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

Abstract

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.

1. Introduction

Proper oral care is fundamental for well-being, as it influences nutrition, communication, and the overall quality of life. Poor oral hygiene can lead to both local (tooth decay, halitosis, gingivitis, periodontitis) and systemic (cardiovascular, neurological, diabetes, respiratory) disorders [1,2,3]. Moreover, inadequate mouth and dental care has been proposed to be a contributing factor to the development of head and neck cancers (HCNs), which are regarded as the 7th most common cancer worldwide [4]. HCNs include malignancies of the oral cavity, oropharynx, hypopharynx, and larynx [1,4]. Notably, Romania reports the highest incidence rates in the European Union among men for laryngeal and nasopharyngeal cancers, and the second-highest rates for cancers of the lip, oral cavity, and oropharynx [5,6]. Despite the recognized link between oral health and systemic outcomes, access to and adherence to adequate oral care remain limited, especially among vulnerable populations such as children, the elderly, hospitalized individuals, people with special needs, and those from low socioeconomic backgrounds [1,2]. The oral microbiome is crucial for maintaining oral health, and its dysbiosis has been strongly associated with chronic inflammatory diseases, including gingivitis, periodontitis, peri-implantitis, and oral mucositis. Persistent microbial imbalance promotes inflammation and oxidative stress, creating a microenvironment that could potentially facilitate oral and oropharyngeal carcinogenesis. Therefore, oral care agents that can modulate microbial biofilms while preserving host cell integrity are of particular interest for preventive and supportive oral health strategies [7,8,9,10]. For these reasons, preventive oral care strategies should be prioritized and implemented equitably to ensure accessibility and affordability across all population groups through budget-friendly, evidence-based, and safe bioactive compounds [1,2]. Current treatment options for HCNs include surgery, radiotherapy, and chemoradiation, systemic chemotherapy, targeted therapy, and immunotherapy, although each option comes with limitations [11,12,13,14]. Surgery may impair speech and swallowing, while radiotherapy and chemoradiotherapy frequently lead to oral toxicity (i.e., mucositis, xerostomia, taste disturbances, dental damage, osteonecrosis). Conventional chemotherapy (i.e., cisplatin, carboplatin, 5-Fluorouracil, paclitaxel, docetaxel) increases the risk of nephrotoxicity, myelosuppression, neuropathy, and targeted or immune therapies can induce dermatologic or immune-related adverse effects [11,12,13,14,15]. Even metronomic regimens like S1 (tegafur, gimeracil, and oteracil potassium), despite seeming promising, show variable efficacy across multiple studies [16]. Besides the mentioned side effects, another major disadvantage of the available treatments is the development of drug resistance, which contributes to treatment failure and poor survival outcomes [17].

In this context, natural products are often preferred over their synthetic analogs due to a lower incidence of adverse effects. Recent studies indicate growing interest in botanical extracts and essential oils in oral care formulations due to their multi-purpose roles, including antimicrobial, anti-inflammatory, antioxidant, biofilm-modulating, and analgesic properties [18,19,20]. Thymol (THY) in particular is a phytocompound that has sparked interest in the dentistry area [21]. THY is a naturally occurring monoterpenoid phenol, which is primarily extracted from Thymus vulgaris (common thyme) from the Lamiaceae family [22]. It is commonly found in commercially available dental products (e.g., mouthwashes, toothpaste, and varnishes) and used for its antibacterial, antifungal, anti-inflammatory, and antioxidant properties, which are largely attributed to its phenolic hydroxyl group [23,24,25]. THY is recommended in dentistry for treating halitosis, tooth decay, aphthous ulcers, gingivitis, and periodontitis [21]. For treating plaque accumulation and gingivitis, THY is used as part of an essential oil mouthrinse at 0.064%, and it is applied for 30 s, twice daily [26,27]. Furthermore, for the prevention of periodontitis and root caries, a combination of chlorhexidine and THY varnish is used, along with scaling and root planning [28]. THY-rich preparations reduce Candida albicans on removable orthodontic appliances using 2% Thymus vulgaris essential oil and are also used for minor mouth and throat infections as diluted gargles [29,30]. Conventional antiseptics such as chlorhexidine are widely used in dentistry; however, they frequently cause adverse effects, including xerostomia, tooth staining, taste disturbances, mucosal irritation, and, rarely, anaphylaxis. These limitations further support the interest in natural alternatives. THY is generally regarded as safe, although mild side effects such as hypersensitive reaction, dyspepsia, and diarrhea have been reported, primarily with internal use [21,31,32].

Beyond its conventional therapeutic roles in oral health, THY has recently attracted attention for its antitumoral effects, particularly in oral squamous cell carcinoma (OSCC). In vitro studies have shown that THY inhibits OSCC cell proliferation through mitochondrial pathways, including ROS production, loss of mitochondrial membrane potential, modulation of the Bax/Bcl-2 ratio, and activation of caspase-9 and caspase-3 [33]. In this context, chemotherapeutic agents are known to induce many toxic effects. For example, 5-Fluorouracil, used to treat a variety of tumors, including colorectal, ovarian, breast, head and neck, and gastrointestinal, does not present selectivity, affecting also healthy cells [34,35]. These limitations have stimulated interest in natural agents such as thymol, especially given that THY is incorporated in various oral care formulations and is in direct contact with oral mucosal tissues during regular use.

Although THY is generally well tolerated at low to moderate concentrations and has known antimicrobial and antibiofilm properties, its safety profile after prolonged contact with the mouth and its specific effects on healthy versus cancerous oral cells are not well understood. Most oral care products are designed for short use, and there is insufficient data on how long or repeated exposure might affect healthy oral cells. At the same time, many studies on antimicrobials and cancer treatments use long exposure times, often lasting 24 h or more, to capture THY’s sustained biological effects. However, there is little information about its safety and selectivity in cell models related to the oral and oropharyngeal area. Therefore, addressing this gap is essential for predicting long-term mucosal tolerance and determining safe concentration levels for oral products containing THY [33,36,37,38,39,40].

In light of these premises, the present study proposes to assess the cytotoxic effects of THY on human primary gingival fibroblasts (HGF-1), representing healthy oral cells, and on Detroit-562 pharyngeal carcinoma cell line, to determine its selectivity and safety limits. Given the importance of minimizing mucosal irritation in products designed for oral care, the potential irritant action of THY was further investigated using the Hen’s Egg Chorioallantoic Membrane (HET-CAM) assay, a well-established in ovo model for evaluating irritation on mucous membranes.

