Whitening toothpastes are ineffective in recovering color from previous dental bleaching - In vitro study

Abstract

The objective of this study was to evaluate the influence of whitening toothpastes on color recovery after dental bleaching and on enamel microhardness. Sixty bovine enamel blocks (6×6×2 mm) were obtained and bleached with 40% hydrogen peroxide (N = 30) ([40% HP] Opalescence Boost 40% PF) or 10% carbamide peroxide (N = 30) ([CP 10%] Opalescence 10% PF). The specimens were subjected to coffee staining for 14 days. The specimens were subdivided into six groups according to whitening treatment and whitening toothpaste (n = 10) and submitted to 14,600 mechanical brushing cycles: 3% HP whitening toothpaste (Colgate Luminous White Glow), whitening toothpaste containing activated charcoal (Colgate Luminous White Activated Charcoal), and conventional toothpaste (Colgate Triple Action). Color changes (ΔE₀₀) and the difference in the Whitening Index for Dentistry (ΔWI_D) were evaluated after whitening, coffee staining, and mechanical toothbrushing using a spectrophotometer CM-3700A (Konica-Minolta), as well as microhardness (HMV-2; Shimadzu). Data were analyzed by 2-way ANOVA (ΔE₀₀, ΔWI_D), 2-way ANOVA with repeated measures (microhardness), and Tukey's test (α = 0.05). No significant differences were observed in ΔE₀₀, regardless of the toothpaste used. The ΔE₀₀ and ΔWI_D mean values were statistically higher for the groups whitened with 40% HP. No difference was observed among the toothpastes when whitened with 40% HP. When whitened with 10% CP, the traditional toothpaste obtained a higher ΔWI_D mean value. For microhardness, traditional toothpaste showed the highest KHN mean value. Whitening toothpastes did not influence the color recovery or reduce the microhardness of dental enamel.

Key Words:
tooth bleaching; tooth bleaching agents; bleaching agents; toothpastes; charcoal

Resumo

Este estudo avaliou a influência de dentifrícios clareadores na recuperação da cor obtida após clareamento dental e na microdureza do esmalte. Sessenta blocos de esmalte bovino (6×6×2 mm) foram obtidos e clareados com 40% de peróxido de hidrogênio (N = 30) ([40% HP] Opalescence Boost 40% PF) ou 10% de peróxido de carbamida (N = 30) ([CP 10%] Opalescence Boost 10% PF). Os espécimes foram submetidos à coloração com café por 14 dias. As amostras foram subdivididas em seis grupos de acordo com o tratamento clareador e o dentifrício clareador (N = 10), e submetidos a 14.600 ciclos de escovação mecânica: dentifrício clareador HP 3% (Colgate Luminous White Glow), dentifrício clareador contendo carvão ativado (Colgate Luminous White Activated Charcoal) e dentifrício convencional (Colgate Triple Action). As alterações de cor (ΔE₀₀) e a diferença no Índice de Clareamento para Odontologia (ΔWI_D) foram avaliadas após clareamento, coloração com café e escovação mecânica usando um espectrofotômetro CM-3700A (Konica-Minolta), bem como a microdureza (HMV-2; Shimadzu). Os dados foram analisados por ANOVA de duas vias (ΔE₀₀, ΔWI_D), ANOVA de duas vias com medidas repetidas (microdureza) e teste de Tukey (α = 0,05). Não foram encontradas diferenças significativas em relação ao ΔE₀₀, independentemente do dentifrício. Os valores médios de ΔE₀₀ e ΔWI_D foram estatisticamente maiores para os grupos clareados com 40% de HP. Não foi observada diferença entre os dentifrícios quando clareados com 40% de HP. Quando clareado com 10% de CP, o dentifrício tradicional obteve um valor médio de ΔWI_D maior. Para microdureza, o dentifrício tradicional apresentou o maior valor médio de KHN. Os dentifrícios clareadores não auxiliaram a recuperação da cor e reduziram a microdureza do esmalte dental.

Introduction

Tooth whitening is one of the most common procedures performed in dental offices to enhance the aesthetic appearance of the smile ⁽¹. This procedure is based on the use of bleaching agents such as hydrogen peroxide ([HP] H₂O₂) in concentrations of 30-44% and carbamide peroxide ([CP] CH₆N₂O₃) in concentrations ranging from 10-22% ^(2,3,4. These materials act by releasing free radicals in the dental structure, and the tooth whitening procedure is considered the gold standard in the literature, as it achieves significant color change due to its high bleaching efficiency and rapid results ^(3,4,5.

