After peroxide was introduced as tooth whitening agent by dentists in the late 60s, night guards for tooth whitening were announced by Heywood et al. [1,2] in the early 90s. The professional teeth whitening techniques in dentistry such as the use of high concentrations of hydrogen peroxide and photo catalysts have been gradually developed [3]. However, the tooth whitening agent, which can be purchased directly by consumers without a dentist’s prescription, was commercialized in the 2000s due to concerns on the side effects by the hydrogen peroxide such as irritation to the gums, tooth hypersensitivity and tooth surface erosion [4,5]. As for whitening efficacy of hydrogen peroxide, as many papers as fluoride of toothpaste have been published [6-8]. There is no doubt about the efficacy of hydrogen peroxide. However, the side effects still remain controversial. Götz et al. [9] evaluated whether a tooth whitening agent containing up to 16% hydrogen peroxide alters the tooth surface via an electron microscope image. It was reported that the tooth whitening effect was clearly increased, but the structure and fine strength of the tooth surface remained unchanged. In addition, Delfino et al. [10] evaluated a tooth whitening agent containing 6.5% hydrogen peroxide in teeth extracted from bovine, and reported that it did not affect the change in hardness of the tooth surface and subsurface. Meanwhile, Mahony et al. [11] reported that even if 6% hydrogen peroxide was used, the highest concentration of hydrogen peroxide in the oral remained at a very low concentration of 0.03%. Gerlach et al. [12] evaluated the content of hydrogen peroxide remaining after 5 minutes applying a patch containing 10% hydrogen peroxide. As a result, 7.3% in the patch, 6.4% in the teeth, 0.7% in the gums, and less than 0.014% in saliva. However, in the study of professional whitening agents with high concentration of hydrogen peroxide and light treatments, it was reported that changes in surface properties such as hardness reduction and acid erosion were observed on the extracted human teeth [13]. Lilaj et al. [14] reported that the concentration of hydrogen peroxide, pH, and contact time influence the surface change of enamel. In addition, in the clinical studies of Donly et al. [15] and Gerlach et al. [16] on the product containing 6.5% and 10% hydrogen peroxide, mild and transient tooth sensitivity and oral irritation were the most representative side effects. However, it was reported that there was no case of discontinuing use. Nevertheless, hydrogen peroxide is a strong oxidizing agent and when it is high in concentration, it can cause serious irritation to soft tissues such as gums. When the pH is very low, it can cause acid erosion on the tooth surface. For this reason, in the case of nonprescription over-the-counter (OTC) tooth whitening agents, which can be purchased and used by consumers directly without a doctor’s prescription, the regulation related with the concentration of hydrogen peroxide varies by country.

In this study, the concentrations of hydrogen peroxide which is widely used in OTC tooth whitening agents around the world were referred. The calcium elution and the surface damage of bovine enamel according to the concentration of hydrogen peroxide and pH were compared with commercial beverages.

1. Preparation of experimental solution

The 3.0%, 6.0% and 10.0% hydrogen peroxide solutions used in the experiment were prepared by diluting with distilled water using a 35% hydrogen peroxide solution reagent. Sepa-rately, for a 6% hydrogen peroxide solution, a diluted phosphoric acid solution (0.1 M) and a sodium hydroxide solution (0.1 M) were used to prepare the pH to be 3.0, 5.0, 7.0, and 9.0, respectively. As control solutions, pH 3.0, 5.0, 7.0 and 9.0 solutions were prepared in the same method using only distilled water without using a hydrogen peroxide solution reagent. As comparative commercial beverages, 3 types of carbonated beverages, orange juice, and black coffee in cans were purchased and used while refrigerated (Table 1).

Table 1 . Experimental solutions

Solutions	Items
Solution 1	Hydrogen peroxide 3.0%
Solution 2	Hydrogen peroxide 6.0%
Solution 3	Hydrogen peroxide 10.0%
Solution 4	Hydrogen peroxide 6.0% (pH 3.0)
Solution 5	Hydrogen peroxide 6.0% (pH 5.0)
Solution 6	Hydrogen peroxide 6.0% (pH 7.0)
Solution 7	Hydrogen peroxide 6.0% (pH 9.0)
Solution 8	Distilled water
Solution 9	Distilled water (pH 3.0)
Solution 10	Distilled water (pH 5.0)
Solution 11	Distilled water (pH 7.0)
Solution 12	Distilled water (pH 9.0)
Solution 13	Carbonated beverage 1
Solution 14	Carbonated beverage 2
Solution 15	Orange juice
Solution 16	Black coffee
Solution 17	Carbonated beverage 3

