Induction of characteristic hepatocyte proliferative lesion with dietary exposure of Wistar Hannover rats to tocotrienol for 1 year

Tocotrienol is an antioxidant which has found commercial application as a food additive and health supplement all over the world. Since there have been no reports regarding toxicological effects of long-term exposure, we performed a 52-week chronic study using Wistar Hannover rats of both sexes given the compound at doses of 0, 0.08, 0.4 or 2% in powdered basal diet. Since 6 animals in the 2% male group died of hemorrhage of several organs by week 50, the maximum dose level was changed to 1% in both sexes for the last 2 weeks. Decrease of body weight gain was observed in the 2% males from week 5 and females from week 10, this persisting to the end of the study. With the high dose, prolongation of prothrombin time and increase of serum ALT in males, and increase of serum ALP in both sexes were observed with statistical significance. In male and female rats receiving 0.4% or less, there were no toxicological changes in any of the parameters examined. At necropsy, multiple cyst-like nodules on the liver surface were macroscopically pronounced in both sexes receiving 2%. On histopathological examination, hepatocellular nodules were evident with distortion of hepatic cords and compression of the surrounding tissue, almost all including areas of spongiosis hepatis. The constituent hepatocytes were immunohistochemically stained with proliferation cell nuclear antigen at high rates. Nevertheless, they did not exhibit overt atypia and the basic lobular architecture remained intact. Additionally, they were consistently negative for glutathione S-transferase placental form (GST-P). Accordingly, we propose the newly categorized but previously used name ‘nodular hepatocellular hyperplasia’, which may not necessarily have a neoplastic or regenerative nature. However, quantitative GST-P analysis of the liver sections overall showed numbers of GST-P foci in the high dose females to be significantly elevated as compared to the control value. Based on the present data demonstrating nodular liver lesions only at the high dose of both sexes, we conclude that the no-observed-adverse-effect level (NOAEL) is 0.4% (303 mg/kg/day for males, and 472 mg/kg/day for females).