National Toxicology Program

National Toxicology Program
https://ntp.niehs.nih.gov/go/14894

Abstract for TR-508 - Riddelliine (CASRN 23246-96-0)

ABSTRACT

Toxicology and Carcinogensis Studies of Riddelliine (CAS No. 23246-96-0) in F344/N Rats and B6C3F1 Mice (Gavage Studies)

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Two Dimensional Chemical Structure

Chemical Formula: C18H23NO6

Riddelliine belongs to a class of toxic pyrrolizidine alkaloids and is isolated from plants of the genera Crotalaria, Amsinckia, and Senecio that grow in the western United States. Cattle, horses, and sheep that ingest these plants succumb to their toxic effects. Riddelliine residues have been found in meat, milk, and honey, and the plants may contaminate human food sources. Riddelliine was nominated for study by the Food and Drug Administration because of its potential for human exposure and its economic impact on the livestock industry and because the toxicity of other pyrrolizidine alkaloids suggests riddelliine may be carcinogenic. Male and female F344/N rats and B6C3F1 mice received riddelliine (approximately 92% pure) by gavage. Female rats and male and female mice were dosed for 2 years; due to high mortality, the study in male rats was terminated at week 72. In vitro genetic toxicology studies were conducted in Salmonella typhimurium and in cultured Chinese hamster ovary (CHO) cells. In addition, riddelliine was evaluated in vivo for induction of micronuclei in mouse bone marrow and peripheral blood erythrocytes and for induction of S-phase DNA synthesis and unscheduled DNA synthesis in the liver of rats and mice. Riddelliine-induced DNA adduct levels were determined in liver tissue obtained from female rats admininstered riddelliine for 3 or 6 months.

2-Year Study in Rats

Groups of 50 male and 50 female rats were administered 0 or 1 mg riddelliine/kg body weight in sodium phosphate buffer by gavage 5 days per week; additional groups of 50 female rats received 0.01, 0.033, 0.1, or 0.33 mg/kg. A wide dose range was used in female rats to better characterize the dose-response curve. Females were dosed for 105 weeks; due to high mortality, male rats were terminated at week 72.

All but three 1 mg/kg males died before week 70, and all 1 mg/kg females died before week 97. Mean body weights of 1 mg/kg males and females were less than those of the vehicle controls throughout most of the study. The only clinical finding related to riddelliine administration was a general debilitation of the animals prior to death.

Hemangiosarcomas were present in the liver of 86% of males and 76% of females in the 1 mg/kg groups, and this neoplasm was considered the cause of the large number of early deaths in these groups. The incidences of hepatocellular adenoma and mononuclear cell leukemia in 1 mg/kg males and females were significantly increased. Nonneoplastic lesions related to riddelliine treatment occurred in the liver and kidney of males and females.

Analyses of liver tissue from female rats treated with riddelliine for 3 or 6 months yielded eight DNA adducts; these were the same as DNA adducts formed in vitro by the metabolism of riddelliine by human liver microsomes in the presence of calf thymus DNA.

2-Year Study in Mice

Groups of 50 male and 50 female mice were administered riddelliine in sodium phosphate buffer by gavage at doses of 0 or 3 mg/kg, 5 days per week, for 105 weeks; additional groups of 50 male mice received 0.1, 0.3, or 1 mg/kg for 105 weeks. A wide dose range was used in male mice to better characterize the dose-response curve.

Survival of males and females administered 3 mg/kg was significantly less than that of the vehicle controls. Mean body weights of 3 mg/kg mice were less than those of the vehicle controls throughout most of the study.

Hemangiosarcomas of the liver were present in 62% of males in the 3 mg/kg group. The incidences of hepatocellular neoplasms occurred with negative trends in male mice and were significantly decreased in 3 mg/kg females. The incidences of alveolar/bronchiolar neoplasms in 3 mg/kg females were significantly increased. Nonneoplastic lesions related to riddelliine administration occurred in the liver and kidney of males and females and in the lung and arteries (multiple tissues) of females.