Although the free radicals provide a satisfactory aesthetic result, these molecules can simultaneously induce adverse biological reactions, such as gingival irritation, dentin sensitivity (during and after whitening treatment), and morphological changes in the dental structure ^(1,6. Aiming to reduce the adverse effects of this technique and to facilitate the population's access to the benefits of the whitening procedure, so-called over-the-counter (OTC) products for dental bleaching have been developed ^(6,7. OTC products are widely accessible to the general population, offering greater affordability than procedures performed under professional supervision. Among the OTC products available, whitening toothpastes are particularly notable ^6,7,8.

Multiple factors, including intrinsic tooth coloration and external staining, can influence the color change in dental structures ⁽⁶. Intrinsic tooth color is affected by light absorption and the optical properties of enamel and dentin. At the same time, extrinsic coloration is influenced by the formation and presence of the acquired salivary pellicle, dietary habits, tobacco use, dark substances (e.g., coffee and red wine), and brushing practices ⁽⁶.

Previous studies ^(1,9 have shown that OTC products, particularly whitening toothpastes, do not achieve the same color change level as traditional whitening gels when used independently ^(1,9. However, as these products primarily work through surface abrasion or chemical interactions to remove surface stains, it can be inferred that whitening toothpastes can effectively help maintain and recover whitening results achieved under dental supervision ⁽¹⁰. Additionally, it is well known that whitening treatment induces changes in tooth structure after interacting with stain molecules ⁽¹¹. According to Kwon & Wertz ⁽¹¹, HP interacts significantly with both the organic and inorganic components of enamel and dentin, reducing the calcium and phosphate content of hydroxyapatite. These modifications can lead to porosities on the enamel surface, creating a new area that is prone to accumulating extrinsic pigments, mainly when staining solutions are consumed after the whitening procedure. In this context, whitening toothpastes would aid in restoring the color achieved through the previous bleaching treatment.

The whitening activity of toothpastes is promoted by the inclusion of abrasives, adsorbent particles, peroxides, enzymes, or optical agents in their formulations ⁽¹². As a result, the bleaching effect may differ depending on the type of active agent, the size and amount of abrasive particles, the concentration of hydrogen peroxide, and the presence of pigments ⁽¹². It is well known that the whitening effect of these products mainly results from the abrasive removal of surface stains, although some formulations also depend on additional agents ⁽¹³. Because the composition of whitening toothpastes varies among manufacturers, their effectiveness and potential effects on enamel structure are also inconsistent ⁽¹². As noted, significant structural changes to enamel may occur during standard bleaching procedures, and these can be exacerbated by the use of highly abrasive dentifrices, as toothbrushing in the oral cavity is primarily responsible for tooth abrasion ^(13,14.

Thus, this study aimed to evaluate the color recovery initially achieved by take-home (10% CP) or in-office (40% HP) whitening treatments, as well as the microhardness of dental enamel after mechanical brushing with whitening toothpastes, following the staining of dental enamel through immersion in a coffee solution. The null hypotheses tested were: [1] there would be no difference between the whitening protocols carried out in terms of color change (ΔE₀₀) and Whitening Index for Dentistry (WID); [2] there would be no difference among the toothpastes in terms of color change (ΔE₀₀) and Whitening Index for Dentistry (WID); [3] there would be no difference in the enamel microhardness regardless of the bleaching treatment.

Materials and methods

Study design

This study followed the design of two factors: [1] teeth whitening gel (in two levels): 40% hydrogen peroxide (in-office approach); and 10% carbamide peroxide (take-home approach); [2] toothpastes (in three levels): whitening toothpaste containing 3% of hydrogen peroxide; whitening toothpaste containing activated charcoal; traditional toothpaste, used as a control (Table 1).

Table 1
Commercial brand, manufacturers, and composition of the materials used in the study.

