IN VITRO PENETRATION OF PETROLATUM IN STRATUM CORNEUM 251 of dotriacontane in the compartment normalized by the total applied dose of dotriacon- tane (total applied radioactivity in 5 μL from the pipette). The amount of petrolatum normalized by the skin area was calculated by the amount of dotriacontane in each com- partment, specifi c activity of dotriacontane for petrolatum, weight of petrolatum in the 5-μL dose bodywash formulation, and the diffusion area of the Franz diffusion cell (i.e., the amount of petrolatum in μg = the amount of dotriacontane divided by the specifi c activity of dotriacontane and 0.71 cm2). Total recovery was calculated using mass balance. Figure 1B summarizes the compartments to be investigated in the present petrolatum penetration study. All experiments were performed with at least four skin donors for each condition. Figu r e 1. (A) Schematic diagram of the experimental procedure to evaluate petrolatum deposition on the skin surface and its penetration into the stratum corneum from the bodywash formulation, and (B) the amount of petrolatum measured in different compartment of the rinsed-off solution, the net applied dose, and the total 5-μL applied dose in the experiments.

JOURNAL OF COSMETIC SCIENCE 252 STATISTICAL ANALYSIS The m eans ± standard error s of the means (SEM) of the data are presented. Data analyses and statistical tests (one-way Analysis of variance, ANOVA) were performed using Microsoft Excel (Redmond, WA) and GraphPad InStat (San Diego, CA), respectively, and a difference of p 0.05 was considered statistically signifi cant. Outlier data points (~9% of the data) of the amounts of dotriacontane (as % applied dose) in the tapes and epidermis in each set of experiments were identifi ed using an outlier test (Outlier calculator, GraphPad Software, La Jolla, CA). RESULTS DEPOSITION AND PENET RATION O F PETROLATUM INTO THE STRATUM CORNEUM The average weight of bodywa sh formulation (5 μL) applied in vitro on the skin (a diffu- sion area of 0.71 cm2) was 2.38 ± 0.05 mg (mean ± SEM, n = 54 total samples, from n = 3 in each set of experiments). Dotriacontane was used as the model compound for the penetration of petrolatum into the stratum corneum. Table I shows that the majority, 61–66% (mean values), of the dotriacontane in the bodywash formulation was removed by the rinse-off protocol. Approximately 8–14% (mean values) of the dotriacontane in the bodywash remained on the skin surface that was removed by the fi lter wipes before the tape-stripping evaluation. Figure 2 shows the amounts of dotriacontane as % applied dose and amounts of petrolatum (μg/cm2) in the fi rst fi ve tapes after the application o f the bodywash system and rinsing. The amounts of dotriacontane in the stratum corneum, collected by tape-stripping, decreased from 2.9% ± 0.3% in the fi rst tape to 0.33% ± 0.05% (mean ± SEM) in the fi fth tape at 1 h postapplication. At 24 and 72 h after bodywash TABLE I The Amount o f Dotriacontane (Petrolatum Probe) as Percent of Bodywash Net Applied Dose Recovered from Different Compartments at 1, 24, and 72 h after Application and Rinsing in the Skin Penetration Study. Mean ± SEM, n = 7–11 Compartment 1 h 24 h 72 h Rinse-offa 61 ± 5 61 ± 4 66 ± 5 Skin surface wipe 14 ± 1 8 ± 1 10 ± 1 Tape-strippingb 8 ± 1 9 ± 1 10 ± 1 Epidermis below Tape 5 (cut out epidermis) 0.7 ± 0.1 3.0 ± 0.4 5 ± 1 Remaining skin (after tape-stripping and cut out epidermis) 0.8 ± 0.2 1.6 ± 0.3 2.0 ± 0.3 Buffer in receptor 0 ± 0c 0 ± 0c 0 ± 0c Donor washd 10 ± 2 10 ± 2 9 ± 1 Total 95 ± 5 92 ± 3 103 ± 4 Range (82–129) (84–104) (87–112) a Rinse-off solution was collected immediately after rinsing, which was performed at 1 min after dosing. b Amounts of dotriacontane on the fi xing tapes used in the tape-stripping procedure are less than 1% applied dose of dotriacontane. c Mean values of dotriacontane in the receptor chamber 0.1% applied dose. d Residual dotriacontane on the glass surface of the donor chamber. The residual amount was determined by washing the donor chamber cap with hexane and collecting the wash solution. This amount was only used for recovery calculation.