Re‐Evaluating Acceptable Intake: A Comparative Study of N‐Nitrosomorpholine and N‐Nitroso Reboxetine Potency

Establishing regulatory limits for Drug Substance-Related Impurities (NDSRIs) is challenging due to the limited genotoxicity and carcinogenicity data available for many of these impurities, often leading to conservative approaches. In this study, we evaluated the genotoxic potential of two structurally related nitrosamines: N-nitrosomorpholine (NMOR) and N-nitroso reboxetine. Compared to the well-studied NMOR, there is little toxicological information available for N-nitroso reboxetine. Currently, both compounds have an acceptable intake value of 127 ng/day, based on a read-across using the available carcinogenicity data of NMOR. While both compounds tested positive in a series of in vitro and in vivo assays, we found that the mutagenic potential of N-nitroso reboxetine was significantly lower than that of NMOR. The benchmark dose (BMD) analysis of in vivo mutagenicity data supports an acceptable intake of 24,000 ng/day for N-nitroso reboxetine. Computational studies, carried out using the quantum-mechanical CADRE program, were consistent with in vitro and in vivo outcomes, suggesting an acceptable intake at or above 1500 ng/day for N-nitroso reboxetine. In comparison to NMOR, this prediction is supported by lower computed reactivity in the hydroxylation step, greater steric hindrance of the alpha carbons, and more facile proton transfer in the heterolysis toward the aldehyde metabolite. The data presented in this work can be used to refine and improve the Carcinogenic Potency Categorization Approach (CPCA). It also underscores the importance of collaboration between regulatory authorities, the pharmaceutical industry, and scientific researchers to address potential risks while avoiding overestimation of the acceptable intake limits for certain NDSRIs.