176 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS generally used a single probe molecule, usually water, as an index of skin barrier integrity. More definitive results may be obtained by using two probe compounds, differing in polarity. The flux of each compound, as well as the ratio between the two, provides information about the barrier. We chose [3H]water and [•4C]SA as the two probe molecules and measured them simultaneously. In this study, the effect of several storage conditions on the permeability of pig skin was investigated. Three different storage conditions were studied, each at two different temperatures, 4øC and -15øC. Skin was stored before dermatoming, after dermatoming, and after dermatoming and drying. The flux of water and SA and the water/SA flux ratio were monitored over time. EXPERIMENTAL MATERIALS Sailcyclic acid, dibasic sodium phosphate, monobasic potassium phosphate, sodium chloride, potassium chloride, scintillation fluid, and glacial acetic acid were obtained from Fisher Scientific (Fair Lawn, NJ). Micro-Yucatan pig skin was received from Charles River Laboratories (Wilmington, MA). [•4C]SA-56.1 mCi/mmol and [3H]H20- 108.3 mCi/mmol were purchased from NEN Products (Boston, MA). Skin-digesting fluid (Solvable) was obtained from Packard Instrument Company, Inc. (Meriden, CT). Aqueous detergent (Palmolive dishwashing liquid) manufactured by Colgate Palmolive (New York) was purchased from a local store. All reagents were either HPLC or ACS grade and were used without any further purification. FORMULATION A 0.1% w/w SA aqueous solution was prepared. The formulation was buffered at pH 2.4. IN VITRO PERMEATION Experimental conditions. Circular pieces (12 mm in diameter) of pig skin were mounted on flow-through diffusion cells (Amie Systems, Riegelsville, PA). The diffusion cells were clamped, and the receptor fluid was pumped at a rate of 3.57 ml/min. The membrane was allowed to equilibrate for one hour before applying the material to be tested. The cells' temperature was maintained at 32øC throughout the experiment using a water bath/circulator (Haake, Paramus, NJ). Sample collection took place with a fraction collector (Isco, Inc., Lincoln, NE) every two hours from the start of the experiment up to ten hours. Samples were collected directly into scintillation vials. All samples were tested in six replicates. The permeation of SA and water was measured upon receipt of the skin and then weekly thereafter for a period of four weeks. Preparation of the skin. Skin stored before dermatoming. Upon receipt, the skin was washed gently with 1% (v/v) aqueous detergent solution, rinsed with distilled water, and patted dry with a paper towel. The skin was then placed in plastic bags and stored either at 4øC or at -15øC. Before starting the experiment, the skin stored at - 15 øC was removed and placed at 4øC overnight. Two hours before being dermatomed, the two pieces of skin were removed

ASSESSING SKIN BARRIER INTEGRITY 177 from the 4øC station and placed at room temperature to equilibrate. A 250-300-pro- thick layer of the skin was dermatomed with a Padgett Electrodermatome (Padgett Dermatome, Division of Kansas City Assemblage Co., Kansas City, MO). Then the skin was cut into circular pieces (12 mm in diameter) and used in permeation experiments. Skin stored after dermatoming. The skin was handled as described in the previous paragraph, except that it was dermatomed before storage. Skin stored after dermatoming and drying. Upon receipt, the skin was washed, rinsed, and dermatomed as described above. The skin was then placed in a dessicator for 24 hours. The dried skin was then removed and stored at either 4øC or -15øC. Before starting the experiment, the skin stored at -15øC was removed and placed at 4øC overnight. Two hours before the beginning of the experiment, the two pieces of skin were removed from the 4øC station and placed in an isotonic buffer solution to hydrate. The skin was then cut into circular pieces and used in the experiment. Receptor fluid. Phosphate-buffered saline (PBS) was used. The buffer was based on Dul- becco's PBS (6) and contained the following ingredients: 0.008 M sodium phosphate, 0.002 M potassium phosphate, 0.14 M sodium chloride, and 0.01 M potassium chloride. This isotonic buffered saline has a pH of 7.4, which is an ideal pH for maintaining sink conditions for SA. Dosing. A 500-pl sample was applied, and the cells were covered throughout the ex- periment. In the preliminary work, this dose was adequate to study the permeation of water and SA over a period of 12 hours. Analysis. Each milliliter of formulation was spiked with 1 lnl of [•4C]SA and 3.3 !•l of [3H]H20. Each lnl of [•4C]SA and [3H]H20 contained 0.1 lnCi (2.2 x 105 DPM) and 1 !•Ci (2.2 x 10 6 DPM), respectively. The receptor fluid from all permeation experi- ments was collected directly into scintillation vials. Ten milliliters of scintillation fluid were added to each vial, and all samples were analyzed in a scintillation counter (Beck- man Instruments, Inc., Fullerton, CA). STATISTICAL ANALYSIS OF DATA Analysis of variance was performed on the permeation rates obtained. The rates obtained over time were compared to the initial ones for any significant differences using Dun- nett's test (95% confidence level). A statistical package (SigmaStat 2.0, Jandel Scientific, San Rafael, CA) was used to process the data. RESULTS EFFECT OF STORAGE TEMPERATURE ON THE PERMEATION OF WATER AND SALICYLIC ACID THROUGH MICRO-YUCATAN PIG SKIN WHEN THE SKIN WAS STORED BEFORE DERMATOMING Plots of the cumulative amounts of water and SA permeating through micro-Yucatan pig skin over time were linear during the four-week period of testing (r 0.997 in all cases). A typical example of such plots is presented in Figure 1. Water and SA flux values were calculated for all cases and plotted as a function of time in Figures 2 and 3, respectively. Most of the flux values obtained at the third and fourth