2. Bovine enamel sample preparation

About 50 bovine teeth without dental caries and tartar were extracted and cleaned for calcium elution experiment. The enamel part of the crown was cut using a diamond hand piece and only the pure enamel was selected. After grinding again with a metal mortar, it was sorted into 18 and 20 mesh so that the average particle size was about 1.0 mm. The selected bovine enamel particles were sufficiently washed with distilled water, immersed in 0.1% thymol solution, and stored in a refrigerator. The bovine teeth were obtained from the cattle slaughtered in accordance with the Korean animal slaughter regulations at permitted market. The reason for grinding and mixing enamel is to enlarge the contact surface to greatly expand the calcium elution and to minimize the influence by teeth characteristics.

The specimens used for surface observation were the recently extracted crown part of bovine enamel without cavities and tartar. Bovine enamel was cut into a round shape with a diameter of 3 mm with a drilling machine, and the surface of the enamel was exposed to the outside using an epoxy resin to form a round column shape. In order to make the enamel surface flat and smooth, the enamel surface was smoothed by polishing using a lapping machine and silicon carbamide papers (#200 to #1200). The measured value of the smoothness using Surface Profile Meter (SV-4000; Mitutoyo, Kawasaki, Japan) was 0.1 µm or less.

3. Measurement of the eluted calcium amount

The crushed bovine enamels were weighed exactly 1.0 g and placed in a 100 ml plastic container with a cap, and then exactly 24.0 g of each test solution was added. After shaking at 36℃ for 3 hours, the crushed bovine enamels were removed, and the eluted calcium amount was analyzed with an inductively coupled plasma atomic emission spectrometer, ICP (Optima 5300 DV; PerkinElmer, Connecticut, USA) at the wavelength of 317.93 nm.

4. Surface observation of bovine enamel

The 3 bovine enamel specimens were immersed in 25 ml of each test solution for 3 hours at 36℃, shaken, thoroughly washed with distilled water, and dried at room temperature. Specimens coated with platinum using a sputter coater were observed at 20,000 and 130,000 times magnification with a field emission scanning electron microscope, SEM (S-4800; Hitachi, Ibaraki, Japan) and an elemental analysis of bovine enamel surface was performed with an energy dispersive X-ray spectroscopy, EDS (E-max; Horiba, Kyoto, Japan).

5. Statistical analysis

Confidence interval estimates were analyzed with 95% confidence rate (α=0.05) using SPSS version 19 (IBM Co., Armonk, NY, USA), a statistical analysis program, for the eluted calcium concentration.

1. Eluted calcium amount on hydrogen peroxide concentration dependence

The eluted calcium amount from bovine enamel increased slightly to 0.41, 0.79 and 1.76 µg/ml when hydrogen peroxide was 3.0%, 6.0% and 10.0%, respectively and distilled water without hydrogen peroxide was analyzed to be 0.04 µg/ml (Table 2). The standard deviation value that appears in the instrument analysis was very small, so it was shown in each result table as a relative standard deviation value.

Table 2 . Eluted calcium amount by hydrogen peroxide solution

Solution	Items	Eluted calcium amount (µg/ml) confidence-interval estimate (α=0.05)	RSD (%)
Solution 1	Hydrogen peroxide 3.0%	0.41 (0.39-0.42)	4.35
Solution 2	Hydrogen peroxide 6.0%	0.79 (0.78-0.80)	1.94
Solution 3	Hydrogen peroxide 10.0%	1.76 (1.72-1.80)	3.75
Solution 8	Distilled water	0.04 (0.04-0.04)	2.20

RSD: relative standard deviation.

2. Eluted calcium amount on pH change dependence

The results of the eluted calcium amount according to pH change at 6% hydrogen peroxide concentration were 37.29, 1.16, 1.10 and 1.06 µg/ml respectively when the pH values (measured) were 3.02, 5.09, 7.05, and 9.08. When the pH values of distilled water without hydrogen peroxide were 3.09, 5.01, 7.00 and 9.04, the eluted calcium amounts were 23.31, 0.48, 0.36, and 0.15 µg/ml, respectively (Table 3).

