Comparison of the cytotoxic and apoptotic effects of mouthwashes containing different active ingredients on human gingival fibroblasts and their antimicrobial efficacy

Abstract

Background

The use of biocompatible materials on oral tissues is important for maintaining tissue health. Therefore, this study compared, in vitro, the antimicrobial effects of seven different mouthwashes (BenriDent, Listerine Total Care, Splat White Plus, Dkdent, Klorhex, Oral-B, Colgate Plax) containing various active ingredients, as well as their apoptotic and cytotoxic effects on human gingival fibroblast (hGF) cells and DNA damage.

Methods

Cytotoxic effects were evaluated using 2,3-Bis(2-Methoxy-4-Nitro-5-Sulfophenyl)-2 H-Tetrazolium-5-Carboxanilide (XTT) and lactate dehydrogenase (LDH) assays. Annexin V assay was applied to detect apoptotic effects. Levels of 8-hydroxy-2’-deoxyguanosine were analyzed to determine DNA damage. Antimicrobial activity against Streptococcus mutans, Candida albicans, Aggregatibacter actinomycetemcomitans, and Streptococcus salivarius was assessed using the broth microdilution method, and minimum inhibitory concentration values were determined. For the XTT assay, cells were analyzed at five concentrations (10%, 25%, 50%, 75%, 100%) and four incubation periods (1 min, 30 min, 2 h, 24 h).

Results

Mouthwashes demonstrated significant differences in terms of cytotoxicity, apoptosis, and DNA damage. The highest cell viability rate was recorded for BenriDent, and the lowest for Splat White Plus. Splat White Plus demonstrated the highest antimicrobial effect against Streptococcus mutans, while Colgate Plax and Oral-B showed the highest effects against Candida albicans. BenriDent, Splat White Plus, and Colgate Plax mouthwashes showed the highest effects against Aggregatibacter actinomycetemcomitans. All mouthwashes demonstrated similar efficacy against Streptococcus salivarius.

Conclusion

It was demonstrated that the mouthwashes caused varying levels of antimicrobial, cytotoxic, and apoptotic effects, as well as DNA damage, except against Streptococcus salivarius.

Introduction

Polimicrobial biofilm is one of the primary etiological factors of dental caries and periodontal disease [1]. The use of mechanical and chemical plaque control methods is of critical importance in the prevention and management of dental caries and periodontal diseases [2, 3]. The primary methods of plaque control include mechanical cleaning procedures such as toothbrushing, interdental brushing, and flossing [4]. However, factors such as time constraints, poor manual dexterity, or lack of motivation may lead to inadequate or improper brushing and flossing, resulting in plaque accumulation [5, 6]. In a previous study, plaque removal efficiency was reported to be 57% in 6-year-olds, 67% in 8-year-olds, and 82% in 12-year-olds [7]. Since low motivation and poor hand–eye coordination commonly observed in children may prevent them from accessing interproximal areas, children may require support from chemotherapeutic agents, such as therapeutic mouthwashes, for mechanical plaque control [8, 9].

Therapeutic mouthwashes contain active ingredients designed to control or reduce conditions such as halitosis, gingivitis, plaque accumulation, and dental caries [10]. The primary active ingredients commonly found in mouthwashes include antimicrobial agents such as chlorhexidine, hydrogen peroxide, essential oils, triclosan, amine fluoride, chlorine dioxide, stannous fluoride, cetylpyridinium chloride, and hyaluronic acid [11]. Additionally, antiviral components present in mouthwashes—such as hydrogen peroxide, β-cyclodextrin, flavonoids, essential oils, cetylpyridinium chloride or povidone-iodine—may be effective in defending against the SARS-CoV-2 [12–14].

Mouthwashes interact with teeth and oral tissues during and after use. Consequently, these products must be both effective and safe for oral tissues [15]. An ideal mouthwash is one that prevents plaque formation, possesses antibacterial properties [16] and exhibits low cytotoxicity toward cells [17–19]. The chemical composition of mouthwashes can lead to harmful effects on the oral mucosa with daily use [20]. Mouthwashes can cause damage to the buccal cell membranes and may lead to deoxyribonucleic acid (DNA) damage through disruption of the double helix structure [21]. When selecting an ideal mouthwash, both its antimicrobial efficacy and biocompatibility must be considered [22].

Although studies evaluating the cytotoxicity of mouthwashes on human gingival fibroblast (hGF) cells exist [23, 24], there are limited studies that comprehensively address biocompatibility parameters, including cytotoxicity, DNA damage, apoptosis, and antimicrobial activity [25]. This study aimed to compare the antimicrobial activities of seven different mouthwashes containing various active substances against Candida albicans (C. albicans), Streptococcus mutans (S. mutans), Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), and Streptococcus salivarius (S. salivarius), as well as their cytotoxic and apoptotic effects on hGF cells and DNA damage levels.

Materials and methods

This study was conducted in accordance with the Declaration of Helsinki and was approved by the Sivas Cumhuriyet University Health Ethics Committee (Approval No. 2024-02/56).

Culture of human gingival fibroblast (hGF) cells

A commercially obtained human gingival fibroblast (hGF) cell line (Cytion, Catalog No: 300703) was used in this study. Therefore, the cells used in the present study represent a commercial hGF cell line rather than primary human gingival fibroblasts. The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco™, Catalog No: 41966-029, Thermo Fisher Scientific, USA) supplemented with 1% L-glutamine, 1% penicillin/streptomycin, 10% fetal bovine serum, 10 ng/mL fibroblast growth factor (FGF), and 10 µg/L insulin. The cells were incubated at 37 °C in a humidified atmosphere containing 5% CO₂.

Before each experiment, cell counting and viability assessment were performed using Trypan Blue staining at a 1:1 ratio. The cell seeding density and plate configuration were adjusted according to the requirements of each assay. For the XTT assay, cells were seeded into 96-well plates at a density of 1 × 10⁴ cells per well. For the LDH and 8-OHdG ELISA assays, cells were prepared and analyzed according to the respective kit protocols under assay-specific conditions. For the Annexin V apoptosis assay, cells were cultured in 6-well plates and then treated with the calculated IC50 concentrations of the tested mouthwashes for 24 h. All experiments were performed in triplicate unless otherwise stated.

