Effect of whitening toothpastes and activated charcoal powder on enamel wear and surface roughness

Abstract This study aimed to evaluate surface roughness (Sa), roughness profile (Rv), and enamel wear after brushing with different whitening toothpastes and charcoal powders. Sixty (n = 10) bovine enamel blocks (6 × 6 × 3 mm) were randomly distributed into six groups according to toothpaste type: regular toothpaste (CONT), toothpaste containing 2% hydrogen peroxide (HP), toothpaste containing titanium dioxide (TiO2), toothpaste containing charcoal (COAL), toothpaste containing charcoal and TiO2 (COAL+TiO2), and activated charcoal powder (COAL_PWD). Each block was subjected to 30,000 reciprocal cycles at a 1:3 proportion slurry. After brushing, the blocks were analyzed using an optical profilometer to determine Sa, Rv, and enamel wear. In addition, representative 3D images of each group and wear profiles were obtained. Sa was analyzed using generalized linear models followed by Bonferroni correction, whereas Rv was analyzed using one-way analysis of variance. After brushing, COAL and COAL+TiO2 showed higher Sa values than COAL_PWD. However, no significant difference was observed in Sa between whitening toothpaste and COAL_PWD, and CONT (p > 0.05). In addition, no differences were observed among the groups in Rv (p > 0.05). Conversely, enamel wear was higher for TiO2, COAL, COAL+TiO2, and COAL_PWD than for CONT. CONT showed the least enamel wear, whereas HP showed intermediate values. Representative 3D images and line profiles showed lower step-height and lower mean surface losses for the CONT and HP groups than for the other groups. Whitening toothpastes and COAL_PWD did not increase Sa or Rv compared with CONT, while CONT demonstrated lower enamel wear.


Introduction
Tooth discoloration can affect self-esteem and quality of life. 1 Some lifestyle factors such as smoking, drinking colored beverages such as tea, coffee, and red wine, and insufficient dental hygiene may cause tooth discoloration. 2 Tooth bleaching with hydrogen peroxidebased agents is a conservative and effective approach to resolve tooth discoloration and render optimal esthetic outcomes. 1Nevertheless, alternative oral care products with claims of a dental whitening effect Declaration of Interests: The authors certify that they have no commercial or associative interest that represents a conflict of interest in connection with the manuscript.
have gained attention in recent years due to their convenient over-the-counter access and lower costs. 1 Toothpastes with a whitening effect usually contain abrasive substances and/or chemical or optical agents.Regular toothpastes have highly complex formulations that include humectants as solvent, abrasives, surfactants, thickening agents, fluorides, opacifying agents, sweeteners, colorants, flavorings, and buffering agents. 34][5] Furthermore, pigments such as blue covarine or titanium dioxide (TiO 2 ), or even hydrogen peroxide have been added to whitening toothpastes. 4,5s abrasives are harder than the stain, removal of extrinsic stains adherent to the enamel surface is successful during tooth brushing without affecting the underlying intrinsic discoloration or natural shade of the tooth. 6Blue covarine may cause an optical illusion after its deposition on the tooth surface, which can shift the color perception from yellow to blue. 7A similar optical effect may be obtained with TiO 2 based on its intense white aspect. 8,9ydrogen peroxide is another agent used to achieve the so-called whitening effect.The mechanism of action is similar to in-office or at-home bleaching procedure: oxidation of organic chromophores to non-colored organic compounds. 4These modifications would turn stains into imperceptible compounds by the human eye. 10Since a very low concentration of hydrogen peroxide (0.1-2%) is added into toothpastes allied to the inherent instability of peroxide, it may directly compromise the whitening action. 10The abrasive property and chemical composition of toothpastes may cause wear and lead to tooth sensitivity. 11s for charcoal, its abrasive potential varies depending on the source and methods of preparing and milling the charcoal. 12It can be extracted from different natural sources such as nutshells, coconut husks, bamboo and peat, and even wood or coal. 12ately, charcoal products used as whitening agents are available in powder forms, incorporated into several toothpastes, and diluted into mouthrinses. 13,14 is important to note that using the powder adds an additional step in the toothbrushing habit since the powder cannot replace the use of fluoridecontaining toothpastes. 8The fine powder used alone or incorporated into toothpastes is activated charcoal, which has been oxidized by controlled reheating or chemical means. 15Activated charcoal has a high surface area, thereby absorbing pigments and chromophores and removing stains. 16However, the whitening effect of activated charcoal products and their impact on the enamel surface remains questionable. 8,13For instance, charcoal powder alone was found to increase enamel surface roughness and not whiten teeth in vitro, 8 while a charcoalcontaining toothpaste powder did not promote greater surface loss compared with brushing with no toothpaste. 17herefore, this study aimed to evaluate the effects of five different toothpastes and charcoal powder on enamel surface roughness and enamel wear of bovine teeth after simulated tooth brushing.The five products included regular toothpaste (CONT), toothpaste containing 2% hydrogen peroxide (HP), toothpaste containing titanium dioxide (TiO 2 ), toothpaste containing charcoal (COAL), toothpaste containing charcoal and TiO 2 (COAL+TiO 2 ),and activated charcoal powder (COAL_PWD).The null hypotheses were as follows: a) whitening toothpastes and charcoal powder do not increase enamel surface roughness compared with regular toothpaste after brushing cycles and b) whitening toothpastes and charcoal powder do not cause more enamel wear compared with regular toothpaste after brushing cycles.

