NTP toxicology and carcinogenesiss studies of citral (microencapsulated) (CAS No. 5392-40-5) in F344/N rats and B6C3F1 mice (feed studies).
Citral is used primarily as lemon flavoring in foods, beverages, and candies. It is also used as a lemon fragrance in detergents, perfumes, and other toiletries. Citral was nominated by the National Cancer Institute for study because of its widespread use in foods, beverages, cosmetics, and other consumer products and its structure as a representative beta-substituted vinyl aldehyde. Male and female F344/N rats and B6C3F1 mice were exposed to microencapsulated citral (greater than 96% pure) in feed for 14 weeks or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, cultured Chinese hamster ovary cells, mouse bone marrow cells, and mouse peripheral blood erythrocytes. 14-WEEK STUDY IN RATS: Groups of 10 male and 10 female F344/N rats were fed diets containing starch microcapsules with a load of 31.3% citral. The concentration of citral in the diet was 3,900, 7,800, 15,600, or 31,300 ppm microencapsulated citral (equivalent to average daily doses of approximately 345, 820, 1,785, and 1,585 mg citral/kg body weight to males and 335, 675, 1,330, and 2,125 mg/kg to females) for 14 weeks. Additional groups of 10 male and 10 female rats received untreated feed (untreated controls) or feed containing placebo microcapsules (vehicle controls). In the second week of the study, all rats in the 31,300 ppm groups were killed moribund. Mean body weights of exposed males and females that survived to the end of the study were generally significantly less than those of the vehicle controls. Feed consumption by 15,600 and 31,300 ppm males and females was less than that by the vehicle controls during the first week of the study. Males and females in the 31,300 ppm groups exhibited listlessness, hunched posture, absent or slow paw reflex, and dull eyes. Exposure of rats to citral may have been associated with forestomach epithelial hyperplasia and hyperkeratosis, bone marrow atrophy and hemorrhage, and nephrotoxicity. 14-WEEK STUDY IN MICE: Groups of 10 male and 10 female B6C3F1 mice were fed diets containing 3,900, 7,800, 15,600, or 31,300 ppm microencapsulated citral (equivalent to average daily doses of approximately 745, 1,840, 3,915, and 8,110 mg/kg to males and 790, 1,820, 3,870, and 7,550 mg/kg to females) for 14 weeks. Additional groups of 10 male and 10 female mice received untreated feed (untreated controls) or feed containing placebo microcapsules (vehicle controls). In the second week of the study, four males in the 31,300 ppm group were killed moribund. Mean body weights of all exposed groups of males and females were significantly less than those of the vehicle controls. Feed consumption by females exposed to 7,800 ppm or greater was less than that by the vehicle controls during the first week of the study. By the end of the study, feed consumption by all exposed groups was greater than that by the vehicle controls. Mice in the 15,600 and 31,300 ppm groups were generally thin and lethargic; a few males in the 7,800 ppm group were also thin. The incidences of ovarian atrophy were significantly increased in females exposed to 15,600 or 31,300 ppm. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female F344/N rats were fed diets containing 1,000, 2,000, or 4,000 ppm microencapsulated citral for 2 years. Additional groups of 50 male and 50 female rats received untreated feed (untreated controls) or feed containing placebo microcapsules (vehicle controls). Dietary concentrations of 1,000, 2,000, and 4,000 ppm delivered average daily doses of approximately 50, 100, and 210 mg/kg to males and females. Survival of all exposed groups of males was significantly greater than that of the vehicle control group. Mean body weights of rats exposed to 4,000 ppm were generally less than those of the vehicle controls from week 49 (males) or 25 (females) to the end of the study. Feed consumption by exposed groups was similar to that by the vehicle controls. No neoplasms or nonneoplastic lesions were attributed to exposure to citral. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female B6C3F1 mice were fed diets containing 500, 1,000, or 2,000 ppm microencapsulated citral for 2 years. Additional groups of 50 male and 50 female mice received untreated feed (untreated controls) or feed containing placebo microcapsules (vehicle controls). Dietary concentrations of 500, 1,000, and 2,000 ppm delivered average daily doses of approximately 60, 120, and 260 mg/kg to males and females. Survival of exposed males and females was similar to that of the vehicle control groups. Mean body weights of mice exposed to 1,000 or 2,000 ppm were generally less than those of the vehicle controls throughout the study, and mean body weights of 500 ppm females were less from week 30 to the end of the study. Feed consumption by the exposed groups was similar to that by the vehicle controls. The incidences of malignant lymphoma occurred with a positive trend in female mice, and the incidence in 2,000 ppm females was significantly greater than that in the vehicle control group. Tissues most commonly affected by malignant lymphoma were the spleen, mesenteric lymph node, thymus, and, to a lesser extent, the ovary.


RESULTS
Citral was not mutagenic in S. typhimurium strain TA98, TA100, TA1535, or TA1537 with or without induced rat or hamster liver S9 enzymes. In cytogenetic tests with cultured Chinese hamster ovary cells, citral induced sister chromatid exchanges with and without S9, but chromosomal aberrations were not significantly increased after exposure to citral, with or without S9. Negative results were obtained in an in vivo bone marrow micronucleus test in male B6C3F1 mice treated by intraperitoneal injection with 250 to 750 mg/kg daily for 3 days. Likewise, no increases in the frequencies of micronucleated erythrocytes were observed in peripheral blood samples collected from male and female mice within 24 hours of the final exposure in the 14-week study. In conclusion, citral gave negative results in in vitro and in vivo tests for genotoxicity, with the exception of the in vitro mammalian cell test for sister chromatid exchange induction


CONCLUSIONS
Under the conditions of these 2-year feed studies, there was no evidence of carcinogenic activity of citral in male or female F344/N rats exposed to 1,000, 2,000, or 4,000 ppm. There was no evidence of carcinogenic activity of citral in male B6C3F1 mice exposed to 500, 1,000, or 2,000 ppm. There was equivocal evidence of carcinogenic activity in female B6C3F1 mice based on increased incidences of malignant lymphoma.
Research Triangle Park, NC 27709, USA
D000080462:Acyclic Monoterpenes; D000284:Administration, Oral; D000818:Animals; D001835:Body Weight / Q000187:drug effects; D016466:CHO Cells; D015197:Carcinogenicity Tests; D002273:Carcinogens / Q000008:administration & dosage / Q000633:toxicity*; D006224:Cricetinae; D004032:Diet; D004305:Dose-Response Relationship, Drug; D004339:Drug Compounding; D005260:Female; D005503:Food Additives / Q000008:administration & dosage / Q000633:toxicity*; D008136:Longevity / Q000187:drug effects; D008223:Lymphoma / Q000209:etiology / Q000473:pathology; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D039821:Monoterpenes / Q000008:administration & dosage / Q000633:toxicity*; D009152:Mutagenicity Tests; D009153:Mutagens / Q000633:toxicity; D009374:Neoplasms, Experimental / Q000209:etiology* / Q000473:pathology; D051381:Rats; D011916:Rats, Inbred F344; D012854:Sister Chromatid Exchange / Q000187:drug effects
D000080462:Acyclic Monoterpenes; D002273:Carcinogens; D005503:Food Additives; D039821:Monoterpenes; D009153:Mutagens; C007076:citral