Genetic Toxicology

Riddelliine was mutagenic in S. typhimurium strain TA100 with, but not without, S9 activation; no significant mutagenic activity was detected in strain TA98 or TA1535, with or without S9. A small, dose-related increase in mutant colonies seen in strain TA97 with S9 was judged to be equivocal. Riddelliine induced sister chromatid exchanges in cultured CHO cells with and without S9. Chromosomal aberrations were induced in CHO cells only in the presence of S9. Following 4 or 13 weeks of daily gavage treatment with riddelliine, no increases in the frequency of micronucleated erythrocytes were noted in the peripheral blood of male or female B6C3F1 mice. Use of a single intraperitoneal injection protocol, however, produced a small but significant increase in the frequency of micronucleated eryth-rocytes in peripheral blood of male Swiss mice 48 hours after injection; bone marrow analysis 24 hours after injection demonstrated a small but insignificant increase in the frequency of micronuclei. Unscheduled DNA synthesis was detected in cultured hepatocytes from male and female rats and mice following 5 or 30 days of riddelliine treatment by gavage. In addition, an S-phase DNA synthesis was observed in cultured hepatocytes of male and female rats treated for either time period.

Conclusions

Under the conditions of these studies, there was clear evidence of carcinogenic activity of riddelliine in male and female F344/N rats based primarily on increased incidences of hemangiosarcoma in the liver. The increased incidences of hepatocellular adenoma and mononuclear cell leukemia in male and female rats were also considered to be treatment related. There was clear evidence of carcinogenic activity of riddelliine in male B6C3F1 mice based on increased incidences of hemangiosarcoma in the liver. There was clear evidence of carcinogenic activity in female B6C3F1 mice based on increased incidences of alveolar/bronchiolar neoplasms.

Administration of riddelliine by gavage resulted in nonneoplastic lesions in the liver and kidney of male and female rats; the liver and kidney of male and female mice; and the lung and arteries (multiple tissues) of female mice.

Decreased incidences of hepatocellular neoplasms in male and female mice were related to riddelliine administration.

Synonyms: 13,19-Didehydro-12,18-dihydroxy senecionan-11,16-dione; 3-ethylidine-3,4,5,6,9,11,13,14,14a,14b-decahydro-6-hydroxy-6- (hydroxymethyl)-5-methylene(1,6)di-oxacyclododecino(2,3,4-gh)-pyrrolizidine-2,7-dione; trans-15-ethylidine-12b-hydroxy-12a- hydroxymethyl-13-methylenesenec-1-enine

Summary of the 2-Year Carcinogenesis Studies of Riddelliine

 

Male
F344/N Rats

Female
F344/N Rats

Male
B6C3F1 Mice

Female
B6C3F1 Mice

Doses in sodium phosphate buffer by gavage

Vehicle control or 1 mg/kg

Vehicle control, 0.01, 0.033, 0.1, 0.33, or 1 mg/kg

Vehicle control, 0.1, 0.3, 1, or 3 mg/kg

Vehicle control or 3 mg/kg

Body weights

1 mg/kg group less than the vehicle control group

1 mg/kg group less than the vehicle control group

3 mg/kg group less than the vehicle control group

3 mg/kg group less than the vehicle control group

Survival rates

49/50, 3/50

33/50, 22/50, 28/50, 22/50, 29/50, 0/50

39/50, 41/50, 40/50, 38/50, 20/50

34/50, 17/50

Nonneoplastic effects

Liver: regenerative hepatocyte hyperplasia (0/50, 49/50); hepatocyte cytomegaly (0/50, 32/50); focal necrosis (0/50, 23/50); eosinophilic focus (3/50, 15/50); mixed cell focus (3/50, 7/50); hemorrhage (0/50, 4/50)

Kidney: renal tubule necrosis (0/50, 6/50)

Liver: regenerative hepatocyte hyperplasia (0/50, 0/50, 0/50, 0/50, 8/50, 50/50); hepatocyte cytomegaly (0/50, 0/50, 7/50, 23/50, 32/50, 29/50); focal necrosis (4/50, 2/50, 3/50, 4/50, 4/50, 15/50); eosinophilic focus (1/50, 2/50, 6/50, 4/50, 12/50, 13/50); mixed cell focus (8/50, 10/50, 10/50, 11/50, 23/50, 5/50); clear cell focus (9/50, 8/50, 9/50, 13/50, 22/50, 2/50); bile duct hyperplasia (2/50, 1/50, 4/50, 4/50, 3/50, 10/50); hemorrhage (0/50, 0/50, 2/50, 0/50, 1/50, 7/50)

Kidney: renal tubule necrosis (0/50, 0/50, 0/50, 1/50, 1/50, 6/50); transitional epithelium hyperplasia (1/50, 1/50, 1/50, 1/50, 0/50, 5/50)