2. Materials and Methods

2.1. Reagents and Instruments

The evaluated compound, thymol, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability kit, trypsin–EDTA solution, phosphate-buffered saline (PBS), Eagle’s Minimum Essential Medium, and trypan blue solution 0.4%, Acridine Orange hydrochloride solution, and Propidium iodide were obtained from Sigma Aldrich, Merck KGaA (Darmstadt, Germany). The specific cell culture media Eagle’s Minimum Essential Medium (EMEM) and Dulbecco’s Modified Eagle’s Medium (DMEM), and fetal bovine serum (FBS) were purchased from ATCC (American Type Cell Collection, Lomianki, Poland). Crystal violet 1% was obtained from Electron Microscopy Sciences (Hatfield, PA, USA). Paraformaldehyde 4% was delivered by Santa Cruz Biotechnology (Dallas, TX, USA). The Caspase-Glo^® 3/7 and Caspase-Glo^® 9 assay kits were sourced from Promega Corporation (Madison, WI, USA). Hoechst 33342 dye and MitoTracker™ Red CMXRos were supplied from Thermo Fisher Scientific (Waltham, MA, USA). JC-1 Dye was bought from Invitrogen, Carlsbad, CA, USA. The antibiotic solution (penicillin 100 U/mL–streptomycin 100 µg/mL) and dimethyl sulfoxide (DMSO) were obtained from PanBiotech (Aidenbach, Germany). The Cytation 5 plate reader, Lionheart FX automated microscope, and Gen5™ Microplate Data Collection and Analysis Software (v3.14) were sourced from BioTek Instruments Inc. (Winooski, VT, USA). The Olympus IX73 inverted microscope, together with the cellSens Dimensions software (v.1.8), was obtained from Olympus (Tokyo, Japan).

2.2. Cell Culture Protocol

The present study was conducted on two cell lines, HGF-1 (human primary gingival fibroblasts) and Detroit-562 (pharyngeal carcinoma) cells, obtained from the American Type Cell Collection, Lomianki, Poland, which were grown in their specific cell culture media (EMEM and DMEM, respectively). Penicillin–streptomycin mixture (1%) and FBS (10%) were used to supplement both media. The cells were maintained at 37 °C in a humidified incubator with 5% CO₂ during all experiments. The morphology of the cells was microscopically examined daily during the experiments. Thymol was dissolved in DMSO to prepare a stock solution, and all final working concentrations were obtained by dilution of the stock solution in the appropriate cell culture media. Consequently, the final DMSO concentration varied with THY’s dose but was maintained below 0.5% (v/v).

2.3. Cell Viability Assay (The MTT Test)

The cell viability was evaluated by performing the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test to determine the effects of thymol (100, 150, 200, 250, and 300 μM) on HGF-1 and Detroit-562 cell lines. Cells were seeded in flat-bottom 96-well plates at a density of 1 × 10⁴ cells/well and allowed to adhere until the desired confluence was reached. Subsequently, cells were treated with THY at 100–300 μM for 24 h. Following treatment, 10 µL of MTT solution was added to each well, and the plates were incubated for 3 h at 37 °C in a humidified atmosphere containing 5% CO₂. After incubation, 100 µL of MTT solubilization solution (isopropanol with 0.04 N HCl) was added to dissolve the formazan crystals, and the plates were kept for an additional 30 min at room temperature. Finally, the absorbance of each well was measured at 570 nm and 630 nm using a Cytation 5 microplate reader. Cell viability was expressed as a percentage relative to the untreated control cells cultured in their respective complete growth media [41].

2.4. Bright-Field Analysis of Cell Morphology

To examine the effects of thymol (100, 150, 200, 250, and 300 μM) on cellular morphology after 24 h of treatment, HGF-1 and Detroit-562 cells were seeded in flat-bottom 96-well plates at a density of 1 × 10⁴ cells/well. Following the treatment period, the cells were observed and imaged using the Lionheart FX automated microscope and the Gen5™ Microplate Data Collection and Analysis Software (Version 3.14) at 20× magnification [42].

2.5. Mitochondrial Immunofluorescence Staining (MitoTracker™ Red CMXRos) and Nuclear Morphology Assessment (Hoechst 33342)

To visualize mitochondrial and nuclear morphology, HGF-1 and Detroit-562 cells were seeded in 96-well flat-bottom plates at a density of 1 × 10⁴ cells/well and maintained under standard culture conditions until reaching the appropriate confluence. The cells were then exposed to thymol (100, 200, and 300 μM) for 24 h. MitoTracker™ Red CMXRos was dissolved in DMSO to obtain a 1 mM stock solution, which was subsequently diluted in the corresponding culture medium to achieve a final working concentration of 300 nM. Then, the protocol was conducted according to the method described by Dahma et al. [43].

$$\mathrm{A}\mathrm{p}\mathrm{o}\mathrm{p}\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{i}\mathrm{c} \mathrm{i}\mathrm{n}\mathrm{d}\mathrm{e}\mathrm{x}={\displaystyle \frac{\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{a}\mathrm{p}\mathrm{o}\mathrm{p}\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{i}\mathrm{c} \mathrm{n}\mathrm{u}\mathrm{c}\mathrm{l}\mathrm{e}\mathrm{i}}{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{n}\mathrm{u}\mathrm{c}\mathrm{l}\mathrm{e}\mathrm{i}}}\times 100$$

2.6. Mitochondrial Membrane Potential

The JC-1 dye was used to investigate the mitochondrial potential in HGF-1 and Detroit-562 cell lines after 24 h of exposure to THY (100, 200, and 300 µM). For this experiment, the cells were seeded in 96-well flat-clear-bottom plates at a density of 1 × 10⁴ cells/well and incubated until they reached the desired confluence. After the treatment, the cells were washed with PBS and stained with JC-1 dye at a concentration of 5 μM (100 μL/well). The cells were then incubated for a period of 45 min at 37 °C and 5% CO₂. Following the incubation, the cells were washed again with PBS and imaged using the Lionheart FX automated microscope and the Gen5™ Microplate Data Collection and Analysis Software (Version 3.14). Afterwards, the fluorescence was read at 590 and 530 nm on Cytation 5 [44].

