j. Soc. Cosmet. Chem., 48, 175-186 (July/August 1997) Dual-probe method for assessing skin barrier integrity: Effect of storage conditions on permeability of micro-Yucatan pig skin HANI M. FARES and JOEL L. ZATZ, Rutgers University College of Pharmacy, P.O. Box 789, Piscataway, NJ 08855-0789. Accepted iCt publication September 30, 1997. Synopsis The effect of several storage conditions on the permeability of pig skin was investigated. Three different storage conditions were studied, each at two temperatures: 4øC and -15øC. Skin was stored before der- matoming, after dermatoming, and after dermatoming and drying. The variability in the flux of water and salicylic acid (SA), and their ratios (water/SA) over time, served as indicators for the change in the barrier properties of skin. It was found that storing the skin after dermatoming at 4øC kept the permeability of the skin unchanged for four weeks. Storing the skin at a temperature of -15øC altered its permeation to water and SA significantly, however when the skin was dried before storage, its stability at -15 øC was improved. Monitoring the fluxes of two compounds of different polarities and their flux ratios over time is a more rigorous way of testing retention of the barrier integrity of skin than studying the change in flux of one molecule. INTRODUCTION The similarities in histology and barrier properties between porcine and human skin led researchers to use porcine skin in studying permeation. The use of fresh skin in every permeation experiment is very expensive and impractical. Thus, there is a need to store the skin for future use. A wealth of information is found in the literature on the effect of storage on the barrier properties of human skin (1,2). However, similar data on porcine skin is very limited. May and Wainwright (3) studied the structural and meta- bolic degeneration of porcine skin stored in Eagle's minimum essential medium (MEM) at 4øC. During the first week, the authors reported a significant loss of cellular enzyme activity and integrity as well as a shrinkage of the epithelium. The effect of storing animal skin in Roswell Park Memorial Institute (RPMI) 1640 tissue culture media on skin's viability was investigated by Rosenquist et al. (4). The results indicated that the viability of rabbit and pig skin was similar to that of human skin, whereas dog, rat, and mouse skin were significantly different in viability as compared to httman skin. Limited information on the effect of storage on pig skin permeability was found in the literature. Hawkins and Reinfenrath (5) showed that freezing pig skin at -80øC changed its permeability to N,N-diethyl-m-toluamide after one week. Previous investigators have 175

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