Fluoride Release from Children's Toothpastes as an Indicator of BioavailabilityABSTRACT
Objective: To evaluate the fluoride release from Colgate Zero Kids® dentifrice compared to two children’s dentifrices, Tandy® and Sorriso Kids®.
Material and Methods: All products, from the same manufacturer, had identical fluoride formulations (NaF/silica; 1,100 ppm F) but differed in rheological properties. A 4-g sample of each dentifrice (4.4 mg F) was agitated in ultrapure water and centrifuged; the supernatant was collected for fluoride analysis using a fluoride ion-selective electrode, calibrated with standards (0.25-16.0 µg F/mL) in 50% TISAB II. Fluoride release was expressed as a percentage of total fluoride, and data (n=6) were statistically analyzed using ANOVA and Tukey’s test (α = 5%).
Results: Mean fluoride release was: Tandy® (71.4 ± 3.2%), Sorriso Kids® (70.3 ± 2.1%), and Colgate Zero Kids® (37.7 ± 9.7%), with Colgate differing significantly (p < 0.05). To investigate the lower fluoride release from Colgate Zero Kids®, the flow and viscosity curves of the toothpastes were obtained using a Brookfield R/S Plus rheometer. Colgate Zero Kids® showed significantly higher thixotropy compared to the other dentifrices.
Conclusion: The results suggest that the reduced fluoride availability in Colgate Zero Kids® may be attributed to its higher viscosity, potentially limiting fluoride ion diffusion. These findings highlight the importance of considering not only fluoride content but also physicochemical properties such as viscosity in dentifrice formulations for effective fluoride bioavailability.
Keywords:
Fluorides; Dentifrices; Biological Availability; Rheology.
Introduction
The use of fluoride toothpastes for caries control in early childhood is widely recommended due to their efficacy and safety [1]. The World Health Organization (WHO) classifies fluoride toothpaste as essential medicine [2], underscoring its role in combating dental caries. Besides facilitating the mechanical removal of biofilm, brushing with these products effectively increases fluoride availability in the oral cavity [3]. Fluoride works locally through physicochemical properties that reduce demineralization and promote dental remineralization [3]. For this to be effective, fluoride must be soluble and bioavailable during brushing [4].
To ensure fluoride bioavailability, especially during children's brief brushing sessions [5], formulations must exhibit appropriate rheological properties [6]. Rheology, which examines how materials deform and flow under external forces, significantly influences the efficient release of fluoride [7,8]. The toothpaste's structure must become sufficiently disorganized during brushing to release its therapeutic agents into the oral cavity [6,9].
However, the viscosity of some formulations can impair fluoride release [8]. Binding agents, such as carboxymethyl cellulose, are crucial for regulating rheological properties and viscosity [10]. Higher viscosity toothpastes tend to have lower fluoride bioavailability, potentially compromising their therapeutic efficacy [8,11].
Laboratory evaluations of rheological behavior can assess anticaries potential. The American Dental Association (ADA) recommends an in vitro test to measure fluoride release from toothpaste in 1 minute, though this test has not been clinically validated [12]. Xavier-Queiroz et al. [13] validated a laboratory model for indicating fluoride bioavailability in toothpastes, finding that gel toothpastes released less fluoride than paste toothpastes. This observation was supported by Ricomini-Filho et al. [14], where differences in fluoride release were attributed to silica presence rather than binding agents. Hydrated silica’s gel behavior may contribute to the cohesion of toothpaste components, hindering fluoride release.
Most children's toothpastes in Brazil are silica-based gels [1,15]. Studies by Ricomini-Filho et al. [14] and Xavier-Queiroz et al. [13] indicated differences in fluoride release among silica-containing toothpastes, but the fluoride release by the majority of these products found in the market [1,15] has not been evaluated.