2.7. Microscopic Evaluation of Cell Viability and Apoptotic Morphological Alterations by Acridine Orange/Propidium Iodide (AO/PI) Double Staining

Apoptotic and necrotic morphological changes in HGF-1 and Detroit-562 cells were examined using acridine orange/propidium iodide (AO/PI) double staining following treatment with THY (100, 200, and 300 µM) for 24 h. Cells were seeded in 96-well plates at 1 × 10⁴ cells per well and allowed to adhere under standard culture conditions. After treatment, each well received 100 µL of staining solution prepared in the corresponding growth medium and containing 10 µg/mL AO and 10 µg/mL PI (final mixture 10%). Plates were incubated for 10 min at room temperature in the dark. Fluorescent images were then captured using a Lionheart FX automated microscope at 20× magnification, with 100 ms exposure time and a 2048 × 2048-pixel resolution. Image acquisition and quantitative analysis were performed using Gen5 Microplate Data Collection and Analysis Software [45].

2.8. Colony Formation Assay

The ability of thymol (100, 150, 200, 250, and 300 μM) to inhibit colony formation in HGF-1 and Detroit-562 cells was assessed using the colony formation test. The cells were cultured in 96-well plates at a density of 1 × 10²/well and left to attach. Then, the cells were treated with thymol for 24 h, and in the course of 7–10 days, the specific cell culture medium was regularly changed with a fresh one. Paraformaldehyde 4% was used to fix the cells at the end of the experiment, and then the cells were stained with crystal violet (0.2%) diluted in PBS for 10 min. The representative images were acquired after rinsing the wells with water. The absorbance was read at 550 nm on Cytation 5 after lysing the cells with SLS 1% [46].

2.9. In Ovo Irritation Assessment —HET-CAM Assay

The potential irritant effect of thymol was evaluated using the hen’s egg chorioallantoic membrane (HET-CAM) assay at 300 μM, representing the highest concentration tested in this study. Fertilized chicken eggs (Gallus gallus domesticus) were incubated at 37 °C and 60% relative humidity. On incubation day 4, approximately 6–7 mL of albumen was carefully removed from each egg to detach the developing membrane. On day 5, a small window was created on the eggshell to expose the chorioallantoic membrane (CAM). On day 10 of incubation, the HET-CAM assay was performed by topically applying thymol at a concentration of 300 μM, prepared by dilution of a DMSO stock solution in distilled water, resulting in a final DMSO concentration below 0.5% (v/v). Distilled water served as the negative control, while a 1% sodium lauryl sulfate (SLS) solution was used as the positive control. After the sample application, the CAM surface was observed for five minutes under a stereomicroscope (Discovery V8, Zeiss, Oberkochen, Germany) equipped with an Axio CAM 105 color camera. Microscopic changes, including hemorrhage (H), lysis (L), and vascular coagulation (C), were recorded at T₀ (before treatment) and T₅ (five minutes post-application). Images were processed using ZEN Core software (v.3.8; Zeiss). The irritant potential of each sample was quantified using the calculated irritation score (IS), determined according to the standard HET-CAM evaluation formula [47]:

$$\mathrm{I}\mathrm{S}=5+{\displaystyle \frac{301-H}{300}}+{\displaystyle \frac{301-\mathrm{L}}{300}}+{\displaystyle \frac{301-C}{300}}$$

2.10. Statistical Analysis

Statistical analyses were performed using GraphPad Prism software (v.9.3.1; GraphPad Software, San Diego, CA, USA; www.graphpad.com). Differences between the THY and the untreated control cells were evaluated using one-way ANOVA followed by Dunnett’s multiple comparison test. Statistically significant differences were indicated with asterisks as follows: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. All experiments were performed in triplicate. The results were normalized to control (untreated cells 100%, cultured in their respective complete growth media).

3. Results

3.1. Cell Viability Assay (The MTT Assay)

The viability of HGF-1 primary gingival fibroblasts and Detroit-562 pharyngeal carcinoma cells was assessed using the MTT method after 24 h of stimulation with THY at concentrations of 100, 150, 200, 250, and 300 μM. HGF-1 cells exhibited a dose-dependent response to THY treatment (Figure 1), although this response was not statistically significant at the lowest concentration tested (100 µM). However, at higher dosages, treatment with THY resulted in a progressive decrease in cell viability, from 82% at 150 µM to 80% at 200 µM, 78% at 250 µM, and approximately 76% at 300 µM.

Similarly, Detroit-562 cells suffered a clear dose-dependent decrease in cell viability, although the reduction at the highest concentration tested (300 μM) was more obvious than in the healthy cells (Figure 2). A significant decrease was noted starting at 150 μM, with viability gradually declining at higher concentrations as follows: 90% at 150 μM, 85% at 200 μM, 84% at 250 μM, and reached its lowest value at 300 μM (approximately 68%), indicating the most pronounced effect occurred at the highest dose tested. At the lowest dosage (100 μM), the cell number slightly decreased, but in a non-significant manner.

3.2. Bright-Field Analysis of Cell Morphology

The cytotoxicity of the test samples was evaluated by imaging both HGF-1 and Detroit-562 cell lines after 24 h of treatment to assess potential changes in cellular morphology. In the case of the healthy cell line, HGF-1, a dose-dependent effect was observed after 24 h of stimulation with THY at 100, 150, 200, 250, and 300 μM (Figure 3). At 100 μM, the cells largely maintained their characteristic elongated, spindle-shaped fibroblast morphology, with only a slight reduction in confluence. As the concentration increased, a gradual decrease in cell confluence was observed, with no notable dysmorphologies. The cells remained adherent and preserved their fibroblast-like appearance, with no dysmorphologies such as cell rounding, shrinkage, or membrane blebbing.

In the Detroit-562 cell line, treatment with THY influenced cell confluence and morphology dose proportionally (Figure 4). At the lowest dosage tested (100 μM), the morphology of the cells did not change significantly, nor did the cell confluency. At intermediate concentrations (150, 200, and 250 μM), the cell confluence decreased slightly, but with no obvious signs of alterations, such as cell rounding, shrinkage, loss of adherence. Nevertheless, at the highest dosage tested (300 μM), the number of cells appeared to drop more drastically, with noticeable signs of dysmorphologies, including cell rounding and shrinkage, as well as membrane blebbing. These morphological features are characteristic of early apoptotic or cytotoxic processes and are in accordance with those obtained in the viability test.