Liver: hepatocyte cytomegaly (4/50, 4/50, 16/50, 33/50, 43/50); hepatocyte karyomegaly (4/50, 4/50, 15/50, 33/50, 43/50); centrilobular hepatocyte necrosis (0/50, 1/50, 3/50, 4/50, 10/50)hemorrhage focal (0/50, 2/50, 1/50, 6/50, 21/50); severity of focal hepatocyte necrosis (1.3, 1.3, 1.8, 2.2, 2.6)

Kidney: severity of nephropathy (1.3, 1.5, 1.8, 2.1, 2.8); glomerulosclerosis (0/49, 1/49, 0/50, 42/50, 41/50); renal tubule karyomegaly (0/49, 0/49, 0/50, 0/50, 12/50); renal tubule dilatation (16/49, 17/49, 24/50, 29/50, 22/50)

Liver: hepatocyte cytomegaly (0/49, 49/50); hepatocyte karyomegaly (0/49, 49/50); bile duct hyperplasia (0/49, 28/50); mixed cellular infiltration (29/49, 41/50)

Kidney: nephropathy (18/49, 47/50); severity of nephropathy (1.3, 3.4); glomerulosclerosis (0/49, 40/50); renal tubule hyaline droplet accumulation (2/49, 14/50); renal tubule pigmentation (2/49, 27/50)

Lung: alveolar epithelial hyperplasia (1/50, 6/50)

Arteries, chronic arterial inflammation (includes focal): small intestine (duodenum) (0/47, 13/46); large intestine (cecum) (0/48, 18/47); kidney (1/49, 16/50); mesentery (1/23, 19/29); ovary (0/49, 26/48); spleen (0/49, 6/50); uterus (0/49, 21/50)

Neoplastic effects

Liver: hemangiosarcoma (0/50, 43/50); hepatocellular adenoma (0/50, 4/50)

Mononuclear cell leukemia (all organs): (2/50, 9/50)

Liver: hemangiosarcoma (0/50, 0/50, 0/50, 0/50, 3/50, 38/50); hepatocellular adenoma (1/50, 0/50, 0/50, 0/50, 1/50, 7/50)

Mononuclear cell leukemia (all organs): (12/50, 8/50, 13/50, 18/50, 18/50, 14/50)

Liver: hemangiosarcoma (2/50, 1/50, 0/50, 2/50, 31/50)

Lung: alveolar/bronchiolar adenoma (1/50, 9/50); alveolar/bronchiolar carcinoma (1/50, 4/50); alveolar/bronchiolar adenoma or carcinoma (2/50, 13/50)

Decreased incidences

None

None

Liver: hepatocellular adenoma (16/50, 18/50, 14/50, 5/50, 0/50); hepatocellular carcinoma (23/50, 21/50, 19/50, 20/50, 3/50); hepatocellular adenoma or carcinoma (36/50, 39/50, 33/50, 23/50, 3/50)

Liver: hepatocellular adenoma (9/49, 0/50); hepatocellular carcinoma (8/49, 0/50); hepatocellular adenoma or carcinoma (16/49, 0/50)

Level of evidence of carcinogenic activity

Clear evidence

Clear evidence

Clear evidence

Clear evidence

Genetic Toxicology of Riddelliine
Assay Test System Results
Bacterial Mutagenicity Salmonella typhimurium gene mutation Positive in strain TA100 with rat and hamster S9, negative without S9; negative in strains TA98 and TA1535 with and without S9; negative in strain TA97 without S9, equivocal with S9
Sister chromatid exchanges Cultured chinese hamster ovary cells in vitro: Positive with and without S9
Chromosomal aberrations Cultured Chinese hamster ovary cells in vitro: Positive with S9, negative without S9
Micronucleus test in mice Single injection study, bone marrow Negative
Micronucleus test in mice Single injection study, peripheral blood Positive, but not confirmed with a repeat test
Micronucleus test in mice 4-week gavage study, peripheral blood Negative
Micronucleus test in mice 13-week gavage study, peripheral blood Negative
Unscheduled DNA synthesis 5-day gavage study Positive in male and female rats and mice
Unscheduled DNA synthesis 30-day gavage study Positive in male rats and male and female mice; negative in female rats
S-Phase DNA synthesis 5-day gavage study Positive in male and female rats; negative in male and female mice
S-Phase DNA synthesis 30-day gavage study Positive in male and female rats and male mice; negative in female mice

Report Date: May 2003

Pathology Tables, Survival and Growth Curves from NTP 2-year Studies

Target Organs & Incidences from 2-year Studies


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