In response to the demand for naturalistic products, new children's fluoride toothpastes have been launched, often without considering the impact of rheological properties on fluoride release. This discrepancy is evident when extruding different children's toothpastes, as observed in laboratory tests with Tandy®, Sorriso Kids®, and Colgate Zero Kids®, where distinct physicochemical properties were noted (Cury JA, personal communication). Colgate Zero Kids® exhibited characteristics akin to a medicated ointment, leading to the hypothesis that it would release fluoride differently compared to the others.
Thus, the objective of this study was to evaluate the in vitro fluoride release of Colgate Zero Kids® toothpaste in comparison to Tandy® and Sorriso Kids®, using a validated laboratory model to indicate fluoride's oral bioavailability during brushing.
Material and Methods
Sampling
Convenience samples of fluoride toothpastes marketed for children, Colgate Zero Kids®, Sorriso Kids®, and Tandy® (Table 1), were selected because they are produced by the same manufacturer, share the same formulation type (NaF/Silica gels; 1100 ppm F; mg F/kg), and appear to have distinct rheological characteristics. Six samples from each brand were purchased and were within their respective expiration dates.
Fluoride Analysis in Toothpastes
The simplified protocol described by Quiroz-Torrez et al. [16] was utilized, as the toothpastes contain NaF/Silica, ensuring that all fluoride is chemically soluble. A sample of 90 to 110 mg was weighed in a test tube (± 0.01 mg) and vortexed in 10.0 mL of purified water. A 1.0 mL aliquot of this suspension was then collected, and 1.0 mL of TISAB II (acetate buffer 1 M, pH 5.0, containing 0.4% CDTA and 1 M NaCl) was added.
Fluoride Release from Toothpastes
To assess fluoride release, a modified laboratory model regarding stirring speed and duration was employed [13]. The proportion toothpaste:water was 1:3 (w/v) simulating the dilution during toothbrushing. Four grams (±0.01) of each toothpaste, equivalent to 4.4 mg of F, were placed at the bottom of a collection bottle (lower external diameter: 45.0 mm; upper external diameter: 50.9 mm; height: 57.1 mm; volume: 80 mL), ensuring that the toothpaste did not touch the bottle's side walls. The homogenizer's metal rod was positioned in the center of the toothpaste mass, gently touching the bottom of the bottle. Then, 12 mL of purified water was added slowly. The mixture was stirred for 40 seconds at 100 rpm. After stirring, the setup was disassembled, and the resuspended content was carefully poured into a test tube. The collected volume was measured, and the tube was centrifuged at 5000 g for 5 min. The supernatant was collected and diluted tenfold for fluoride analysis (Figure 1). Six repetitions (n=6) were conducted for each toothpaste. The fluoride concentration released during the test was determined using a specific electrode by the simplified technique, as all the toothpastes contained NaF/Silica. After stirring each sample, the metal spatula was rinsed with purified water and dried with paper towels.
Determination of Fluoride in Toothpastes and Calculation of the Percentage of Released Fluoride
A calibration curve was established using fluoride standards ranging from 0.25 to 16.0 µg F/mL, prepared in 50% TISAB II (v/v). The fluoride standards were made from NaF 99.99% (Sigma-Aldrich, St. Louis, MO, USA). The accuracy of the analysis was confirmed with a standard fluoride solution (Orion 940907, Thermo Fisher Scientific Inc., Boston, MA, USA), and the mean coefficient of variation of the triplicates was assessed.
Fluoride concentrations were measured using a fluoride-specific ion electrode [17] (Orion 96-09, Thermo Fisher Scientific Inc., Boston, MA, USA), connected to an ion analyzer (Orion Star A214; Thermo Fisher Scientific Inc., Boston, MA, USA). The linear regression coefficient between the fluoride concentrations of the standards and their corresponding mV values was calculated using Excel® (Microsoft Corporation, Chicago, USA), yielding an r2 value of 1.00. The precision of the calibration curves was verified, with percentage variation between observed and expected values ranging from -0.1 to 0.9%. Results were expressed in ppm F (µg F/g; mg F/kg).