3.3. Mitochondrial Immunofluorescence Staining (MitoTracker™ Red CMXRos) and Nuclear Morphology Assessment (Hoechst 33342)

To explore the potential mechanism by which THY induces cytotoxicity, the mitochondrial and nuclear morphologies of both HGF-1 and Detroit-562 cell lines were examined using immunofluorescence staining with MitoTracker and Hoechst 33342 after 24 h of treatment. In untreated HGF-1 cells (Figure 5A), nuclei displayed typical morphology. However, cells exposed to THY showed nuclear and mitochondrial changes, but only at higher concentrations (200 and 300 μM). These changes included chromatin and mitochondrial condensation, as well as nuclear shrinkage (indicated by white arrows). Additionally, the apoptotic index was calculated, revealing a concentration-dependent increase in apoptotic nuclei, with the highest percentage (>8%) observed at the highest dose (Figure 5B).

On the Detroit-562 pharyngeal carcinoma cells (Figure 6A), THY at 100 μM caused no obvious signs of alterations. Exposure to 200 μM THY produced mild changes, including nuclear and mitochondrial condensation, whereas at the maximum concentration (300 μM), mitochondrial clustering, as well as shrunken nuclei, was observed (marked with white arrows), which is suggestive of the initiation of the apoptotic process following treatment. Untreated cell nuclei have a characteristic morphology, being round or oval in shape, with no signs of fragmentation or condensation. These effects were also concurrent with a dose-dependent reduction in confluency. The apoptotic index was calculated for Detroit-562 cells and illustrated in Figure 6B. The percentages of nuclei with apoptotic features increased in a concentration-dependent manner, with the highest percentage (>20%) resulting from the highest dosage applied.

3.4. Mitochondrial Membrane Potential (ΔΨm)

Since elevated intracellular ROS levels are known to impair mitochondrial function [48], we proceeded to assess mitochondrial membrane integrity using the JC-1 assay. In the case of the HGF-1 cell line, THY treatment induced a concentration-dependent alteration of mitochondrial polarization. At 100 µM, red fluorescence remained predominant (Figure 7A), and the aggregate/monomer ratio increased slightly from the control value of 39.33% to 39.92% (Figure 7B), indicating a modest enhancement of mitochondrial activity. At 200 µM, red fluorescence was still visible, but the ratio decreased to 32.79%, marking the onset of mitochondrial membrane depolarization. This effect became pronounced at 300 µM, where a strong shift toward green fluorescence and a marked reduction in the aggregate/monomer ratio to 13.05% suggested substantial loss of mitochondrial membrane potential and progression toward cell death.

Detroit-562 pharyngeal carcinoma cells also presented a dose-dependent response to THY treatment, although the pattern differed slightly from that observed in the healthy cells (Figure 8A,B). The lowest concentration (100 µM) slightly decreased the aggregate/monomer ratio from 4.92% to 4.70%. Cells treated with 200 µM THY exhibited mixed red and green fluorescence and a decrease in the ratio to 4.40%, suggesting partial mitochondrial depolarization, while at 300 µM, the ratio fell to 3.90%, and the predominance of green fluorescence confirmed marked mitochondrial depolarization and loss of ΔΨm, consistent with advanced apoptotic events.

3.5. Microscopic Evaluation of Cell Viability and Apoptotic Morphological Alterations by Acridine Orange/Propidium Iodide (AO/PI) Double Staining

The AO/PI double staining was conducted to visualize and quantify the influence of THY (100, 200, and 300 µM) on HGF-1 and Detroit-562 cell viability and apoptosis after 24 h. Following the AO/PI test, cells without treatment displayed a uniform and intense green fluorescence, reflecting intact morphology because of the AO penetration into viable cells (Figure 9A). The red fluorescent cells, corresponding to PI as a marker of necrosis, appeared in a dose-dependent manner in both cell lines. In the case of the HGF-1 fibroblasts, they displayed greater resistance to THY-induced cell death. Green fluorescence predominated across all treatments, indicating mainly viable cells, with only a minor presence of red-stained cells at elevated concentrations. Survival rates gradually decreased from 81% at 100 µM to 73% at 200 µM and 65% at 300 µM, while the apoptotic fraction increased modestly from 5% to 20% (Figure 9B).

In Detroit- 562 cells, a dose-dependent shift in fluorescence occurred after THY treatment, consistent with progressive apoptosis and necrosis (Figure 10A). The initially strong and uniform AO green signal diminished as THY concentration increased, indicating a gradual loss in viability and the onset of early apoptotic events. Concurrently, PI-positive cells’ red fluorescence began to appear and intensify, reflecting membrane disruption implicated in necrosis and late apoptosis processes. Quantitatively, cell survival decreased from 69% at 100 µM to 65% at 200 µM and 61% at 300 µM, while apoptotic rates increased from 15% to 42%, confirming a concentration-dependent cytotoxic effect (Figure 10B).

3.6. Colony Formation Test

The effect of THY (100, 150, 200, 250, and 300 µM) on the clonogenic potential of HGF-1 and Detroit-562 was evaluated through the colony formation assay. In HGF-1 human gingival fibroblasts (Figure 11A), treatment with low concentrations of THY (100 µM) increased colony formation compared with the control represented by cells cultured in their respective complete growth media (116% vs. 100%), suggesting a mild stimulatory effect on cell proliferation. However, at higher THY concentrations, a progressive reduction in clonogenic potential was observed, with colony formation rates declining to 92%, 90%, 79%, and 75% at 150–300 µM (Figure 11B), although these differences were not statistically significant.

In Detroit-562 cells (Figure 12A), THY exposure significantly reduced both the colony formation rate and number of colonies in a dose-dependent manner across the tested concentrations (100–300 µM). THY markedly suppressed clonogenic survival, as evidenced by a gradual decline in the colony formation rate from 68% at 100 µM to 40% at 300 µM compared with untreated controls (Figure 12B). This reduction was accompanied by visibly smaller and fewer colonies, indicating impaired proliferative potential and long-term survival.

