Clinical Research Associates

3. Assay to Determine Decrease in Active Ingredient During Use

Buffer made of Sodium Acetate (2N) and Acetic Acid (2N)
Leuco Crystal Violet Solution
Horseradish Peroxidase
Deionized water

Volunteer/trays
1. Obtain volunteers for study- consent is only criteria.
2. Have impression made of upper arch and then pour stone into the impression.
3. Make vacuum-formed tray. Trim off excess plactic to within 1mm of tooth/gingiva margin.
4. Check tray for fit. If tight (uncomfortable), start over.

To generate standard curve make 10% solution of CP in DI H2O. A 10% solution (as per assay) can be obtained by combining the following weights:
CP: 5.8168 g
H2O: 50.5292
10% solution needs to be diluted ~ 1:10^7 to hit assay window (absorbance @ 596nm 0.000 - 3.334)

Use Proxigel (Reed & Carnrick, Piscataway, JN 08855) to establish the fraction of gel applied to tray that can be recovered for each person. This eliminates differences in mouth and tooth size, and dentition arrangement. Proxigel chosen becuase:
1) It is highly water soluble
2) CP concentration is very simular from batch to batch
3) It is a moderately thick gel.

1. To the trimmed vacuum-formed tray add a total of 0.25 g of gel to the area corresponding to the labial surface of the 6 maxillary anterior teeth and spread using a pasteur pipet.
2. Have the individual put the tray in mouth and immediately wipe out all gel that squishes up onto gums using Kimwipes.
3. Immediately remove tray and place tray in a 250 ml tared beaker.
4. Rinse mouth 2 times using a total (final) volume of 25 ml deionized H2O. Spit water into beaker containing tray.
5. Rinse tray w/DI water thoroughly.
6. Weigh beaker again and record weight of water/gel solution.
7. Add stir bar to solution, cover with a piece of foil and mix on stir plate at low speed until gel dissolves (~5 minutes for most gels).
8. Tare 10 ml volumetric flasks on analytical balance.
9. To tared 10 ml flasks add drops of the CP solution from step 7. Weigh again on analytical balance and calculate weight of solution added.
10. Repeat step 1-9 using different weights (#s of drops) of the water/gel solution.
11. To each flask containing some of the sample to be assayed, add 1.0 ml Leuco Crystal Violet, followed by 0.5 ml of horseradish peroxidase. Mix and add 4.0 ml buffer. Dilute to volume with distilled water. Cover with parafilm and mix thoroughly. Place volumetric flasks in a 20 degrees C water bath for 10 min. Transfer a portion of solution to a 1cm cell cuvette and measure absorbance at 596nm against a reagent blank prepared in the same manner but containing no CP.
12. Repeat tests three times for each individual.
13. Calculate average Baseline for each individual:
Baseline = (({first Wt. CP recovered / Wt. CP added to tray} + {second Wt. CP recovered / Wt. CP added to tray} + {third Wt. CP recovered / Wt. CP added to tray}) / 3) X 100

1. Assay gels using USP method for determination of carbamide peroxide. (U.S. Pharmacopeia, USPXXII NFXVII, 1990 pp 223-224)
2. Dilute and assay by oxidase method. (Chemical Analysis, Vol.8, Second Edition, Colorimetric Determination of Nonmetals, pp 309- 310)

Solutions of Leuco Crystal Violet and Horseradish Peroxidase should already be prepared and stored at 4 degrees C.
1. In a tared vacuum-formed tray apply 0.25 g of gel to areas corresponding to 6 maxillary anterior teeth and spread using a Pasteur pipet being certain there is 0.25 g of gel in tray after spreading gel.
2. Put tray into mouth, being certain tray is on all the way.
3. Prior to end of wear time, label and tare a 250 ml beaker and several 10ml voumetrics.
4. At end of wear time, remove tray and place in 250 ml beaker. Gently wash gel off teeth using tongue and a total of 80ml deionized H2O for moderate to thin gels and up to 150 ml for thick gels. Spit wash water into beaker holding vacuum formed tray. Visually inspect teeth to be certain gel has been washed out from between teeth. Using forceps the deionized water in a squeeze bottle, wash gel from tray. Put rinsed tray into warm soapy water.
5. Weigh 250 ml beaker containing gel. Subject may now wash and expectorate.
6. Add stir bar to 250ml beaker, cover, then stir at low speed until dissolved at room temperature.
7. Add to the tared 10 ml volumetrics various weights of solution from 250 ml beaker. Weight to be sampled depends on concentration of CP in gel and thickness of gel. As thickness of gel increases, weight tested decreases and as concentration of CP increases, weight tested decreases.

8. Assay by Oxidase test method. (Chemical Analysis, Vol. 8, Second Edition, Colormetric Determination of Non Metals, pp 309 - 310)
Initial Testing
Time 0 - when Leuco Crystal Violet added
Time 10 minutes - when sample added to cuvette for reading - abs596nm
Subsequent Testing
Time 0 - when Lueco Crystal Violet added
Time 5 minutes - when sample added
Solution in 10 ml volumetric held at 20 degrees C during 10 minute reaction time in Lauda RM 20 refrigerated circulating water bath.
9. Calculate % CP remaining after test time.

