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Development of an analysis method to determine peroxides in or released from tooth-whitening/dental-bleaching products.

4 4 Tests on products

On the basis of the method development and the tests on the pure substances used in tooth-whitening products, a protocol for analysis of the peroxide content in tooth-whitening products was set up.

Based on the fact that the analysis uses a sealed analysis chamber of about 100 ml in accordance with the description in section 3.1, the following standard conditions can be established:

1. The analysis is conducted at 22°C.
2. The quantity of active substance (peroxide) in the sample is limited, so that the maximum oxygen development corresponds to 80% of the buffer’s oxygen saturation. This means (where the chamber volume is 100 ml and the temperature is 22°C) a maximum oxygen development of 0.88 mg O₂.
3. 5,000 units of catalase per reaction are used in order to ensure an adequate reaction rate.
4. Results are read after 5 minutes (providing the signal is stable).
5. pH in the reaction chamber is adjusted with a 50 mM phosphate buffer to 7.0.

A number of tooth-whitening products already on the market in Denmark were examined using this protocol.

The products involved in the validation represent:

1. Different concentrations of peroxide
2. Different contents of active substances
3. Different formulations of the product

The procedure for measuring active substances in tooth-whitening agents includes two types of controls:

1. Parallel determination on a pure substance in a known quantity
2. Adding a pure substance in a known quantity as a spikeafter the reaction with the tooth-whitening agent has ended

A total of seven products were analysed in repetitions of at least 14:

Table 5: List of products tested (anonymised)

The Danish Environmental Protection Agency has been provided with further information about the products, (such as retailer, product no., batch no. etc.).

In parallel with the catalase method, all the products were also analysed traditionally using redox titrations. All the products and the two pure substances, hydrogen peroxide and urea peroxide, were titrated with potassium permanganate, while the products containing urea peroxide were also titrated with sodium thiosulfate.

The results of both titrations and test of the catalase bioassay are shown in Table 6, along with the manufacturers’ specifications of contents. For three of the products, information on the quantity of active substance in the product was not available.

Table 6: Peroxide content in 17 tooth-whitening products as well as hydrogen peroxide and urea peroxide found from the two titration methods and the catalase bioassay, respectively.

In Annex 4, the data of Table 6 is shown with standard deviations.

In general, there was good correspondence between the concentrations found through titration and those found using the catalase bioassay. The deviations were particularly small between the concentrations found through KMnO₄ titrations and the catalase bioassay.

Products “E” and “F” were analysed twice, as no hydrogen peroxide could be detected in product “E” by neither titration or by the catalase bioassay. Similarly, only an insignificant, small quantity of peroxide was found in product “F” using the catalase bioassay, while none could be detected by titration.

In order to determine whether this lack of peroxide content was due to a flaw in the assay or in the product, another set of products was purchased and analysed. This time around, peroxide was found in product “E” as well as “F” by both methods.

Determination of the peroxide concentration in the tooth-whitening products was conducted on several different sample concentrations using the catalase bioassay, and in at least 14 repetitions per product. Correspondingly, the assay was tested on the pure substances, hydrogen peroxide and urea peroxide, in a number of different concentrations.

The coefficient of variation for measurements of products using the catalase bioassay was 0.9 – 3%. Correspondingly, the variation for measurements of the pure, active substances was 0.9 – 1.9%. The redox titrations were conducted in repetitions of three on both products and pure substances. For the potassium permanganate titrations of the tooth-whitening products, the coefficient of variation was 0.4 – 2.4%. For the pure substances, the variation of the measurements was 0.4 – 1%. For the sodium thiosulfate titrations, the variation was very high for the products, namely 9.4 – 23.8%. However, the variation for sodium thiosulfate titration of urea peroxide in pure form was only 0.33%.

With regard to the tested products, the variation of (accuracy of) the catalase bioassay was therefore comparable to the variation of the chemical analysis based on potassium permanganate titration, while the catalase bioassay appeared more accurate than the sodium thiosulfate titrations.

With regard to the manufacturers’ specifications of the peroxide content in the products, the determinations of concentration using the catalase bioassay deviated by 3.3 – 5.9%. The concentrations found from potassium permanganate titration deviated from the manufacturers’ specifications by 3.4 – 7.7% and those from sodium thiosulfate titration by 4.6 – 28.9%. Therefore, the variability of the catalase bioassay was comparable to the variability of the potassium permanganate titration, while the variability of the sodium thiosulfate titration was higher.

Results of control experiments in which a known quantity of urea peroxide was added to the product assays are shown for all products in Annex 3, 3a-i. These confirm that the tooth-whitening products tested do not contain substances that inhibit the catalase activity. Hence, the catalase bioassay developed is suitable for the analysis of tooth-whitening products.

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Version 1.0 Marts 2009, © Danish Environmental Protection Agency