2,4-Hexadienal, a colorless to yellow liquid with a pungent "green" or citrus odor, is used as a food additive for flavor enhancement, as a fragrance agent, as a starting material or intermediate in synthetic reactions in the chemical and pharmaceutical industries, as a fumigant, and as a corrosion inhibitor for steel. 2,4-Hexadienal was nominated for study by the National Cancer Institute because of the potential for carcinogenicity based on its a,b-unsaturated aldehyde structure and the potential link between exposure to lipid peroxidation products in the diet and human malignancies. The commercial product is a mixture containing chiefly trans,trans-2,4-hexadienal in equilibrium with cis,trans-2,4-hexadienal. Male and female F344/N rats and B6C3F1 mice received 2,4-hexadienal (89% trans,trans; 11% cis,trans) in corn oil by gavage for 16 days, 14 weeks, or 2 years. Tissues and plasma from dosed rats were examined for malondialdehyde and glutathione concentrations, and DNA adducts were characterized in liver and forestomach samples from dosed rats and mice. Genetic toxicology studies were conducted in Salmonella typhimurium, rat and mouse bone marrow cells, and mouse peripheral blood erythrocytes.
Sixteen-day study in rats
Groups of five male and five female rats were administered 0, 3, 9, 27, 80, or 240 mg 2,4-hexadienal/kg body weight in corn oil by gavage, 5 days per week, for 16 days. Three male and three female 240 mg/kg rats died before the end of the study. Mean body weight gains of 240 mg/kg rats were significantly less than those of the vehicle controls. Clinical findings included diarrhea, ataxia, lethargy, and nasal/eye discharge in males, and lethargy, paleness, and abnormal breathing in females in the 240 mg/kg groups. Liver weights of 240 mg/kg females were significantly greater than those of the vehicle controls. Gross and microscopic lesions indicative of forestomach necrosis and ulceration were present in most 240 mg/kg rats, and forestomach epithelial hyperplasia was microscopically evident in most 80 mg/kg rats.
Sixteen-day study in mice
Groups of five male and five female mice were administered 2,4-hexadienal in corn oil by gavage at doses of 0, 3, 9, 27, 80, or 240 mg/kg, 5 days per week, for 16 days. Chemical-related deaths occurred in one male and one female in the 240 mg/kg groups. Female mice in the 240 mg/kg group lost weight during the study. Gross and microscopic lesions indicative of forestomach necrosis and ulceration were present in all 240 mg/kg mice, and forestomach epithelial hyperplasia and hyperkeratosis were microscopically evident in 80 mg/kg mice.
Fourteen-week study in rats
Groups of 10 male and 10 female rats were administered 2,4-hexadienal in corn oil by gavage at doses of 0, 7.5, 15, 30, 60, or 120 mg/kg, 5 days per week, for 14 weeks. All rats survived to the end of the study. Mean body weights of 30, 60, and 120 mg/kg males were significantly less than those of the vehicle controls. The only clinical finding attributed to 2,4-hexadienal administration was hypersalivation in 30 and 120 mg/kg males and females. The incidences of forestomach hyperplasia and nasal olfactory atrophy or necrosis were significantly increased in 120 mg/kg rats. Nasal lesions occurred in most 120 mg/kg male rats.
Fourteen-week study in mice
Groups of 10 male and 10 female mice were administered 2,4-hexadienal in corn oil by gavage at doses of 0, 7.5, 15, 30, 60, or 120 mg/kg, 5 days per week, for 14 weeks. No deaths were attributed to administration of 2,4-hexadienal. Mean body weights of males and females were similar to those of the vehicle controls throughout the study. Clinical findings included salivation and anal wetness in males and females. Kidney weights of 60 and 120 mg/kg males and liver weights of 60 mg/kg males and females were significantly greater than those of the vehicle controls. The incidences of forestomach hyperplasia and/or nasal olfactory atrophy or necrosis were significantly increased in 120 mg/kg mice.
Two-year study in rats
Groups of 50 male and 50 female rats were administered 2,4-hexadienal in corn oil by gavage at doses of 0, 22.5, 45, or 90 mg/kg, 5 days per week, for up to 105 weeks. Survival of all dosed groups of rats was similar to that of the vehicle control groups. The mean body weights of 90 mg/kg males were generally less than those of the vehicle controls throughout the study.
The incidences of squamous cell papilloma of the forestomach occurred with positive trends in male and female rats. This neoplasm was found in 58% of males and 34% of females in the 90 mg/kg groups. In the forestomach of male rats, papilloma multiplicity was increased in the 90 mg/kg group, and squamous cell carcinomas were found in one 45 mg/kg male and two 90 mg/kg males. Epithelial hyperplasia of the forestomach occurred in most 45 and 90 mg/kg rats.
Two-year study in mice
Groups of 50 male and 50 female mice were administered 2,4-hexadienal in corn oil by gavage at doses of 0, 30, 60, or 120 mg/kg, 5 days per week, for up to 105 weeks. Survival of dosed mice was similar to that of the vehicle controls. The mean body weights of all dosed groups were generally similar to those of the vehicle controls throughout the study. The incidences of squamous cell papilloma of the forestomach occurred with positive trends in male and female mice; squamous cell carcinomas were present in 120 mg/kg males and females. Epithelial hyperplasia of the forestomach occurred in many 120 mg/kg mice. Two 120 mg/kg males had uncommon squamous cell carcinoma of the oral cavity (tongue).
Genetic toxicology
2,4-Hexadienal was mutagenic in S. typhimurium strain TA100 with and without induced hamster or rat liver enzymes; no mutagenic activity was detected with strains TA1535 or TA98, with or without S9. Results of bone marrow tests in male rats and male mice given intraperitoneal injections of 2,4-hexadienal showed a small increase in the induction of micronucleated erythrocytes. However, neither test was repeated, and the test results were judged to be inconclusive. Results of peripheral blood micronucleus tests in male and female mice treated with 2,4-hexadienal by gavage for 14 weeks were negative.
Conclusions
Under the conditions of these 2-year gavage studies, there was clear evidence of carcinogenic activity of 2,4-hexadienal in male and female F344/N rats and male and female B6C3F1 mice based on increased incidences of squamous cell neoplasms of the forestomach. The occurrence of squamous cell carcinoma of the oral cavity (tongue) in male B6C3F1 mice may have been related to the administration of 2,4-hexadienal.
Hyperplasia of the forestomach in male and female rats and mice was associated with administration of 2,4-hexadienal.