3.7. In Ovo Irritation Assessment —HET-CAM Assay

The irritant potential of THY at the highest concentration tested (300 µM) was investigated in ovo through the HET-CAM test (Figure 13). Distilled water (H₂O) that served as the negative control did not induce any alteration in the vascular structure. Similarly, the compound of interest, THY, did not alter in any way the vascular structure at the maximum concentration tested. On the other side, sodium lauryl sulfate (SLS) 1% (positive control) significantly affected the vascular structure shortly after its application on the CAM through hemorrhage, lysis, and coagulation.

In

Table 1. Calculated irritation score (IS) for THY (300 µM), Positive Control (SLS 1%), and Negative Control (H2O) using the HET-CAM assay.

Sample	Irritation Score (IS)	Irritant Potential
Positive Control (SLS 1%)	19.91	Strong irritant
Negative Control (H2O)	0.07	Non-irritant
THY 300 μM	0.07	Non-irritant

, the calculated IS for the tested samples is presented. The highest IS value was obtained post-treatment with SLS 1%. THY at the highest concentration tested (300 µM) presented the same IS as H₂O. For this reason, THY at 300 µM was classified as non-irritant on the CAM, lacking vascular toxicity.

4. Discussion

THY is a popular phytocompound often used in everyday oral care products for its antimicrobial, anti-inflammatory, and antioxidant properties [24,25]. A key factor to consider when evaluating active ingredients for oral health is their ability to control microbial colonization and biofilm development. THY is effective in killing planktonic microbes, but most importantly, it inhibits biofilm formation, reducing plaque accumulation. Numerous studies have demonstrated its antibiofilm activity against periodontitis-associated microorganisms like Streptococcus spp., Lactobacillus spp., Actinomyces spp., and Candida albicans [36,37,49,50]. By preventing biofilm formation, it lowers the incidence and severity of periodontitis, a chronic inflammatory condition that affects almost 540 million people globally. Besides representing a major cause of tooth loss, persistent periodontal inflammation has been shown to reshape the oral microenvironment in ways that favor carcinogenesis and tumor progression, including the development of HCNs [7,51]. THY has been previously shown to exert anticancer effects across multiple cancer types, including oral squamous cell carcinoma [52].

Another important aspect to acknowledge when testing a compound is if it alters the functional integrity of gingival fibroblasts, which are crucial stromal cells that maintain tissue architecture, modulate wound-healing processes, immune responses, and influence both inflammatory and neoplastic processes within the oral cavity [7,53,54,55]. From a safety perspective, the behavior of THY on these healthy stromal cells is therefore central when considering its long-term use in oral care products.

Literature data indicate that THY is generally well-tolerated at low to moderate concentrations, but toxicity emerges at higher doses or under specific exposure conditions. Case reports have shown that excessive ingestion of THY-containing mouthwashes can contribute to metabolic acidosis and systemic toxicity, while locally, concentrated THY may cause skin corrosion and irritation [56,57]. At the same time, in vitro data on fibroblasts and keratinocytes suggest that viability is maintained above the ISO 10993-5 non-cytotoxic threshold (≥ 70%) up to around 0.090 mg/mL (~600 µM) [49], supporting a relatively broad safety window when THY is used within recommended limits.

For these reasons, in this paper, a healthy human gingival fibroblast line (HGF-1) was used to assess THY’s cytocompatibility. Furthermore, Detroit-562 pharyngeal carcinoma cell line was chosen to see whether THY exerts selective cytotoxicity. The concentrations (100–300 µM) were carefully selected based on previous studies, remaining below the reported toxic limits [49]. Since extended exposure intervals (24 h) are frequently used to evaluate THY’s biological activity in vitro, the same exposure duration was applied to assess cytocompatibility and potential differences between healthy and malignant cells under comparable conditions [33,36,37,38,39,40].

Although oral care products that contain THY (e.g., mouthwashes, toothpaste) are in contact with the oral mucosa for only a short period (from a few seconds to several minutes), a 24 h incubation period was chosen to mimic prolonged exposure. The rationale behind the extended time frame was to reflect possible cumulative or residual effects that may occur with daily use, since THY-containing products are also used by children or by individuals with compromised buccal tissue. This consideration is supported by studies reporting that approximately 30% of mouthwashes contain THY. Although ethanol is considered the most dangerous and toxic ingredient, THY itself can induce metabolic acidosis with overexposure and locally can produce skin corrosion or irritation [51].

Furthermore, previous studies have demonstrated that THY manifests antimicrobial and antibiofilm activities for prolonged periods (24 h). Accordingly, a 24 h incubation time was used to match the established duration of THY’s biological activity and to evaluate its potential cytotoxic effects under comparable exposure conditions in both healthy fibroblast and pharyngeal carcinoma cells [36,37,39,49].

In this regard, this paper addressed a relevant gap in the current literature by evaluating the effects of THY after a 24 h exposure period, which is longer than typical application times, making it possible to better capture possible cumulative toxicity and selectivity under extended contact conditions, particularly in oropharyngeal cell models, where such data is limited. Together, these tests were conducted to provide a comprehensive safety profile of THY in oral health-related contexts.

As a first step, the impact of THY on the cell viability was assessed in HGF-1 human primary gingival (Figure 1) and Detroit-562 pharyngeal carcinoma (Figure 2) cell lines using the MTT assay, following 24 h treatment at increasing concentrations (100, 150, 200, 250, and 300 μM). The MTT assay is a widely used method for assessing cytotoxicity, relying on the ability of viable cells to reduce MTT into insoluble formazan crystals. The amount of produced formazan is directly proportional to the number of viable cells, allowing for a quantitative evaluation of cell metabolic activity and the number of viable cells [58,59]. THY exerted a dose-dependent response in HGF-1 cells, where the viability decreased from 82% at 150 μM to 75% at 300 μM. Similarly, in Detroit-562 cells, THY induced a concentration-dependent reduction in cell viability, reaching statistical significance from 150 μM. However, according to ISO 109930-5 standards, which define cytotoxicity as a reduction in cell viability of more than 30%, THY demonstrated cytotoxic potential only at the highest concentration tested (300 μM). Although these results indicate that THY was not cytotoxic to the healthy cells within the tested range, its comparable effect on both cell types suggests limited selectivity between normal and malignant cells. Similar results were observed in other fibroblast models. Dashtaki et al. showed that THY at more than 100 µg/mL (~665 µM) increased t-BHP-induced cytotoxicity, reducing viability in both MCF-7 and foreskin-derived fibroblast cells. Though fibroblasts presented greater viability than the malignant cells. Interestingly, THY pre-treatment protected fibroblasts from oxidative damage while remaining toxic to the breast cancer cells [60]. In contrast, Bowen et al. reported that an essential oil mouthrinse containing THY caused severe cytotoxicity to normal gingival fibroblast (HGF-1) with a 93% loss of viability after only 30 s of exposure, and that alcohol removal did not attenuate this effect [61]. Conversely, under controlled-released conditions, THY has been shown to promote fibroblast proliferation [62,63]. Similarly, Guimarães et al. further showed a dose response in human gingival fibroblasts with EO from Lippia origanoides (containing THY methyl ether 5.2%). A clear dose-dependent effect was observed, as confirmed by the MTT assay, after 1 h of exposure from 90% at 600 μg/mL to 36% at 650 μg/mL, indicating that lower concentrations were non-toxic, while cytotoxicity increased drastically beyond 630 μg/mL, which is equivalent to approximately 4.2 mM [64]. In vitro and in vivo, a 10% EO of Plectranthus tenuiflorus, containing approximately 85.3% THY, accelerated wound closure, improved reepithelialization, stimulated skin appendages, and stimulated fibroblast growth at low dosages, while higher dosages of EO and pure thymol have inhibitory effects toward the proliferation of fibroblasts [65].

