Evaluation of the Cytotoxicity, Genotoxicity and Acute Oral Toxicity of Thymus longicaulis subsp. chaubardii (Rchb.f.) Jalas

<b>Background/Objectives</b>: <i>Thymus longicaulis</i> subsp. <i>chaubardii</i> (TL) (Rchb.f.) Jalas is widely used in traditional Turkish medicine for respiratory, digestive and uro-genital disorders. The aim of this study was to determine its phytochemical profile and to evaluate its cytotoxic, genotoxic and acute oral toxicity effects. <b>Methods</b>: The phenolic composition of the methanolic extract was determined by HPLC-DAD. Cytotoxicity and genotoxicity were evaluated in NIH3T3 cells using MTT, comet and micronucleus assays. Acute toxicity was evaluated in rats at doses of 300 and 2000 mg/kg body weight according to the OECD Guideline 420. <b>Results</b>: Rosmarinic acid (87.37 ± 5.39 µg/mg) was the major phenolic compound. TL extract showed >90% cell viability at 50-200 µg/mL, indicating no cytotoxicity. Comet assay revealed a slight increase in DNA damage at 100-200 µg/mL (<i>p</i> < 0.001), though significantly lower than the H₂O₂ group (<i>p</i> < 0.001). No significant (<i>p</i> > 0.05) effect was observed in the micronucleus assay between the treated groups. In rats, TL extract caused no mortality or behavioral changes over 14 days. No significant differences were observed in body or organ weights. Hematologically, platelet count increased (<i>p</i> < 0.001) and eosinophils decreased (<i>p</i> < 0.01 and <i>p</i> < 0.001). Biochemical tests showed lower ALT and AST levels (<i>p</i> < 0.01 and <i>p</i> < 0.05, respectively) and significantly decreased triglycerides in the high-dose group (<i>p</i> < 0.001). Histopathological examination showed no organ damage. <b>Conclusions</b>: The results of this study indicate that TL methanol extract is non-toxic up to 2000 mg/kg and exhibits no significant cytotoxic or genotoxic effects. These findings support its safe use and traditional medicinal value.