Table 3 . Eluted calcium amount by pH change with and without hydrogen peroxide

Solution	Items	Measured pH	Eluted calcium amount (µg/ml) confidence-interval estimate (α=0.05)	RSD (%)
Solution 4	Hydrogen peroxide 6.0% (pH 3.0)	3.02	37.29 (36.96-37.61)	1.41
Solution 5	Hydrogen peroxide 6.0% (pH 5.0)	5.09	1.16 (1.15-1.17)	0.81
Solution 6	Hydrogen peroxide 6.0% (pH 7.0)	7.05	1.10 (1.07-1.12)	3.27
Solution 7	Hydrogen peroxide 6.0% (pH 9.0)	9.08	1.06 (1.05-1.06)	0.37
Solution 9	Distilled water (pH 3.0)	3.09	23.31 (23.14-23.48)	1.17
Solution 10	Distilled water (pH 5.0)	5.01	0.48 (0.47-0.48)	1.44
Solution 11	Distilled water (pH 7.0)	7.00	0.36 (0.35-0.36)	1.47
Solution 12	Distilled water (pH 9.0)	9.04	0.15 (0.14-0.15)	5.77

RSD: relative standard deviation.

3. Eluted calcium amount on commercial beverages

The measured pH values of five commercial beverages and the eluted calcium amount from bovine enamel after treatment were 47.70 µg/ml at pH 2.43 for carbonated beverage 1, 33.09 µg/ml at pH 2.44 for carbonated beverage 2, 65.69 µg/ml at pH 3.59 for orange juice, 46.17 µg/ml at pH 5.83 for black coffee, and 46.17 µg/ml at pH 3.01 for carbonated beverage 3 (Table 4). The above results are the amounts obtained by subtracting the calcium content of each beverage itself from the calcium content of each beverage solution treated with bovine enamel.

Table 4 . The pH and the eluted calcium amount by marketed beverages

Solution	Items	Measured pH	Eluted calcium amount (µg/ml) confidence-interval estimate (α=0.05)	RSD (%)
Solution 13	Carbonated beverage 1	2.43	47.70 (47.28-48.11)	1.40
Solution 14	Carbonated beverage 2	2.44	33.09 (32.14-34.03)	4.60
Solution 15	Orange juice	3.59	65.69 (64.47-66.91)	3.00
Solution 16	Black coffee	5.83	4.13 (4.02-4.23)	4.09
Solution 17	Carbonated beverage 3	3.01	46.17 (45.37-46.96)	2.78

RSD: relative standard deviation.

4. Surface observation and elemental analysis of bovine enamel

The 6 Solutions (3, 5, 8, 9, 13, 17) among 17 experimental solutions used in this study were selected for the analysis of bovine enamel surface. Bovine enamel specimens treated with each of the selected solutions were examined with an SEM and the elements on the surface of the enamel were analyzed with an EDS to check whether there was any change in the chemical composition of bovine enamel surface (Figure 1, 2).

Figure 1. Field-Emission Scanning Electron Microscope pictures after treatment (Left; 20,000 magnifications, right: 130,000 magnifi-cation). (A) Distilled water: Solution 8, (B) Hydrogen peroxide 10%: Solution 3, (C) Hydrogen peroxide 6% (pH 5.0): Solution 5, (D) Carbonated beverage 1: Solution 13, (E) Carbonated beverage 3: Solution 17, (F) Distilled water (pH 3.0): Solution 9.

Figure 2. Energy Dispersive X-ray Spectroscopy pictures after treatment. (A) Distilled water: Solution 8, (B) Hydrogen peroxide 10%: Solution 3, (C) Carbonated beverage 1: Solution 13, (D) Carbonated beverage 3: Solution 17, (E) Hydrogen peroxide 6% (pH 5.0): Solution 5, (F) Distilled water (pH 3.0): Solution 9.

The limited concentrations of hydrogen peroxide for OTC tooth whitening products in the several countries are 0.1% or less in Europe, 3.0% or less in Korea and China, and 6.0% or less in Taiwan. Since there is no regulation on the concentration of hydrogen peroxide in the United States, the analyzed concentration of OTC tooth whitening products sold in the United States was 3.0% to 10.0%. In this study, the concentration of hydrogen peroxide was set to 3.0%, 6.0% and 10.0% by referring to the concentrations of hydrogen peroxide used by countries. Al-Salehi et al. [17] compared and analyzed hardness of bovine teeth and quantity of calcium and phosphorus eluted from bovine teeth at 0, 3, 10 and 30% of hydrogen peroxide concentration. The results of this study were comparable to those of our study. The eluted calcium amount in bovine enamel gradually increased in proportion to the concentration, but the amount was very small. It slightly increased at 30% hydrogen peroxide concentration, which is the concentration of the professional tooth whitening product. In the study of Tezel et al. [18] the eluted calcium amount was evaluated with a whitening product with high concentration of hydrogen peroxide and relatively low concentration of 10% carbamide peroxide. The eluted calcium amount of carbamide peroxide group and the control group were 19.53±4.03 µg/ml and 18.35±4.00 µg/ml. There was no statistically significant difference between groups.