Preparation of mouthwashes

Seven different commercially available mouthwashes were used in the study. These mouthwashes are: Listerine Total Care, Oral-B, Dkdent, Splat White Plus, Colgate Plax, Klorhex, and Benri Dent (Table 1). The mouthwashes were diluted using DMEM at concentrations of 10%, 25%, 50%, 75%, and 100% (v/v).

Table 1.

Composition of mouthwashes used in the study

Mouthwashes	Ingredients	Manufacturer
Splat White Plus	Aqua, Hydrogenated Starch Hydrolysate, PVP, Polyglyceryl-4 Laurate/Sebacate, Polyglyceryl-6 Caprylate/Caprate, Sodium Coco-Sulfate, Aroma, Cyclodextrin, Zinc Gluconate, Citrus Limon Peel Oil, Ananas Sativus Fruit Extract, Maltodextrin, Thymus Serpillum Oil, Glycyrrhiza Glabra Root Extract, Stevia Rebaudiana Leaf Extract, Glycerin, Pentylene Glycol, Bifida Ferment Lysate, Phthalimidoperoxycaproic Acid, Potassium Thiocyanate, Lactoferrin, Lactoperoxidase, Glucose Oxidase, Glucıse Pentaacetate, Sodium Benzoate, Potassium Sorbate, Benzyl Alcohol, Citric Acid, Limonene, Citral, Linalool.	Splat Global, UK
Colgate Plax	Aqua, Glycerin, Propylene Glycol, Sorbitol, Poloxamer 407, Aroma, Cetylpyridinium Chloride, Potassium Sorbate, Sodium Fluoride (225 ppm), Sodium Saccharin, Menthol, CI 42,051.	Colgate-Palmolive, Polonya
Listerine Total Care	Aqua, Alcohol, Sorbitol, Chloride, Poloxamer 407,Benzoic Acid, Sodium Flouride (450 ppm), Eucalyptol, Aroma, Zinc Chloride, Sodium Saccharin, Methyl Salicylate, Thymol, Mentol, Sodium Benzoate, Sucralose, CI 16,035, CI42090.	JNTL Turkey Tüketici Sağlığı Limited Şirketi, Türkiye
BenriDent	Aqua, Glycerin, Sorbitol, PEG-40 Hydrogenated Castor Oil, Aroma, Sodium Benzoate, Clorhexidine Digluconate, Cetylpyridinium Chloride,2-Bromo-2-Nitropropane-1,3-Diol, CI 42,090	Gratis, EU
Oral-B	Aqua, Glycerin, polysorbate 20, Aroma, Methylparaben, Cetylpyridinium Chloride, Sodium Fluoride (225 ppm), Sodium Saccharin, Sodium Benzoate, Propylparaben, CI 42,051, CI 47,005	Procter&Gamble, Almanya
Dkdent	Aqua, Glycerin, Sorbitol, Clorhexidine Digluconate, Propylene Glycol, Poloxamer 407, Saccharin, Sodium Benzoate, Morus Nigra Fruit Extract, Aroma, Menthol.	Ezel Kozmetik, Türkiye
Klorhex	Aqua, Sorbitol, Propylene Glycol, Peg-40 hydrogenated castor oil, Ascorbic acid, Clorhexidine Digluconate, Aroma, Sodium metabisulfite, Sodium phosphate, Mentha arvensis herb oil, Sodium hydroxide, Sodium fluoride (500 ppm), Sucralose, CI47005, CI 42,051.	Drogsan, Türkiye

Preparation of microorganisms tested in the study

The microorganisms tested in the study (S. mutans (ATCC 25175), S. salivarius (ATCC 25975), A. actinomycetemcomitans (ATCC 29522), and C. albicans (ATCC 10231)) were obtained from the American Type Culture Collection (Rockville, MD, United States). Bacterial cultures were obtained after incubation in Mitis Salivarius Agar (HIMEDIA, United States) in a 5% CO₂ environment. The fungal culture was produced after 24-hour incubation at 37 °C in Sabouraud Dextrose Agar (MERCK, Germany).

Determination of the cytotoxicity of mouthwashes on the human gingival fibroblast (hGF) cell Line

In this study, an 2,3-Bis(2-Methoxy-4-Nitro-5-Sulfophenyl)-2 H-Tetrazolium-5-Carboxanilide (XTT) kit (ATCC, Catalog No: 30–1011 K) and an lactate dehydrogenase (LDH) ELISA kit (REED BİOTECH, Catalog No: ESD0117H) were used to determine the cytotoxicity of mouthwashes on the hGF cell line.

XTT assay

For the XTT assay, hGF cells were seeded into 96-well plates at a density of 1 × 10⁴ cells/well and allowed to attach under standard culture conditions. The cells were then exposed to the tested mouthwashes at concentrations of 10%, 25%, 50%, 75%, and 100% for 1 min, 30 min, 2 h, and 24 h. Untreated hGF cells cultured in complete medium were used as the negative control, while cells treated with 0.12% chlorhexidine were used as the positive control. The experiment was performed in three independent replicates.

Cell viability was assessed using an XTT assay kit according to the manufacturer’s instructions. The XTT reagents were stored at − 20 °C in the dark and brought to 37 °C before use. The XTT working solution was prepared by adding 0.1 mL of activation reagent to 5 mL of XTT reagent. After the mouthwash exposure period, 50 µL of XTT working solution was added to each well, and the cells were incubated at 37 °C for 2–4 h. The plates were gently tapped to ensure homogeneity in the wells. After incubation, absorbance was measured at 475 nm using an ELISA reader (Multiskan FC Microplate Photometer, Thermo Fisher Scientific, Boston, USA). Cell viability was calculated relative to the untreated negative control group.

LDH assay

For the LDH assay, hGF cells were seeded into 6-well plates at a density of 0.5 × 10⁶ cells/well and allowed to attach for 24 h under standard culture conditions. After attachment, the cells were treated with the tested mouthwashes at their calculated IC50 concentrations for 24 h. Untreated hGF cells cultured in complete medium served as the negative control, whereas cells treated with the lysis solution provided in the LDH assay kit were used as the positive control for maximum LDH release. The assay was performed in three independent replicates.

