NTP Toxicology and Carcinogenesis Studies of EMODIN (CAS NO. 518-82-1) Feed Studies in F344/N Rats and B6C3F1 Mice.
Emodin is a naturally occurring anthraquinone present in the roots and bark of numerous plants of the genus Rhamnus. Extracts from the roots, bark, and/or dried leaves of buckthorn, senna, cascara, aloe, frangula, and rhubarb have been used as laxatives since ancient times and currently are widely used in the preparation of herbal laxative preparations. Anthraquinone glycosides are poorly absorbed from the gastrointestinal tract but are cleaved by gut bacteria to produce aglycones (such as emodin) that are more readily absorbed and are responsible for the purgative properties of these preparations. There is extensive exposure to emodin and other anthraquinones resulting from the use of herb-based stimulant laxatives. Reports that 1,8-dihydroxyanthraquinone, a commonly used laxative ingredient, caused tumors in the gastrointestinal tract of rats raised the possibility of an association between colorectal cancer and the use of laxatives containing anthraquinones. Because emodin is a hydroxyanthraquinone structurally similar to 1,8-dihydroxyanthraquinone, is present in herbal laxatives, and was reported to be mutagenic in bacteria, it was considered a potential carcinogen and was selected for in-depth evaluation. Male and female F344/N rats and B6C3F1 mice were exposed to emodin (at least 94% pure) in feed for 16 days, 14 weeks, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, cultured Chinese hamster ovary cells, rat and mouse bone marrow cells, and mouse peripheral blood erythrocytes. 16-DAY STUDY IN RATS: Groups of five male and five female rats were fed diets containing 0, 600, 2,000, 5,500, 17,000, or 50,000 ppm emodin (equivalent to average daily doses of approximately 50, 170, 480, 1,400, or 3,700 mg emodin/kg body weight to males and 50, 160, 460, 1,250, or 2,000 mg/kg to females) for 15 (males) or 16 (females) days. Three female rats died before the end of the study. Mean body weights of males and females exposed to 5,500 ppm or greater were significantly less than those of the controls. Feed consumption by males and females receiving 17,000 or 50,000 ppm was decreased throughout the study. Macroscopic lesions were present in the kidney of rats exposed to 17,000 or 50,000 ppm. 16-DAY STUDY IN MICE: Groups of five male and five female mice were fed diets containing 0, 600, 2,000, 5,500, 17,000, or 50,000 ppm emodin (equivalent to average daily doses of approximately 120, 400, 1,200, or 3,800 mg/kg to males and 140, 530, 1,600, or 5,000 mg/kg to females; 50,000 ppm equivalents were not calculated due to high mortality) for 15 (males) or 16 (females) days. All mice exposed to 50,000 ppm died before the end of the study. Mice in the 17,000 ppm groups lost weight during the study. Feed consumption by 5,500 ppm females was greater than that by the controls throughout the study. Macroscopic lesions were present in the gallbladder and kidney of mice exposed to 17,000 ppm. 14-WEEK STUDY IN RATS: Groups of 10 male and 10 female rats were fed diets containing 0, 312.5, 625, 1,250, 2,500, or 5,000 ppm emodin (equivalent to average daily doses of approximately 20, 40, 80, 170, or 300 mg/kg to males and females) for 14 weeks. Mean body weights of males exposed to 2,500 ppm or greater and females exposed to 1,250 ppm or greater were significantly less than those of the controls. During the first week of the study, feed consumption by males exposed to 2,500 or 5,000 ppm and females exposed to 5,000 ppm was less than that by the controls. Feed consumption by these groups was similar to that by the controls for the remainder of the study. In rats exposed to 2,500 or 5,000 ppm, there were increases in platelet counts in males and females and segmented neutrophil counts in females. Total serum protein and albumin concentrations were decreased in females exposed to 2,500 or 5,000 ppm. Relative kidney weights of rats exposed to 1,250 ppm or greater and relative lung weights of rats exposed to 625 ppm or greater were significantly increased compared to the control groups. Relative liver weights were incree increased in females exposed to 625 ppm or greater. The estrous cycle length wassignificantly increased in females exposed to 1,250 or 5,000 ppm. All male rats exposed to 1,250 ppm or greater and all exposed female rats had pigment in the renal tubules; and the severity of pigmentation generally increased with increasing exposure concentration. The incidences of hyaline droplets in the cortical epithelial cytoplasm were increased in all groups of exposed males and in females exposed to 312.5, 625, or 1,250 ppm. 14-WEEK STUDY IN MICE: Groups of 10 male and 10 female mice were fed diets containing 0, 312.5, 625, 1,250, 2,500, or 5,000 ppm emodin (equivalent to average daily doses of approxi mately 50, 100, 190, 400, or 800 mg/kg to males and 60, 130, 240, 500, or 1,100 mg/kg to females) for 14 weeks. All mice survived to the end of the study. Mean body weights of males exposed to 2,500 or 5,000 ppm were significantly less than those of the controls. Feed consumption by exposed groups was generally similar to that by the controls. Relative kidney weights of male mice exposed to 1,250 ppm or greater, relative lung weights of males exposed to 625 ppm or greater, and relative liver weights of female mice exposed to 625 ppm or greater were increased. The incidences and severities of nephropathy were increased in males and females exposed to 1,250 ppm or greater. The incidences of renal tubule pigmentation were significantly increased in males exposed to 625 ppm or greater and in females exposed to 1,250 ppm or greater. 