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BreathRx Toothpaste Effect on Caries Formation in the Rat
Study Number
Oral Health Research Institute Number 1281 IACUC Number DS0000721 R
Study Sponsor Discus Dental
8550 Higuera Street
Culver City, California 90232
Study Site
Indiana University School of Dentistry Bioresearch Facility
1121 W. Michigan Street Indianapolis, IN 46202-5186
Conducting Agency
Indiana University School of Dentistry Oral Health Research Institute
415 Lansing Street
Indianapolis, Indiana 46202-2876
Purpose
The purpose of this study was to observe the effect of the Sponsor's experimental dentifrice on dental caries in the rat. The three groups were comprised of a positive control (USP Reference Standard), one negative control, and one experimental dentifrice.
Test Substances
The test substances were three coded products supplied by the Sponsor. To perform this study, 500 grams of each dentifrice were required. The sponsor was responsible for the necessary evaluation related to the composition, purity, strength, stability, storage requirements, expiration dates and any other applicable requirements.
Test Design
The test design was similar to FDA Method #37. The major variations were the diet used (MIT 200 rather than #469), the caries scoring method (Keyes method rather than HMA; see Appendix A) and treatment frequency. Experimental procedures were conducted according to the FDA regulations Part 58.
Test products were given a code by the Sponsor and decoded by the Research Compliance Officer at study completion
** Treatments were administered seven days per week, with a single daily treatment on weekends.
Justification for Animal Use
For a variety of reasons governmental and professional review agencies have agreed to accept a battery of pre-clinical tests as a means of documenting the caries-preventive potential of certain types of fluoride dentifrices in lieu of long-term clinical trials in children. This battery of tests includes the use of a rat caries model with a minimal test design consisting of a negative control (placebo dentifrice) group, a positive control group involving the use of a similar fluoride dentifrice whose caries-preventive benefits have been demonstrated in a controlled clinical trial, and an experimental group similarly treated with the experimental fluoride dentifrice.
Using litters as a covariate, the use of between 50 and 58 (depending on type of fluoride) animals per treatment group satisfies the most stringent power requirements of the ADA's Council on Dental Therapeutics 20% clinical difference between treatments at 80% power. However, we have been routinely using 40 animals per treatment group and these tests have consistently been accepted by both the ADA's COT and the FDA. This required initiating the study with 40 animals per group. These animals were provided by 21 litters.
IACU Approval
The protocol was reviewed and approved by the Institutional Animal Care and Use Committee prior to receipt of the animals.
Animals
1. Type of Animals
Weanling mixed-sex Sprague Dawley rats; approximately 40-50 grams at study initiation.
2. Source
Harlan Sprague Dawley, Inc. P.O. Box 29176 Indianapolis, Indiana 46229
3. Housing
The litters were maintained in large solid-bottom (box-type) cages with dams until the pups were weaned at 18 days of age. Starting at 9 days of pup age, the dams were rotated daily among the litters. The pups were maintained in the box cages until 21 days of age. At this time the pups were stratified and housed in pairs in suspended wire-bottomed cages which had been cleaned and sanitized prior to usage. The cages were arranged so that all animals of the individual groups were together and the cages were labeled with group designation and treatment (treatment code) that the animals received.
4. Identification and Stratification
When the animals were 21 days old they were given unique numbers by ear-punch with records kept of littermates. Animals were assigned to groups in such a manner that groups were balanced for litter; weight and sex, there were 40 animals per group.
Animal Care
1. Diet
Upon receipt, dams and litters were provided rodent lab diet until the pups were 8 days of age. On day 8 (pup age) dams and litters were provided Diet MIT 305 (composition in Appendix B). Animals were provided diet MIT 200 (Composition in Appendix C) ad libitum at day 18 (pup age) and throughout the test period.
2. Water
All animals were provided with deionized water ad libitum.
3. Care
Box caging was changed at day 13 and again at day 18 of pup age. Cage board was changed three times a week at the time when fresh food and water were given (Monday, Wednesday and Friday). Clean and sanitized water bottles and food jars were provided weekly. Suspended caging and banks were sanitized bi-weekly. The animals were observed daily by a staff member and weekly by the attending veterinarian for any signs of health problems.
4. Room Environment
The animals were housed in an AAALAC-accredited facility. Room temperatures were maintained at 72°F (±6°F) with 10-15 air changes per hour and a 12-hour light cycle.
Inoculation. On day 15 (pup age), the pups received an oral inoculation of streptomycin-resistant S. sobrinus 6715 culture (Appendix D). This involved flooding the mouth with 0.2 ml of culture/animal. On day 18 (pup age) the animals were provided Diet MIT 200 and were inoculated with S. sobrinus for three consecutive days (days 18, 19 and 20). This involved placing 0.1 ml of the S. sobrinus culture on the occlusal surfaces of each of the mandibular molar quadrants, putting 10 cc of this concentration-adjusted culture into each water bottle, and lightly spraying the bedding with remaining culture. All water bottles were removed and sanitized 24 hours after inoculum had been added. The inoculum was administered to the animals with a 200 micro pipetter.
Experimental Treatment Initiation
The treatment phase began at day 22 of pup age.
Experimental Procedures
1. Preparation and Labeling
Each treatment had a labeled plastic beaker which was designated for that treatment only. Fresh materials (Le., obtained from stock supply) were used for each treatment. The dentifrices were mixed in a 1:1 ratio (by weight) with deionized water. Specifically, 10 grams of dentifrice were weighed into a 30 ml beaker; 10 grams of deionized water were then weighed and added to the dentifrice. The mixture was then stirred by hand (30 seconds) with a clean microspatula for the purpose of creating a smooth mixture. The beaker containing the slurry and a small magnetic stirring bar was placed on a magnetic stirrer which was set at the lowest speed and allowed to stir for 3 minutes. The slurry was prepared immediately prior to each treatment.