After treatment, culture supernatants were collected and LDH release was measured using a human LDH ELISA kit (REED BIOTECH, Catalog No: ESD0117H) according to the manufacturer’s instructions. Briefly, 100 µL of standard, blank, and sample solutions were added to the appropriate wells and incubated at 37 °C for 90 min. After washing, 100 µL of biotin-labeled detection antibody working solution was added to each well and incubated at 37 °C for 1 h. The wells were then washed, followed by the addition of 100 µL of HRP-conjugated working solution and incubation at 37 °C for 30 min. After subsequent washing, 100 µL of substrate reagent was added and incubated at 37 °C for approximately 15 min. Finally, 50 µL of stop solution was added to each well, and the optical density was measured at 450 nm using a microplate reader.

Determination of DNA damage in the human gingival fibroblast (hGF) cell line using mouthwashes

Oxidative DNA damage was evaluated by measuring 8-hydroxy-2’-deoxyguanosine (8-OHdG) levels using a commercially available ELISA kit (Catalog No.: E-EL-0028, Elabscience, USA). For this assay, hGF cells were seeded into 6-well plates at a density of 0.5 × 10⁶ cells/well and allowed to attach for 24 h under standard culture conditions. After attachment, the cells were treated with the calculated IC50 concentrations of the tested mouthwashes for 24 h. Untreated hGF cells cultured in complete medium were used as the negative control to determine baseline oxidative DNA damage. Hydrogen peroxide-treated hGF cells were used as the positive control to induce oxidative DNA damage. The assay was performed in three independent replicates.

The kit had a sensitivity of 0.94 ng/mL and a detection range of 1.56–100 ng/mL. After treatment, samples were collected and prepared according to the manufacturer’s instructions. Briefly, 50 µL of each standard, negative control, positive control, and sample was added to the appropriate wells. Then, 50 µL of biotinylated detection antibody working solution was added to each well and incubated at 37 °C for 45 min. After washing, 100 µL of HRP-conjugate working solution was added and incubated at 37 °C for 30 min. Following the final washing steps, 90 µL of substrate reagent was added to each well and incubated at 37 °C for approximately 15 min. Finally, 50 µL of stop solution was added, and the optical density was measured at 450 nm using a microplate reader.

Determination of the apoptotic effects of mouthwashes on a human gingival fibroblast (hGF) cell line

Apoptotic and necrotic cell populations were evaluated using a Merck Millipore Annexin V-FITC/PI apoptosis detection kit according to the manufacturer’s instructions. For this assay, hGF cells were seeded into 6-well plates at a density of 0.5 × 10⁶ cells/well and allowed to attach for 24 h under standard culture conditions. The cells were then treated with the calculated IC50 concentrations of the tested mouthwashes for 30 min to simulate short-term clinical exposure to mouthwash formulations. Untreated hGF cells cultured in complete medium were used as the negative control to determine the baseline distribution of viable, apoptotic, and necrotic cells. Apoptosis-induced hGF cells were used as the positive control to validate the gating strategy during flow cytometric analysis. The experiment was performed in three independent replicates.

After the 4-min exposure period, both floating cells in the culture supernatant and adherent cells were collected to minimize the loss of detached apoptotic or necrotic cell populations. The culture supernatant was first collected, and the adherent cells were then washed with PBS, detached using Trypsin/EDTA, and combined with the previously collected supernatant fraction. The combined cell suspension was centrifuged at 800 rpm for 5 min. The harvested cells were counted using Trypan Blue staining and stained with Annexin V-FITC and propidium iodide according to the Merck Millipore kit protocol. After incubation in the dark at room temperature, binding buffer was added, and the samples were analyzed using a Muse flow cytometer. Viable, early apoptotic, late apoptotic, and necrotic cell populations were determined according to Annexin V-FITC/PI staining patterns.

Evaluation of the antimicrobial activity of mouthwashes

In this study, the minimum inhibitory concentration (MIC) method was used to evaluate the antimicrobial activity of the tested commercial mouthwash formulations. Since the formulations are complex liquid preparations rather than pure chemical compounds, stock solutions were prepared directly from the undiluted commercial products. The concentration of the active ingredient in each formulation was calculated based on the manufacturer’s label (% w/v), and converted to µg/mL units using the formula: % w/v × 10,000 = µg/mL. Accordingly, an initial stock concentration of 100 µg/mL was prepared by diluting the commercial formulation in sterile distilled water, and this concentration was used as the starting point for serial dilutions.

Ten microliters (10 µL) of each mouthwash solution at 100 µg/mL were added to the first row of U-bottom 96-well plates containing 10 µL of sterile Cation-adjusted Mueller-Hinton Broth (CAMHB) for bacteria or RPMI-1640 medium for C. albicans, yielding a total well volume of 100 µL following subsequent additions. Two-fold serial dilutions were then performed across the plate to generate a total of 10 consecutive concentrations (ranging from 100 µg/mL to 0.195 µg/mL). Fifty microliters (50 µL) of microbial suspensions, adjusted to a 0.5 McFarland turbidity standard (5 × 10⁵ CFU/mL for bacterial strains and 0.5–2.5 × 10³ CFU/mL for C. albicans), were added to each well. A growth control well (containing microbial suspension without any antimicrobial agent) and a sterility control well (containing medium only, without microorganisms) were included on each plate to ensure assay validity.

Standard antibiotics to which the respective microorganisms are known to be susceptible — imipenem and ampicillin for bacterial strains (S. mutans, S. salivarius, and A. actinomycetemcomitans) and fluconazole for C. albicans — were added at 50 µL to the corresponding microbial cultures in the last row of each plate, serving as positive controls to confirm the susceptibility of the test strains and validate the experimental conditions. Bacterial plates were incubated at 37 °C for 24 h, while yeast plates were incubated at 37 °C for 48 h under aerobic conditions.

Following incubation, 20 µL of 2 mg/mL 2,3,5-triphenyltetrazolium chloride (TTC; AFG Scientific, 284261) solution was added to each well to assess microbial viability. The plates were further incubated at 37 °C for 2 h. Metabolically active (viable) microorganisms reduce TTC to the red-colored formazan compound, whereas the absence of red coloration indicates growth inhibition. The MIC was defined as the lowest concentration of the mouthwash formulation at which no visible red formazan formation was observed, indicating complete inhibition of microbial growth. All experiments were performed in triplicate, and MIC values were reported as the modal value across the three independent replicates.

Statistical analysis

Statistical analyses were performed using IBM SPSS Statistics for Windows, version 23.0 (IBM Corp., Armonk, NY, USA). Descriptive statistics for continuous variables were presented as mean, standard deviation, median and minimum–maximum values, while categorical variables were expressed as percentages (%).Since the data did not follow a normal distribution, comparisons among three or more groups were conducted using the Kruskal-Wallis test and the Bonferroni-corrected Dunn test was used for pairwise comparisons when significant differences were detected. A p-value of < 0.05 was considered statistically significant.