Methodology Experimental design
Bovine enamel blocks (n =1 0/group) were subjected to brushing with six different toothpaste types: regular toothpaste (control), hydrogen peroxide-containing toothpaste, TiO 2 -containing toothpaste, two activated charcoal-containing toothpaste, and activated charcoal powder.Surface roughness (Sa), roughness profile (Rv), and enamel wear were measured after simulated tooth brushing.

Specimen preparation
Bovine enamel blocks were obtained to measure the effects of different whitening toothpastes on Sa, Rv, and enamel wear.Bovine incisors were cleaned and stored in 0.1% thymol solution at 4°C for no longer than 60 days.Teeth without cracks and defects in the enamel and with at least 3.5-mm thickness of the buccal hard tissue were selected (60 incisors).Roots were removed 2 mm below the cementoenamel junction using a low-speed precision cutting machine (Isomet, Buehler, Lake Bluff, USA) under water refrigeration.One block (6 × 6 × 3 mm) of each tooth was obtained from the central region of the crown.The enamel surface of each block was flattened, finished with silicon carbide sandpaper (#600, #1200, and #2000 grit), and polished using a diamond aqueous suspension (1/4 mm) with polishing cloths and a polishing machine (Arotec Ind. Com., São Paulo, Brazil).Dentin was flattened using a polishing machine equipped with #600 silicon carbide sandpaper.After finishing and polishing, the blocks were cleaned in an ultrasonic bath for 10 min and stored in distilled water at 4°C until use.

Group division
The blocks were randomly divided into six groups (n = 10) according to the brushing agent used: a) Colgate Triple Action (CONT): a regular toothpaste, b) Colgate Luminous Advanced Expert (HP): 2% hydrogen peroxide-containing toothpaste, c) Colgate Optic White Stain Fighter (TiO 2 ): TiO 2 -containing toothpaste, d) Colgate Optic White with Charcoal Teeth Whitening (COAL): activated charcoalcontaining toothpaste, e) Crest 3D White Whitening Therapy Charcoal Deep Clean (COAL+TiO 2 ): activated charcoal-and TiO 2 -containing toothpaste, and f) activated charcoal powder (COAL_PWD).The sample size was calculated with a desired power of 95% and α = 0.05, and at least eight samples were obtained for each group.The brushing agents used in this study are listed in Table 1.

Brushing protocol
Adhesive tape (Floor and Safety Marking Tape, Scotch ® 3M, Austin, USA) was applied to only half of the enamel surface of each block to protect this region from brushing and to serve as a control area for enamel wear and Rv.The blocks were then attached to a mechanical brushing machine (MEV 4-10XY -Odeme Dental research, Luzerna, Brazil) with the enamel surface positioned upward.The uncovered area was subjected to 30,000 reciprocal strokes (150 cycles/min) with a load of 200 g, simulating approximately 2 years of toothbrushing. 18The same soft toothbrushes (Colgate Classic Clean Toothbrush, Colgate-Palmolive Company) were used for all groups.The blocks were then immersed in a 1:3 slurry prepared using toothpaste or charcoal powder in distilled water.The pH of the slurry was measured using a pH meter (Ms Tecnoponon Instrumentação, Piracicaba, SP, Brazil).After brushing, the samples were washed in running water and stored in distilled water at 37°C.