The fluoride concentrations in the toothpastes were determined to calculate the amount of released fluoride. The fluoride concentration in the toothpastes is expressed as μg F/g, indicating that 'x' μg F can be found in 1 g of toothpaste. Since 4 g of toothpaste was used, the calculation was based on the fluoride concentration found. Using the amount of fluoride in the toothpaste used for testing and the amount released into the water, the percentage of fluoride released was calculated as follows: (mg F released x 100) / (mg F in the paste).
Rheological Analysis of the Toothpastes
Flow and viscosity curves were obtained using a Brookfield R/S Plus rheometer equipped with a 25 mm parallel-plate geometry spindle with a 0.5 mm gap under controlled shear rate. Using a Teflon spatula, approximately 0.5 g of toothpaste gel was placed at the center of the rheometer plate, where the spindle was subsequently positioned. Ascending and descending flow curves were then obtained by applying a shear rate ascending from 1 to 60 s⁻1 with 60-second intervals. All experiments were conducted in triplicate at 25 °C with temperature regulation provided by the PTR-1 thermal control system [18]. The hysteresis represents the area between the ascending and descending curves and was calculated using Rheo v2.8 software to obtain the thixotropy and apparent viscosity of the evaluated dentifrices [19].
Statistical Analysis
The data for the percentage of fluoride released were compared using one-way ANOVA followed by Tukey's test, with a significance level of 5%. Analyses were conducted using SPSS® Statistics for Windows, version 21.0 (SPSS Inc., Chicago, IL, USA). For viscosity comparisons, ANOVA was also used. The calculated hysteresis areas were compared by using one-way ANOVA followed by Tukey’s multiple comparison test with a significance level set at 5%.
Results
According to the manufacturers, all the analyzed toothpastes were formulated with NaF and contained 1,100 ppm F; mg F/kg. The fluoride concentration in the tested toothpastes was determined (Figure 2). Tandy toothpaste showed a fluoride concentration of 1110.0 ppm TF, Sorriso Kids had 1095.4 ppm TF, and Colgate Zero Kids presented 1133.6 ppm TF.
The percentage (%) of fluoride release from the toothpastes was determined (mean; ± SD; n=6) (Figure 3). Tandy toothpaste (71.4 ± 3.2A) and Sorriso Kids (70.3 ± 2.1A) exhibited similar fluoride release, while Colgate Zero Kids released a significantly lower percentage of fluoride (37.7 ± 9.7B). Distinct letters indicate a statistical difference (p<0.05).
The flow curves show that Colgate Zero Kids toothpaste exhibits significantly higher thixotropy compared to Sorriso Kids and Tandy, indicating greater resistance of Colgate Zero Kids to increasing shear rates. On the other hand, Sorriso Kids and Tandy presented significantly smaller hysteresis areas, reflecting lower apparent viscosity (Figure 4).
Rheological Properties of the evaluated toothpastes. A: Flow curves of the evaluated toothpastes. These curves represent stepped shear tests at increasing shear stress (Tau[Pa]) and shear rate (D[1/s]) in parallel-plate rheometric analysis. B: Thixotropy of the evaluated toothpastes calculated from the area of hysteresis of the flow curves. *Indicates statistical significance (p < 0.05), One-way ANOVA followed by Tukey’s test.
Discussion
Fluoride release can vary significantly among different formulations of silica- NaF/based gel toothpastes [1,13,14]. In Brazil, most commercial children's toothpastes utilize this composition [15], yet the bioavailability of fluoride in these formulations still requires further investigation. This study analyzed the in vitro fluoride release from three silica-based gel children's toothpastes with distinct rheological characteristics.
According to the labels, the selected toothpastes were formulated with sodium fluoride (NaF), declaring a total fluoride concentration (TF) of 1,100 ppm, using silica as an abrasive. Given that NaF/Silica formulations present all their TF in a soluble form [3], the fluoride concentration was determined using the simplified technique described by Quiroz-Torrez et al. [16]. This protocol does not involve acid, unlike conventional methods [18], since there are no insoluble calcium salts to dissolve, and the fluoride salt used is not sodium monofluorophosphate (Na2FPO3). The results indicated that the fluoride found was indeed soluble and at concentrations close to those declared (Figure 2). From these concentrations, the amount of fluoride in the 4 g of toothpaste used for the release test was calculated.
