development, National Center for Environmental Assessment, Washington, DC. Online.
http://www.epa.gov/iris/.
Voronin, V., I. Bel'gova, L. Voronkova et al. 1982. Korrektur der toxizitatsbefunde uber
crotonaldehyde (CA; Technische Vorschriften TU 6-09-3667-74). Gig. TrudaProf. Zabol.
26(8):53-54 (abstract).
WHO (World Health Organization). 2008. Concise International Chemical Assessment
Document 74. 2-Butenal. WHO, Geneva.
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APPENDIX A. DETAILS OF BENCHMARK DOSE MODELING
FOR SUBCHRONIC p-RfD
Model Fitting Procedure for Quantal Noncancer Data
The model fitting procedure for dichotomous noncancer data is as follows. All available
dichotomous models in the EPA BMDS (version 2.1) are fit to the incidence data using the extra
risk option. The multistage model is run for all polynomial degrees up to n-1 (where n is the
number of dose groups including control). Adequate model fit is judged by three criteria:
goodness-of-fit p-value (p > 0.1), visual inspection of the dose-response curve, and scaled
residual at the data point (except the control) closest to the predefined benchmark response.
Among all the models providing adequate fit to the data, the lowest BMDL is selected as the
point of departure when the difference between the BMDLs estimated from these models is more
than 3-fold (unless it appears to be an outlier); otherwise, the BMDL from the model with the
lowest Akaike Information Criterion (AIC) is chosen. In accordance with EPA (2000) guidance,
benchmark doses (BMDs) and lower bounds on the BMD (BMDLs) associated with an extra risk
of 10% are calculated for all models.
Model Fitting Results for Forestomach Hyperplasia in Male F344 Rats (Hazleton
Laboratories, 1986a; NTP-PWG, 1987)
Applying the procedure outlined above to the data for forestomach hyperplasia in male
rats, adequate model fit was achieved with all models. Table A-l shows the modeling results.
BMDLs from models providing adequate fit differed by less than 3-fold. In accordance with
EPA (2000) guidance, the lowest AIC was selected from among models providing adequate fit.
For this data set, the resulting benchmark dose (BMDio) and associated 95% lower confidence
limit (BMDLio) were 7.88 and 4.77 mg/kg-day, respectively, based on the log probit model.
Figure A-l shows the fit of the log probit model to the data.
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Table A-l. Model Predictions for the Incidence of
Forestomach Hyperplasia in Male F344 Rats"
Model
Degrees of
Freedom
x2
X2 Goodness-of-Fit
p-V alueb
AIC
BMD10
(mg/kg-day)
BMDL10
(mg/kg-day)
Gamma0
4
2.89
0.58
41.55
7.94
3.69
Logistic
4
5.13
0.27
44.13
11.46
7.91
Log logisticd
4
3.00
0.56
41.68
7.93
3.95
Log probitd
4
2.73
0.60
41.34
7.88
4.77
1-degree multistage6
5
3.84
0.57
42.22
4.06
2.66
2-degree multistage6
4
3.18
0.53
41.91
7.98
3.40
3-degree multistage6
4
3.18
0.53
41.91
7.98
3.35
4-degree multistage6
4
3.18
0.53
41.91
7.98
3.31
5-degree multistage6
4
3.18
0.53
41.91
7.98
3.28
Probit
4
4.85
0.30
43.60
10.74
7.49
Weibulf
4
2.93
0.57
41.67
7.82
3.59
Quantal-linear
5
3.84
0.57
42.22
4.06
2.66
aHazleton Laboratories, 1986a; NTP-PWG, 1987
bValues <0.10 fail to meet conventional goodness-of-fit criteria
°Power restricted to >1
dSlope restricted to >1
"Betas restricted to >0
AIC = Akaike Information Criterion; BMDi0 = maximum likelihood estimate of the dose/concentration associated
with the selected benchmark response; BMDLio = 95% lower confidence limit on the BMD
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LogProbit Model with 0.95 Confidence Level
Dose
09:47 01/22 2009
Figure A-l. Fit of Log Probit Model to Data on Forestomach Hyperplasia in Male F344
Rats (Hazleton Laboratories, 1986a; NTP-PWG, 1987)
BMDs and BMDLs indicated are associated with an extra risk of 10% and are in units of mg/kg-day.
Model Fitting Results for Forestomach Hyperplasia in Female F344 Rats (Hazleton
Laboratories, 1986a; NTP-PWG, 1987)
Applying the procedure outlined above to the data for forestomach hyperplasia in female
rats, adequate model fit was achieved with all models. Table A-2 shows the results. BMDLs
from models providing adequate fit differed by approximately 3-fold, with the quantal-linear and
1-degree multistage model that gave the low BMDL estimate having relatively poor fit and
appearing to be an outlier. The BMDL estimates from the remaining models differed by only
1.5-fold. In accordance with EPA (2000) guidance, the lowest AIC was selected from among
these remaining models. The resulting BMDio and BMDLio were 9.98 and 5.45 mg/kg-day,
respectively, based on the 2-degree multistage model. Figure A-2 shows the fit of the 2-degree
polynomial multistage model to the data.
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Table A-2. Model Predictions for the Incidence of
Forestomach Hyperplasia in Female F344 Rats"
Model
Degrees of
Freedom
x2
X2 Goodness-of-Fit
p-V alueb
AIC
BMD10
(mg/kg-day)
BMDL10
(mg/kg-day)
Gamma0
4
0.27
0.99
34.39
10.63
5.92
Logistic
4
2.26
0.69
37.00
13.06
8.96
Log logisticd
4
0.21
1.00
34.32
10.75
6.18
Log probitd
4
0.10
1.00
34.13
10.55
6.28
1-degree multistage6
5
4.09
0.54
38.05
4.39
2.83
2-degree multistage6
5
0.48
0.99
32.78
9.98
5.45
3-degree multistage6
5
0.48
0.99
32.78
9.98
5.35
4-degree multistage6
5
0.48
0.99
32.78
9.98
5.35
5-degree multistage6
5
0.48
0.99
32.78
9.98
5.35
Probit
4
1.79
0.77
36.31
12.51
8.57
Weibulf
4
0.51
0.97
34.76
10.38
5.56
Quantal-linear
5
4.09
0.54
38.05
4.39
2.83
"Hazleton Laboratories, 1986a; NTP-PWG, 1987
bValues <0.10 fail to meet conventional goodness-of-fit criteria
°Power restricted to >1
dSlope restricted to >1
"Betas restricted to >0
AIC = Akaike Information Criterion; BMDi0 = maximum likelihood estimate of the dose/concentration
associated with the selected benchmark response; BMDLio = 95% lower confidence limit on the BMD
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Multistage Model with 0.95 Confidence Level
Dose
13:20 01/22 2009
Figure A-2. Fit of Multistage (2-Degree Polynomial) Model to Data on Forestomach
Hyperplasia in Female F344 Rats (Hazleton Laboratories, 1986a; NTP-PWG, 1987)
BMDs and BMDLs indicated are associated with an extra risk of 10% and are in units of mg/kg day.
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