3D Optical profilometer analysis
A non-contact 3D optical profilometer (Zygo New ViewÔ 7300, Zygo Corp., Middlefield, USA) with 0.1-nm height resolution was used to scan the enamel surface and determine Sa, Rv, and wear resistance.Three 1-mm 3D images of the center of each block were obtained, with one half of the blocks being the control area and the other half the brushed area.The 3D images were analyzed using ProfilmOnline (Filmetrics Inc., San Diego, USA).For Sa, which describes the mean arithmetic height in three dimensions, two 300 mm × 300 mm measurements for each area (control and brushed) were performed for each 3D image.Rv (2D), which corresponds to the maximum valley depth deviation from the mean line, was calculated using the mean of three measurements for each image (1 mm length).The depth of the brushed surface (surface wear) was assessed using the two-point height tool (step-height measurement in line) of the ProfilmOnline.The mean step-heights of the control and brushed areas were calculated to determine the surface loss for each image.Representative images based on the average values were obtained for each group.Data were exported to software (Origin-Pro 2022, OriginLab, Northampton, USA), and representative line profile graphs were obtained for each group.Enamel wear and Sa (within-subject factor: brushing; betweensubject factor: group) data were analyzed using generalized linear models corrected for multiple testing by the Bonferroni correction, whereas Rv was analyzed using one-way analysis of variance (p < 0.05).

Results pH, Sa, and Rv
The pH of the slurry varied from 6.5 (HP) to 10 (COAL_PWD) and is presented in Table 1.Table 2 lists the Sa values of the control and brushed areas.Generalized linear models indicated that the "group" factor significantly influenced Sa (p = 0.018), while brushing and the interaction group*brushing did not (p = 0.912 and p = 0.255, respectively).No significant differences were observed among the groups when only the control areas were compared, indicating a homogeneous distribution of the samples among the groups before brushing.After brushing, TiO 2 (p = 0.006) and COAL+TiO 2 (p = 0.005) toothpastes demonstrated higher Sa values than COAL_PWD.The other toothpastes presented intermediate Sa values, with no significant differences among them.When comparing Sa of the brushed side with that of the corresponding unbrushed side, no significant differences were observed for any of the brushing agents (p > 0.05).Numerically, half of the groups presented a decrease in Sa values (from 2.9% to 21.9% among the brushing agents), while the other half presented an increase (from 4.7% to 13.8%).In addition, no significant difference was observed in Rv among the groups (Table 3).

Enamel wear
Figure 1 shows the enamel wear values for each tested group.Unlike Sa and Rv, enamel wear was higher for TiO 2 , COAL, COAL+TiO 2 , and COAL_PWD than for regular toothpaste (CONT; p < 0.001) and HP (p < 0.001).In addition, HP presented a higher enamel wear than CONT (p = 0.038).Representative surface 3D images are shown in Figure 2, and Figure 3 shows representative graphs of the line profiles for each brushing agent tested.

