Highly cross-linked starch (HXLS) is distarch phosphate made from tapioca starch and is a resistant starch type 4. To assess the safety of HXLS，acute toxicity，subchronic toxicity and mutagenicity were examined. The acute oral LD50 in male and female rats has been estimated to be greater than 2000 mg/kg of body weight. In a 28-day subchronic study，oral administration of HXLS at concentrations of 500 and 1000 mg/kg of body weight for 4 weeks did not cause any significant toxicologic symptoms. No treatment-related changes were noted in the general condition and appearance of the rats，body weights，food consumption，urinalysis，hematological and blood chemical findings，necropsy findings，organ weights，or histopathological findings. The no observed adverse effect level (NOAEL) has been estimated to be greater than the highest level of 1000 mg/ kg of body weight，for both male and female rats. HXLS did not show any potential to induce gene mutations in reverse mutation tests using Salmonella typhimurium TA98，TA1537，TA100，TA1535 and Escherichia coli WP2uvrA strains. The studies were conducted as part of an investigation to examine the safety of HXLS. The results of these studies suggested that HXLS is safe for use in food.. Key words : highly cross-linked starch，acute，subchronic，mutagenicity，safety.