Material	Commercial brand	Composition
Opalescence Boost 40% PF	Ultradent, South Jordan, Utah, USA	40% Hydrogen peroxide, 3% potassium nitrate, 1% sodium fluoride, chemical activator, water, carbopol.
Opalescence PF 10%	Ultradent, South Jordan, Utah, USA	10% Carbamide peroxide, potassium nitrate, fluoride ions 0.11% weight/weight (1,100 ppm), water.
Colgate Luminous White Activated Charcoal	Colgate Palmolive, São Bernardo do Campo, SP, Brazil.	Water, hydrated silica, sorbitol, calcium pyrophosphate, glycerin, PEG-12, pentasodium triphosphate, tetrapotassium pyrophosphate, aroma, sodium lauryl sulfate, sodium monofluorophosphate, cellulose gum, sodium saccharin, xanthan gum, cocamidopropyl betaine, CI 77266 (Charcoal Powder), CI 16035, CI 42090, CI 19140, limonene.
Colgate Luminous White Glow	Colgate Palmolive, São Bernardo do Campo, SP, Brazil.	Calcium pyrophosphate, propylene glycol, PEG/PPG 116/66 copolymer, PEG-12, glycerin, PVP, sodium lauryl sulfate, sodium saccharin, phosphoric acid, sucralose, butylated hydroxytoluene, water, sodium monofluorophosphate, tetrasodium pyrophosphate, silica, 3% hydrogen peroxide.
Colgate Triple Action	Colgate Palmolive, São Bernardo do Campo, SP, Brazil.	Water, calcium carbonate, sorbitol, sodium lauryl sulfate, sodium monofluorophosphate, flavor, cellulose gum, tetrasodium pyrophosphate, sodium bicarbonate, benzyl alcohol, sodium saccharin, xanthan gum, sodium hydroxide, CI 74260, CI 74160, limonene.

The response variables were: (i) color change (ΔE₀₀) measurement with spectrophotometer; (ii) the difference (Δ) in the Whitening Index for Dentistry (WID), both comparing the baseline, the result after 14 days of whitening and after the toothbrushing cycle ¹⁵; (iii) enamel microhardness of the baseline, the result after 14 days of whitening treatment and after the mechanical toothbrushing ⁴.

The sample size was defined based on previous studies ^4,16 and a pilot test with 10 specimens per group (n = 10). The calculation aimed to ensure a statistical power of at least 0.8 (β = 0.2) at a significance level of 0.05. Thus, six groups were obtained:

(I) 10% CP + 3% HP TP: Whitening treatment was done using 10% carbamide peroxide (CP), following tooth brushing with toothpaste (TP) containing 3% HP, after staining.

(II) 10% CP + Activated Charcoal TP: A whitening treatment was created using 10% CP, followed by tooth brushing with TP containing activated charcoal after staining.

(III) 10% CP + Traditional TP: A whitening treatment was performed using 10% CP, followed by tooth brushing with traditional TP after staining.

(IV) 40% HP + 3% HP TP: A whitening treatment was performed using 40% hydrogen peroxide (HP) after brushing with toothpaste (TP) containing 3% HP, following staining.

(V) 40% HP + Activated Charcoal TP: A whitening treatment was made using 40% HP, followed by tooth brushing with TP containing activated charcoal after staining.

(VI) 40% HP + Traditional TP: A whitening treatment was performed using 40% HP, followed by tooth brushing with traditional toothpaste after staining.

Figure 1 illustrates the flowchart of the study design, and Figure 2 provides a detailed view of the study design.

Figure 1
Flowchart of the study design.

Figure 2
Illustration of the study design. Created in BioRender. Detogni, A. (2025) "https://biorender.com/s67o632"https://BioRender.com/s67o632

Specimen preparation

Thirty bovine teeth were selected and sectioned from the middle third of each crown using a diamond disc attached to a universal cutting machine (Isomet, Buehler, Lake Bluff, IL, USA) to obtain the specimens. These samples consisted of 6 mm × 6 mm blocks containing enamel on the upper surface and dentin on the lower surface. Two samples were obtained from each dental crown (N = 60). Underwater cooling, the enamel surface was polished with abrasive SiC paper of increasing grit sizes (#600, #1200, and #2000) using an automatic polishing machine (Aropol 2V, Arotec, Cotia, SP, Brazil). The thickness of each sample was standardized to 2 mm (1 mm of enamel and 1 mm of dentin). The samples were stored in distilled water at 37 °C throughout all the experimental procedures.