The release test (Figure 3) showed that Tandy and Sorriso Kids toothpastes had similar percentages of fluoride release, while Colgate Zero Kids released significantly less, approximately half of the amount observed in the others. This result may be attributed to the significantly higher thixotropy of Colgate Zero Kids compared to Sorriso Kids and Tandy (Figure 4), which tends to hinder the diffusion of fluoride ions during brushing. These findings align with existing literature, which indicates that the consistency and rheological properties of formulations directly affect the effective release of fluoride [8].
This finding is particularly relevant in the current context, where there is increasing interest in products with natural appeal, prompting the development of new fluoridated children's toothpaste formulations. However, these formulations are often created without adequately considering how rheological properties impact fluoride bioavailability. For instance, Colgate Zero Kids toothpaste, designed to meet the demand for more "natural" compositions, exhibits high viscosity. As shown in the results (Figure 4), Colgate Zero Kids toothpaste exhibits high thixotropy and a strong internal gel structure, requiring more force to initiate flow (Figure 4A). This implies greater effort to extrude product from the tube and lower product spreadability during brushing. Additionally, the high thixotropy (Figure 4B), as indicated by the hysteresis area, suggests that the product may take longer to recover its structure after being subjected to shear (or agitation). Smaller hysteresis areas were found for Tandy and Sorriso Kids toothpastes, indicating lower thixotropy, apparent viscosity, and shear resistance, demonstrating that these toothpastes might be easier to dispense from the tube, spread on the toothbrush, and during brushing. The reason for this difference is unknown and cannot be fully explained based on the compositions declared on the toothpaste labels (Table 1). Therefore, Colgate Zero Kids showed reduced fluoride release compared to toothpastes previously used by the pediatric population [17]. This emphasizes the importance of considering the interaction between physicochemical composition and expected clinical performance in toothpaste. To the best of our knowledge, the rheological characteristics of Colgate Zero Kids are unique among toothpastes currently available, whether intended for children or for the whole family.
Regarding the technical aspects of the study, the in vitro model was adjusted based on the time and stirring speed used [13], highlighting the need for methodological adjustments for different toothpaste formulations. The appropriate dimensions of the collection vessel (lower external diameter: 45.0 mm; upper external diameter: 50.9 mm; height: 57.1 mm; volume: 80 mL) are essential for proper analysis execution. Furthermore, the position of the spatula during the agitation of toothpaste and water must be correctly placed near the bulge at the bottom of the vessel to ensure contact with the entire sample being analyzed.
Conclusion
This research demonstrates that the reduced in vitro fluoride release observed in Colgate Zero Kids toothpaste is likely attributable to its higher viscosity, a consequence of its distinct rheological properties. This increased viscosity may hinder the prompt availability of fluoride during brushing, potentially compromising its anticaries efficacy. These findings highlight the critical need to evaluate not only the total fluoride content in pediatric toothpastes, but also the physicochemical factors that govern its bioavailability. Future in vivo studies are warranted to confirm the clinical relevance of these in vitro observations.
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Financial Support
This study was financed in part by the Coordination for the Improvement of Higher Education-Brazil (CAPES)-Finance Code 001 and the National Council for Scientific and Technological Development (CNPq) (Finances 314765/2020-4 and 422626/2021-3). The authors also thank Coordination for the Improvement of Higher Education-Brazil CAPES (grant number 88887.806283/2023-00) for the scholarship provided to the first author.
Acknowledgements
We are thankful to the Laboratory of Biochemistry at Piracicaba Dental School, UNICAMP, for providing the necessary facilities.
Data Availability
The data used to support the findings of this study can be made available upon request to the corresponding author.
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Edited by
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Academic Editor:
Alessandro Leite Cavalcanti
