Thujone is a monoterpene ketone that exists in two stereoisomeric forms: (–)-3-isothujone, or α-thujone, and (+)-3-thujone, or β-thujone. Thujone occurs in nature as a mixture of α- and β-isomers and is the primary constituent of essential oils derived from a variety of plants. Although thujone itself is banned as a food additive in the United States, it appears in many other natural flavoring substances that are approved food additives and flavorings. α-Thujone was nominated for study by the National Cancer Institute based on the potential for widespread human exposure through consumption of herbs and essential oils containing thujone. Two-week toxicity studies were conducted with α-thujone and an α,β-thujone mixture (71%, 12%, respectively). The α,β-thujone mixture (70%, 11%, respectively) was selected for subsequent 3-month and 2-year studies because thujone occurs naturally as a mixture and human exposure is to the mixture. Male and female F344/N rats and B6C3F1 mice were administered α-thujone or α,β-thujone for 2 weeks and α,β-thujone for 3 months or 2 years by gavage. Genetic toxicology studies of α-thujone and α,β-thujone were conducted in Salmonella typhimurium and Escherichia coli and mouse peripheral blood erythrocytes.
Two-week study of alpha-thujone in rats
Groups of five male and five female rats were administered 0, 1, 3, 10, 30, or 100 mg α-thujone/kg body weight in 0.5% methylcellulose by gavage for 16 days. All male rats survived to the end of the study; three 100 mg/kg female rats died before the end of the study. Mean body weights of dosed rats were similar to those of the vehicle controls. The thymus weights of 100 mg/kg females were significantly less than those of the vehicle controls. Clinical findings included convulsions/seizures in three 100 mg/kg females. No gross or histologic findings were attributed to administration of α-thujone.Two-week study of alpha,beta-thujone in rats
Groups of five male and five female rats were administered 0, 1, 3, 10, 30, or 100 mg α,β-thujone/kg body weight in 0.5% methylcellulose by gavage for 16 days. One 100 mg/kg male rat died before the end of the study. Final mean body weights and body weight gains of 10 mg/kg male rats were significantly less than those of the vehicle control group. No convulsions/seizures were observed in rats administered α,β-thujone. No gross or histologic findings were attributed to α,β-thujone administration.Two-week study of alpha-thujone in mice
Groups of five male and five female mice were administered 0, 1, 3, 10, 30, or 100 mg α-thujone/kg body weight in 0.5% methylcellulose by gavage for 17 days. Four 100 mg/kg males and all five 100 mg/kg females died before the end of the study. Mean body weights of surviving mice were similar to those of the vehicle controls. Clinical findings included seizures and tremors in 100 mg/kg males and hyperactivity in 100 mg/kg male and female mice. No gross or histologic findings were attributed to administration of α-thujone.Two-week study of alpha,beta-thujone in mice
Groups of five male and five female mice were administered 0, 1, 3, 10, 30, or 100 mg α,β-thujone/kg body weight in 0.5% methylcellulose by gavage for 17 days. All 100 mg/kg male mice and two 100 mg/kg female mice died before the end of the study. Final mean body weights of surviving dosed mice were similar to those of the vehicle control groups; the mean body weight gains of 3 mg/kg males and 100 mg/kg females were significantly greater than those of the vehicle control groups. No seizures or tremors were observed in mice administered α,β-thujone. There were no gross or histologic findings associated with α,β-thujone administration.Three-month study of alpha,beta-thujone in rats
Groups of 10 male and 10 female rats were administered 0, 12.5, 25, 50, 75, or 100 mg α,β-thujone/kg body weight in 0.5% methylcellulose by gavage for 14 weeks. Administered doses of α,β-thujone were lower than target concentrations as indicated by after dosing sample analysis. Additional clinical pathology groups of 10 male and 10 female rats were administered the same doses for 3 or 24 days. Two male and eight female 75 mg/kg rats and eight male and nine female 100 mg/kg rats died before the end of the study. Final mean body weights and body weight gains of 50 and 75 mg/kg females were significantly increased. Seizures were observed in male rats administered 50 mg/kg or greater and in female rats administered 25 mg/kg or greater. All but one of the early deaths occurred in animals that had previously been observed to have seizures. Thymus weights were significantly decreased in 75 and 100 mg/kg males.Incidences of congestion and/or hemorrhage of the brain were increased in 75 mg/kg females and 100 mg/kg males and females. The incidences of pigmentation in the brain were also increased in 50 mg/kg or greater females. Changes in the pituitary gland were increased with α,β-thujone administration in female rats, and included atrophy of the pars distalis and dilatation of Rathke’s cleft. Congestion, hemorrhage, and/or edema were observed in several other organs of rats that died before the end of the study. The incidences of renal tubule mineralization were significantly increased in all dosed groups of female rats.
