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Abstract for TR-584

Toxicology Studies of Indole-3-Carbinol in F344/N Rats and B6C3F1/N Mice and Toxicology and Carcinogenesis Studies of Indole-3-Carbinol in Harlan Sprague Dawley Rats and B6C3F1/N Mice (Gavage Studies)

CASRN: 700-06-1
Chemical Formula: C9H9NO
Molecular Weight: 147.18
Synonyms/Common Names: 3-(Hydroxymethyl)indole; indole-3-methanol; 3-indolemethanol; 3-indolylcarbinol; 3-indolylmethanol
Report Date: July 2017

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Abstract

Indole-3-carbinol is sold as a sole ingredient in dietary supplements or as a combination nutraceutical along with a variety of herbs and/or vitamins. It is marketed for its potential ability to prevent cancer and provide other health benefits, such as detoxifying the liver and boosting the immune system. Indole-3-carbinol is a naturally formed breakdown product of glucosinolate glucobrassicin, a component found in cruciferous vegetables of the Brassica genus, including broccoli, brussels sprouts, cauliflower, cabbage, kale, kohlrabi, and turnips. Exposure to indole-3-carbinol occurs through the oral route through the ingestion of Brassica vegetables or dietary supplements. Indole-3-carbinol was nominated by the National Cancer Institute for toxicity and carcinogenicity testing because of its occurrence in natural products and for its potential use as a breast cancer chemopreventive agent. Male and female F344/N rats and Harlan Sprague Dawley rats received indole-3-carbinol in corn oil by gavage for 3 months or 2 years, respectively. Male and female B6C3F1/N mice received indole-3-carbinol in corn oil gavage for 3 months or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium and Escherichia coli, rat bone marrow cells, and mouse peripheral blood erythrocytes.

3-Month Study in F344/N Rats

Groups of 10 male and 10 female core study rats were administered 0, 18.75, 37.5, 75, 150, or 300 mg indole-3-carbinol/kg body weight in corn oil by gavage, 5 days per week for 14 weeks. Groups of 10 male and 10 female clinical pathology study rats were administered the same dose for 25 days. All rats survived to the end of the study. The mean body weight gain of 300 mg/kg males was significantly less than that of the vehicle controls. The absolute and relative liver weights of all dosed groups of males and females were significantly increased compared to the vehicle controls. The relative kidney weights of 75 mg/kg or greater males and all dosed groups of females were significantly increased, as were the absolute kidney weights of 75 mg/kg males and 18.75, 37.5, and 300 mg/kg females. The absolute and relative thymus weights of 75 mg/kg or greater females were significantly decreased. There were significant and dose-dependent increases in CYP1A1-associated 7-ethoxyresorufin-O-deethylase (EROD) and CYP1A2-associated acetanilide-4-hydroxylase (A4H) activities in the liver of all dosed groups of male and females rats. Pulmonary EROD activity was significantly increased in males administered 75 mg/kg or greater and in all dosed groups of females. Indole-3-carbinol exhibited the potential to be a reproductive toxicant in female rats based on a significantly increased probability of extended diestrus and an increase in overall estrous cycle length (approximately 1 day) observed at 300 mg/kg. In the small intestine, significantly increased incidences of lamina propria lipidosis and lymphatic ectasia occurred in the duodenum of 150 and 300 mg/kg males and females and in the jejunum of 75 mg/kg or greater males and females. In the mesenteric lymph node, significantly increased incidences of dilatation of the lymphatic vessels associated with lipidosis occurred in 150 and 300 mg/kg males and in 300 mg/kg females.

3-Month Study in B6C3F1/N Mice

Groups of 10 male and 10 female mice were administered 0, 15.6, 31.25, 62.5, 125, or 250 mg indole-3-carbinol/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 groups of males and females were similar to those of the vehicle controls. Liver weights of 125 and 250 mg/kg males and all dosed groups of females were significantly increased compared to the vehicle controls. There were significant and dose-dependent increases in A4H activities in the liver of all dosed groups of males, and hepatic EROD activities were significantly increased in males administered 31.25 mg/kg or greater. Hepatic A4H and EROD activities were significantly increased in 125 and 250 mg/kg females. Indole-3-carbinol exhibited the potential to be a reproductive toxicant in male and female mice based on significantly decreased sperm motility in all dosed groups of males and a significantly increased probability of extended diestrus in females administered 250 mg/kg. No histopathologic lesions were observed that could be attributed to the administration of indole-3-carbinol.