Color measurement

The color analyses were performed against a white background using the spectrophotometer CM-3700A (Konica Minolta, Tokyo, Japan), connected to the software CyberChrome on Color 2006, based on the CIEDE2000 system ¹⁵. After calibration, the equipment was set to reflectance mode, utilizing a light source with a wavelength range of 400-700 nm, a 2-degree standard observer, and the CIE Standard Illuminant D65. For each analysis, the color of the specimens was measured in triplicate, and the average was calculated from these values. The software provided color information for all coordinates: L* (black-white), a* (green-red), and b* (blue-yellow). In addition to the color change analysis (ΔE₀₀), these values were also used to calculate the Whiteness Index for Dentistry (WID), as shown below.

Initial microhardness measurement

A microhardness machine (HMV-2; Shimadzu, Tokyo, Japan), equipped with a Knoop indenter, was used to measure the microhardness of the enamel surface on the specimens. Specimens were measured with a vertical static load of 25 g for 5 s at 40x magnification. Three measurements were taken per specimen: one at the center and two at 1 mm to the left and right, respectively. The average of these three values was considered the initial microhardness value. After whitening and brushing treatments, the changes in microhardness (before and after each treatment) were calculated by:

$\mathrm{K}\mathrm{H}\mathrm{N}\mathrm{}=\mathrm{}\mathrm{K}\mathrm{H}\mathrm{N}\mathrm{f}\mathrm{}-\mathrm{}\mathrm{K}\mathrm{H}\mathrm{N}\mathrm{i}$

Where KHNf represents the final microhardness measurement and KHNi the initial measurement ¹⁰.

Whitening treatment

The specimens were randomly divided into two groups according to the whitening protocol. Half of the specimens (N = 30) were submitted to in-office whitening treatment with 40% hydrogen peroxide ([40% HP] Opalescence Boost 40% PF, Ultradent, South Jordan, Utah, USA), applied in three sessions of 45 min each with 7 7-day interval between sessions (manufacturer instructions). The take-home whitening treatment (N = 30) was performed with 10% carbamide peroxide ([10% CP] Opalescence PF 10%, Ultradent, South Jordan, Utah, USA) for eight hours per day over a period of 10 days (manufacturer instructions) ^17,18. In both treatments, the gel was applied by placing a small drop (0.2 mL) in the center of the specimen using a syringe, then spreading over the entire surface with a disposable micro applicator (Microbrush Tube Series Regular 2.0 mm, Blue, Microbrush International, Grafton, Massachusetts, USA) to form an even layer. Following the whitening treatments, new color and microhardness measurements were performed.

Staining

Following the tooth whitening, the specimens were submitted to staining by immersing in soluble coffee (3 Corações tradicional, Varginha, MG, Brazil): powder = 16.0 g, water 200 mL; 3 x daily, 15 min/each, 37 ◦C for 14 days ^4). The specimens were stored in distilled water at 37 ºC during the staining intervals. After, specimens were subdivided into three groups (N = 10) according to the toothpaste used for the mechanical toothbrushing test: 3% HP (whitening toothpaste containing 3% hydrogen peroxide, Colgate Luminous White Glow, Colgate Palmolive, São Bernardo do Campo, SP, Brazil); activated charcoal (activated charcoal-based whitening toothpaste, Colgate Luminous White Activated Charcoal, Colgate Palmolive, São Bernardo do Campo, SP, Brazil); and traditional (conventional toothpaste, Colgate Triple Action, Colgate Palmolive, São Bernardo do Campo, SP, Brazil).

Simulated tooth brushing

Simulated toothbrushing (MSet; Elquip, São Carlos, São Paulo, Brazil) was made using toothbrushes (Colgate Classic Clean, Colgate Palmolive, São Bernardo do Campo, SP, Brazil) according to ISO/DTS 145692 under a load of 200 g at 356 rpm, with a back-and-forth stroke length of 3.8 cm. The toothpastes (5 g) were diluted in distilled water (5 mL) at a 1:1 ratio. The toothbrushing machine simulated the equivalent of one year of tooth brushing by a healthy individual (14,600 cycles) ^17,19. Afterward, the specimens were washed in running water, dried with absorbent papers, and stored separately in plastic tubes with 3 mL of distilled water per specimen at 37 °C until the final color and microhardness measurements were obtained.

