Preliminary Assessment of the Mutagenic Potential of Hydroethanolic Syzygium cumini Seed Extract using Salmonella Typhimurium TA102: An In-vitro Study

Introduction: Syzygium cumini seed extract has been widely investigated for its pharmacological potential, particularly for the treatment of metabolic disorders. Before developing it further for broader therapeutic applications, its genotoxic risk needs to be evaluated using validated screening tests. As an initial hazard-identification step, the present study used a single-strain bacterial reverse mutation screen rather than a full, multistrain regulatory battery. Aim: To assess the mutagenic potential of hydroethanolic S. cumini seed extract (SCE) using a TA102-based bacterial reverse mutation screen, benchmarked against the principles of the Ames assay. Materials and Methods: This was a laboratory-based, singleexperiment, quantitative in-vitro screening study conducted at Vydehi Institute of Medical Sciences and Research Centre (VIMS and RC) and Bugworks Research India Pvt., Ltd., Bengaluru, Karnataka, India. Screening for mutagenicity potential was performed using the histidine-dependent mutant strain Salmonella enterica serovar Typhimurium TA102. The bacterial culture was grown in M9 medium and then exposed to hydroethanolic SCE at final concentrations of 50, 100, and 200 µg/mL. A bacterial culture containing 1% DMSO served as the negative control, whereas a similar culture exposed to 0.02% Methyl Methanesulfonate (MMS) served as the positive control. Approximately, 250 µL of a concentrated bacterial suspension (approximately 109 CFU/mL) was plated on histidine-deficient M9 agar in triplicate. Mutations were assessed by growth of revertant colonies on agar plates with and without amino acid supplementation. His+ revertant colonies were enumerated after incubation, and the triplicate plate counts were summarised as arithmetic mean±sample SD. The assay was performed without exogenous metabolic activation (S9 mix). Results: The culture control showed confluent growth on amino acid-supplemented medium, whereas the positive control produced increased revertant colonies on histidine-deficient plates, confirming assay responsiveness. For TA102, the mean revertant counts±SD were 292.0±24.6 for the 1% DMSO control; 293.0±67.8, 332.0±34.9, and 291.0±11.3 for S. cumini extract at 50, 100, and 200 µg/mL, respectively; and approximately 2,700±400 for 0.02% MMS. The extract produced revertant counts of 1.00-, 1.14-, and 1.00-fold the DMSO-control mean at 50, 100, and 200 µg/mL, respectively, with no concentrationdependent increase observed. MMS produced an approximately 9.25-fold response, confirming assay responsiveness. Conclusion: Hydroethanolic Syzygium cumini seed extract did not produce a biologically meaningful or concentration-related increase in TA102 revertant colonies at 50-200 µg/mL. The responsive MMS positive control confirmed the responsiveness of the quantitative TA102 assay. These findings provide preliminary evidence supporting further genotoxicity evaluation of this extract batch, rather than establishing an absence of genotoxic risk, and justify additional preclinical studies using a broader, multistrain, S9-inclusive genotoxicity assay panel.