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Abstract for TR-565

Toxicology and Carcinogenesis Studies of Milk Thistle Extract in F344/N Rats and B6C3F1 Mice (Feed Studies)

CASRN: 84604-20-6
Synonyms/Common Names: Bull thistle; cardo blanco; cardui mariae fructus; cardui mariae herba; Cardum marianum L.; Carduus marianus L.; chardon-marie; emetic root; frauendistel; fructus silybi mariae; fruit de chardon marie; heal thistle; holy thistle; isosilibinin; kanger; kocakavkas; kuub; lady's thistle; lady's thistle extract; marian thistle; mariana mariana; mariendistel; Marienkrörner; Mary thistle; mild thistle; milk ipecac; pig leaves; royal thistle; Saint-Mary thistle; shui fei ji; silidianin; silybi mariae fructus; silybin; silybinin; Silybum marianum extract; silychristin; snake milk; sow thistle; St. Mary's thistle; variegated thistle; venue thistle; wild artichoke Trade names for milk thistle extract (silymarin): Legalon®, Thisilyn®
Report Date: May 2011

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Abstract

Milk thistle extracts have been used as medicinal herbs in the treatment of liver cirrhosis, chronic hepatitis (liver inflammation), and gallbladder disorders. Treatment claims also include lowering cholesterol levels; reducing insulin resistance; reducing the growth of cancer cells in breast, cervical, and prostate gland cancers; and antiviral activity. Other reported uses of milk thistle in folk medicine include as a treatment for malarial fever, bronchitis, gallstones, jaundice, peritonitis, uterine congestion, varicose veins, and as a milk production stimulant for nursing mothers. The roots soaked in water overnight are used in food, and the despined leaves are added to salads. Roasted milk thistle fruit has been used as a coffee substitute. Milk thistle extract was nominated for study by the National Institute of Environmental Health Sciences because it is one of the most widely used herbs in the United States. Male and female F344/N rats and B6C3F1 mice were exposed to an ethanol/water extract of milk thistle fruit (milk thistle extract) containing approximately 65% silymarin in feed for 3 months or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium and Escherichia coli and mouse peripheral blood erythrocytes.

3-Month Study in Rats

Groups of 10 male and 10 female rats were fed diets containing 0, 3,125, 6,250, 12,500, 25,000, or 50,000 ppm milk thistle extract (equivalent to average daily doses of approximately 260, 525, 1,050, 2,180, or 4,500 mg milk thistle extract/kilogram body weight to males and 260, 510, 1,050, 2,150, or 4,550 mg/kg to females) for 14 weeks. All rats survived to the end of the study. Mean body weights of exposed groups were within 10% of those of the controls. Feed consumption by exposed and control groups was similar. The sperm motility in 12,500, 25,000, and 50,000 ppm males was decreased by 5%, 11%, and 9%, respectively, relative to that of the controls; the total number of spermatid heads per testis decreased by 11%, 21%, and 9% in 12,500, 25,000, and 50,000 ppm males. No significant differences in estrous cyclicity were observed between exposed and control groups of female rats. No exposure-related histopathologic lesions were observed.

3-Month Study in Mice

Groups of 10 male and 10 female mice were fed diets containing 0, 3,125, 6,250, 12,500, 25,000, or 50,000 ppm milk thistle extract (equivalent to average daily doses of approximately 640, 1,340, 2,500, 5,280, or 11,620 mg/kg to males and 580, 1,180, 2,335, 4,800, or 9,680 mg/kg to females) for 14 weeks. All mice survived to the end of the study. Mean body weights and feed consumption of all exposed groups were similar to those of the controls. Absolute and relative thymus weights were significantly decreased in 25,000 and 50,000 ppm males. No significant differences were observed between exposed and control groups, for sperm parameters of male mice, for estrous cyclicity of female mice, or for reproductive organ weights of male or female mice, when mice were administered milk thistle extract in feed at 12,500, 25,000, or 50,000 ppm. No exposure-related histopathologic lesions were observed.

2-Year Study in Rats

Groups of 50 male and 50 female rats were fed diets containing 0, 12,500, 25,000, or 50,000 ppm milk thistle extract (equivalent to average daily doses of approximately 570, 1,180, or 2,520 mg/kg to males and 630, 1,300, or 2,750 mg/kg to females) for 105 to 106 weeks. Exposure to milk thistle extract had no effect on survival of male or female rats. Mean body weights of all exposed groups were similar to those of the controls throughout the study. Feed consumption by exposed groups of males and females was generally similar to that by the controls throughout the study.

Significantly decreased incidences of mammary gland fibroadenoma, adenoma, or carcinoma (combined) occurred in females exposed to 25,000 or 50,000 ppm.

Significantly increased incidences of clear cell and mixed cell focus of the liver occurred in 25,000 and 50,000 ppm females. The incidences of bile duct hyperplasia were significantly decreased in 50,000 ppm males and in all exposed groups of females, and the incidence of mixed inflammatory cell infiltration was significantly decreased in 50,000 ppm males.

