Several essential oils contain pulegone and are used for flavoring foods, drinks, and dental products, as fragrance agents, and in herbal medicines. Pulegone was nominated for study by the National Institute of Environmental Health Sciences based on the potential for human exposure and the absence of carcinogenicity data. Male and female F344/N rats and B6C3F1 mice received pulegone (approximately 96% pure) by gavage for 2 weeks, 3 months, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, Escherichia coli, and mouse peripheral blood erythrocytes.
Two-week study in rats
Groups of five male and five female rats were administered 0, 37.5, 75, 150, 300, or 600 mg pulegone/kg body weight in corn oil by gavage, 5 days per week for 16 days. All male rats and nearly all female rats in the 300 and 600 mg/kg groups died prior to the end of the study. All moribund sacrifices and early deaths were attributed to liver toxicity. Mean body weight gains of males administered 37.5 or 150 mg/kg were significantly less than that of the vehicle controls. Clinical findings in 300 and 600 mg/kg rats included nasal/eye discharge, thinness, lethargy, and ruffled fur. Liver and kidney weights of dosed groups of females were generally significantly greater than those of the vehicle control group. The incidences of necrosis and cytoplasmic vacuolization of the liver in 300 and 600 mg/kg males and females were significantly greater than those in the vehicle control groups.
Two-week study in mice
Groups of five male and five female mice were administered 0, 18.75, 37.5, 75, 150, or 300 mg pulegone/kg body weight in corn oil by gavage, 5 days per week for 16 days. Four females and one male in the 300 mg/kg groups died by study day 5. All early deaths were attributed to liver toxicity. Mean body weights of the dosed groups were similar to those of the vehicle controls. Clinical findings were observed only in 300 mg/kg mice and included thinness, lethargy, and ruffled fur. Liver weights of 300 mg/kg males were significantly greater than those of the vehicle controls. The incidences of cytoplasmic vacuolization and diffuse fatty change in 300 mg/kg females and necrosis in 300 mg/kg males were significantly greater than those in the vehicle controls.
Three-month study in rats
Groups of 10 male and 10 female rats were administered 0, 9.375, 18.75, 37.5, 75, or 150 mg pulegone/kg body weight in corn oil by gavage, 5 days per week for 14 weeks. All rats survived until the end of the study except for one female in the 150 mg/kg group that died on day 9. Mean body weights of 75 and 150 mg/kg males and 150 mg/kg females were significantly less than those of the vehicle controls. At the end of the study, there was a small dose-related decrease in the erythron, evidenced by decreases in the hematocrit and hemoglobin values and the erythrocyte counts. An apparent erythroid response to the decreased erythron was evidenced by increased reticulocyte counts. Reduced and oxidized glutathione levels were generally increased in 75 and 150 mg/kg males and in 37.5 mg/kg or greater females. Absolute and relative liver weights of 75 and 150 mg/kg females and relative liver weights of males administered 18.75 mg/kg or greater were significantly greater than those of the vehicle controls. The absolute kidney weight of 150 mg/kg females and the relative kidney weights of all dosed groups, except 9.375 mg/kg males, were significantly greater than those of the vehicle controls. Absolute and relative thymus weights of 150 mg/kg males and females and the absolute thymus weight of 75 mg/kg males were significantly less than those of the vehicle controls.
In the kidney, there was hyaline glomerulopathy in 75 mg/kg males and 150 mg/kg males and females. The incidence of renal tubule protein casts was significantly increased in the 150 mg/kg females. In the liver, incidences of bile duct hyperplasia and hepatocyte hypertrophy in 75 and 150 mg/kg males and 150 mg/kg females, hepatocyte focal necrosis in 150 mg/kg males, and oval cell hyperplasia and periportal fibrosis in 150 mg/kg males and females were increased. Incidences of bone marrow hyperplasia in 37.5 mg/kg males and 75 and 150 mg/kg males and females, heart mineralization in 150 mg/kg males, glandular stomach mineralization in 75 and 150 mg/kg females, and cellular histiocytic infiltration in the lung and ovarian cyst in 150 mg/kg females were significantly increased.
