IN VITRO PENETRATION OF PETROLATUM IN STRATUM CORNEUM 249 was in contact with 5 mL receptor medium, which consisted of PBS containing 2% w/v BSA and 0.02% NaN3. The use of BSA in the receptor chamber increased the solubility of lipophilic compounds in the chamber and resembled the condition of dermis in vivo for the penetration study (5). Each diffusion cell was placed on a thermostated heating and stirring module and maintained at 37°C at the receptor, resulting in a stratum corneum surface temperature of 34 ± 1°C. A micro magnetic stir bar was placed in the receptor chamber to ensure stirring throughout the experiments. The relative humidity level in the room during the penetration experiments was approximately 20–30%. The skin sam- ple was equilibrated for 2 h, followed by a prescreening water permeability assay to con- fi rm the skin integrity before the penetration experiments. Briefl y, 0.15 mL of 3 H-water was added onto each skin in the diffusion cells. Five minutes postdosing, cotton swabs were used to remove excess 3 H-water from the donor chambers. After 1 h, 2-mL samples from the receptor chamber were collected in scintillation vials and the solutions were mixed with 10 mL scintillation cocktail and analyzed using a liquid scintillation counter (Beckman Coulter LS 6500 multipurpose scintillation counter, Fullerton, CA). Skin sam- ples with water permeation values greater than 1.6 μL/cm2 were discarded. Following the prescreening procedure, the receptor solution was replaced with fresh receptor medium twice to remove the residual radioactivity. The skin sample was then allowed to equili- brate in the heating and stirring module overnight. FINITE DOS E SKIN PENETRATION STUDY WITH SPLIT-THICKNESS SKIN SAMPLES Bodywash preparation. The experimental bodywash was prepared by mixing the bodywash base (ingredients include water, sodium trideceth sulfate, sodium chloride, cocamidopro- pyl betaine, trideceth-3, fragrance, guar hydroxypropyltrimonium chloride, sodium ben- zoate, xanthan gum, disodium ethylenediaminetetraacetic acid, citric acid, sodium hydroxide, acrylates/C10-30 alkyl acrylate crosspolymer, methylchloroisothiazolinone, and methyl- isothiazolinone) with 9.8% petrolatum, 0.2% glyceryl monooleate, and 14 C-dotriacontane (C32 alkane, as the model permeant for petrolatum). Specifi cally, 29.4 mg of petrolatum and 0.6 mg glyceryl monooleate were mixed with 30 μL hexane containing the radiola- beled dotriacontane in a small glass vial by hand using a stainless steel spatula for 3 min. The mixture was then left in a fume hood to allow hexane evaporation for approximately 50 min. After that, 270 mg bodywash base was added to the mixture and mixed by the spatula for 3 min. The size of petrolatum particles in the mixture was examined by light microscopy to ensure uniform sizes in the range of 0.10 ± 0.05 mm. The fi nal concentra- tion of petrolatum was 9.8% as particles dispersed in the bodywash base with 10 μCi 14 C-dotriacontane as the tracer in 300 mg bodywash. Applying and ri nsing method. To simulate a skin washing condition, 5 μL dose of body- wash was applied to the stratum corneum side of the skin sample using a positive dis- placement pipette. The bodywash was spread evenly on the skin with a glass rod (5 mm diameter, with a smooth ground glass fl at tip). The fl at surface of the glass rod tip was moved over the skin in circular motion for 30 s. After the bodywash was applied and left on the skin for another 30 s, i.e., a total of 1-min contact, 0.5 mL water was added to the donor chamber of the Franz diffusion cell. A pipette was used to create water motion in the donor chamber by pipetting the water “in and out” of the chamber three times over 10 s (approximately one “in and out” action every 3 s). Immediately after rinsing, the solution in the donor chamber was removed and placed into a collection

JOURNAL OF COSMETIC SCIENCE 250 vial. This rinsing step was repeated three times to simulate a washing scenario in the rinse-off protocol. After the rinsing step, the pipette tip used in this step was rinsed twice with 0.5 mL hexane and twice with 0.5 mL water. The used glass rod was also rinsed twice with 1 mL hexane and twice with 2 mL water. The rinse-off solution, the pipette tip rinsing solution, and glass rod rinsing solution were then combined and mixed with scintillation cocktail and analyzed using the liquid scintillation counter. To check the weight and amount of 14 C-dotriacontane in the applied dose, 5 μL of the bodywash was placed in a container and weighed with an analytical balance, and the content in the container was mixed with scintillation cocktail and analyzed using the liquid scintillation counter. Sample collectio n and tape-stripping. In the penetrati on study, 2-mL samples of receptor medium were collected at 1, 24, or 72 h postapplication of the bodywash. After sampling, the dif- fusion cells were disassembled and each skin sample was wiped twice with Whatman fi lter paper soaked with 0.5% Tween 20 in PBS and once with fi lter paper soaked with 70%/30% ethanol/deionized water (v/v) to remove the remaining bodywash on the skin surface. After the fi lter wipes, tape-stripping was performed to remove a portion of the stratum corneum. To determine the penetration profi le of petrolatum in the stratum cor- neum, tape-stripping was performed up to 12 times at 1, 24, or 72 h postapplication of the bodywash: in the 1-h experiments, fi ve times tape-stripping were applied, and in selected skin samples, 12 times tape-stripping were applied in the 24 and 72 h experi- ments, 5 times tape-stripping were applied. For the tape-stripping, the edges of the skin were covered with adhesive tapes (fi xing tapes), leaving a central square hole of 1 × 1 cm2 (the available area for tape-stripping). Standard D-Squame® disc (Cuderm Corporation, Dallas, TX), a diameter of 2.2 cm and an area of 3.8 cm2, was pressed onto the skin for 5 s using the D-Squame® pressure instrument, and the D-Squame® disc was quickly re- moved. After tape-stripping, the skin was cut to isolate the diffusion region of the epidermis using a cork borer, and the epidermis was separated from the remaining skin using a pair of forceps. Each skin section was dissolved separately in 1 mL Solvable (PerkinElmer Life and Analytical Sciences, Boston, MA): each tissue was mixed with Solvable in a scintillation vial and kept in an oven at 50°C overnight for the solubiliza- tion of the sample. The donor chamber was washed with hexane to remove the residual cosmetic ingredients on its surface. The receptor collections, fi lter paper wipes, tapes from tape-stripping, solubilized skin sections, and donor chamber wash solutions were mixed with scintillation cocktail and analyzed separately using the scintillation counter. The samples were discarded when one of the following occurred: tearing of the skin be- fore Tape 3 in the tape-stripping procedure or an outlier from the statistical analysis in an experiment. DATA ANALYSIS Figure 1A summarize s the experimental procedure in the present study. The net applied dose of dotriacontane was the remaining amount of dotriacontane on the skin before the rinse- off and was calculated by the difference between the total applied dose and the amount remaining on the glass rod (i.e., the net applied dose of dotriacontane = the total applied dose of dotriacontane minus the amount in the glass rod rinse). The total applied dose of dotriacontane was determined by mixing 5-μL dose of bodywash directly with the scintil- lation cocktail. The % applied dose in each compartment was calculated by the amount