Genotoxicity risk assessment of a 7-hydroxymitragynine-enriched Kratom preparation: An integrated in silico and in...

The increasing global use of Mitragyna speciosa (Kratom) necessitates a thorough safety assessment, particularly regarding the genotoxic potential of its key alkaloids. This study employed an integrated in silico and in vitro approach to evaluate the genotoxicity of a well-characterized, semi-synthetically enhanced Kratom preparation (SKP) enriched in 7-hydroxymitragynine (7-OHMG; 56.31 % of total composition). Computational predictions using the OECD QSAR Toolbox and VEGA-QSAR platform indicated a lack of genotoxic activity for the major Kratom alkaloids (mitragynine, paynantheine, speciogynine, 7-hydroxymitragynine, and speciociliatine) across various in vitro and in vivo endpoints. However, several DNA-binding structural alerts were identified, particularly under metabolic activation, and prediction reliability ranged from low to moderate. To empirically verify these findings, an in vitro cytokinesis-block micronucleus (CBMN) assay was conducted in human TK6 cells following OECD Test Guideline 487. The extract induced concentration-dependent cytotoxicity. A statistically significant increase in micronucleus frequency was observed only at the highest concentration tested (125 µg/mL) under short-term (4 h) exposure conditions, both with and without S9 metabolic activation. Excessive cytotoxicity prevented analysis at high concentrations during long-term (24 h) exposure. Importantly, a complementary bacterial reverse mutation assay (Ames test) conducted on the identical extract showed no mutagenic activity up to 5000 µg/plate across five strains. In conclusion, while a weak positive chromosomal effect was noted at a highly cytotoxic concentration, the overall weight of evidence-including negative in silico predictions, negative Ames results, and limited in vitro micronucleus response-suggests that this 7-OHMG-enriched Kratom preparation does not present a significant genotoxic hazard under the conditions tested. This study underscores the value of a combined computational and experimental workflow for the robust genotoxicity assessment of complex natural products.