Bleaching procedure

To receive the bleaching treatment, the animals were anesthetized with an intramuscular injection of ketamine (40 mg/kg) (Francotar; Virbac do Brazil Industria e Comércio, Roseira, SP, Brazil) and xylazine (5 mg/mL) (Virbaxil, Virbac do Brazil Industria e Comércio). The buccal surfaces of the teeth were cleaned with a piece of sterile gauze before application of the bleaching agent containing 35% hydrogen peroxide (HP, Whiteness HP Maxx, FGM, Joinville, SC, Brazil). The product was applied to the buccal surfaces of the first right mandibular molars twice for 5 minutes each, to simulate in-office therapy. To prevent any possible damage to oral mucosal tissues caused by the bleaching agent, small cotton rolls were used for relative isolation, and aspiration tips were used to remove the product from tooth surfaces. The non-bleached left mandibular molars were used as negative controls.

In the clinical situation, the 35% HP gel used in the present study is applied for up to three 15 minute periods at each bleaching session. However, in the present study, the application period was shortened to compensate for the differences between enamel/dentin thicknesses in human and rat teeth. In this way, a relationship between the dentin thickness of mandibular incisor human teeth and time of exposure to the bleaching agent was identified, and a similar proportion was used to determine the time of application of the bleaching agent to the rat molars.7,28