Final color measurement

After the treatments, a final color measurement was performed using previously described parameters. The values obtained after tooth brushing were compared with those obtained after the whitening treatments. The differences between the measurements were calculated for each coordinate (ΔL, Δa, Δb). The color difference was calculated using the CIEDE2000 formula ^15,20:

${\mathrm{\Delta }\mathrm{{\rm E}}}_{00}=\surd \mathrm{}̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅\mathrm{}\left[\right(\mathrm{}\mathrm{\Delta }\mathrm{L}\mathrm{\text{'}}\mathrm{}/\mathrm{}\mathrm{K}\mathrm{L}\mathrm{S}\mathrm{L})2\mathrm{}+\mathrm{}(\mathrm{}\mathrm{\Delta }\mathrm{C}\mathrm{\text{'}}\mathrm{}/\mathrm{}\mathrm{K}\mathrm{C}\mathrm{S}\mathrm{C})2\mathrm{}+\mathrm{}(\mathrm{}\mathrm{\Delta }\mathrm{H}\mathrm{\text{'}}\mathrm{}/\mathrm{}\mathrm{K}\mathrm{H}\mathrm{S}\mathrm{H})2\mathrm{}+\mathrm{}\mathrm{R}\mathrm{T}\mathrm{}\mathrm{}(\mathrm{}\mathrm{\Delta }\mathrm{C}\mathrm{\text{'}}\mathrm{}/\mathrm{}\mathrm{K}\mathrm{C}\mathrm{S}\mathrm{C}\left)\right(\mathrm{}\mathrm{\Delta }\mathrm{H}\mathrm{\text{'}}\mathrm{}/\mathrm{}\mathrm{K}\mathrm{H}\mathrm{S}\mathrm{H}\left)\right]$

The formula uses the differences of three CIELAB metrics: ΔL* (lightness), ΔC* (chroma), and ΔH* (hue). The RT function was added to improve the performance of the equation by adjusting the chromatic differences in the blue region. The formula also considers the weighting functions for lightness (SL), chroma (SC), and hue (SH), along with their parametric factors (KL, KC, KH). The difference (Δ) in the Whitening Index for Dentistry (WID) was calculated as it correlates the data obtained from CIEDE2000 with color perception. Positive results indicate values of lightning or whitening, while negative values demonstrate darkening (6). The WID was calculated according to the following formula ²¹:

Equation:

${WI}_D\mathrm{}=\mathrm{}0.511\mathrm{L}\mathrm{*}\mathrm{}-\mathrm{}2.324\mathrm{a}\mathrm{*}\mathrm{}-\mathrm{}1.100\mathrm{b}\mathrm{*}$

The ΔWI_D values were determined by the differences between the values obtained after tooth whitening and the baseline, and after toothbrushing and tooth whitening.

Statistical analysis

Statistical analysis was performed using SPSS software (version 15.0; Statistical Package for the Social Sciences, SPSS Inc., Chicago, IL, USA). The normality of the data was assessed using the Shapiro-Wilk test, and homoscedasticity was checked using Levene’s test. Statistical analyses were conducted using various experimental designs, with a significance level of α = 0.05. Two-way ANOVA was used to analyze the color change data (ΔE₀₀ and ΔWI_D) obtained after different whitening treatments and simulated tooth brushing protocols, according to the whitening treatments (in-office and take-home) and toothpastes (3% HP, activated charcoal, and traditional). A two-way ANOVA with repeated measures (evaluation periods: baseline, after whitening treatment, and after tooth brushing) was employed to evaluate the data obtained from the microhardness analysis of dental enamel according to the whitening treatments (in-office and take-home) and toothpastes (3% HP, activated charcoal, and traditional). Tukey post-hoc test (α = 0.05) was used to perform multiple comparisons following the ANOVA.

Results

Table 2 presents the mean values and standard deviations for the color change ΔE₀₀ across the different experimental groups, categorized by whitening treatment and toothpaste used. The in-office whitening treatment (40% HP) demonstrated a higher color change, as indicated by the pooling mean value, than the take-home whitening performed with carbamide peroxide (10% CP). No statistically significant differences were observed among the toothpastes.

Table 2
Color change mean values and standard deviation (in ΔE₀₀) for the different groups according to the whitening agent and toothpaste used.

