Whitening mouthwash containing hydrogen peroxide decreases enamel microhardness in vitro

Aim: To assess the effect of a mouthwash containing hydrogen peroxide (HP) on Knoop microhardness (KMH) of bovine enamel. Methods: Fifty-one enamel slabs were polished and divided into groups (n=17), according to the product used during 28 days: HP – mouthwash containing 1,5% of HP (4 min, once/day); CP 10% carbamide peroxide gel (2 hours/day); AS no treatment (kept in artificial saliva (AS). Each fragment was submitted to KMH test (three indentations/fragment, with a 50 g load for 5 sec) four times: before (baseline); during (14 and 28 days) and after (7 days immerged in AS) the bleaching treatment. The data were submitted to repeated-measures two-way ANOVA (α=0.05). Results: There was no effect of the interaction between the time and treatment factors (p=0.327). No significant effect was observed from the time factor (p = 0.054). The factor treatment showed significant effect (p =0.002). Regardless of time, the KMH of the enamel submitted to HP was lower than the value observed with the use of CP, which did not differ significantly from the control group (AS). Conclusion: Although there was a trend of decreasing enamel microhardness over time, only the mouthwash containing hydrogen peroxide had a significant effect.


Introduction
One of the most important factors for the aesthetic balance of the smile in our society is the color of our teeth, for being quickly noticed even before many other aesthetic anomalies.Tooth discoloration or stains can be located on the surface of dental enamel (extrinsic stains) or in dental structure (intrinsic stains) 1,2 .
Extrinsic stains are obtained after the dental eruption in the oral cavity, from food or oral use products with great staining potential such as tea, coffee, tobacco, red wine, cola based beverages, etc. Intrinsic stains have a multifactorial etiology, both pre-and post-eruptive 3 .In vital teeth, discoloration can be natural (teeth with yellow or gray shades), caused by the ingestion of tetracycline or fluoride, or defects of congenital formations (amelogenesis and dentinogenesis imperfecta) 4,5 .
Dental bleaching is being a highly requested procedure by patients seeking cosmetic dentistry resources as a result of a poor aesthetic, caused by dental discoloration 6,7 .Hydrogen peroxide has been used for dental bleaching in the late 1800s, firstly with the aim of removing pigments from non vital and later from vital teeth 8 .Hydrogen peroxide is a vehicle of oxygen radical, which promotes oxidation and reduction of pigments.These pigments are fractionated in to smaller molecular chains, being entirely or partially removed from the dental structure by diffusion 9 .
For vital teeth, the techniques are the in-office technique, the over-the-counter bleaching, and the home-use nightguard vital bleaching technique, being each technique driven by different peroxide concentrations, time and mode of whitening product application.The home-use technique consists in applying a 10 or 16% carbamide peroxide gel that can be applied every night for 6 to 8 hours, or during the day, for one or 2 hours, with less tooth sensitivity within the decreased application time 10 .In the in-office technique the soft tissues are protected, and a hydrogen peroxide gel in a higher concentration is applied several times in a short period of time over the dental surface 8 .
The over-the-counter technique uses products that are bought in supermarkets or drugstores, such as bleaching toothpastes and mouthwashes, aimed to whiten teeth in short periods of time with a low cost.These mouthwashes have in addition to water, antimicrobials, salts, and sometimes alcohol, and also contain hydrogen peroxide in its composition 11 , but in lower peroxide concentrations (around 1,5%) than those observed in in-office and home-use gels.Although the effectiveness of over-the-counter products in dental bleaching has been reposted, it is less than the one obtained from home-use carbamide peroxide at 10% 12 .Besides this, controversies are known about the effects of bleaching agents on the microhardness of dental structures, especially the enamel 13 .
The over-the-counter products has been known to reduce the hardness of dental enamel 14 and resin composites 15 when used for 14 days, depending on the trade mark being used, and whether or not the product contains hydrogen peroxide (active component).The literature is still scarce to affirm the relation of mouthwashes recently launched on the market, which contains the bleaching agent (hydrogen peroxide) with the possible loss of minerals, and consequently a decrease of enamel microhardness.This issue is especially important if one considers that patients can use this product without the supervision of a professional and that manufacturer indicate that they can be used for 28 consecutive days, a relatively long time.
Considering that peroxide-containing mouthwashes are available for purchase without the need of a dentist recommendation and that they can affect enamel structure in someway, it is necessary to conduct experimental studies to verify this possible association.Thus, the aim of this study was to monitor the effect of a mouthwash containing 1.5% of hydrogen peroxide, compared to 10% carbamide peroxide gel, by means of Knoop microhardness test in bovine enamel, in the treatment periods (for 28 days) and post-bleaching (7 days).The null hypothesis was that neither bleaching agents nor treatment periods would affect enamel Knoop microhardness.

Materials and Methods
Ethical aspects -This study was approved by the Animal Research Ethical Committee (protocol #2013/0143).