In this study, the eluted calcium amount tended to gradually increased by the increase of hydrogen peroxide (Table 2). This result was presumably due to the decrease of pH caused by the increased hydrogen peroxide. The eluted calcium amount after treatment showed that the pH effect was dominant over hydrogen peroxide effect (Table 3). In particular, the eluted calcium amount increased significantly at pH 3.0 but it was very small at pH 5.0 or higher. Hannig et al. [19] analyzed calcium and phosphorus eluted from bovine enamel in the high acidity region using various acids. It can be seen that the elution of calcium and phosphorus in bovine enamel increased significantly as the pH decreased. Barbour et al. [20] evaluated the hardness and elasticity after treatment for 120 seconds by considering the drinking time of beverages in the pH range of 2.3 to 6.3 using human enamel. The hardness showed a linear result, and the modulus of elasticity showed a quadratic curve with a gentle inflection point around pH 5.0. Although treatment time in our study was much longer than that in Barbour’ study, the change caused by acidity shows a similar pattern. Meanwhile, looking at the measured pH and the eluted calcium amount of the commercial beverages, all of them had low pH. Black coffee with the highest pH had the lowest eluted calcium amount of 4.13 µg/ml at pH 5.83. The three carbonated beverages and orange juice had a low pH of 2.43 to 3.59 and the eluted calcium amounts were about 8 to 16 times higher than black coffee. Compared to a solution containing 6% hydrogen peroxide and adjusted to pH 5.0, the eluted calcium amount was about 28 to 56 times higher. In particular, orange juice, which has a higher pH than carbonated drinks, was able to confirm the highest amount of calcium elution, which was thought to be the influence of various additives used to increase the marketability of orange juice (Table 4).

These results can also be seen from the results studied by Zimmer et al. [21] after bovine enamel and dentin were immersed in several kinds of acidic beverages at 37℃ for 7 days, the weight of lost tooth was evaluated. Orange juice showed more weight loss than coke with lower acidity and it was thought that not only the pH but also the substances used in the beverages are related to the calcium elution of teeth. Caneppele et al. [22] exposed bovine dentin to acidic beverages and analyzed the damaged depth and content of calcium and phosphorus at the tooth surface using a surface analyzer and energy- dispersive X-ray fluorescence spectrometry. It was concluded that although all beverages caused damage to the teeth, the extent was not proportional to the pH of the beverage. The pH range was 2.16 to 3.55 similar to our study. It can be seen that the above results are supported by the SEM photographs of bovine enamel specimen used in the experiment (Figure 1).

There was little change in bovine enamel surface treated with only water as a control group and bovine enamel surface treated with hydrogen peroxide. On the other hand, surface damage was observed even when only the pH of water was lowered to 3.0, and surface damage was evident in two types of carbonated drinks with low pH. Another peculiarity here is that the surface damage of bovine enamel treated with carbonated drinks (pH 3.01) was observed to be greater than treated with the water only which is almost the same pH (pH 3.09). It seems to be that not only pH but the ingredients influenced calcium elution. In a study by Ren et al. [23] which compared orange juice (pH 3.8) and 6% hydrogen peroxide tooth whitener (pH 5.5), there were enamel hardness reduction, surface roughness, and enamel surface damage in orange juice. However, the tooth whitening agent containing 6% hydrogen peroxide was similar to the control group treated with water. These results indicate that pH takes precedence in damage to the enamel, and the effect of hydrogen peroxide itself is insignificant. The results of the atomic detector using the EDS connected to the SEM showed that all experimental groups were similar to bovine enamel treated with water only (Figure 2). The changes in the atomic level of the enamel surface due to hydrogen peroxide need to be studied in the future using more precise analytical instruments, and the effects of various additives used in beverages need to be studied. In addition, the next study aims to find the pH point that can significantly damage teeth by performing at the pH 3.0 to 5.0 range in tooth whitening agents containing hydrogen peroxide.

The following conclusions were obtained as a result of the eluted calcium amount and surface change of bovine enamel according to changes in hydrogen peroxide concentration and pH with comparing commercial beverages. The change in the eluted calcium amount from the bovine enamel according to the concentration of hydrogen peroxide was very small and the surface change by the SEM and elemental analysis by the EDS showed no difference from the surface treated with water. Regardless of hydrogen peroxide, calcium elution and surface damage of bovine enamel were small at pH 5 or higher, but strong calcium elution and surface damage of bovine enamel were observed near pH 3. Carbonated beverages and orange juice caused higher calcium elution and surface damage to bovine enamel than 10% hydrogen peroxide solution.

No potential conflict of interest relevant to this article was reported.