	3% HP	Activated Charcoal	Traditional	Pool Mean
40% HP	3.86 (1.2) A	2.43 (1.4) A	2.50 (1.4) A	2.93 a
10% CP	1.87 (1.0) A	1.78 (0.9) A	2.13 (1.3) A	1.93 b

• Different uppercase letters indicate differences among toothpastes for the same whitening agent (lines) (p < 0.05). Different lowercase letters in the column indicate differences between the pool means of whitening agents regardless of the toothpaste (p < 0.05).

Table 3 presents the mean values and standard deviations for the Whitening Index for Dentistry (WID) after the evaluated periods and treatments using different whitening approaches and toothpastes. For 3% HP toothpaste, no statistically significant difference was observed between the two whitening gels (40% HP and 10% CP). For the groups brushed with activated charcoal and traditional toothpaste, the whitening with 40% HP showed statistically significantly higher mean values when compared with the take-home approach using 10% CP, indicating a greater perception of whiteness. When comparing the toothpastes used for the same whitening approach, it was noted that there were no statistically significant differences among the toothpastes after the whitening in the take-home approach (10% CP). Nonetheless, when used after in-office treatment (40% HP), the toothpaste containing 3% HP showed the lowest effectiveness compared to the traditional toothpaste and activated charcoal, which did not differ statistically from each other.

Table 3
CIELAB-based Whiteness Index for Dentistry (ΔWI_D) mean values and standard deviation of the different groups according to the whitening agent and toothpaste used.

	3% HP	Activated Charcoal	Traditional
40% HP	15.03 (5.3) a, B	18.26 (4.2) a, AB	22.29 (7.3) a, A
10% CP	14.67 (4.8) a, A	12.35 (5.3) b, A	13.56 (4.3) b, A

• Different uppercases indicate differences among toothpastes (lines) (p<0.05). Different lowercases indicate differences between whitening agents for the same toothpaste (columns) (p <0.05).

Table 4 shows the microhardness values obtained according to the evaluation period (baseline, after whitening protocols, and after tooth brushing), the toothpastes used, and the previous whitening treatment. It was observed that regardless of the whitening agent used and the evaluation period, the toothpastes demonstrated statistically significant mean values (p = 0.00001), with the highest microhardness value observed in the group using traditional toothpaste, followed by the activated charcoal and the 3% HP toothpastes, respectively. Regarding the whitening agents, when used in conjunction with toothbrushing and the 3% HP toothpaste, the lowest mean value was obtained in the group where the 10% CP was applied (p = 0.00001). There was no statistically significant difference between the whitening agents used in the group that applied traditional toothpaste and the group that applied toothpaste with activated charcoal. Furthermore, when comparing the values obtained across different evaluation periods, the highest microhardness value was observed at baseline, regardless of the treatment applied, followed by the groups post-toothbrushing and then post-whitening, respectively.

Table 4
Knoop microhardness mean values and standard deviation (in KHN) according to the evaluation period (baseline, post-whitening, and post-brushing), toothpaste used, and previous whitening protocol.

Toothpaste	Whitening agent	Baseline	Post-whitening	Post-brushing	Pool Mean
3% HP (296.59) *	40% HP	348.36 (33.63)	273.39 (41.76)	297.40 (53.40)	286.91 a
	10% CP	367.46 (50.20)	225.62 (44.34)	267.34 (46.44)	254.06 b
Activated Charcoal (318.10) *	40% HP	3’70.73 (35.43)	277.30 (35.20)	298.71 (46.31)	316.55 a
	10% CP	397.33 (32.67)	250.50 (39.38)	314.00 (41.07)	309.17 a
Traditional (346.36) *	40% HP	379.86 (53.80)	310.05 (35.80)	353.53 (41.86)	366.32 a
	10% CP	402.50 (44.07)	286.06 (48.18)	346.18 (45.70)	389.10 a
Pool Mean		377.71 A	270.49 C	312.86 B

• Uppercase letters indicate significant differences among the evaluation periods (pooling mean values). Lowercase letters indicate significant differences between the whitening agents evaluated. HP = hydrogen peroxide; CP = carbamide peroxide (pooling mean values). Asterisks (*) indicate statistically significant differences among the assessed toothpastes (p < 0.05).