The anticancer potential of THY against HNCs has been previously suggested in various experimental models. De la Chapa et al. analyzed THY’s antiproliferative effect using the MTS test (a similar method to the MTT) at various concentrations on various OSCC cell lines (Cal27, SCC4, SCC9), finding GC₅₀ values between 300 and 550 μM, well below the ~4.3 mM concentration in commercial mouthwashes. THY showed non-selective cytotoxicity across multiple cancer cell lines, including cervical (HeLa), lung (H460), breast (MDA-MB-231), and prostate (PC3), with similar GC₅₀ values of 350–500 μM [33]. In our study, THY reduced Detroit-562 cell viability to 68% at 300 µM, indicating slightly higher resistance to THY compared with OSCC lines, which exhibited GS₅₀ values between 300–550 µM. In another study, THY also proved dose-dependent cytotoxic effect on Cal27 cells, as determined by the MTS cell viability assay. In vivo intra-tumor administration of THY (4.3 mM) daily for two weeks significantly inhibited Cal27 tumor growth in mice (p < 0.001) [66]. Similarly, in KYSE-30 esophageal cancer cells, THY produced time- and dose-dependent cytotoxicity with minimal toxicity to normal fibroblasts [67]. Together with our findings, these data suggest that THY displays a concentration-dependent dual behavior. In healthy cells, it presents good tolerance and even pro-healing potential at moderate dosages, while also having antiproliferative effects toward malignant cells. However, at higher concentrations, its cytotoxicity extends to normal cells, thus reducing selectivity.

The morphology and confluence of HGF-1 and Detroit-562 cells were further evaluated after 24 h, as these parameters represent an important component of the cytotoxic profile. The healthy HGF-1 cell line exposed to THY (100–300 µM) for 24 h also showed a dose-dependent decrease in confluence (Figure 3). However, even at the highest dose, these cells maintained their typical fibroblast-like shape without obvious morphological changes. In Detroit-562 cells, THY treatment led to a dose-dependent reduction in confluence, becoming noticeable at 250 µM and significantly more pronounced at the highest tested concentration of 300 µM (Figure 4). This was accompanied by notable morphological changes, such as cell rounding, shrinkage, and membrane blebbing. In Caco-2 colorectal carcinoma cells, THY induced cytotoxic effects even at 75 µM, showing a time-dependent response, with pronounced morphological alterations occurring after 48 h of exposure at 250 µM [68]. In another malignant experimental model, THY treatment was more sensitive for LNCaP prostate cancer cells, even at a lower dosage (50 µM). It showed a clear dose-dependent response to THY (50–250 µM) after 24 h, with progressive cell shrinkage, reduced cell density, and a marked decline in viability (94.38% to 20.58%). In contrast, normal HaCaT keratinocytes exhibited minimal reduction in viability under the same conditions [69]. In another study, osteoblast-like cells (MC3T3-E1) exhibited shape alterations, such as rounding, cytoskeletal disorganization, and detachment after a brief exposure of 30–270 s with a commercial mouthwash containing THY [70]. Together, these findings indicate that THY exerts dose-dependent cytotoxic and morphological effects on both malignant and normal cells. While previous studies reported a stronger selectivity toward cancerous lines, our results suggest only partial selectivity, as THY affected Detroit-562 and HGF-1 cells to a comparable extent at moderate concentrations (100–250 µM). However, at the highest concentration tested (300 µM), Detroit-562 cells exhibited slightly greater sensitivity, indicating a modest preference of THY toward malignant cells.

In continuation, to explore the possible mechanism of THY-induced cytotoxicity, the mitochondrial and nuclear morphologies of HGF-1 (Figure 5A) and Detroit-562 (Figure 6A) cells were analyzed by immunofluorescence staining with MitoTracker and Hoechst 33342 after 24 h of treatment. Mitochondria, while primarily recognized for their role in cellular energy generation, are highly versatile organelles involved in various essential functions. They contribute to biosynthetic processes, maintain intracellular calcium homeostasis, and serve as a major source of reactive oxygen species. In addition to their metabolic roles, mitochondria function as central signaling centers that coordinate cellular stress responses and store pro-apoptotic molecules, thereby acting as pivotal regulators of both cell survival and programmed cell death. Because of their role, mitochondria are often investigated in cancer research [71].

The immunofluorescence results indicate that THY induces apoptosis through mitochondrial and nuclear damage in both cell types, with slightly greater sensitivity in Detroit-562 cells. At 200 and 300 μM, both lines exhibited chromatin and mitochondrial fragmentation together with shrunken nuclei, while mitochondrial clustering was evident only in Detroit-562 cells at the highest concentration, indicating a more pronounced apoptotic response. These structural alterations corresponded with the apoptotic index, which increased dose-dependently, reaching 8% in HGF-1 (Figure 5B) and 20% in Detroit-562 (Figure 6B) at 300 μM, consistent with the observed decline in confluence. Overall, these findings confirm that THY activates mitochondria-mediated apoptosis in a concentration-dependent manner, showing only modest selectivity toward malignant cells. Similar mitochondrial and nuclear apoptotic features post-THY exposure have been reported in other malignant models, including colorectal and leukemia cell lines, supporting the relevance of our observations [72,73,74].