Discussion
The first null hypothesis was accepted because all whitening toothpastes and charcoal powder presented similar Sa and Rv compared with regular toothpaste after brushing.However, COAL_PWD presented lower Sa than the TiO 2 and COAL+TiO 2 toothpastes, indicating that charcoal powder can promote more homogenous surface wear, acting similarly to polishing materials.Although no statistically significant difference was detected, COAL_PWD reduced Sa after brushing (21.9% reduction), corroborating the expected polishing effect.In contrast to other toothpastes, TiO 2 and COAL+TiO 2 toothpastes also contain hydrated silica.Both these toothpastes showed a slight increase in Sa (13.8% and 7.2%, respectively), with no significant difference.Hydrated silica is a medium-hard abrasive added to toothpastes to remove stains. 4However, its abrasiveness depends on the amount, size, shape, and percentage of the silica added, 4,19 which is not disclosed by the manufacturer.
It is unclear whether Sa plays an important role in bacterial adhesion.Some studies have shown that roughened surfaces can significantly enhance bacterial attachment, 20,21 while others have failed to report roughness as a determining factor for bacterial adhesion 22 and biofilm formation. 23Regarding microbiological aspects, Rv seems to be more relevant to the growth and development of Streptococcus mutans than Sa.The morphology of the depressed areas helped to avoid the displacement of S. mutans colonies, improving consolidation of the acquired pellicle. 24Although no significant differences were observed for Rv among the groups in this study, deeper valleys were observed for all whitening toothpastes and charcoal powder than for CONT (Figure 3).In contrast, in a recent systematic review and meta-analysis, the majority of studies (five of seven) revealed an increase in Sa, which could be attributed to the use of whitening toothpastes.Consequently, the meta-analysis showed a positive correlation between Sa and the intervention group (whitening  toothpastes). 25Additionally, an in vitro study observed surface morphology changes when a whitening toothpaste containing charcoal was tested.The whitening toothpaste groups presented large and deep craters in conjunction with other flaws on the surface, despite the initial smooth aspect. 14Even with the benefit of stain removal, abrasives might increase the risk of superficial damage to dental tissues and restorative materials. 26lthough no difference in surface roughness was observed between the unbrushed and brushed sides for the same toothpaste, differences in enamel wear were observed, and thus, the second null hypothesis had to be rejected.The main finding of this study was the lowest wear after 30,000 brushing cycles for the CONT group.This toothpaste contains sodium bicarbonate as an abrasive, which is reported to have a low hardness. 4We suggest that this softer abrasive prevents the increase in enamel wear following brushing cycles compared with the other toothpastes.HP exhibited an intermediate wear value.It contained silica as an abrasive and had the lowest slurry pH of 6.5.Although an increase in enamel wear was observed for HP compared with the CONT group, TiO 2 , COAL, COAL+TiO 2 , and COAL_PWD presented the highest wear values.The last four groups also presented a high variation of values within the same group (from 1-2 to 3.5-4 mm of enamel wear) and had abrasives with medium hardness. 4We hypothesize that the slurry was not always homogenous for these groups, which affected the maintenance of abrasives between toothbrush bristles.However, these groups exhibited surface wear that was always greater than 1 mm, which was twice that observed for CONT.The COAL_PWD group presented further surface loss when sequential brushing was performed with any of the whitening toothpastes.As the manufacturer indicates powder use prior to regular brushing and the patients decide which product to purchase, surface loss might be exacerbated in a clinical setting depending on the powder and toothpaste combination.
One reason for concern is that most of these toothpastes do not include the abrasiveness index (RDA) on the label.According to the American Dental Association (ADA), the maximum RDA value to avoid structural damage to dental tissues is 250. 4 Further, ADAapproved toothpastes should also contain fluoride. 27owever, these so-called natural, ecofriendly, and organic toothpastes have gained visibility, as shown in a recent study that evaluated the labels of 50 dental products containing activated charcoal. 13Another main concern is the absence of fluoride (in powder or toothpaste) and charcoal's potential to inactivate fluoride due to it absorptive capability. 13,28ll tested toothpastes contained fluoride, except for COAL_PWD, which is a powder containing carbon, kaolin, Citrus Aurantium Dulcis oil (orange oil), and flavor, according to the manufacturer.Fluoride can be found in different formulations such as NaF, Na 2 FPO 3 , C 2 7H 60 F 2 N 2 O 3 , SnF 2 , or a combination of these. 29Since no erosive challenge was performed with low-pH substances in this study, we could not evaluate the protective effect of fluoride.However, the beneficial effects of fluoride are well described in the literature.It acts as a catalyst in the de-remineralization process, accelerating remineralization by around five times.This is possible because of reservoir formation in saliva, which remains for a few hours, and aggregation in the dental biofilm, which lasts for an extend period. 30or this reason, toothpastes (or any product designed to substitute toothpastes) without fluoride should be discouraged.However, it should be kept in mind that a pH higher than 5.5 along with the presence of fluoride in all toothpastes do not rule out the possibility of decrease in enamel mineral content.An in situ study indicated that toothbrushing with fluoride-containing toothpaste was not capable of maintaining the enamel surface microhardness. 31herefore, abrasion may play a fundamental role in the regulation of enamel mineral loss.
Phosphoric acid can also be found in toothpaste and claims to control pH in TiO 2 toothpastes.However, it is unclear whether the presence of phosphoric acid has a microabrasion effect (similar to products designed for microabrasion).The microabrasion uses phosphoric acid to change the optical characteristics of the enamel and increase the polishing aspect, resulting in different light refractions and the ability to camouflage stains. 32Moreover, restorations might also be affected, losing the polish, brightness, and volume, as well as increasing Sa.Recently, charcoal-based toothpaste and powder were shown to significantly increase surface loss with a conventional composite by means of a profilometry method. 33hus, the use of whitening toothpastes and powders requires caution, especially in patients with risk factors for gingival recession, non-carious lesions, and hypersensitivity 3,34 and should not be used for a prolonged time.
Based on this, it is important to highlight that even though systematic reviews and metaanalyses have concluded that whitening toothpastes indeed promote a higher color change than regular toothpastes, 35,36 clinical evidence indicates that the whitening effect of these toothpastes is not longlasting. 37In other words, patients would have to prolong their use, thereby increasing the risk of enamel surface loss, as shown in this study.Instead, patients could seek professionally supervised bleaching with long-term efficacy and safety, which are well-established in the literature. 38,39In addition, even though these systematic reviews were conducted prior to the massive launch of activated charcoal products, in vitro studies have already reported their inefficacy in promoting color change. 8,40Therefore, it becomes even clearer that the use of such products should be discouraged by dentists.
It is worth mentioning that this study presents the inherent limitation of an in vitro design, not ruling out the action of human saliva on upholding or recovering Sa, Rv, and enamel wear.Additionally, no temporal evaluation of the variables was conducted.In other words, analyses could be performed at different time points to determine whether a certain brushing time was detrimental to the enamel surface.Finally, the lack of colorimetric evaluation could be questioned, but previous studies have shown that charcoal-based products are not as effective as peroxide-based agents in resolving tooth discolation. 8,15