Three-month study of alpha,beta-thujone in mice
Groups of 10 male and 10 female mice were administered 0, 6.25, 12.5, 25, 50, or 75 mg α,β-thujone/kg body weight in 0.5% methylcellulose by gavage for 14 weeks. Administered doses of α,β-thujone were lower than target concentrations as indicated by after dosing sample analysis. All 75 mg/kg mice and nine male and seven female 50 mg/kg mice died before the end of the study. Mean body weights of surviving dosed mice were similar to those of the vehicle controls. Seizures were observed in 50 and 75 mg/kg male mice and in female mice administered 25 mg/kg or greater. Early deaths of 50 and 75 mg/kg mice occurred in animals that had previously been observed to have seizures. The incidences of lung congestion in 75 mg/kg males and females and lung hemorrhage in 50 mg/kg males were generally significantly greater than those in the vehicle control group.Two-year study of alpha,beta-thujone in rats
Groups of 50 male and 50 female rats were administered 0, 12.5, 25, or 50 mg α,β-thujone/kg body weight in 0.5% methylcellulose by gavage for up to 105 weeks. All of the 50 mg/kg male and female rats died before the end of the study, and survival of 25 mg/kg males and females was significantly less than that of the respective vehicle control group. Mean body weights of all dosed groups were generally within 10% of those of the vehicle control groups throughout the study. Seizures occurred in all 50, most 25, and a few 12.5 mg/kg rats.Incidences of preputial gland carcinoma and adenoma or carcinoma (combined) in male rats occurred with positive trends, and the combined incidence in 25 mg/kg males was significantly greater than that in the vehicle control group. The incidence of benign pheochromocytoma of the adrenal medulla was significantly increased in 25 mg/kg male rats.
The incidences of necrosis and pigmentation of the brain were significantly increased in 50 mg/kg males, and the incidence of pigmentation was significantly increased in 50 mg/kg females. Pigmentation was localized to macrophages and was consistent with hemosiderin. Lower incidences of these brain lesions also occurred in rats administered 12.5 or 25 mg/kg. In female rats, increased incidences of atrophy of the pars distalis and dilatation of Rathke’s cleft in the pituitary gland were observed in the 50 mg/kg and 25 and 50 mg/kg groups, respectively. In the spleen, the incidences of pigmentation in 25 and 50 mg/kg males and 50 mg/kg females were significantly increased. The incidences of mineralization of the kidney were significantly increased in all dosed groups of males.
Two-year study of alpha,beta-thujone in mice
Groups of 50 male and 50 female mice were administered 0, 3, 6, 12, or 25 mg α,β-thujone/kg body weight in 0.5% methylcellulose by gavage for up to 105 weeks. Survival of male and female mice in the 25 mg/kg groups was significantly less than that of the vehicle controls. Mean body weights of all dosed groups of males and of females administered 12 mg/kg or less were within 10% of those of the vehicle control groups throughout the study. Mean body weights of 25 mg/kg females were less than those of the vehicle controls after week 29. Most male and all female 25 mg/kg mice had seizures. No neoplasms or nonneoplastic lesions were attributed to α,β-thujone administration.Single-dose toxicokinetic studies of alpha-thujone and alpha,beta-thujone
Single-dose toxicokinetic studies of α-thujone and α,β-thujone were conducted in male and female F344/N rats and B6C3F1 mice following intravenous and oral gavage administration. Intravenous doses of α-thujone and α,β-thujone, respectively, were 1.6 and 3.0 mg/kg for rats and 3.2 and 6.0 mg/kg for mice. Gavage doses of α-thujone and α,β-thujone, respectively, were 25 and 50 mg/kg for rats and 40 and 80 mg/kg for mice. α-Thujone absorption was rapid and independent of dose, species, and sex following gavage administration of either formulation. In general, elimination was faster in mice than in rats. The bioavailability was higher in female rats compared to male rats following administration of either formulation although a sex difference was not observed in mice. Female rats and male and female mice showed greater than proportional increases in bioavailability with increasing dose following administration of α-thujone and α,β-thujone, respectively, which is likely due to saturation of elimination kinetics. Following administration of both formulations, α-thujone was distributed to the brain; females generally had higher brain/plasma ratios than males in both species.Genetic toxicology
Neither α,β-thujone nor α-thujone was mutagenic in bacterial tester strains (S. typhimurium and E. coli) when testing was conducted with or without exogenous metabolic activation provided by rat or hamster liver S9 mix. In vivo, daily exposure by gavage to α,β-thujone for 3 months did not result in an increase in micronucleated erythrocytes in the peripheral blood of male mice. However, female mice had a small but significant increase in micronucleated erythrocytes in the peripheral blood at the end of the 3-month study. No significant changes in the percentage of reticulocytes among total erythrocytes was seen in either male or female mice at the end of the 3-month study, suggesting that α,β-thujone did not induce bone marrow toxicity.Conclusions
Under the conditions of these 2-year gavage studies, there was some evidence of carcinogenic activity of α,β-thujone in male F344/N rats based on increased incidences of preputial gland neoplasms; increased incidences of benign pheochromocytoma of the adrenal medulla may have been related to administration of α,β-thujone in male F344/N rats administered 12.5 or 25 mg/kg. There was no evidence of carcinogenic activity of α,β-thujone in female F344/N rats administered 12.5 or 25 mg/kg. There was no evidence of carcinogenic activity of α,β-thujone in male or female B6C3F1 mice administered 3, 6, or 12 mg/kg.
Administration of α,β-thujone for 2 years resulted in increased incidences of seizures in F344/N rats and B6C3F1 mice and increased incidences of nonneoplastic lesions in the brain and spleen of male and female F344/N rats, the kidney of male F344/N rats, and the pituitary gland of female F344/N rats.