Three-month study in mice
Groups of 10 male and 10 female mice were administered 0, 9.375, 18.75, 37.5, 75, or 150 mg pulegone/kg body weight in corn oil by gavage, 5 days per week for 14 weeks. All mice survived to the end of the study. Mean body weights of dosed mice were similar to those of the vehicle controls. Reduced and oxidized glutathione levels were generally greater than vehicle control levels in 150 mg/kg males and in 75 and 150 mg/kg females. Liver weights of 150 mg/kg males and 75 and 150 mg/kg females were significantly greater than those of the vehicle controls. No histopathologic lesions were observed that could be attributed to the administration of pulegone.
Two-year study in rats
Groups of 50 male and 50 female rats were administered 0, 18.75 (males only), 37.5, 75, or 150 (females only) mg pulegone/kg body weight in corn oil by gavage, 5 days per week for up to 104 weeks. Due to excessive morbidity and mortality, 75 mg/kg males and 150 mg/kg females were not administered pulegone after week 60 (stop-exposure); these groups were administered the corn oil vehicle until the end of the study. Survival of 37.5 mg/kg males was significantly less than that of the vehicle controls; only two 75 mg/kg stop-exposure males survived, and no 150 mg/kg stop-exposure females survived to the end of the study. Compared to those of the vehicle controls, mean body weights were less in 75 mg/kg stop-exposure males after week 13 and in 75 mg/kg and 150 mg/kg stop-exposure females after weeks 21 and 9, respectively. Clinical findings included thinness, lethargy, and ruffled fur in the 75 mg/kg stop-exposure males and 150 mg/kg stop-exposure females.
The incidences of urinary bladder papilloma and of papilloma or carcinoma (combined) were significantly increased in 150 mg/kg stop-exposure females.
In the kidney, incidences of hyaline glomerulopathy were significantly increased in 37.5 mg/kg and 75 mg/kg stop-exposure males and in all dosed groups of females. The severity of chronic progressive nephropathy was increased in 37.5 mg/kg and 75 mg/kg stop-exposure males and in 75 mg/kg and 150 mg/kg stop-exposure females; the incidences of nephropathy were significantly increased in 75 mg/kg and 150 mg/kg stop-exposure females. The incidence of renal cyst was significantly increased in 75 mg/kg stop-exposure males.
In the liver, incidences of diffuse hepatocyte cellular alteration were significantly increased in 37.5 mg/kg and 75 mg/kg stop-exposure males and 75 mg/kg and 150 mg/kg stop-exposure females. There were significant increases in the incidences of other liver lesions including fatty change, bile duct cyst, hepatocyte necrosis, oval cell hyperplasia, bile duct hyperplasia, and portal fibrosis.
In the nose, 37.5 mg/kg and 75 mg/kg stop-exposure males and all dosed groups of females had significantly increased incidences of olfactory epithelium degeneration. All dosed groups of females had significantly increased incidences of respiratory metaplasia of the olfactory epithelium and nasal inflammation.
In the forestomach, incidences of inflammation and ulcer were significantly increased in 37.5 mg/kg and 75 mg/kg stop-exposure males, and incidences of epithelial hyperplasia and perforation were increased in 75 mg/kg stop-exposure males. In the glandular stomach, the incidence of inflammation was significantly increased in 75 mg/kg stop-exposure males.
Two-year study in mice
Groups of 50 male and 50 female mice were administered 0, 37.5, 75, or 150 mg pulegone/kg body weight in corn oil by gavage, 5 days per week for 105 weeks. Survival of all dosed groups was similar to that of the vehicle controls. Mean body weights of 150 mg/kg males and females were less than those of the vehicle controls after weeks 25 and 33, respectively.