Results

XTT assay results of mouthwashes at different concentrations after 1-minute incubation

The distribution of cell viability values following the application of the mouthwashes is shown in Fig. 1. No statistically significant differences in cytotoxicity were observed among the mouthwashes at any concentration (10%: p = 1,000; 25%: p = 0.905; 50%: p = 0.468; 75%: p = 0.362; 100%: p = 0.557).

Fig. 1.

Boxplot representation of cell viability after 1-minute incubation with mouthwashes at different concentrations: a 10%, b 25%, c 50%, d 75%, e 100%

XTT assay results of mouthwashes at different concentrations after 30-minute incubation

The distribution of cell viability is shown in Fig. 2. At 10% concentration, the cytotoxic effects of Oral-B and Splat White Plus were significantly greater than those of Dkdent, BenriDent, Klorhex, Listerine Total Care, and Colgate Plax (Oral-B: p = 0.006; Splat White Plus: p = 0.013). At 25% concentration, cell viability in cells exposed to Dkdent and Splat White Plus was significantly lower compared to that in the BenriDent, Klorhex, Listerine Total Care, and Colgate Plax groups (all p = 0.029).

Fig. 2.

Boxplot representation of cell viability after 30-minute incubation with mouthwashes at different concentrations: a 10%, b 25%, c 50%, d 75%, e 100%

At 50% and 100% concentrations, the cytotoxic effects of Listerine Total Care (p = 0.035 and p = 0.012, respectively) and Dkdent (p = 0.025 and p = 0.041, respectively) were significantly greater than those of Klorhex. Additionally, in cells treated with Listerine Total Care at 100% concentration, cell viability was significantly higher than Colgate Plax (p = 0.048). The cytotoxic effects of BenriDent at 50%, 75%, and 100% concentrations were significantly higher than those of Klorhex (p = 0.001, p = 0.002, and p = 0.001, respectively) and Colgate Plax (p = 0.014, p = 0.001, and p = 0.005, respectively). In addition, the cytotoxic effect of Splat White Plus at 75% concentration was significantly higher than those of Klorhex (p = 0.041) and Colgate Plax (p = 0.025).

BenriDent reduced cell viability significantly more than Oral-B at 50% and 75% concentrations, and more than Splat White Plus at 100% concentration (Oral-B at 50% and 75%: p = 0.014 and p = 0.048, respectively; Splat White Plus at 100%: p = 0.018). Additionally, at a 50% concentration, Klorhex exhibited significantly lower cytotoxic effect compared to Splat White Plus (p = 0.041). Cell viability was lowest in the BenriDent group at 100% concentration (87.33%).

XTT assay results of mouthwashes at different concentrations after 2-hour incubation

The distribution of cell viability values is shown in Fig. 3. At all concentrations, at least one mouthwash exhibited a cytotoxic effect significantly different from the others (10%: p = 0.046, 25%: p = 0.016; 50%: p = 0.015; 75%: p = 0.010; 100%: p = 0.006) At the 10% concentration, Oral-B exhibited significantly greater cytotoxicity than Colgate Plax (p = 0.032), Listerine Total Care (p = 0.027) and BenriDent (p = 0.016). Similarly, Splat White Plus showed significantly greater cytotoxicity than Colgate Plax (p = 0.032), Listerine Total Care (p = 0.027) and BenriDent (p = 0.016).

Fig. 3.

Boxplot representation of cell viability after 2-hour incubation with mouthwashes at different concentrations: a 10%, b 25%, c 50%, d 75%, e 100%

The cytotoxic effects of Splat White Plus were significantly greater than those of BenriDent (p = 0.002) and Colgate Plax (p = 0.002) at 25% concentration. At the 50% concentration, Splat White Plus exhibited significantly greater cytotoxicity than Colgate Plax (p = 0.002) and Klorhex (p = 0.002). At the 75% concentration, Splat White Plus exhibited significantly greater cytotoxicity than Colgate Plax (p = 0.021) and Klorhex (p = 0.001). At the 100% concentration, Splat White Plus exhibited significantly greater cytotoxicity than Colgate Plax (p = 0.018), Klorhex (p = 0.015), and Oral-B (p = 0.021).

At the 100% concentration, BenriDent significantly reduced cell viability compared to Oral-B (p = 0.004), Colgate Plax (p = 0.003), and Klorhex (p = 0.002). At the 75% concentration, BenriDent significantly reduced cell viability compared to Colgate Plax (p = 0.048) and Klorhex (p = 0.002). At the 25% concentration, BenriDent reduced cell viability significantly more than Oral-B (p = 0.030). At the 100% concentration, Dkdent reduced cell viability significantly more than Klorhex (p = 0.035).Cell viability was lowest in the BenriDent at 100% concentration (72.37%).

XTT assay results of mouthwashes at different concentrations after 24-hour incubation

The distribution of cell viability is shown in Fig. 4. At all concentrations, cell viability after mouthwash application showed statistically significant differences among the mouthwashes (10%: p = 0.009; 25%: p = 0.004; 50%: p = 0.007; 75%: p = 0.004; 100%: p = 0.004). At 10% concentration, the cytotoxic effects of Dkdent, Colgate Plax, and BenriDent were significantly greater than those of Klorhex (p = 0.030, p = 0.012 and p = 0.005, respectively) and Oral-B (p = 0.035, p = 0.015 and p = 0.006, respectively). At the 10% concentration, Colgate Plax and BenriDent showed significantly lower cytotoxicity than Splat White Plus. At the 25% concentration, Colgate Plax and BenriDent also showed significantly lower cytotoxicity than Splat White Plus (p = 0.018 and p = 0.003, respectively) and Dkdent (p = 0.004 and p = 0.000, respectively).

Fig. 4.

