Genotoxic Potential of Metodesnitazene and Etodesnitazene: Insights with and Without S9 Metabolic Activation

: The ongoing emergence of New Psychoactive Substances represents a growing threat to public health, as newly synthesized compounds continuously enter the illicit drug market, evading standard detection methods and challenging regulatory frameworks. Among New Psychoactive Substances, nitazenes are potent non-fentanyl opioids associated with severe cases of intoxication. This study evaluated the genotoxic potential of metodesnitazene and etodesnitazene in the human TK6 cell line. Cells were exposed to increasing concentrations of studied compounds, with and without S9 metabolic activation system. Preliminary assessments and micronuclei frequency analyses were performed by flow cytometry in at least three independent experiments. Metodesnitazene induced an increase in micronuclei frequency starting from 12.5 μM (p < 0.05), whereas etodesnitazene induced an effect only at 50 μM. Metabolic activation increases micronuclei formation at higher concentrations of metodesnitazene 25 μM, but did not substantially affect the response to etodesnitazene. Both compounds also induced intracellular reactive oxygen species production, measured through a chemiluminescent-based bioassay, suggesting oxidative stress as a potential contributing mechanism. These findings highlight the need for compound-specific toxicological profiling to better anticipate the acute and long-term risks associated with nitazene consumption.