2-Year Study in Mice

Groups of 50 male and 50 female mice were fed diets containing 0, 12,500, 25,000, or 50,000 ppm milk thistle extract (equivalent to average daily doses of approximately 1,610, 3,530, or 7,770 mg/kg to males and 1,500, 3,175, or 7,180 mg/kg to females) for 105 to 106 weeks. Exposure to milk thistle extract had no effect on survival of male or female mice. The mean body weights of the 25,000 ppm groups were less than those of controls after week 25; mean body weights of 50,000 ppm groups were less than those of controls after week 12. Feed consumption by exposed groups of males and females was generally similar to that by the controls throughout the study.

Significantly decreased incidences of hepatocellular adenoma and hepatocellular carcinoma occurred in 50,000 ppm males, and decreased incidences of hepatocellular adenoma or carcinoma (combined) occurred in 25,000 and 50,000 ppm males.

Genetic Toxicology

Five milk thistle extracts were tested independently in bacterial mutagenicity studies using a variety of S. typhimurium tester strains and one E. coli strain. Results were negative in three of the five studies, with and without exogenous metabolic activation. In two studies, milk thistle extract was mutagenic in S. typhimurium strain TA98 in the presence of exogenous metabolic activation enzymes. Silymarin, a major constituent of milk thistle extract, was positive in S. typhimurium strains TA98 and TA100, when testing occurred in the presence of exogenous metabolic activation enzymes. Silybin, another component of milk thistle extract, was negative in a S. typhimurium gene mutation assay, with and without liver S9 activation enzymes.

Administration of milk thistle extract in feed for 3 months did not increase the frequencies of micronucleated normochromatic erythrocytes, an indication of chromosomal abnormalities, in the peripheral blood of male or female B6C3F1 mice.

Conclusions

Under the conditions of these 2-year feed studies, there was no evidence of carcinogenic activity of milk thistle extract in male or female F344/N rats or B6C3F1 mice exposed to 12,500, 25,000, or 50,000 ppm.

Exposure to milk thistle extract resulted in increased incidences of clear cell and mixed cell foci in the liver of female rats and decreases in body weights of exposed groups of male and female mice.

Decreased incidences of mammary gland neoplasms occurred in exposed groups of female rats, and decreased incidences of hepatocellular neoplasms occurred in exposed groups of male mice.

Studies

Summary of the 2-Year Carcinogenesis and Genetic Toxicology Studies of Milk Thistle Extract
  Male
F344/N Rats
Female
F344/N Rats
Male
B6C3F1 Mice
Female
B6C3F1 Mice
Concentrations in feed 0, 12,500, 25,000, or 50,000 ppm 0, 12,500, 25,000, or 50,000 ppm 0, 12,500, 25,000, or 50,000 ppm 0, 12,500, 25,000, or 50,000 ppm
Body weights Exposed groups similar to the control group Exposed groups similar to the control group 25,000 ppm group 10% less than the control group after week 17; 50,000 ppm group 10% less than the control group after week 9 25,000 ppm group 11% less than the control group after week 25; 50,000 ppm group 10% less than the control group after week 12
Survival rates 36/50, 36/50, 35/50, 38/50 38/50, 36/50, 34/50, 37/50 45/50, 46/50, 43/50, 49/50 40/50, 43/50, 42/50, 45/50
Nonneoplastic effects None Liver: clear cell focus (9/50, 10/50, 17/49, 21/50); mixed cell focus (3/50, 6/50, 11/49, 10/50) None None
Neoplastic effects None None None None
Decreased incidences None Mammary gland: fibroadenoma (28/50, 27/50, 17/50, 18/50); fibroadenoma, adenoma, or carcinoma (28/50, 27/50, 19/50, 20/50) Liver: hepatocellular adenoma (12/50, 13/50, 5/50, 1/50); hepatocellular carcinoma (17/50, 15/50, 14/50, 7/50); hepatocellular adenoma or carcinoma (26/50, 22/50, 16/50, 8/50) None
Level of evidence of carcinogenic activity No Evidence No Evidence No Evidence No Evidence
Genetic Toxicology of Milk Thistle Extract
Assay Test System Results
Bacterial Mutagenicity: Salmonella typhimurium Milk thistle extract: Negative in strains TA97, TA100, TA102, TA104, and TA1535 with and without S9; positive in strain TA98 with S9; negative in E. coli strain WP2 uvrA/pKM101 with and without S9
Bacterial Mutagenicity: Salmonella typhimurium Silymarin: Positive in strains TA98 and TA100 with S9; negative without S9
Bacterial Mutagenicity: Salmonella typhimurium Silybin: Negative in strains TA97, TA98, TA100, and TA1535 with and without S9
Micronucleated erythrocytes Mouse peripheral blood in vivo: Negative