The incidences of multiple hepatocellular adenoma were significantly increased in all dosed groups of males, and the incidences of hepatocellular adenoma (includes multiple) and hepatoblastoma (includes multiple) were significantly increased in the 75 mg/kg males. The combined incidences of hepatocellular adenoma, hepatocellular carcinoma, or hepatoblastoma occurred with positive trends and were significantly increased in 75 mg/kg males and 150 mg/kg females. The incidence of hepatocellular adenoma was significantly increased in 150 mg/kg females. The incidences of several nonneoplastic liver lesions were significantly increased, primarily in the 75 and 150 mg/kg groups. These nonneoplastic lesions included clear cell, eosinophilic, and mixed cell foci; focal fatty change; centrilobular hepatocyte hypertrophy; intravascular hepatocyte; necrosis; pigmentation; bile duct cyst and hyperplasia; and oval cell hyperplasia.
In the kidney, incidences of hyaline glomerulopathy were significantly increased in all dosed groups of males and 75 and 150 mg/kg females. The incidence of mineralization was significantly increased in 150 mg/kg females, and the incidence of nephropathy in 150 mg/kg females and severity of nephropathy in 150 mg/kg males were increased. Incidences of congestion of the glomerulus were increased in 150 mg/kg males and females.
The incidence of osteoma or osteosarcoma (combined) in all organs of 75 mg/kg females exceeded the historical control ranges. One 150 mg/kg male and one 75 mg/kg female had nasal osteoma; no nasal osteomas have been observed in historical control mice.
The incidences of olfactory epithelial degeneration of the nose were significantly increased in all dosed groups of females and in 75 and 150 mg/kg males. Incidences of inflammation, nerve atrophy, and olfactory epithelium metaplasia of the nose were significantly greater in 150 mg/kg males and females than in the vehicle control groups.
In the forestomach, incidences of squamous hyperplasia and inflammation were significantly increased in 75 mg/kg males and 150 mg/kg males and females, and the incidences of ulcer were significantly increased in 75 and 150 mg/kg males.
Genetic toxicology
Pulegone was tested in three independent bacterial mutagenicity assays. Results from two of the assays were negative, with and without exogenous metabolic activation enzymes (S9). One of these assays used the same lot of pulegone that was tested in the 2-year rodent bioassay. Bacterial strains tested in these two assays included S. typhimurium strains TA97, TA98, TA100, and TA1535 as well as E. coli strain WP2 uvrA/pKM101. Results of the third test, also conducted with the same lot of pulegone as the 2-year bioassay, were clearly positive in Salmonella typhimurium strain TA98 and in Escherichia coli strain WP2 uvrA/pKM101 in the presence of rat liver S9. In vivo, no significant increases in the frequencies of micronucleated erythrocytes were seen in peripheral blood of male or female mice in the 3-month study.
Conclusions
Under the conditions of these 2-year gavage studies, there was no evidence of carcinogenic activity of pulegone in male F344/N rats administered 18.75, 37.5, or 75 (stop-exposure) mg/kg. There was clear evidence of carcinogenic activity of pulegone in female F344/N rats based on increased incidences of urinary bladder neoplasms. There was clear evidence of carcinogenic activity of pulegone in male and female B6C3F1 mice based on increased incidences of hepatocellular neoplasms (adenomas in both sexes and hepatoblastomas in males). Osteomas and osteosarcomas in female B6C3F1 mice may have been related to pulegone administration.
A unique renal lesion, hyaline glomerulopathy, was observed in all dosed groups of male and female mice and female rats and in 37.5 mg/kg and 75 mg/kg stop-exposure male rats. In rats, renal failure secondary to hyaline glomerulopathy and nephropathy contributed to the decreased survival in the 75 mg/kg stop-exposure males and 150 mg/kg stop-exposure females.
Pulegone administration was also associated with the occurrence of nonneoplastic lesions in the liver and nose of rats and mice and in the forestomach of male and female mice and male rats.