Conclusion
Within the limitations of this in vitro study, it can be concluded that a. Different agents added to the toothpastes tested did not affect the enamel Sa. b.Rv was similar for all the toothpastes tested.c.Brushing with the regular toothpaste resulted in the lowest enamel wear after 30,000 cycles.
Figure1shows the enamel wear values for each tested group.Unlike Sa and Rv, enamel wear was higher for TiO 2 , COAL, COAL+TiO 2 , and COAL_PWD than for regular toothpaste (CONT; p < 0.001) and HP (p < 0.001).In addition, HP presented a higher enamel wear than CONT (p = 0.038).Representative surface 3D images are shown in Figure2, and Figure3shows representative graphs of the line profiles for each brushing agent tested.Figure2A(CONT) and Figure2B(HP) show a lower and subtle step-height of the brushed surface than the other

Figure 1 .
Figure 1.Box-plot graph of enamel wear of each toothpaste and charcoal powder.Different lower-case letters indicate significant differences between groups.CONT presented the lowest wear values, followed by HP.COAL, COAL+TiO 2 , and COAL_PWD presented the highest wear values, with no difference between them.

Figure 2 .
Figure 2. Representative 3D surface images of unbrushed (left side) and brushed (right side) areas of each group tested.(A) CONT.(B) HP. (C) TiO 2 .(D) COAL.(E) COAL+TiO 2 .(F) COAL_PWD.Height axes indicate that COAL, COAL+TiO 2 , and COAL_PWD presented a more evident and deeper step on the brushed side compared with CONT and HP.

Table 1 .
Commercial name, manufacturer, composition, and fluoride concentration of each toothpaste tested.
*According to the manufacturer.**The composition is described as per the Brazilian version of toothpastes.The composition may vary by country.

Table 2 .
Mean (95% CI) surface roughness (Sa in µm) before and after brushing.Percentages were calculated using the mean Sa for each side.Different letters indicate significant differences between the groups.Lower-case letters compare different toothpaste types within the same brushing condition (control x brushed).Upper-case letters compare different brushing condition within the same toothpaste type. *