To further explore the mitochondrial involvement in THY’s mechanism of action, mitochondrial membrane potential (ΔΨ) was assessed in HGF-1 and Detroit-562 cells using JC-1 dye. This parameter serves as a crucial indicator of mitochondrial function and overall cell health. In this assay, red fluorescence detected at 590 nm corresponds to JC-1 aggregates, which are characteristic of polarized mitochondria found in healthy, non-apoptotic cells. Conversely, green fluorescence observed at 530 nm reflects the presence of JC-1 in its monomeric form, indicative of depolarized mitochondria typical of apoptotic or dead cells. Monitoring these fluorescence signals provides valuable insight into mitochondrial integrity and cellular viability [42].

In the case of the HGF-1 cell line, THY treatment induced a concentration-dependent alteration of mitochondrial polarization (Figure 7A). At 100 µM, red fluorescence remained predominant, and the aggregate/monomer ratio increased slightly from the control value of 39.33% to 39.92%, indicating a modest enhancement of mitochondrial activity. At 200 µM, red fluorescence was still visible, but the ratio decreased to 32.79%, marking the onset of membrane depolarization. This effect became pronounced at 300 µM, where a strong shift toward green fluorescence and a marked drop in the ratio to 13.05% suggested substantial loss of ΔΨm and progression toward apoptosis (Figure 7B). These results confirm that mitochondrial function in healthy fibroblasts is generally preserved at low-to-moderate concentrations, but impaired at higher doses. In contrast, Detroit-562 pharyngeal carcinoma cells showed earlier and more pronounced ΔΨm disruption (Figure 8A). Both the control and 100 µM-treated groups exhibited mostly red fluorescence, reflecting intact mitochondrial polarization. At 200 µM, cells displayed a mixed red-green signal, indicating partial depolarization, while at 300 µM, green fluorescence became dominant, consistent with strong mitochondrial dysfunction and apoptosis (Figure 8B). Previous studies have also demonstrated THY’s role in mitochondrial metabolism [74,75]. In HL-60 leukemia cells, THY induced a red-to-green shift in JC-1 fluorescence at concentrations up to 100 µM, with no such effect observed in PBMCs, indicating selective action toward malignant cells [74]. These comparative findings support the idea that THY-induced apoptosis involves mitochondrial depolarization and occurs more readily in malignant cells. In our models, however, this selectivity was only partial. The loss of ΔΨm at 200–300 µM in both cell lines corroborates the MitoTracker and Hoechst findings and reinforces THY’s dose-dependent mitochondrial mechanism, with Detroit-562 cells exhibiting greater sensitivity than HGF-1.

To further confirm the observed effects and discover whether mitochondrial dysfunction resulted in cell death, the membrane integrity and overall cell viability were subsequently assessed using acridine orange/propidium iodide (AO/PI) staining. The AO/PI double-staining technique is a reliable method used for differentiating viable from non-viable cells. It allows for visualization and quantification of apoptotic and necrotic cells through distinct fluorescence signals. Acridine orange (AO) easily gets into viable cells and binds to nucleic acids, emitting green fluorescence. Propidium iodide (PI), on the other hand, can only get into cells with damaged membranes, producing red fluorescence in dead or dying cells. The contrasting colors provide a simple yet efficient way to evaluate the integrity of the membrane and observe how different treatments affect cells [76]. Due to these reasons, AO/PI staining was used to gain insight into the cytotoxic and apoptotic effects of THY on both normal (HGF-1) and malignant (Detroit-562) cell lines at 100, 200, and 300 µM after 24 h. In controls, both cell lines displayed pronounced green fluorescence, indicative of elevated viability and preserved cellular morphology. After exposure to THY, the proportion of red fluorescent (PI⁺) cells increased in a dose-specific manner. This was especially apparent for Detroit-562 cells (Figure 10A,B), which showed a substantial decrease in survival (from ~69% at 100 µM to ~61% at 300 µM) along with a rise in apoptosis (from ~15% to ~42%). HGF-1 fibroblasts, on the contrary, had smaller drops in survival (Figure 9A,B), especially at lower dosages (from about 81% to 73% at 100 and 200 µM, respectively) and 65% at 300 µM. The apoptotic fraction was also significantly lower (from about 5% to 20%) compared to that in the malignant cell line, which means the normal cells were more resistant to THY-induced cell death.

The colony formation (clonogenic) assay provides critical insight into THY’s long-term effects on cell proliferation and survival. Unlike short-term viability tests, this assay measures the ability of single cells to grow into colonies, reflecting sustained proliferative capacity [77]. In our study, healthy HGF-1 gingival fibroblasts showed robust colony-forming ability after THY exposure, with only a modest decline at the highest dose and a slight increase at the lowest dose (Figure 11A). This mild stimulatory effect at low concentrations is consistent with previous reports describing hormetic or pro-proliferative responses of fibroblasts to low doses of THY or THY-rich essential oils [65,78]. In contrast, Detroit-562 pharyngeal carcinoma cells were much more sensitive, with fewer and smaller colonies observed as THY concentration increased (Figure 12A,B). This differential outcome, relatively preserved colony formation in normal fibroblasts but stronger inhibition in carcinoma cells, highlights a degree of selective cytotoxicity by THY toward malignant cells over the 10-day incubation period.

These findings are in line with literature reports that THY’s effects on cells are highly dose-dependent. At low concentrations, THY tends to spare or even benefit normal cells while targeting cancerous cells, whereas at higher concentrations, its selectivity diminishes [78]. Güneş-Bayır et al. observed that sub-cytotoxic doses of THY could harm tumor cells without affecting healthy cells, but high doses began to affect both cell types [79]. Likewise, Khorshid et al. noted a hormetic pattern in which pure THY at 0.0005% w/v (~33 µM) stimulated human skin fibroblast proliferation over 48–72 h, whereas higher concentrations became inhibitory [65]. Our observation of a slight proliferative boost in fibroblast colonies at 100 µM THY is consistent with this hormetic behavior. However, it must be emphasized that THY’s therapeutic window can be narrow. Some normal cell types show considerable sensitivity at elevated doses. For example, Pathania et al. reported an IC₅₀ of ~86 µg/mL (~0.57 mM) for THY on a normal epithelial cell line, indicating that concentrations above the low hundreds of micromolar can compromise the viability of non-malignant cells [80]. In the context of oral cells, Guimarães et al. found that an essential oil with THY derivatives had little toxicity on human gingival fibroblasts up to ~600 µg/mL, but viability dropped sharply beyond ~650 µg/mL (approximately 4–5 mM) as fibroblast proliferation was inhibited [64]. Thus, maintaining THY levels below cytotoxic thresholds is key. Because of that, the concentrations used in our assays (100–300 µM) were chosen based on reported safety margins and remained below the limit associated with fibroblast toxicity [49]. Within this range, HGF-1 cells retained at least 75% viability, indicating acceptable compatibility with normal gingival fibroblasts. Together with the clonogenic outcomes, these findings suggest that THY can hinder the long-term proliferative capacity of carcinoma cells while maintaining a substantially better tolerance profile in healthy gingival cells, supporting its suitability as an oral care active when used at appropriate concentrations.

