Abstract
D-Allulose is a rare monosaccharide structurally similar to D-fructose, characterized by low caloric content and relatively high sweetness. This study aimed to conduct a 90-day oral toxicity test to systemically evaluate the potential toxicological effects of D-allulose produced via a novel one-step fermentation process using genetically engineered Escherichia coli AS10 strain, which was genetically modified to express enzymes involved in the biosynthesis of D-allulose from D-glucose. The objectives were to determine its NOAEL, provide scientific evidence for the safety evaluation of this innovative food ingredient, and meet the regulatory requirements for the safety assessment of a novel process-derived food product. A total of 96 SD rats were randomly divided into four groups (24 rats per group, with equal numbers of males and females): a control group (basal feed) and three treatment groups fed with feed containing 2.5%, 5.0%, and 10.0% D-allulose (corresponding to estimated dietary intakes of 2000, 4000, and 8000 mg/kg body weight per day, respectively, based on an assumed daily feed intake of 8% of body weight) for 90 consecutive days. A comprehensive battery of toxicological assessments was performed on each animal, including measurements on body weight, food consumption, feed efficiency ratios, hematological metrics, serum biochemical profiles, organ weights, and histopathological examinations. No treatment-related mortality or overt toxic symptoms were observed in any group during the study period. Statistically significant differences were noted in partial metrics (e.g., body weight, feed consumption, HCT%, PLT, ALP, TC, and absolute/relative weight of kidney) between treatment groups and control group, but these changes were deemed nontoxicologically significant due to consistency with normal physiological variability (within the historical in-house reference ranges) or lack of corresponding pathological lesions. The NOAEL of D-allulose was established at 8000 mg/kg BW/day in rats, confirming its safety for use as a food ingredient.