Experimental Design
The factors under study were the bleaching agents, at three levels, and times of treatment, at four levels: 1. Treatment agents: CP -10% carbamide peroxide.HP -Mouthwash containing 1.5% hydrogen peroxide.
The experimental units were composed of 51 enamel slabs randomly distributed into the three levels of treatment agents (n=17).The response variables were Knoop microhardness (KMH).Analysis in each period was performed in the same specimen, which consisted of a block.
The bleaching agents, as well as the artificial saliva, are shown in Table 1.

Preparation of Dental Slabs
In this experiment, 51 bovine incisors stored in thymol (0.1%, pH 7.0) were used.The teeth were debrided with scalpel blades and periodontal curettes and had their roots separate from the dental crowns using a diamond disc of high concentration (Extec Corp, Enfield, CT, EUA) in a precision cutter (Isomet 1000, Buehler Lake Bluff, Illinois, EUA).Longitudinal sections were cut to obtain 3mm x 3mm enamel slabs, and those with stains or cracks were excluded after visual observation under a stereomicroscope loupe (EK3S3, São Paulo, São Paulo, Brazil) at 30x magnification.Before (baseline), during (14 and 28 days) and 7 days after the treatment, all specimens were submitted to the microhardness tests, using a microhardness tester (Pan-Tec Digital microhardness tester HVS-1000/Panambra, São Paulo, São Paulo, Brazil) with a Knoop penetrator, performing three indentations with a 50-g load for 5 seconds.
As KMH is a non-destructive testing, analysis in each period was performed in the same specimen.

Treatment agent procedures
The treatment agents used in this study were described in Table 1, according to composition, manufacturer and pH values.
Group CP: 10% Carbamide peroxide gel (Opalescense PF, Brazil´s Ultradent, Indaiatuba, São Paulo, Brazil), simulating the home-use bleaching, applying 0.02 mL of the product over each specimen for two hours/day 12 .Then they were rinsed with distilled water and stored in 5 mL of artificial saliva, and kept in a bacteriological stove (Odontobrás Ind. e Com.Equip.Med.Odont.LTDA Model: ECB 1.3 digital, Ribeirão Preto/SP/Brazil), at 37°C.This procedure was repeated for four weeks.Hypodermic syringes were used for the accuracy of quantity of gel over each fragment.
Group HP: The specimens were immersed in the mouthwash containing 1 4 mL of the solution and subjected to agitation on an agitator platform (Cientec®, CT 158) simulating a mouth rinse, and rinsed with distilled water after the procedure and immersed in 5 mL of artificial saliva, kept in a bacteriological stove (Odontobrás Ind. e Com.Equip.Med.Odont.LTDA Model: ECB 1.3 digital, Ribeirão Preto/SP/Brazil), at 37°C.This procedure was repeated for four weeks.
Group AS: No bleaching agent was applied, the specimens were kept in 5 mL of artificial saliva that were renewed in every two days and the specimens were rinsed with distilled water and kept in a bacteriological stove (Odontobrás Ind. e Com.Equip.Med.Odont.LTDA Model: ECB 1.3 digital, Ribeirão Preto, SP, Brazil), at 37°C.There was no evident calculus or material over specimens stored in saliva for 28 days.

Post-Treatment Period
After the treatment, the fragments were kept in their individual receptacles with 5 mL of artificial saliva, in a bacteriological stove (Odontobrás Ind. e Com.Equip.Med. Odont.LTDA Model: ECB 1.3 digital, Ribeirão Preto/SP/Brazil), at 37°C for 7 days to evaluate the post-treatment period and a possible remineralizing effect of this solution.The solution was changed every two days.Knoop microhardness was also measured after 7 days of post-treatment.

Statistical Analysis
After checking normality (Kolmogorov-Smirnov; p>0.05) and homoscedasticity (Levene; p>0.05) of data, the repeated measures two-way analysis of variance was performed, followed by Tukey's test for multiple comparisons.Statistical calculations were performed with SPSS 20 (SPSS Inc., Chicago, IL, USA).The significance level was set at 5%.

Results
Table 2 shows the averages and standard deviations of Knoop microhardness for the tested groups, in different times.The two-way analysis of variance for repeated measurements showed no significant interaction between the factors under study (p =0.327).No significant effect was observed from the time factor (p = 0.054).The treatment factor showed significant effect (p =0.002).Tukey's test revealed that regardless of time, the microhardness mean of the enamel submitted to HP treatment was lower than that observed with the use of CP, which did not differ significantly from the control group (artificial saliva).Figure 1 illustrates a line chart of Knoop microhardness of experimental groups over time.It can be observed that although there was a trend of decrease in microhardness within time, no statistical difference was observed among the different periods of evaluation.

