title: NTP Toxicology and Carcinogenesis Studies of Isobutyraldehyde (CAS No. 78-84-2) in F344/N Rats and B6C3F1 Mice (Inhalation Studies).
abstract: Isobutyraldehyde, a branched alkyl aldehyde, is used as a chemical intermediate and flavoring agent. It was nominated by the National Cancer Institute for toxicity and carcinogenicity studies by the NTP. Reasons for nomination and selection of isobutyraldehyde for study included its high potential for human exposure as suggested by its high production volume, its use as a chemical intermediate and food flavoring agent, suspicion of carcinogenicity due to an increased incidence of cancer at an aldehyde manufacturing plant where workers were exposed to a variety of aldehydes, its structural relationship to formaldehyde (a nasal carcinogen in rats), and the lack of toxicity and carcinogenicity studies on isobutyraldehyde in animals. Although human exposure occurs orally, dermally, or via inhalation, the inhalation route of exposure was selected for these animal studies because of the instability of isobutyraldehyde in water and feed. Male and female F344/N rats and B6C3F1 mice were exposed to isobutyraldehyde (approximately 99% pure) by inhalation for 13 weeks or 2 years. Genetic toxicology studies were conducted in vitro in Salmonella typhimurium, L5178Y mouse lymphoma cells, and cultured Chinese hamster ovary cells; in vivo tests were conducted in Drosophila melanogaster germ cells and bone marrow cells of rats and mice. 13-WEEK STUDY IN RATS: Groups of 10 male and 10 female F344/N rats were exposed to 0, 500, 1,000, 2,000, 4,000, or 8,000 ppm isobutyraldehyde by inhalation, 6 hours per day, 5 days a week, for 13 weeks. All rats exposed to 8,000 ppm died before the end of the study. Three male rats and six female rats in the 4,000 ppm groups and one female in the 500 ppm group died before the end of the study. The final mean body weight of male rats in the 4,000 ppm group and the body weight gains of 4,000 ppm males and females were significantly less than those of the chamber controls. Clinical findings in rats exposed to 4,000 or 8,000 ppm included abnormal respiratory sounds, decreased activity, nasal discharge, prostration, and slowed respiration. A minimal mature neutrophilia, evidenced by increased segmented neutrophil numbers, occurred in exposed groups of male and female rats. Exposure to isobutyraldehyde resulted in minimal increases in alanine aminotransferase activity in all groups of male and female rats. Spermatozoal motility in 500 and 1,000 ppm males was significantly reduced and females exposed to 4,000 ppm differed significantly from the chamber control females in the relative time spent in the estrous stages. No gross lesions were observed at necropsy that could be associated with isobutyraldehyde exposure. In the 8,000 ppm groups, severe necrosis of the epithelium, and occasionally of the entire mucosa, of the nasal turbinates accompanied by an acute inflammatory reaction was observed. Increased incidences of squamous metaplasia and mild acute inflammation occurred in male and female rats exposed to 4,000 ppm. Minimal to mild degeneration of the olfactory epithelium was observed in all male rats in the 2,000 and 4,000 ppm groups. Male rats exposed to 4,000 or 8,000 ppm and females exposed to 4,000 ppm had mild osteodystrophy of the turbinate bone. The incidences of necrosis/degeneration of the larynx and trachea were increased in male rats in the 8,000 ppm group. The incidences of mild to moderate lymphoid depletion of the spleen and thymus and lymphoid necrosis of the thymus were significantly increased in male and female rats exposed to 8,000 ppm. 13-WEEK STUDY IN MICE: Ten male and 10 female B6C3F1 mice were exposed to 0, 500, 1,000, 2,000, 4,000, or 8,000 ppm isobutyraldehyde by inhalation, 6 hours per day, 5 days per week, for 13 weeks. One male in the chamber control group, one male in the 1,000 ppm group, nine males and all females in the 4,000 ppm groups, and all males and females in the 8,000 ppm groups died before the end of the study. The final mean body weight and body weight gain of female mice in the 1,000 ppm group were significantly less than those of the chamber controls. Clinical findal findings included decreased activity, tremors, prostration, and slower and labored respiration. The absolute and relative kidney weights of males in the 1,000 and 2,000 ppm groups were significantly increased. There were no gross lesions observed at necropsy that could be associated with isobutyraldehyde exposure. Histopathologically, the nasal cavity and lymphopoietic tissues were considered target organs, with changes similar, but not identical, to those observed in rats. Increased incidences of nonneoplastic lesions of the nasal cavity were observed in male and female mice exposed to 1,000 ppm or greater. These lesions included necrosis, inflammation, hyperplasia, and squamous metaplasia of the epithelium; serous and suppurative exudate within the nasal passages; olfactory epithelial degeneration; and osteodystrophy of the turbinate bone. Mild to moderate lymphoid depletion and/or lymphoid necrosis were observed in the thymus of male and female mice exposed to 8,000 ppm. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female F344/N rats were exposed to 0, 500, 1,000, or 2,000 ppm isobutyraldehyde by inhalation, 6 hours per day, 5 days per week, for 105 weeks Survival, Body Weights, and Clinical Findings No differences in survival rates between exposed and chamber control rats were found. The mean body weights of male and female rats were generally similar to those of the chamber controls throughout the study. Pathology Findings No increase in neoplasm incidences that could be attributed to exposure to isobutyraldehyde was observed in male or female rats. Nonneoplastic lesions related to isobutyraldehyde exposure were limited to the nose and consisted of squamous metaplasia of the respiratory epithelium, degeneration of the olfactory epithelium, and suppurative inflammation. Incidences of minimal to mild squamous metaplasia in 1,000 and 2,000 ppm males and females and in 500 ppm females were significantly greater than those in the chamber controls. Another lesion associated with isobutyraldehyde exposure was minimal to mild degeneration of the olfactory epithelium in 2,000 ppm males and females. The incidences of suppurative inflammation (rhinitis) in male and female rats exposed to 2,000 ppm were increased compared to the chamber controls. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female B6C3F1 mice were exposed to 0, 500, 1,000, or 2,000 ppm isobutyraldehyde by inhalation, 6 hours per day, 5 days per week, for 105 weeks. Survival, Body Weights, and Clinical Findings There was an exposure-related decrease in survival of male mice, and the survival of males exposed to 2,000 ppm was marginally lower than that of the chamber controls. The mean body weights of female mice exposed to 1,000 or 2,000 ppm were lower than those of the chamber controls during the second year of the study. Pathology Findings No neoplasms that could be attributed to iso butyraldehyde exposure were observed in mice. Non neoplastic lesions related to isobutyraldehyde exposure were limited to the nose. The incidences of olfactory epithelial degeneration in 1,000 and 2,000 ppm males and females were significantly greater than in the chamber controls. GENETIC TOXICOLOGY: Isobutyraldehyde is mutagenic in vitro and in vivo, with the strongest responses observed in mammalian cell assays that measured chromosomal damage. Results of an initial mutagenicity test in S. typhimurium were negative; a second test, con ducted with different strains and varying concentrations of induced S9 activation enzymes, gave equivocal results. Strongly positive responses were obtained in the mouse lymphoma assay for mutation induction in L5178Y cells without S9 and in cytogenetic tests for induction of sister chromatid exchanges and chromosomal aberrations in cultured Chinese hamster ovary cells. Sister chromatid exchanges were significantly increased with and without S9, but induction of chromosomal aberrations was noted unequivocally only in the absence of S9. No induction of sex-linked recessive lethal mutations was observed in germ cells of male D. melanogaster administered isobutyraldehyde by feeding or by injection. In vivo, isobutyraldehyde was demonstrated to induce chromosomal aberrations in bone marrow cells of male mice, but no increases in micronuclei were observed in bone marrow cells of mice or rats after administration of isobutyraldehyde. All these in vivo cytogenetic studies used doses that reached lethality CONCLUSIONS: Under the conditions of these 2-year inhalation studies, there was no evidence of carcinogenic activity of isobutyraldehyde in male or female F344/N rats or male or female B6C3F1 mice exposed to 500, 1,000, or 2,000 ppm isobutyraldehyde. In male and female rats, exposure to isobutyraldehyde induced squamous metaplasia and suppurative inflammation of the nasal respiratory epithelium and degeneration of the nasal olfactory epithelium. In male and female mice, exposure to isobutyraldehyde caused degeneration of the nasal olfactory epithelium. Synonyms: Dimethylacetaldehyde; 2-formylpropane; isobutanal; isobutylcarboxaldehyde; isobutyral; isobutyric aldehyde; isobutyrylaldehyde; isopropylformaldehyde; 2-methylpropanal; 2-methyl-1-propanal; a-methylpropionaldehyde; 2-methylpropionaldehyde; valine aldehyde