Boxplot representation of cell viability after 24-hour incubation with mouthwashes at different concentrations: a 10%, b 25%, c 50%, d 75%, e 100%

At a 50% concentration, Listerine Total Care reduced cell viability significantly more than Klorhex, Colgate Plax, Oral-B, and Dkdent (p = 0.006, p = 0.007, p = 0.025 and p = 0.041, respectively). At the 50% concentration, the cytotoxic effect of Klorhex was significantly greater than that of Splat White Plus (p = 0.006). BenriDent exhibited significantly lower cytotoxic effect compared to Colgate Plax (p = 0.005) and Klorhex (p = 0.015) at 75% concentration; compared to Colgate Plax (p = 0.015) at 100% concentration and compared to Oral-B (p = 0.018) at 25% concentration.

At the 100% concentration, Dkdent and Splat White Plus exhibited significantly higher cytotoxicity than Oral-B (p = 0.005 and p = 0.015, respectively) and BenriDent (p = 0.001 and p = 0.002, respectively). Dkdent caused significantly lower cell viability than Listerine Total Care (p = 0.018) and Splat White Plus (p = 0.004) at 75% concentration, and than Klorhex at 25% concentration (p = 0.021). At the 100% concentration, Dkdent exhibited significantly greater cytotoxicity than Listerine Total Care (p = 0.030).

Results of the LDH cytotoxicity test on human gingival fibroblast cells

The results of the LDH cytotoxicity test for hGFs are presented in Table 2. Colgate Plax caused significantly lower LDH release from the cells compared to BenriDent (p = 0.018), Klorhex (p = 0.000), and Listerine Total Care (p = 0.003). Splat White Plus caused significantly lower LDH release compared to Listerine Total Care (p = 0.018) and Klorhex (p = 0.003), while Dkdent caused significantly lower LDH release compared only to Klorhex (p = 0.018).

Table 2.

Comparison of LDH release levels in cells

Mouthwashes	Med (Min-Max)
Dkdenta	29.30 (29.05–29.54)
Splat White Plusb	27.28 (27.17–27.39)
BenriDentc	34.86 (34.68 − 35.03)
Klorhexd	36.90 (36.55 − 37.26)
Listerine Total Caree	33.50 (33.33–33.66)
Colgate Plaxf	26.77 (26.61 − 26.92)
Oral-Bg	35.39 (35.27–35.51)
Test Statistic	19.636
p* value	0.003
**Mouthwashes showing significant differences	f-c, f-d, f-e, b-e, b-d, a-d

Med medyan, Min: minimum, Max: maxsimum

*Kruskal-Wallis Test, ^**Dunn-Bonferroni Test

p*<0.05

Results of the 8-OHdG ELISA test in human gingival fibroblast cells

The results of the 8-OHdG ELISA test for hGF cells are presented in Table 3. Splat White Plus caused a significant increase in 8-OHdG formation in cells compared to BenriDent (p = 0.003), Klorhex (p = 0.006), and Oral-B (p = 0.007). Dkdent caused a significant increase in 8-OHdG formation compared to Klorhex (p = 0.048).

Table 3.

Comparison of 8-OHdG (ng/mL) levels in cells

Mouthwashes	Med (Min-Max)
Dkdenta	4.8 (4.66 − 4.94)
Splat White Plusb	5.56 ( 5.33 − 5.79)
BenriDentc	3.58 (3.41 − 3.75)
Klorhexd	3.56 (3.24 − 3.88)
Listerine Total Caree	3.6 (3.42 − 3.78)
Colgate Plaxf	4.56 (4.11 − 5.01)
Oral-Bg	3.57 (3.21 − 3.93)
Test Statistic	15.896
p* value	0.014
**Mouthwashes showing significant differences	a-d, b-c, b-d, b-g

Med medyan, Min minimum, Max maxsimum

*Kruskal-Wallis Test, ^**Dunn-Bonferroni Test

p*<0.05

Apoptosis and necrosis findings in human gingival fibroblast cells induced by mouthwashes

The Annexin V-FITC/PI assay was performed after a 4-min exposure to the XTT-derived IC50 concentrations of the tested mouthwashes. Therefore, the flow cytometry findings represent the short-term apoptotic and necrotic responses of hGF cells to mouthwash exposure. The percentage of viable cells remained relatively high across all groups, ranging from 93.90% to 99.40%. The highest percentage of viable cells was observed in the BenriDent group (99.40%), followed by Oral-B (97.90%), Listerine Total Care (97.90%), Klorhex (97.60%), Colgate Plax (96.40%), Dkdent (96.10%), and Splat White Plus (93.90%).

Among the tested mouthwashes, Splat White Plus showed the lowest viable-cell percentage and the highest early apoptotic cell percentage (5.50%). The lowest early apoptotic cell percentage was observed in the BenriDent group (0.40%). Late apoptotic cell percentage was highest in the Dkdent and Colgate Plax groups (1.70%), whereas the lowest value was observed in the BenriDent group (0.20%). The necrotic cell population was highest in the Dkdent group (0.80%). These findings indicate that short-term exposure to the IC50 concentrations of the tested mouthwashes resulted in limited apoptotic and necrotic changes, with Splat White Plus showing the most prominent early apoptotic response among the tested formulations Fig. 5.

Fig. 5.

Flow cytometric analysis of cell populations (viable, early/late apoptotic, and necrotic). Panels represent: a Dkdent, b Klorhex, c Oral-B, d Listerine Total Care, e BenriDent, f Colgate Plax and g Splat White Plus

Findings on the antimicrobial activity of mouthwashes

The MIC values of the mouthwashes against tested microorganisms are presented in Table 4. Compared to standard antibiotics, the highest antimicrobial effect against S. mutans was observed with Splat White Plus (MIC = 1.56 µg/mL), followed by BenriDent and Colgate Plax (MIC = 6.25 and 12.5 µg/mL, respectively). The highest antimicrobial activity against C. albicans was observed with Colgate Plax and Oral-B (MIC = 1.56 and 3.125 µg/mL, respectively). The lowest MIC values against A. actinomycetemcomitans were observed with BenriDent, Splat White Plus, and Colgate Plax (50 µg/mL). The MIC values of all mouthwashes tested against S. salivarius were determined to be 100 µg/mL [26–29].

Table 4.

