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.

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.

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-1 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-1 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 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 (NTP, 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 Laboratories, 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 (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.

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 Laboratories, 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.

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.

Chung et al. (1986)

In a peer-reviewed study, groups of male F344 rats (23–27/group) were exposed to trans-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 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.

Acute Oral Toxicity

Acute oral lethality studies with commercial crotonaldehyde reported median lethal dose (LD₅₀) values in rats ranging from 165 to 300 mg/kg (Kennedy and Graepel, 1991; Borriston, 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

trans-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 Graepel, 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 trans-crotonaldehyde at a dose of 14 mg/kg, but sperm abnormalities were not observed at 7 mg/kg (Jha and Kumar, 2006).