Synthesis and Toxicological Evaluation of N‐Nitroso Vildagliptin Amide Impurity in BALB 3T3 Clone Cells

Nitrosamine impurities in pharmaceuticals have become a significant regulatory concern because of their established carcinogenic and genotoxic potential. N-nitroso vildagliptin amide impurity (NNVI), associated with the antidiabetic drug vildagliptin, raises potential safety concerns, yet its biological toxicity remains inadequately explored. To address this gap in our study, we synthesized and characterized NNVI and evaluated its cytotoxic, oxidative, and genotoxic effects using BALB 3T3 cell lines, within the context of the US FDA's acceptable daily intake (ADI) limit for NNVI. NNVI was synthesized under mild acidic conditions via nitrosation of the vildagliptin amide intermediate and confirmed structurally using NMR, IR, and mass spectrometry. BALB 3T3 cells were treated with NNVI at concentrations ranging from 5 to 125 ppm. Cytotoxicity was assessed with the MTT assay, whereas oxidative stress was measured via levels of superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), nitric oxide synthases (iNOS), reduced glutathione (GSH), and Nrf2. Genotoxicity was determined through the alkaline comet assay. The results showed a dose-dependent decrease in cell viability, with significant cytotoxicity observed at concentrations ≥ 50 ppm. Oxidative stress markers indicated adaptive antioxidant responses at lower concentrations (5-25 ppm), but higher concentrations caused oxidative imbalance, antioxidant depletion, and increased lipid peroxidation. Genotoxic effects were evident at 100 ppm, with increased DNA tail intensity. These findings confirm that NNVI presents a toxicological risk at concentrations above regulatory limits. The study underscores the importance of strict impurity control in pharmaceutical formulations and contributes to the wider effort of ensuring drug safety by limiting nitrosamine exposure to acceptable thresholds.