Minimum inhibitory concentration values (µg/mL) of mouthwashes against microorganisms

	Microorganisms MIC Values (µg/mL)
Mouthwashes	Streptococcus mutans	Streptococcus salivarius	Aggregatibacter actinomycetemcomitans	Candida albicans
Dkdent	100	100	100	50
BenriDent	6.25	100	50	25
Oral-B	100	100	100	3.125
Splat White Plus	1.56	100	50	50
Listerine Total Care	100	100	100	50
Colgate Plax	12.5	100	50	1.56
Klorhex	50	100	100	25
Antibiotics (Control)	Imipenem	Imipenem	Ampicillin	Fluconazole
MIC (µg/mL)	2	2	2	4

Discussion

In this study, seven different mouthwashes containing various active ingredients were evaluated for their effects on Candida albicans (C. albicans), Streptococcus mutans (S. mutans), Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), and Streptococcus salivarius (S. salivarius), as well as for their cytotoxic, apoptotic, and DNA-damaging effects on hGF cells.

In this study, BenriDent, which contains 2-bromo-2-nitropropan-1,3-diol, exhibited statistically significant cytotoxic effects on hGF cells compared to Colgate Plax and Klorhex at 75% and 100% concentrations at both 30 min and 2 h. At a 100% concentration at 2 h, BenriDent also showed significantly higher cytotoxicity than Oral-B (p < 0.05). In their study, Kajimura et al. [30] reported that the test solution containing 2-bromo-2-nitropropane-1,3-diol and homemade cosmetic product formulations could cause formaldehyde release under different application conditions (pH, temperature, and duration). Souto-Lopes et al. [31] stated in their study that formaldehyde reduced the viability of hGF cells in a dose-dependent manner. Formaldehyde is a colorless, toxic gas that can rapidly convert to a gaseous form at room temperature [32, 33]. Consequently, in this study, it is hypothesized that the formaldehyde released as a result of the degradation of the 2-bromo-2-nitropropane-1,3-diol compound contained in BenriDent may be responsible for the lower cell viability values observed at 30 min and 2 h compared to Colgate Plax, Klorhex, and Oral-B.

In our study, at the 24-hour mark, Dkdent exhibited significantly greater cytotoxicity than Listerine Total Care and Splat White Plus at 75% concentration, and than Oral-B, BenriDent, and Listerine Total Care at 100% concentration (p < 0.05). Additionally, the lowest cell viability was observed in Dkdent at a 100% concentration (30.30%). The Dkdent mouthwash used in the study contains the active ingredient chlorhexidine. Mariotti et al. [34] reported that incubation of fibroblasts in a medium containing chlorhexidine at different concentrations for 1, 5, and 15 min led to a decrease in protein production and, in a concentration-dependent manner, reduced cell proliferation after a 24-hour recovery period. The long-term effect of chlorhexidine on cells stems from its substantivity property [35, 36]. Therefore, the higher cytotoxic effect of Dkdent observed at the 24-hour mark in this study may be related to the chlorhexidine component’s substantivity property, which increases the toxic effect over time.

Following the application of the mouthwash, cell viability was found to be lowest at the 2-hour mark in BenriDent at a 100% concentration (72.37%). This mouthwash contains cetylpyridinium chloride and chlorhexidine compounds. A study in the literature reported that a periodontal gel containing various compounds, including cetylpyridinium chloride, caused a cytotoxic effect on hGF cells [37]. However, Kanno et al. [38] stated that cetylpyridinium chloride causes cell death in the human lung epithelial cell line (A549) via a caspase-3-dependent apoptotic pathway. Additionally, several studies have reported that chlorhexidine exerts a cytotoxic effect on hGFs [39, 40]. Therefore, it is believed that the findings in this study likely stem from cetylpyridinium chloride and chlorhexidine.

Based on the results of the Annexin V assay and flow cytometry analysis, it was determined that the highest live cell rate was 99.40% for BenriDent, while the lowest rate was 93.90% for Splat White Plus. However, in a previous study, it was reported that cell death in hGF cells exposed to chlorhexidine-containing and enzymatic mouthwashes at a 50% concentration primarily occurred via necrosis following a 24-hour incubation period [24]. It is thought that differences in the formulations of the mouthwashes used in the study may have contributed to this discrepancy.

According to the results of the 8-OHdG ELISA test, Dkdent led to a statistically significant increase in 8-OHdG formation compared to Klorhex (p < 0.05). The fact that the Klorhex mouthwash used in the study contains ascorbic acid may be one of the reasons for this difference. Although ascorbic acid is generally known as vitamin C, it is one of the essential compounds necessary for the human body to maintain its healthy functions [41]. The strong antioxidant properties of ascorbic acid facilitate the reduction of Fe³⁺ to Fe²⁺ [42]. Previous studies reported a significant decrease in 8-OHdG levels in the peripheral blood lymphocytes of chronic hemodialysis patients receiving vitamin C supplementation, and in the mononuclear cell DNA of other subjects [43, 44]. Similarly, the lower 8-OHdG formation observed with Klorhex compared to Dkdent in this study may be related to the antioxidant effect of ascorbic acid.

According to the current findings, Splat White Plus was found to induce significantly higher levels of 8-OHdG formation in cells compared to BenriDent, Klorhex, and Oral-B (p < 0.05). The Splat White Plus mouthwash used in the study contains the citral. Duerksen-Hughes et al. [45] reported that citral increases p53 levels in cells. An increase in p53 levels can trigger oxidative stress in cells [46]. Therefore, it is thought that the current finding in this study may be due to the citral found in Splat White Plus and that Splat White Plus may cause higher levels of DNA damage compared to the aforementioned mouthwashes.

Based on the LDH test results, significantly lower levels of LDH release were observed in cells treated with Colgate Plax compared to those treated with BenriDent, Klorhex, and Listerine Total Care (p < 0.05). LDH is an enzyme found in all cells that is released into the extracellular environment as a result of disruption of the cell membrane integrity [47]. Dinu et al. [48] reported that, 24 h after hGFs were exposed to chlorhexidine for 1, 2, or 3 min, cell membrane integrity was disrupted only at the highest tested concentrations (0.02% and 0.2%), regardless of exposure duration. Chlorhexidine can disrupt cell membrane integrity by forming non-specific electrostatic interactions with the negatively charged regions of phospholipids and proteins in the plasma membrane of eukaryotic cells [49]. Quaternary ammonium compounds, on the other hand, form strong interactions with the phospholipid bilayer of the plasma membrane due to their positively charged structures. Additionally, their long alkyl chains may disrupt the structure of these lipid layers, leading to compromised membrane integrity [50]. A previous study noted that LDH activity increased in bronchoalveolar lavage fluid following intratracheal administration of cetylpyridinium chloride to Sprague-Dawley rats [51]. In light of these findings, the chlorhexidine-containing mouthwashes BenriDent and Klorhex may contribute to greater LDH leakage and, consequently, increased cell membrane damage compared with Colgate Plax.