Beyond cell proliferation, the irritation potential was also evaluated in ovo using the Hen’s Egg Test on the Chorioallantoic Membrane (HET-CAM). This assay is a well-established alternative to animal ocular irritation tests, used to predict the irritancy of substances on mucosal or vascularized membranes [81]. Importantly, HET-CAM is highly relevant for oral safety because it models the compound’s effect on a living membrane’s vasculature, thus indicating whether a product might cause inflammation or tissue damage upon contact. In our HET-CAM results, THY at the highest tested concentration (300 µM) was classified as non-irritant, with an irritation score (IS) of 0.07, identical to the negative control (distilled water). Stereomicroscopic examination showed that even 5 min after THY application, the CAM’s blood vessels remained intact and indistinguishable from the untreated embryo, with no signs of hemorrhage, lysis, or coagulation. By contrast, the positive control (1% sodium lauryl sulfate) rapidly triggered severe vascular damage (bleeding, membrane whitening, clotting) and a high IS of 19.9, denoting a strong irritant. According to standard HET-CAM criteria, substances with an IS under 1 are considered non-irritating, whereas SLS falls in the severe irritant range [82]. Thus, our data clearly show that THY lacks the acute irritancy or corrosive effect that harsh detergents exhibit. These findings reinforce THY’s safety profile: even at a concentration exceeding typical oral care use, it did not elicit any vascular inflammatory response on the CAM. This outcome aligns with other reports of THY-containing natural products being well-tolerated in similar assays. For example, an Origanum essential oil hydrogel (rich in thymol and carvacrol) caused no CAM irritation, comparable to water, whereas SLS induced obvious damage, mirroring our results with THY [81].

The novelty of this study lies in its direct comparison of THY’s effects on healthy oral fibroblasts and malignant pharyngeal cells under the same experimental conditions, an aspect barely examined in oral-related models. Our results showed that THY produced a dose-dependent reduction in viability, mitochondrial integrity, and clonogenic capacity in both cell lines, with only modest selectivity. While Detroit-562 cells were slightly more sensitive at the highest dose, the overall similarity in response highlights that THY’s selective cytotoxicity is limited within the tested range. Importantly, the absence of irritation on the CAM at the highest dose tested (300 µM) supports THY’s compatibility with mucosal-like tissues and strengthens its safety profile for oral contact applications. These combined findings are relevant for dentistry because they clarify the boundaries between THY’s beneficial bioactivity and its cytotoxic potential when contact is prolonged.

Looking ahead, future studies should include more complex oral tissue models, such as 3D gingival equivalents or co-culture systems, to better replicate the structure and cellular interactions of the oral mucosa. Repeated short-contact exposures that mimic real oral hygiene routines, would also help determine whether cumulative effects develop over time. This approach is particularly relevant given the relatively narrow margin between THY’s biological activity and cytocompatibility, as short but repeated contact may generate local peak concentrations that are not captured by single prolonged-incubation models. Also, the exploration of various formulation strategies (e.g., encapsulation or controlled-release systems) may help optimize THY delivery and potentially enhance its differential impact on malignant cells while preserving healthy cells’ integrity. These strategies could as well minimize variability in mucosal exposure caused by factors such as saliva dilution, volatility, and user-dependent application, thereby improving dosing predictability and safety.

The present work has several limitations. The findings are based on in vitro and in ovo models, which cannot fully reproduce the structural complexity of the human oral mucosa. Also, only two cell lines were investigated, and the results may not fully capture the heterogeneity of normal oral tissues or head and neck cancers. A further methodological limitation of the present study is that a vehicle-only (DMSO) control was not included. Although DMSO is widely used as a solvent in in vitro assays and concentrations below 0.5% (v/v) are generally considered acceptable and non-cytotoxic in many cell models, it is also recognized that DMSO is not biologically inert and may induce subtle cellular or molecular effects depending on cell type, exposure duration, and endpoint analyzed [83,84]. Therefore, while the final DMSO concentration in all THY-treated conditions was maintained below 0.5% (v/v), a direct comparison between maximal DMSO exposure and DMSO-free controls was not performed, and a minor contribution of the vehicle cannot be fully excluded. Future studies should therefore include vehicle-matched DMSO controls, particularly in repeated short-contact or long-term exposure models, to further strengthen the interpretation of THY-specific effects. Additionally, in vivo studies in animal models or clinical settings would be valuable to confirm that THY does not induce chronic mucosal irritation or toxicity and to explore any chemopreventive benefits.

5. Conclusions

The present work characterized the cellular responses to THY in healthy gingival fibroblasts and pharyngeal carcinoma cells after 24 h of exposure and assessed its irritation potential in ovo. THY produced a concentration-dependent reduction in viability, with fibroblasts showing higher tolerance, whereas carcinoma cells exhibited more pronounced apoptotic features and reduced clonogenic survival, indicating a modest degree of selectivity. THY at 300 µM did not elicit irritation in the HET-CAM model, indicating low acute irritation potential. In summary, this study contributes new evidence that helps contextualize THY’s benefits and limitations in oral health. By tackling the under-explored issue of THY’s selective cytotoxicity and long-term safety on oral cells, we provide a scientific basis for its continued use and future development. Within the limits of the experimental models used, the findings indicate that THY maintains acceptable cytocompatibility in gingival fibroblasts, exerts stronger inhibitory effects on carcinoma cells, and does not induce acute irritation at the tested concentration.