Discussion

This study evaluated the ability of different whitening toothpastes to recover the bleaching effect after staining, both after in-office (40% HP) and take-home approaches (10% CP), to assess whether whitening toothpastes are effective in restoring the color achieved through prior tooth whitening, after pigmentation caused by dietary habits. Therefore, the data used to compare color changes were obtained after tooth whitening and after brushing with whitening toothpastes. Data collected after pigmentation was not included in this analysis, as assessing whether the coffee solution altered the color of whitened samples was not among the study objectives. Additionally, a study has examined the impact of coffee on enamel structure and staining ²². Its findings showed that coffee not only changes the superficial color of enamel but also affects the internal tooth structures, confirming its strong pigmenting ability. This evidence supports our statement that the whitening effect was effectively 'lost' after staining with coffee, explaining why detailed pigmentation data were not the focus of the present study.

The results of the present study demonstrated no significant differences between the various whitening protocols in terms of color change (ΔE₀₀). However, in terms of the Whitening Index for Dentistry (WID), the whitening protocol using 40% HP (in-office approach) showed significantly higher whitening mean values than the protocol using 10% CP (take-home approach) when applied before the whitening toothpaste containing 3% HP. Therefore, the first null hypothesis, that there would be no difference between the bleaching protocols in terms of color change (ΔE₀₀) and Whitening Index for Dentistry (WID), was partially rejected.

The color of the teeth is determined by the optical properties of the enamel and dentin. It is influenced by intrinsic factors (tooth structure) or extrinsic stains (resulting from the deposition of pigments on the tooth's surface) ^9,23,24. Tooth color can be improved through various methods, including external bleaching of vital teeth, internal bleaching of non-vital teeth, professional cleaning, the use of whitening toothpastes, and microabrasion of enamel with abrasives and acids ^23,24,25. Due to its cost-effectiveness and efficiency, tooth whitening is the most used technique for altering tooth color ^11,23.

The process of color change happens in three distinct phases: [1] diffusion of the bleaching agent through the tooth structure; [2] interaction of hydrogen peroxide delivered as carbamide peroxide (CH₆N₂O₃) or hydrogen peroxide (H₂O₂) with organic chromophores within the tooth structure, where free radicals oxidize pigment molecules by breaking carbon-carbon double bonds (C=C) and converting them into single bonds (C-C); and [3] a resulting color change, due to the easy removal of the now low-molecular-weight molecules from the tooth structure ^11,26. This result depends on the duration of exposure and the concentration of the bleaching compound ²⁶.

The usage times of whitening agents range from 30 minutes to 10 hours ^23,26. However, it is known that once hydrogen peroxide degrades, it becomes ineffective, whether used in in-office or take-home protocols (²³⁾. Therefore, increasing exposure time does not enhance the effectiveness of the treatment when lower-concentration products are applied. This explains the more remarkable perceived color change (ΔWI_D) in specimens treated with 40% HP compared to those treated with 10% CP.

Regarding the toothpastes, the present study's results showed no statistically significant differences between the different toothpastes evaluated in terms of color change (ΔE₀₀). When evaluated through the ΔWI_D, there were no statistically significant differences between toothpastes when used after the take-home approach (10% CP). However, after in-office whitening (40% HP), the traditional and activated charcoal toothpastes showed higher bleaching rates than whitening toothpastes containing 3% HP. Thus, the second null hypothesis of this study -that there would be no differences among the toothpastes used in terms of color change (ΔE₀₀) and the Whitening Index for Dentistry (WID) -was partially rejected.

Whitening toothpastes were developed as an alternative to conventional bleaching treatments (in-office or take-home) due to their lower cost and greater accessibility than over-the-counter products ²⁵. These products promise to whiten the tooth structure within 2-4 weeks of use, due to the presence of abrasive components in their composition that are responsible for removing extrinsic stains from the dental structure ^6,25,27. Additionally, some toothpastes contain low concentrations of hydrogen peroxide, ranging from 1.5% to 6%, and optical brighteners, such as blue covarine, which are deposited on the enamel surface, giving the dental structure a lighter and bluish appearance ^27,28.