2-Year Study in Sprague Dawley Rats

Groups of 50 male and 50 female rats were administered 0, 75, 150, or 300 mg indole-3-carbinol/kg body weight in corn oil by gavage, 5 days per week for 104 (males) or 105 (females) weeks. Survival of dosed groups of males and females was similar to that of the vehicle controls. Mean body weights of dosed groups of males and females were similar to those of the vehicle controls throughout the study.

In the standard evaluation of the uterus, the incidences of adenocarcinoma occurred with a positive trend and were increased in all dosed groups. Extended evaluations of the uterus were conducted and additional neoplasms were identified. In the combined standard and extended evaluations, the incidence of adenocarcinoma was significantly increased in 150 mg/kg females. In the standard evaluation, the incidence of squamous metaplasia of the endometrium was significantly increased in the 150 mg/kg group.

The incidences of fibroma or fibrosarcoma (combined) in the skin occurred with a positive trend in females. An increased incidence of fibroma in the skin was observed in 300 mg/kg females. A single incidence of fibrosarcoma occurred in vehicle control and in 300 mg/kg females.

Significantly increased incidences of lymphatic ectasia in the duodenum and jejunum of the small intestine with generally increased severities occurred in 150 and 300 mg/kg males and females.

The incidences of lymphatic ectasia in the mesenteric lymph node were significantly increased in 300 mg/kg males and females compared to the vehicle controls.

In the liver, the incidences of clear cell focus in 300 mg/kg females, eosinophilic focus in 150 and 300 mg/kg females, and bile duct cyst in 300 mg/kg males were significantly increased compared to the vehicle controls.

Gene expression studies in 300 mg/kg females suggested activation of multiple xenobiotic transcription factors in rat liver with the most pronounced activation being associated with AhR and Nrf2. Consistent with these findings was the up-regulation of genes associated with xenobiotic metabolism, which suggests the potential for indole-3-carbinol to modify drug efficacy and safety. These findings are largely similar to results from other transcriptomic studies of indole-3-carbinol.

The incidences of follicular cell hypertrophy in the thyroid gland were significantly increased in all dosed groups of males, and the severities of the lesion increased with increasing dose.

2-Year Study B6C3F1/N in Mice

Groups of 50 male and 50 female mice were administered 0, 62.5, 125, or 250 mg indole-3-carbinol/kg body weight in corn oil by gavage, 5 days per week for 105 weeks. Survival of 250 mg/kg females was significantly greater than that of the vehicle controls. Mean body weights of dosed groups of males were similar to those of the vehicle controls throughout the study; however, those of 250 mg/kg female mice were at least 10% less than those of the vehicle controls between weeks 32 and 92.

Incidences of hepatocellular adenoma occurred with a positive trend in males and the incidence was significantly increased in the 250 mg/kg group. The incidences of multiple hepatocellular adenoma were significantly increased in 62.5 and 250 mg/kg males. There were significantly increased incidences of single and multiple hepatocellular carcinoma in 125 mg/kg males compared to the vehicle controls. In males, the incidences of hepatoblastoma occurred with a positive trend, and the incidences of multiple hepatoblastoma increased with increasing dose. The incidences of hepatoblastoma and multiple hepatoblastoma were significantly increased in 250 mg/kg males. The combined incidences of hepatocellular adenoma, hepatocellular carcinoma, or hepatoblastoma occurred with a positive trend in males and were significantly increased in males administered 125 or 250 mg/kg. The combined incidences of hepatocellular carcinoma and hepatoblastoma were significantly increased in 125 and 250 mg/kg males. The incidences of clear cell focus were significantly increased in all dosed groups of males, and the incidences of eosinophilic focus were significantly increased in 62.5 and 125 mg/kg females.