Timer
Large glass plate sectioned into columns with a horizontal line across the top
Sample of each of 23 bleaching gels
Micrometer
250 ul positive displacement pipette

1. Draw columns on a plate of glass and label each with the bleach brand name.
2. Draw a horizontal line across the top to use as the starting point for the gels.
3. Place 100 ul sample of gel above each horizontal line so the bottom edge of the gel is directly over the horizontal line.
4. Once all the gels are in place raise the glass plate to 90 degrees and start timer.
5. After 1 minute has passed return the glass plate flat to the table and measure the distance (mm) traveled by each of the gels using a micrometer and record values in lab book.

Procedure for Testing pH Values on Orion/901 Meter:
Calibration and Meter Use:
1. Remove screw cap on electrode.
2. Rinse electrode with DI water.
3. Clean off excess salt residue on probe.
4. Discard old filling solution from electrode by compressing the top to the electrode handle.
5. Fill electrode with new filling solution. Invert several times and expel filling fluid once again.
6. Fill probe with new solution about 1inch below the black handle on top.
7. Rinse exterior of electrode with DI water
8. Rinse electrode with pH reference solution of pH 7.
9. Insert electrode into the solution mentioned in #8.
10. Set pH meter to the "pH" setting. Wait for numbers to stabalize.
11. Press "set concentration" button on meter.
12. Wait for reading to stabilize.
13. Remove electrode and rinse with DI water.
14. Rinse electrode with pH reference solution of pH 4.
15. Immerse in second pH solution. Let stabilize.
16. Set slope on meter to the pH value of pH 4.
17. Let stablilize.
18. Remove electrode from buffer solution and rinse with DI water.
19. Immerse probe in storage solution until ready to read.
20. Test samples (see following instructions for method used to test 23 bleach samples).
21. Rinse probe with DI water and blot dry electrode tip between each sample.
22. Replace screw cap on electrode and place electrode in storage solution.
23. Turn pH meter to "stand by".

Pasteur pipettes
Tape and Sharpie marker (for labeling)
16x100 mm test tubes
23 bleach dispensers, one from each gel evaluated

Orion/901 pH Meter
Test tube rack for 20x150 mm test tubes
100 ml beakers
Stir plate
Stir bars
Balances: top loading electronic and analytical
Timers
Squeeze bottle filled with DI water

1. Tare and label three 100 ml beakers.
2. Add 1 g of bleach gel being evaluated to each beaker.
3. Add 9 g of deionized water to each beaker.
4. Allow beakers to stir until bleach is completely dissolved.
5. Label three 16x100 test tubes, one for each corresponding beaker.
6. Transfer dissolved bleach solution to the respective test tube.
7. Calibrated pH meter is inserted into first test tube.
8. Record pH meter readings at .5 minutes, 1 minute, 2 minutes, and 3 minutes.
9. Repeat for each test tube.
10. Repeat procedures 1 - 9 until three acceptable pH values are obtained for each bleach.

Procedure:

1. Volunteers were solicited to participate in the tooth whitening study. A minimum of 3 patients participated and used each product.
2. Each individual was screened and qualified to participate after meeting the following criteria: -Six anterior teeth free of any large restorations -Tooth discoloring of 3 or higher using the Vita Shade Guide.
3. After meeting the criteria each subject signed a consent form.

1. An Alginate impression was taken of the arch to be treated.
2. After an accurate impression was taken a stone model was poured.
3. The model was trimmed for optimal adaptation of tray material.
4. Tray material was then heated using vacuum former until it sagged about 1.0 inch.
5. The material was then adapted to model and allowed to vacuum for 30 seconds.
6 After tray material was thoroughly cooled it was cut and removed from stone model.
7. Tray was then scalloped using scissors to 0.5 mm to 1.0 mm below the gingival margins.
8. Tray was again seated on stone model to assess accurate fit.

1. An intra-oral exam was performed on each subject to map all existing restorations and assure they were intact. Findings were recorded in the their patient file.
2. Full mouth x-rays were then taken and included in the file.
3. Each subject received a prophylaxis cleaning to remove decay, calculus and extrinsic stains.
4. Three photos were taken at different F settings to insure a good picture.
5. Initial shades were taken by an expert lab technician using Vita Shade Guide. Results were recorded in lab book.
6. Subjects were then instructed on bleach use according to the protocol recommend by the manufacturer.
7. Patients were given a journal to keep daily. Journals included product instructions, a product use schedule (to record date, time started, time ended, and observations during use.
8. With strict instructions for compliance to the regimen, subjects were given their bleach kits and sent home to begin treatment.
9. Subjects were contacted periodically during treatment to confirm daily use. In the case that tooth or gum sensitivity occurred subjects were instructed to discontinue use for one day or until sensitivity subsided. The treatment period was extended for the same amount of time missed due to sensitivty so as to complete the treatment.
10. After the specified treatment plan was achieved subjects stopped bleaching and waited at least five days for teeth to rehydrate after coming in for the final recall.
11. At the final recall shades were again taken by John Archibald using 3D Vita Shade Guide.
12. Final photos were taken.
13. Patient journals were returned for assessment.
14. Subjects were surveyed to find out the following items of interest: Satisfaction, sensitivity, tray fit, taste, & convienience.