Although the toothpastes used in this study contain these components, the results demonstrated that the whitening toothpastes exhibited color change values similar to those of the traditional toothpaste used as a control after both bleaching protocols and staining. The traditional toothpaste also presents a superior result than the toothpaste containing 3% HP when used after the whitening through the in-office approach (40% HP) in terms of ΔWI_D. Typically, the whitening provided by toothpastes occurs only on extrinsic stains, resulting from the abrasion of the tooth surface by abrasive particles that become trapped between the toothbrush bristles and the tooth surface ^7,29. Additionally, the activated charcoal in certain products acts similarly to traditional toothpastes, generating abrasion on the dental surface and removing stains ³⁰. Thus, the type, size, and hardness of the abrasive particles, as well as the load applied during the process, influence the result obtained ^29,30.

Hydrated silica and calcium carbonate are the most common abrasives, typically used in concentrations ranging from 8 to 20% (w/w). In contrast, sodium bicarbonate is less abrasive and can be used at concentrations exceeding 50% (w/w) ²⁹. Other abrasives, such as calcium pyrophosphate, can be used. In this study, the toothpaste containing powdered activated charcoal has two other abrasive particles in its composition (hydrated silica and calcium pyrophosphate) (Table 1). The presence of these three different particles, which vary in size and proportion, in the toothpaste generates a more significant abrasive potential compared to toothpaste containing HP 3% (calcium pyrophosphate and silica), but is similar to the abrasive action of conventional toothpaste (calcium carbonate and sodium bicarbonate).

Removing intrinsic stains is challenging to accomplish with toothpastes due to the limitation of the whitening agent's action in surface-bound stains ^7,29. The hydrogen peroxide present in toothpastes is challenging to establish ⁷. Besides, the reduced concentration of this component associated with the short time of contact with the tooth surface implies reduced intrinsic whitening of the dental structure since HP acts through the formation of free radicals and diffusion through the hard tissues ^11,29, which explains the inferior result of the toothpaste containing 3% HP, in terms of ΔWI_D in the present study.

Regarding the microhardness of dental enamel after whitening protocols, the highest microhardness value was observed in the group that used traditional toothpaste (control). Lower microhardness mean values were found in the group where a 10% CP agent was applied previously to the toothpaste containing 3% HP. Overall, the highest microhardness values were observed in the baseline measurements, where no treatments were performed on the dental enamel. Thus, the third null hypothesis, that there would be no differences in enamel microhardness regardless of the treatment, was rejected.

It is well known that the dental bleaching process, which involves applying bleaching gel to the enamel surface for extended periods, may have detrimental effects on the enamel structure, such as changes in microhardness ³¹. Regarding whitening toothpastes, the composition and type of whitening agent might affect the enamel microhardness ³¹. According to two systematic reviews and meta-analyses, a reduction in microhardness of the enamel surface occurs when whitening toothpastes are used ²⁷. The observed alterations are attributed to the presence of whitening agents (such as 3% HP) and the acidic and oxidizing properties of these products, which cause mineral loss ²⁷, in addition to the abrasive action of the particles on the surface.

Thus, based on the results of this study, the use of whitening toothpastes aimed at promoting the recovery of color obtained through a previous bleaching protocol is not effective, and traditional toothpastes yield similar results. Besides, the two whitening toothpastes tested in this study showed reduced enamel microhardness. This study presents some limitations that must be considered. The pH and abrasivity of the tested toothpastes were not assessed, although both parameters are known to directly affect enamel microhardness and whitening performance. In addition, samples were stored in distilled water throughout the experimental procedures, and remineralizing solutions, such as artificial saliva, were not used, which may have influenced the microhardness outcomes. Although the primary focus was on the ability of whitening toothpastes to maintain the color achieved by conventional dental bleaching after a staining challenge, detailed pigmentation data were not included, though the results confirmed that the whitening effect was influenced by staining. Moreover, as an in vitro investigation, the experimental conditions do not fully replicate the dynamic oral environment, where factors such as saliva composition, biofilm formation, and salivary flow may further modulate the results. Thus, the present findings should be interpreted with caution, and further in situ and clinical studies are required to validate these outcomes.

Acknowledgments

The authors would like to thank the Coordination for the Improvement of Higher Education Personnel (CAPES) for supporting this study (cod: 001).

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• Data availability
  The research data are available within the article.

Data availability

The research data are available within the article.

Publication Dates

• Publication in this collection
  15 Dec 2025
• Date of issue
  2025

History

• Received
  14 Apr 2025
• Accepted
  29 Sept 2025