In the glandular stomach, the incidences of epithelium hyperplasia, chronic inflammation, and pigmentation were significantly increased in 125 and 250 mg/kg males and all dosed groups of females compared to the vehicle controls.

In the nose, incidences of nerve atrophy in 250 mg/kg males and females, respiratory metaplasia of the olfactory epithelium in 250 mg/kg males and 125 and 250 mg/kg females, atrophy of the olfactory epithelium in 125 and 250 mg/kg males and 250 mg/kg females, necrosis of the olfactory epithelium in 250 mg/kg males, respiratory epithelium hyaline droplet accumulation in 62.5 and 125 mg/kg males, respiratory epithelium hyperplasia in 250 mg/kg males and females, and inflammation in 250 mg/kg females were significantly greater than the vehicle control incidences.

Genetic Toxicology

Indole-3-carbinol was tested in three independent bacterial mutagenicity assays, and results were varied. Two assays yielded results that were judged to be equivocal in one or more of the tester strains (S. typhimurium strains TA97 and TA100 and E. coli strain WP2 uvrA/pKM101). Weak positive responses were seen in a third assay in S. typhimurium strain TA100, both with and without exogenous metabolic activation. In vivo, no increase in the frequency of micronucleated polychromaticerythrocytes (PCEs) was seen in bone marrow of F344/N rats given three doses of indole-3-carbinol by gavage; however, a significant decrease in the percent PCEs was seen in the bone marrow of treated rats, indicating that indole-3-carbinol (500 to 2,000 mg/kg per day) was toxic to the bone marrow. In addition to the rat study, micronucleus frequencies in normochromatic erythrocytes (NCEs) of male and female mice were assessed in peripheral blood at the end of the 3-month study; no significant increases in micronucleated NCEs were seen and no significant changes in percent PCEs occurred over the dose range tested (15.6 to 250 mg/kg per day).

Conclusions

Under the conditions of these 2-year gavage studies, there was no evidence of carcinogenic activity of indole-3-carbinol in male Harlan Sprague Dawley rats administered 75, 150, or 300 mg/kg. There was some evidence of carcinogenic activity of indole-3-carbinol in female Harlan Sprague Dawley rats based on increased incidences of malignant uterine neoplasms (primarily adenocarcinoma). The occurrences of fibroma and fibro-sarcoma in the skin may have been related to indole-3-carbinol administration. There was clear evidence of carcinogenic activity of indole-3-carbinol in male B6C3F1/N mice based on increased incidences of liver neoplasms (hepatocellular adenoma, hepatocellular carcinoma, and hepatoblastoma). There was no evidence of carcinogenic activity of indole-3-carbinol in female B6C3F1/N mice administered 62.5, 125, or 250 mg/kg.

Administration of indole-3-carbinol caused increased incidences of nonneoplastic lesions in the small intestine, mesenteric lymph node, and liver of male and female rats, the thyroid gland of male rats, the uterus of female rats, and the liver, glandular stomach, and nose of male and female mice.
  