2. Treatment Procedure
A cotton-tipped applicator was dipped into the slurry (for 2 seconds) and was applied to one-half of the rat's mouth in such a way that the sides of the applicator came into contact with both the mandibular and maxillary molars on one side of the mouth. The treatment was accomplished by using a rolling motion of the sides of the applicator over the mandibular and maxillary molar teeth for 15 seconds.
The applicator was dipped Into the slurry for the second time (again, for 2 seconds) and the other side of the rat's mouth similarly treated for 15 seconds. A new applicator was used for each animal.
3. Schedule for Treatment Applications
Treatments were administered twice daily for five days with a single daily treatment on weekends. The first treatment each day began at approximately the same time every day, and the second treatment began no earlier than six hours after the first treatment. Singular treatments were given at a 24 hour interval on weekends.
4. Storage of Material
Treatment materials were stored at room temperature. All treatment products were returned to the sponsor at the study completion.
5. Recovery
One week after the initiation of the inoculation regimen and at study termination, an oral swabbing was taken from each rat using a sterile cotton swab (six-inch, single-tipped applicator). The microorganisms on the mandibular and maxillary molar teeth were sampled, using a rolling motion of the swab for 15 seconds on one side of the mouth, rolled over the tongue, and rolled over the molar teeth on the other side of the mouth for an additional 15 seconds.
Immediately after the applicator was removed from the animal's mouth, it was streaked across half of a 100 mm petri plate containing Mitis Salivarius agar to which 200 units/ml of streptomycin sulfate had been added. The plates were incubated for 48 hours at 37°C with 10% C02' The colony count taken after the 48 hours of incubation was recorded in the logbook.
Experimental Duration of Study
The duration of the experimental phase was three weeks.
Termination of Animal Phase
1. Final Observation and Examination
Immediately prior to termination all animals were observed for any visual signs of ill health or pathology, individually weighed and an oral swabbing taken to confirm S. sobrinus implantation.
Study Completion
1. Tissue Preparation
The cleaned hemljaws (four quadrants) were put into plastic vials with the code numbers taped to the vial. A murexide solution (0.3 g murexide; 300 ml DI H20 and 700 ml of ethanol) was added to each vial and the jaws were allowed to stain overnight. The jaws were then rinsed and allowed to air dry.
2. Tissue Evaluation
The hemijaws were microscopically examined for smooth surface caries, sectioned, and then microscopically examined for sulcal and interproximal caries using the Keyes method as outlined in Appendix A.
3. Data Processing and Analysis
Statistical analyses were performed using the Bartlett-Box F and the Cochran's C tests for homogeneity of variance (at a=0.05). In cases where the variances were homogeneous, a one-way analysis of logarithmic or square root transformation of the data was made according to the relationship between group means and variances, and transformed data reanalyzed. In cases where a significant "F" value was found, Tukey's HSD test and/or Duncan's multiple range tests were used to test for significant differences between the individual means. For extreme variance heterogeneity, the nonparametric Kruskal-Wallis one-way analysis of variance was used.
4. The specific types of data which were tabulated, statistically analyzed, and reported for each group is as follows:
Data Experimental Phase Initial number of animals
Final number of animals
Percent mortality
b. Growth Data Experimental Phase
Initial body weight (mean ± S.E.M.) Final body weight (mean ± S.E.M.) Body weight gain (mean ± S.E.M.)
c. Caries Data
Enamel and dentinal involvement of smooth surface lesions (mean ± S.E.M.).
Enamel and dentinal involvement of interproximal lesions (mean ± S.E.M.).
Enamel and dentinal involvement of sulcal lesions (mean ± S.E.M.).
Total caries involvement combining the scores from the Keyes method of scoring smooth surface, interproximal, and sulcal caries (mean ± S.E.M.). Percent of animals and level of infection in each group infected at both initiation and at termination of study period.
Record Maintenance
All records (protocols, amendments, data sheets and final reports) are maintained in a book designated for this study as part of the OHRI Laboratory Archives. The hard tissue specimens are also maintained in the Archives.
Study Duration
Study initiation began July 14, 2000. The completion of the final report was November 30, 2000.
Results
There was no mortality experienced during the treatment phase of this study.
There were no statistical differences observed among the groups in terms of growth.
All caries data are discussed in terms of enamel involvement only.
Smooth surface caries data are shown in tables 1281-2 through 1281-4. The group treated with the BRX Placebo was significantly greater in buccal-lingual smooth surface caries formation than either of the other two groups. There were no other statistically significant differences observed among the groups. Interproximal caries data are shown in table 1281-3.There were no statistically significant differences in interproximal caries observed among the groups. Total smooth surface caries data are shown in table 1281-4. The group treated with the BRX Placebo was significantly greater in total smooth surface caries formation than either of the other two groups. There were no other statistically significant differences observed among the groups in total smooth surface caries
Information.
Sulcal caries data are shown in table 1281-5. The group treated with the BRX Placebo was significantly greater in total smooth surface caries formation than either of the other two groups. There were no statistically significant differences in sulcal caries observed among the groups.
Total caries formation is shown in table 1281-6. The group treated with the BRX Placebo was significantly greater in total caries formation than either of the other two groups. There were no other statistically significant differences observed among the groups in face caries formation. S. sobrinus data are shown in table 1281-7. The animals were well infected with S. sobrinus at both study initiation and termination.
Conclusion
The BRX dentifrice and USP Reference Standard were equal in efficacy in reducing caries formation as compared to dentifrice BRX Placebo.
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