Discussion
The present in vitro study assessed the effect of a mouthwash containing 1.5% of hydrogen peroxide as an active component in its composition on bovine enamel surface microhardness and compared with 10% carbamide peroxide.
The results obtained by microhardness analysis showed that the specimens of bovine enamel submitted to whitening mouthwash had smaller microhardness values compared to those submitted to 10% carbamide peroxide gel.Therefore, the null hypothesis was rejected.
The pH of the products used were measured and showed the result of 6.8 to the whitening gel and 3.4 for the whitening mouthwash.It can be assumed that the low pH of the whitening mouthwash may have acted on the demineralization of enamel surface favoring the decrease of microhardness, since the pH value is less than the enamel critical pH (5.5).In fact, Lima et al. 16 (2013) demonstrated, by 3D scanning eletronic microscopy, the erosive potential of Plax Whitening associated with toothbrushing.
These results are similar to previous studies which also verified reduction of dental enamel microhardness 14 and resin composite 15 with the use of whitening mouthwashes.It would be interesting to investigate the effect of such products on the morphology of dental enamel by means of scanning electron microscopy and atomic force microscopy, however, literature is still scarce in these evaluations and should be considered in further studies.
Also, the manufacturer of the mouthwash recommends that the product should be used for 1 minute before and 1 minute after brushing the teeth, twice a day, totaling 4 minutes a day.In the present research, brushing was not considered so that only the whitening mouthwash effect was observed.It is speculated that the application of mouthwashes in association with brushing could have brought different results, as the literature has demonstrated that this association may cause enamel loss 16 .
On the other hand, the prolonged immersion time (4 consecutives minutes) can also have exacerbated the results in a more significative manner than fractioning the immersions two times a day, with storage of specimens in saliva between cycles.
Having this in mind, Potgieter et al. ( 2014) 17 demonstrated no statistical reduction in enamel microhardness when Plax whitening was applied twice a day, for 1 minute in each immersion, although they did not perform toothbrushing.
On the other hand, the use of the whitening gel containing 10% of carbamide peroxide did not influence significantly on the bovine enamel microhardness, a fact that may be related to the presence of fluoride ions in its composition, in addition to the pH value, greater than the critical pH for demineralization of dental enamel.In fact, Cavalli et al. 18 (2010) showed that the presence of fluoride in the composition of the carbamide peroxide based whitening gel minimized the loss of ions of the dental surface.In a literature review of Attin et al. 19 (2009) it was observed that 49% of the in vitro studies reviewed did not show microhardness reduction over time with the use of home-use bleaching agents.Previous experimental studies had similar results to those found in the present study 20,21 .Perhaps, the effects of carbamide peroxide gel on the dental structures may also be related to the time of gel in contact with dental structure.The manufacturer indicates the use of 10% carbamide peroxide gel for 8 hours, but, in the present study, the protocol of 2 hours of application was choosed because this time prooved to sufficiently bleach enamel 12 , reduce tooth sensitivity 10 , while does not affect the concentration of enamel minerals, assessed with the technique of enamel microbiopsy 22 .Thus, if the manufacturer's instructions were strictly followed, different results could be achieved.
The results of this study also demonstrated that, although there was a tendency of decrease in microhardness values for the time of 28 days, there was no significant difference in the evaluated times, when considered in conjunction with the bleaching treatments.This may have been attributed due to the presence of artificial saliva in all groups, which may have leveled the results of microhardness for being a ion supersaturated solution 23 that favors the absorption and precipitation of salivary components, such as calcium and phosphate 24 .
Although it has been reported the effectiveness of OTC products to whiten dental enamel 12 , 25 the present study demonstrated reduction in bovine dental enamel microhardness after using the mouthwash containing 1.5% of hydrogen peroxide, showing that such product should be used with caution, especially if considered the carcinogenic potential that such products may have 26 .Still, literature is scarce regarding the effects of such products on the mineral loss of tooth structure, requiring, therefore, more studies on this subject, mainly in situ studies and clinical trials.Also, the use of OTC whitening mouthwashes in conjunction with toothbrushing should be evaluated in further studies.
This study concluded that although there was a trend of decreasing microhardness values over time, only the whitening mouthwash containing hydrogen peroxide had a significant effect.

Figure 1 .
Figure 1.Chart representing Knoop microhardness of experimental groups over time.
dental uncovered by resin.After 24 hours, the specimens were removed from the molds and flattened in a pneumatic polishing machine (Ecomet/Automet 250, Buehler, Lake Bluff, II, EUA) with decreasing granulations (400, 600 and 1.200) (Arotec S/A Ind e Comércio São Paulo/SP/Brazil) of abrasive paper under water cooling, and cleaned in a Ultrasonic washer (Unique Ind. e Com, de Prod.Elet. The enamel slabs were embedded in polyester resin (Maxi Rubber Ind Quím LTDA.Diadema/SP/Brazil) in 2.0-cm diameter polyvinyl chloride (PVC) molds, leaving the exter-nal surface of

Table 1 .
Products used in the experiment.
.5% hydrogen peroxide (Colgate Plax Whitening, Colgate Palmolive Ind e Comércio Ltda.S.B. Campo/SP, Brazil), for four minutes, once a day, each specimen were submerged in * Measured in laboratory, in triplicate.

Table 2 .
Averages and standard deviations of Knoop microhardness for the groups tested, in different times.
Grand means followed by distinct letters statistically differ between each other (lowercase compare times and uppercase compare treatments)