Exploring Thymol’s Cytocompatibility and Potential Selective Cytotoxicity in Human Primary Gingival Fibroblasts and Pharyngeal Carcinoma Cells: An In Vitro and In Ovo Investigation

Background/Objectives: Thymol (THY) is widely used in oral care products for its antimicrobial and anti-inflammatory activity, but data on its cytocompatibility, potential differential effects on oropharyngeal-derived cells, and mucosal irritation under prolonged exposure remain limited. This study evaluated THY’s effects on healthy human gingival fibroblasts (HGF-1) and pharyngeal carcinoma (Detroit-562) cells after 24 h exposure, together with its irritation potential in ovo. Methods: Cells were treated with THY (100–300 µM) for 24 h. Cellular viability (MTT), morphology, mitochondrial alterations (MitoTracker™/Hoechst 33342), mitochondrial membrane potential (JC-1), and apoptosis/necrosis (AO/PI) were assessed. Clonogenic assays evaluated long-term proliferative capacity. Lastly, irritation score was examined using the HET-CAM assay at 300 µM. Results: THY produced a dose-dependent viability decrease in both lines, with HGF-1 viability remaining ≥75% and Detroit-562 reduced to ~68% at 300 µM. Morphology, mitochondrial staining, JC-1 ratios, and AO/PI imaging showed progressive apoptotic features, more evident in Detroit-562 cells. Clonogenic capacity increased slightly in HGF-1 at 100 µM and declined to ~75% at 300 µM, whereas Detroit-562 colonies decreased from ~68% to ~40% across the dose range. Additionally, THY (300 µM) showed no irritation in the HET-CAM assay. Conclusions: THY demonstrated acceptable cytocompatibility in gingival fibroblasts, stronger inhibitory effects on carcinoma cells at higher concentrations, and no acute irritation in ovo. These findings support THY’s safe use within defined concentration limits and justify further evaluation in advanced oral tissue models.