Effect of Commercial Children's Mouthrinses and Toothpastes on the Viability of Neonatal Human Melanocytes: An In Vitro Study

Abstract

In this study, we examined the cytotoxic effects of six commercial children's mouthrinses (designated as #1, #2, #3, #4, #5, and #6) and four commercial children's toothpastes (designated as #1, #2, #3, and #4) on primary human neonatal melanocytes that were used as a representative model for oral melanocytes. Mouthrinses diluted directly with culture medium (1:2, 1:5, 1:10, 1:100, and 1:1000) were added to monolayers of melanocytes for 2 min, followed by 24 h recovery, after which MTS cytotoxicity assay was conducted. The extracts of each toothpaste were prepared (50% w/v), diluted in culture medium (1:2, 1:5, 1:10, 1:50, 1:100, and 1:1000), and added to cell monolayers for 2 min (standard brushing time), followed by an analysis of cell viability after 24 h. Results showed that all mouthrinses except mouthrinse #4 showed significantly greater loss of cell viability, ascribed to cetylpyridinium chloride (CPC) that induced significant cytotoxicity to melanocytes (IC₅₀ = 54.33 µM). In the case of toothpastes, the examination of cellular morphology showed that a 2 min exposure to all toothpaste extracts induced a concentration-dependent decline in cell viability, pronounced in toothpaste containing sodium lauryl sulfate (SLS) detergent. Further results suggested SLS to be the critical driver of cytotoxicity (IC₅₀ = 317.73 µM). It is noteworthy that toothpaste #1 exhibited much lower levels of cytotoxicity compared to the other three toothpastes containing SLS. Taken together, these findings suggest that the melanocytotoxicity of children's mouthrinse (#4) and toothpaste (#1) is comparatively low. To the best of our knowledge, this is the first study to examine the impact of children's toothpastes and mouthrinses on neonatal primary human melanocytes. Future studies to investigate these findings in a realistic scenario replicating oral cavity conditions of the presence of microbiota, pellicle layer and saliva, and other cell types are warranted.

Keywords: cetylpyridinium chloride; children’s mouthrinses; children’s toothpastes; cytotoxicity; gingiva; primary human melanocytes; sodium lauryl sulfate.

Figure 1

Cell viability of HEMn-LP cells estimated 24 h after a 2 min exposure to (A) mouthrinse #1; (B) mouthrinse #2; (C) mouthrinse #3; (D) mouthrinse #4; (E) mouthrinse #5; and (F) mouthrinse #6 at dilutions 1:2, 1:5, 1:10, 1:100, and 1:1000; * p < 0.05, ** p < 0.01, *** p < 0.001, and # p < 0.0001 compared to the control (Ctrl) group; one-way ANOVA with Dunnett’s post hoc test. All data are mean ± SD of at least three independent experiments.

Figure 2

Representative phase-contrast microscopic images (×20 magnification, scale bar = 100 µm) of HEMn-LP cells taken 24 h after a 2 min exposure to the culture medium (control) and conditioned medium prepared with different dilutions (1:2, 1:5, 1:10, 1:10, and 1:1000) of children mouthrinses #1, #2, #3, #4, #5, and #6.

Figure 3

Cell viability of HEMn-LP cells estimated 24 h after a 2 min exposure to the conditioned medium of (A) toothpaste #1; (B) toothpaste #2; (C) toothpaste #3; and (D) toothpaste #4 at dilutions (1:2, 1:5, 1:10, 1:50, 1:100, and 1:1000); * p < 0.05, ** p < 0.01, *** p < 0.001, and # p < 0.0001 compared to the control group; one-way ANOVA with Dunnett’s post hoc test. All data are mean ± SD of at least three independent experiments.

Figure 4

Representative phase-contrast microscopic images (×10 magnification, scale bar = 100 µm) of HEMn-LP cells taken 24 h after a 2 min exposure to the culture medium (control) and conditioned medium prepared with different dilutions (1:2, 1:5, 1:10, 1:50, 1:100, and 1:1000) of children toothpastes #1, #2, #3, and #4.

Figure 5

Viability of HEMn-LP cells estimated 24 h after a 2 min exposure to different concentrations (0.01–3 mM) of cetylpyridinium chloride (CPC); # p < 0.0001 compared to the control (CPC: 0 mM) by one-way ANOVA with Dunnett’s post hoc test. Data are an average of at least three independent experiments.

Figure 6

Viability of HEMn-LP cells estimated 24 h after a 2 min exposure to different concentrations (0.01–3 mM) of sodium lauryl sulfate (SLS); # p < 0.0001 compared to the control (SLS: 0 mM) by one-way ANOVA with Dunnett’s post hoc test. Data are an average of three independent experiments.