Studies

Summary of the 2-Year Carcinogenesis and Genetic Toxicology Studies of Indole-3-carbinol
  Male
Sprague Dawley Rats
Female
Sprague Dawley Rats
Male
B6C3F1/N Mice
Female
B6C3F1/N Mice
Doses in corn oil by gavage 0, 75, 150, or 300 mg/kg 0, 75, 150, or 300 mg/kg 0, 62.5, 125, or 250 mg/kg 0, 62.5, 125, or 250 mg/kg
Survival rates 20/50, 13/50, 17/50, 12/50 21/50, 19/50, 20/50, 30/50 27/50, 31/50, 32/50, 32/50 33/50, 40/50, 26/50, 45/50
Body weights Dosed groups similar to the vehicle control group Dosed groups similar to the vehicle control group Dosed groups similar to the vehicle control group 250 mg/kg group at least 10% less than the vehicle control group between weeks 32 and 92
Nonneoplastic effects Intestine small, duodenum: lymphatic, ectasia (0/43, 0/48, 15/47, 14/48)
Intestine small, jejunum: lymphatic, ectasia (0/40, 2/39, 27/40, 41/42) Lymph node, mesenteric: lymphatic, ectasia (0/50, 0/50, 1/50, 5/50)
Liver: bile duct, cyst (0/50, 0/50, 2/50, 5/50)
Thyroid gland: follicular cell, hypertrophy (21/50, 34/46, 33/48, 36/47)
Uterus: endometrium, metaplasia, squamous (standard evaluation-12/50, 18/50, 20/50, 11/50)
Intestine small, duodenum: lymphatic, ectasia (0/48, 0/47, 16/48, 38/47)
Intestine small, jejunum: lymphatic, ectasia (0/47, 0/46, 30/48, 47/48)
Lymph node, mesenteric: lymphatic, ectasia (0/50, 0/50, 1/50, 15/48)
Liver: clear cell focus (6/50, 7/50, 4/50, 18/48); eosinophilic focus (0/50, 4/50, 5/50, 6/48)
Liver: clear cell focus (7/50, 17/50, 22/49, 20/50)
Glandular stomach: epithelium, hyperplasia (0/50, 1/47, 22/47, 40/49); inflammation, chronic (1/50, 1/47, 18/47, 45/49); pigmentation (0/50, 1/47, 38/47, 48/49)
Nose: nerve, atrophy (0/50, 0/50, 0/50, 8/50); olfactory epithelium, respiratory metaplasia (14/50, 14/50, 20/50, 27/50); olfactory epithelium, atrophy (3/50, 5/50, 11/50, 17/50); olfactory epithelium, necrosis (0/50, 0/50, 0/50, 6/50); respiratory epithelium, accumulation, hyaline droplet (18/50, 34/50, 30/50, 26/50); respiratory epithelium, hyperplasia (35/50, 40/50, 41/50, 45/50)
Liver: eosinophilic focus (16/50, 26/50, 26/50, 21/50)
Glandular stomach: epithelium, hyperplasia (1/48, 7/50, 10/49, 35/50); inflammation, chronic (0/48, 15/50, 29/49, 47/50); pigmentation (0/48, 15/50, 31/49, 49/50)
Nose: nerve, atrophy (0/50, 0/50, 1/50, 50/50); olfactory epithelium, respiratory metaplasia (7/50, 8/50, 16/50, 49/50); olfactory epithelium, atrophy (1/50, 2/50, 3/50, 45/50); respiratory epithelium, hyperplasia (32/50, 31/50, 38/50, 50/50); inflammation (4/50, 1/50, 8/50, 39/50)
Neoplastic effects None Uterus: adenocarcinoma (standard evaluation-0/50, 1/50, 4/50, 4/50; standard and extended evaluations, combined-5/50, 4/50, 13/50, 10/50) Liver: hepatocellular adenoma (26/50, 32/50, 31/49, 41/50); hepatocellular carcinoma (12/50, 11/50, 29/49, 14/50); hepatoblastoma (3/50, 4/50, 4/49, 14/50); hepatocellular adenoma, hepatocellular carcinoma, or hepatoblastoma (36/50, 36/50, 44/49, 45/50) None
Equivocal findings None Skin: fibroma (1/50, 0/50, 0/50, 4/50); fibrosarcoma (1/50, 0/50, 0/50, 1/50); fibroma or fibrosarcoma (2/50, 0/50, 0/50, 5/50) None None
Level of evidence of carcinogenic activity No evidence Some evidence Clear evidence No evidence
Genetic Toxicology of Indole-3-carbinol
Assay Test System Results
Bacterial Mutagenicity Salmonella typhimurium Negative in Salmonella typhimurium strains TA98, TA102, TA104, TA1535, and TA1537 with S9 and without S9; equivocal in strain TA97 without S9; equivocal in one study in strain TA100 with 10% rat S9; weakly positive in a second study in strain TA100 with and without 30% hamster S9; equivocal in Escherichia coli strain WP2 uvrA/pKM101 without S9.
Micronucleated erythrocytes Rat bone marrow in vivo: Negative
Micronucleated erythrocytes Mouse peripheral blood in vivo: Negative