Evaluation of 2-isopropyl-N-2,3-trimethylbutyramide by a comprehensive toxicity study using gpt delta rats

2-Isopropyl-N-2,3-trimethylbutyramide (ITB) is a food-flavoring agent classified as an aliphatic amide. In 2016, the Joint FAO/WHO Expert Committee on Food Additives evaluated ITB and concluded that additional data on toxicity and in vivo genotoxicity are required for its safety evaluation. In this study, we comprehensively investigated ITB toxicity using reporter gene transgenic animals. Male F344 gpt delta rats were administered ITB by oral gavage at doses of 0, 5, 50, or 500 mg/kg/day for 13 weeks. Neurological symptoms were observed in the early phase of treatment at doses ≥50 mg/kg. Periportal hepatocellular vacuolation was observed histopathologically at doses ≥50 mg/kg, along with increased liver weight and serum alanine aminotransferase levels. Kidney weight increased and serum chloride levels decreased at doses ≥5 mg/kg, indicating that ITB exerted potential nephrotoxic effects at lower doses. Accordingly, the lowest observed adverse effect level in the present study was at 5 mg/kg/day. No significant changes in gpt and red/gam mutant frequencies were detected in the liver or kidney, demonstrating a lack of ITB genotoxicity. Immunohistochemical analysis of GST-P-positive foci also suggested that ITB showed no hepatocarcinogenic potential. Overall, our findings demonstrate that ITB induces hepatic and renal toxicity but shows no evidence of in vivo genotoxicity or hepatocarcinogenic potential, providing essential information for safety assessment.