In this manuscript, the toxicology and safety of pomegranate seed oil (PSO) was evaluated by in vitro (Ames, chromosomal aberration), and in vivo toxicity tests (acute toxicity and 28-day toxicity in Wistar rats). No mutagenicity of PSO was observed in the absence and presence of metabolic activation up to precipitating concentrations of 5000 microg/plate (Ames test) or 333 microg/ml (chromosome aberration test). The acute oral toxicity study revealed no significant findings at 2000 mg PSO/kg body weight. In the 28-day dietary toxicity study PSO was dosed at concentrations of 0, 10,000, 50,000 and 150,000 ppm, which resulted in a mean intake of 0-0, 825-847, 4269-4330 and 13,710-14,214 mg PSO/kg body weight per day in males-females, respectively. At 150,000 ppm dietary exposure to PSO, a much higher dose than the level of PSO that elicits antidiabetic and anti-inflammatory efficacy, increased hepatic enzyme activities determined in plasma (aspartate, alanine aminotransferase and alkaline phosphatase) and increased liver-to-body weight ratios were observed. However, these effects might be the result of a physiological response to exposure to a very high level of a fatty acid which is not part of the normal diet, and are most likely not toxicologically relevant. The no observable adverse effect level (NOAEL) was 50,000 ppm PSO (=4.3 g PSO/kg body weight/day).