2-YEAR STUDY IN RATS: Groups of 65 male and 65 female rats were fed diets containing 0, 280, 830, or 2,500 ppm emodin (equivalent to average daily doses of approximately 110, 320, or 1,000 mg/kg to males and 120, 370, or 1,100 mg/kg to females) for 105 weeks. Ten male and ten female rats from each group were necropsied at 6 months. Blood samples from five male and five female rats in each group were evaluated at 3, 6, and 12 months for plasma emodin concentrations; these rats were necropsied at 12 months. Survival, Body Weights, and Feed Consumption: Survival of exposed males and females was similar to that of the controls. The mean body weights of rats in the 2,500 ppm groups were less than those of the controls beginning at week 2 of the study. Feed consumption by exposed groups was similar to that by the controls throughout the study. Pathology Findings: Three Zymbal's gland carcinomas were observed in female rats exposed to 2,500 ppm. This incidence exceeded the range observed for current historical controls and was considered an equivocal finding. At the 6- and 12-month interim evaluations and at 2 years, emodin-related increases in the incidences of renal tubule hyaline droplets occurred in all exposed groups. The incidences of renal tubule pigmentation were significantly increased in all exposed groups of males at 2 years. There were negative trends in the incidences of mononuclear cell leukemia in male and female rats, and the incidences in the 2,500 ppm groups were significantly decreased. In females exposed to 2,500 ppm, the incidence was below the historical control range; the incidence in males exposed to 2,500 ppm was at the lower end of the historical control range. 2-YEAR STUDY IN MICE: Groups of 60 male mice were fed diets containing 0, 160, 312, or 625 ppm emodin (equivalent to average daily doses of approximately 15, 35, or 70 mg/kg) for 105 weeks. Groups of 60 female mice were fed diets containing 0, 312, 625, or 1,250 ppm emodin (equivalent to average daily doses of approximately 30, 60, or 120 mg/kg) for 105 weeks. Ten male and ten female mice from each group were necropsied at 12 months. Survival, Body Weights, and Feed Consumption Survival and mean body weights of exposed males and females were similar to those of the controls. No differences in feed consumption were noted between exposed and control groups. Pathology Findings: Low incidences of renal tubule adenoma and carcinoma occurred in exposed male mice; these incidences included one carcinoma each in the 312 and 625 ppm groups. Renal tubule neoplasms are rare in male mice, and their presence in these groups suggested a possible association with emodin exposure. At the 12-month interim evaluation, the severity of nephropathy was slightly increased in males exposed to 625 ppm. Also at 12 months, the severity of nephropathy increased from minimal in the lower exposure groups to mild in females exposed to 1,250 ppm; the incidence in this group was significantly increased compared to the control group. At 2 years, the severities of nephropathy were slightly increased in males exposed to 625 ppm and females exposed to 1,250 ppm. The incidences of nephropathy were significantly increased in all exposed groups of females. At the 12-month interim evaluation, the incidences of renal tubule pigmentation were significantly increased in all exposed groups of males and in females exposed to 625 or 1,250 ppm. The severities increased with increasing exposure concentration. At 2 years, the incidences of renal tubule pigmentation were significantly increased in all exposed groups; severities increased with increasing exposure concentration. GENETIC TOXICOLOGY: Emodin was mutagenic in Salmonella typhimurium strain TA100 in the presence of S9 activation; no mutagenicity was detected in strain TA98, with or without S9. Chromosomal aberrations were induced in cultured Chinese hamster ovary cells treated with emodin, with and without S9. Three separate in vivo micronucleus tests were performed with emodin. A male rat bone marrow micronucleus test, with emodin administered by three intraperitoneal injections, gave negative results. Results of acute-exposure (intraperitoneal injection) micronucleus tests in bone marrow and peripheral blood erythrocytes of male and female mice were negative. In a peripheral blood micronucleus test on mice from the 14-week study, negative results were seen in male mice, but a weakly positive response was observed in similarly exposed females. CONCLUSIONS: Under the conditions of these 2-year feed studies, there was no evidence of carcinogenic activity of emodin in male F344/N rats exposed to 280, 830, or 2,500 ppm. There was equivocal evidence of carcinogenic activity of emodin in female F344/N rats based on a marginal increase in the incidence of Zymbal's gland carcinoma. There was equivocal evidence of carcinogenic activity of emodin in male B6C3F1 mice based on a low incidence of uncommon renal tubule neoplasms. There was no evidence of carcinogenic activity of emodin in female B6C3F1 mice exposed to 312, 625, or 1,250 ppm. Exposure of rats to emodin resulted in increased incidences of renal tubule hyaline droplets and pigmentation in males, increased incidences of renal tubule hyaline droplets in females, and increased severities of renal tubule pigmentation in males and females. Emodin exposure resulted in increased incidences of renal tubule pigmentation in male and female mice and increased incidences of nephropathy in female mice. Incidences of mononuclear cell leukemia decreased in male and female rats exposed to 2,500 ppm. Synonyms: Archin; C.I. 75440; C.I. Natural Green 2; C.I. Natural Yellow 14; emodol; frangulic acid; frangula emodin; 6-methyl- 1,3,8-trihydroxyanthraquinone; Persian Berry Lake; rheum emodin; schuttgelb; 1,3,8-trihydroxy-6-methyl-9,10- anthracenedione; 1,3,8-trihydroxy-6-methylanthraquinone; 4,5,7-trihydroxy-2-methylanthraquinone.