The current findings indicate that Klorhex induced significantly higher LDH release compared to Splat White Plus and DkDent (p < 0.05). The Klorhex mouthwashes used in the study contain sodium fluoride. Lee et al. [52] reported that bamboo salt (0.01%) and sodium fluoride (0.002%) did not cause a statistically significant change in cell viability in hGF cells based on LDH test results. The presence of sodium fluoride alongside different mouthwash components may contribute to this difference. The current results suggest that the higher LDH release observed in the Klorhex group may be related to differences in chlorhexidine concentration or formulation compared with Dkdent.

S. mutans is considered one of the primary pathogens responsible for the development of dental caries [53]. There are three key characteristics that contribute to the cariogenicity of S. mutans: (i) its ability to produce organic acids through the fermentation of various carbohydrates (acidogenesis), (ii) its ability to survive under environmental stress conditions and at low pH, and (iii) its ability to synthesize extracellular glucan homopolymers from sucrose [54, 55]. This bacterium promotes exopolysaccharide synthesis through the production of glucosyltransferases, thereby enhancing bacterial adhesion and biofilm formation on tooth surfaces [56]. Ballini et al. [57] reported that the use of a mouthwash containing sea salt, xylitol, lysozyme, and menthol for 3 months resulted in a 21% reduction in plaque index, accompanied by inhibition of S. mutans, in adolescents aged 14–17 years. Twetman and Petersson [58] noted that in children treated with a 1% chlorhexidine/thymol varnish, the group that did not achieve significant inhibition of interdental mutans streptococci had higher caries progression scores than the group that showed high levels of inhibition. The researchers reported that the inhibition of mutans streptococci may be an important factor in preventing the formation of interdental caries and halting the progression of existing carious lesions.

C. albicans is considered the most common opportunistic pathogen causing infections in immunocompromised individuals due to its high adaptability to different host niches [59]. Although C. albicans contributes to the development of dental caries, its role in caries pathogenesis stems from its interactions with various bacterial species [60, 61]. The presence of C. albicans contributes to increased production of extracellular polymeric substances by inducing changes in the biofilm environment. Consequently, C. albicans facilitates the formation of microcolonies by S. mutans [62]. Aljaffary et al. [63] reported that in individuals responding to nystatin mouthwash treatment, there was a numerical decrease in S. mutans and C. albicans levels in dental plaque at the 3-month follow-up compared to baseline, along with clinical improvements such as reduced bleeding on probing and decreased plaque accumulation. In their study, Basudan et al. [64] reported that a reduction in oral Candida carriage with chlorhexidine was accompanied by positive clinical outcomes in parameters such as the plaque index, gingival index, and probing depth.

A. actinomycetemcomitans is a Gram-negative pathobiont that colonizes the gingival sulcus and leads to the development of infection by weakening the host response [65–67]. This species plays a significant role in the progression of periodontitis in adolescents and young adults [68]. Renvert et al. [69] reported that a reduction in A. actinomycetemcomitans levels following periodontal treatment was accompanied by a 3.4 mm reduction in probing depth and a 1.2 mm gain in clinical attachment level. Pavičič et al. [70] reported that in patients with A. actinomycetemcomitans-associated periodontitis, periodontal treatment was accompanied by a reduction in probing depth, bleeding index, and plaque index.

S. salivarius is one of the pioneer bacteria that colonize the oral mucosa within the first few days after birth and are found in human saliva and on the dorsal surface of the tongue [71, 72]. S. salivarius has strains with probiotic properties, such as K12 and M18 [73]. The S. salivarius K12 strain is effective in the treatment of halitosis [74, 75]. A previous study reported that the use of the S. salivarius M18 strain in children for 3 months resulted in a significant reduction in dental plaque formation and S. mutans concentrations [76]. Another study reported that the use of the S. salivarius M18 strain for 3 months in children aged 4–10 years reduced black stain formation [77].

In the antimicrobial activity results, MIC values were classified as effective (MIC ≤ 100 µg/mL), moderate (100 < MIC ≤ 625 µg/mL), and weak (MIC > 625 µg/mL) based on reference values. Additionally, these values were compared with standard antibiotics such as imipenem, ampicillin, and fluconazole [26–29]. When MIC values were evaluated, Splat White Plus was found to be the most effective mouthwash against S. mutans (MIC = 1.56 µg/mL).The Splat White Plus mouthwash used in the study contains linalool and lemon peel essential oils. Linalool, generally recognized as safe in most cases, is a monoterpene alcohol commonly found in both monocotyledonous and dicotyledonous plants [78, 79] and possesses antimicrobial, antioxidant, anxiolytic, antiviral, and antidepressant effects [80]. Luo et al. [81] reported that the essential oil derived from lemon peel inhibits S. mutans’ acid production capacity and LDH enzyme activity. The higher antibacterial effect of Splat White Plus against S. mutans compared to other mouthwashes with different active ingredients can be explained by the inhibitory effect of the lemon peel essential oil on S. mutans virulence factors and the antibacterial effect of the linalool compound.

Current findings indicate that Oral-B and Colgate Plax mouthwashes are more effective against C. albicans than other mouthwashes (MIC = 3.125 and 1.56 µg/mL). Consistent with the present study, Olejnik et al. [82] evaluated the antimicrobial effects of six mouthwashes: Colgate Plax Cool Mint, Listerine Professional, Oral-B Pro-Expert, Mint Perfekt Sensitiv, Elmex, and Smile 3D Protection “Multi Care.” They reported that the mouthwashes containing cetylpyridinium chloride and sodium fluoride—specifically Colgate Plax Cool Mint, Oral-B Pro-Expert, and Smile 3D Protection “Multi Care”—exhibited stronger antifungal activity against Candida strains. cetylpyridinium chloride possesses mechanisms of action such as disrupting cell membrane integrity, preventing microorganisms from adhering to surfaces, suppressing extracellular matrix production, and interfering with quorum sensing mechanisms that play a significant role in biofilm formation [83]. On the other hand, although it is known that fluoride inhibits bacterial metabolism, its exact mechanism of action against Candida species has not yet been fully clarified [84, 85]. In light of this information, the higher antifungal activity of Oral-B and Colgate Plax mouthwashes compared to the other mouthwashes used in the study can be attributed to the antifungal effects of cetylpyridinium chloride and sodium fluoride present in their formulations.

