The Genotoxic Ambiguity of 4‐Aminoquinolines: Resolving Hydroxychloroquine's Mechanistic Safety Profile Through Integrated Toxicology Assessment

Hydroxychloroquine sulfate (HCQ), widely prescribed for autoimmune disorders, carries unresolved concerns regarding potential genotoxic risks. This integrated in vitro assessment comprehensively evaluated HCQ's cytogenotoxic profile using bacterial reverse mutation (Ames test in TA98/TA100 strains, 5-80 μg/plate), DNA strand break detection (alkaline Comet assay), chromosomal instability assessment (cytokinesis-block micronucleus test in human lymphocytes, 10-40 μg/mL), oxidative stress biomarkers (TOS/TAR/OSI), plasmid DNA protection (pBR322 under UV/H₂O₂), and molecular docking targeting DNA polymerase δ. Results demonstrated no mutagenicity in TA98. While a statistically significant (p ≤ 0.001) increase in revertants was observed in TA100 at a single concentration, this was transient, nondose-dependent, and biologically insignificant as it remained below the two-fold threshold defined by OECD criteria. No significant DNA damage occurred in mammalian systems, with Genetic Damage Index ≤ 0.14 and micronucleus frequency consistently below 9.75%. Molecular docking revealed weak binding affinity to DNA polymerase δ (ΔG = -5.6 kcal/mol). HCQ induced pronounced dose-dependent cytostasis, evidenced by a 20.5% reduction in Nuclear Division Index at 40 μg/mL, without accompanying genotoxicity. Redox modulation was confirmed through a 15.4% decrease in oxidative stress index. Crucially, HCQ exhibited a complex, biphasic effect on plasmid DNA, paradoxically exacerbating damage at a low concentration (10 μg/mL) while offering significant protection at higher concentrations under combined oxidative stress. These data establish HCQ as a cytostatic agent devoid of genotoxic risk, reinforcing its clinical safety profile while highlighting the necessity for environmental risk validation through in vivo models.