The antibacterial effects of BenriDent, Splat White Plus, and Colgate Plax mouthwashes against A. actinomycetemcomitans were found to be higher than those of the other mouthwashes used in the study (MIC = 50 µg/mL). García-Gargallo et al. [86], in their evaluation of chlorhexidine and cetylpyridinium chloride antibacterial activity against various bacteria including A. actinomycetemcomitans, demonstrated that a 0.12% chlorhexidine and 0.05% cetylpyridinium chloride mouthwash reduced plaque levels after root planing and scaling. Sreenivasan et al. [87] found that cetylpyridinium chloride-containing mouthwashes, with or without ethanol, exhibited greater antimicrobial activity against various bacteria, including A. actinomycetemcomitans, compared to fluoride-containing mouthwashes. Due to its positively charged structure, cetylpyridinium chloride interacts with negatively charged bacterial surfaces to exert an antimicrobial effect [88]. The outer membrane structure of Gram-negative bacteria remains stable through the interaction of divalent cations with the negative charges in the Lipid A region of the lipopolysaccharide on the cell membrane. It is hypothesized that the entry of polycationic molecules, such as chlorhexidine, into the cell is facilitated by their displacement of the divalent cations associated with LPS [89].

One of citral’s key biological properties is its proven antibacterial effect against Gram-negative bacteria [90]. A study has shown that citral causes damage to the cell wall and membrane of Gram-negative bacteria [91]. In the current study, the high antibacterial efficacy of BenriDent against A. actinomycetemcomitans may be attributed to a synergistic effect resulting from the combined use of chlorhexidine and cetylpyridinium chloride. The effect of Splat White Plus on the aforementioned bacterium may be linked to the citral compound present in its formulation. The greater activity of Colgate Plax compared with Oral-B may be related to differences in cetylpyridinium chloride concentration or overall formulation.

According to references cited in the literature [26–29], the mouthwashes Oral-B, Colgate Plax, Klorhex, BenriDent, Splat White Plus, Dkdent, and Listerine Total Care have been found to be effective against S. salivarius (MIC = 100 µg/mL). However, in a study in the literature, the MIC values of chlorhexidine and cetylpyridinium chloride at a 50% concentration for S. salivarius were reported as 1.95 and 0.24 µg/mL, respectively [92]. In another study, the MIC value of sodium fluoride against S. salivarius was reported as 0.93 mg/mL [93]. This discrepancy may be attributed to the fact that the S. salivarius strain tested in the study (ATCC 25975) exhibited similar levels of susceptibility to the mouthwash formulations tested.

Although 0.12% chlorhexidine gluconate is a widely accepted benchmark in oral antimicrobial research, a pure chlorhexidine gluconate solution was not included as a separate positive control. Instead, Klorhex — a commercial mouthwash formulation containing chlorhexidine gluconate as its active ingredient — was incorporated into the test panel to fulfill this reference function. However, as Klorhex represents a complex commercial formulation rather than a chemically defined pure compound, formulation-specific factors such as excipients may introduce variability. Furthermore, the absence of established Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) susceptibility breakpoints for chlorhexidine gluconate against the microorganisms tested in this study (S. mutans, S. salivarius, A. actinomycetemcomitans, and C. albicans) would limit the clinical interpretability of any chlorhexidine gluconate MIC data. Future studies may consider including both standardized chlorhexidine gluconate solutions and chlorhexidine gluconate-containing commercial formulations in parallel for more precise mechanistic comparisons.

This study has several limitations. First, biological factors such as saliva, pH fluctuations, and enzymes are absent in in vitro cell studies. Second, the concentrations of certain active ingredients in the mouthwash formulations are unknown. Third, there is a possibility that excipients in mouthwashes may alter the efficacy of the active ingredient. Fourth, a multispecies biofilm model was not used to evaluate antimicrobial activity in the present study. Therefore, the current findings may not fully reflect the in vivo environment. Further in vitro and in vivo studies are required to generalize the current findings.

Conclusion

In conclusion, this study demonstrated that different mouthwash formulations induce varying degrees of cytotoxic and apoptotic effects on cells. At 2 and 24 h, a decrease in cell viability was observed in all mouthwash groups in proportion to the increase in concentration. Depending on the differences in mouthwash formulations, varying levels of effects were observed on LDH release and 8-OHdG formation in cells. According to the results of the antimicrobial activity test, the highest antimicrobial efficacy against S. mutans was found in Splat White Plus, against C. albicans in Colgate Plax and Oral-B, and against A. actinomycetemcomitans in BenriDent, Splat White Plus, and Colgate Plax mouthwashes. All tested mouthwashes showed similar antimicrobial activity against S. salivarius. The current findings highlight the importance of evaluating cytotoxic, apoptotic, and antimicrobial effects alongside DNA damage levels when assessing cellular effects related to mouthwash formulations. To develop mouthwash formulations with high antimicrobial efficacy and low cytotoxic effects for oral tissues, it is recommended that more comprehensive studies be conducted in this field.

Abbreviations

XTT

2

3-Bis(2-Methoxy-4-Nitro-5-Sulfophenyl)-2 H-Tetrazolium-5-Carboxanilide

LDH

Lactate dehydrogenase

hGF

Human gingival fibroblast

C. albicans

Candida albicans

S. mutans

Streptococcus mutans

A. actinomycetemcomitans

Aggregatibacter actinomycetemcomitans

S. salivarius

Streptococcus salivarius

8-OHdG

8-hydroxy-2′-deoxyguanosine

MIC

Minimum inhibitory concentration

Authors' contributions

A.K. conceived the ideas. A.K. and T.Y. designed the study. C.Ö. and T.T. carried out the experimental procedures. A.K. and T.Y. analysed the data. C.Ö. and T.T. wrote the Materials and Methods section of the paper, while T.Y. wrote the remaining sections. All authors read and approved the final manuscript.

Funding

This study was supported by the Scientific Research Projects Coordination Unit of Sivas Cumhuriyet University (Grant No: DİŞ-2024-320).

Data availability

The datasets generated during and analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Ethical approval for the study was obtained from Sivas Cumhuriyet University Health Ethics Committee (Approval No. 2024-02/56). All methods were carried out in accordance with the Declaration of Helsinki and relevant guidelines and regulations.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.