62 JOURNAL OF COSMETIC SCIENCE in viable tissue. The probe of the Mexameter emits the light of three defined wavelengths: 568nm, 660nm, and 880nm (two wavelengths are used for each endpoint). The receiver measures the light reflected by the tissue and, as the quantity of emitted light is defined, the quantity of light absorbed by the tissue can be calculated (5). This instrument is commonly used in clinical trials and has been adapted for use in the laboratory study to assess differences in melanin production compared to controls. Results obtained using the Mexameter from tissues exposed to test materials were compared to the negative and positive controls using a t-test (p0.05). The amount of melanin present in tissue can also be quantified by extracting with 2 N NaOH (6-7). Tissues were placed in 0.5 ml of 2 N NaOH and incubated for approximately 48 hours with mechanical agitation to exitact the melanin. Aliquots from each replicate were read spectrophotometrically at 410 nm. The mean absorbance of the six replicates was divided by the mean absorbance of the negative control and multiplied by 100 to calculate the mean percent for each test material. The mean percent value of each test material was then compared to the mean percent value of the positive control using the t-test (p0.05). Results The results and conclusion of this study will be presented and discussed at the Society of Cosmetic Chemists' December meeting. Refereuces !. Oslxrne, R. and M.A. Perkins, The Procter & Gamble Company. Evaluation of human skin cell cultures for in vitro skin irritancy testing. In VitroToxicology: Mechanisms and New Technology. New York: Mary Ann Liebert, Inc. 8:317-324. (1991). Carmichael, J., W.G. Degra• A.F. Gazdar, J.D. Minna, and J.B. Mitchell. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chromosensitivity testing. Cancer Res. 47:936-942. (1987). Triglia, D., S.S. Braa, T. Donnelley, I. Kidd, and G.K. Haughton, Marrow Tech, Inc. A three dimensional human derreal model substate for in vitro toxicological studies. In Vitro Toxicology: Mechanisms and New Technology. New York: Mary Ann Liebert, Inc. 8:351-362. (1991). 4. MatTek product literature. 5. Courage+Khazaka (Mexameter) product literature. Talwar, H.S., C. Griffiths, G. Fisher, A. Russman, K. Krach, S. Benrazavi, and J. Voorhees. Differential Regulation of Tyrasinase Activity in Skin of White and Black Individuals In Vivo by Topical Retinoic Acivl J. Invest. Dermat. 100:800-802. (1993). Iozumi, K., G. Hoganson, R. Pennel!a, M. Everett, and B. Fuller. Role of Tyrosinase as the Determinant of Pigmentation in Cultured Human Melanocytes. J. Invest. Dermat. 100:806-808. (199S).

PREPRINTS OF THE 1998 ANNUAL SCIENTIFIC MEETING 63 A COMPARISON OF SKIN VIABILITY ASSAYS FOR IN VITRO SKIN ABSORPTION/METABOLISM STUDIES H.L. Hood • and R.L. Bronaugh z •Bristol-Myers Squibb, Clairol Division, Stamford, CT 2Office of Cosmetics and Colors, Food & Drug Administration, Washington, DC Introduction If metabolism is to be assessed during an in vitro percutaneous absorption study, it is essential that skin viability be maintained. It has been demonstrated that the viability of skin can be maintained for up to 24 hr in flow-through diffusion cells by utilizing a physiological receptor fluid with a carbohydrate energy source, such as HEPES-buffered Hanks' balanced salt solution (HHBSS) •. Water-insoluble compounds may not partition freely from excised skin into the aqueous HHBSS receptor fluid:. Therefore, to increase the aqueous solubility of lipophilic compounds in the receptor fluid 4% bovine serum albumin (BSA) is routinely added to HHBSS in our laboratory. A number of tests have been developed for assessing skin viability in vitro 3. Anaerobic glucose utilization (or the lactate assay) is the method employed most frequently in our laboratory as an indicator of tissue viability. Recently, however, we have observed that the addition of 4% BSA to HHBSS results in lower levels of measurable lactate in the receptor fluid. The objective of this study was to develop an alternate means of measuring skin viability that is unaffected by BSA. The MTr assay 4, which determines the presence of viable cells with metabolically active mitochondria, was evaluated as an alternative to the lactate assay. Viability of skin was measured with both the lactate assay and MTr assay following perfusion with HHBSS and HHBSS + 4% BSA. Species differences in the two assays were also examined using hairless guinea pig, fuzzy rat, and human skin. Methods Perfusion Study. Skin perfusion studies were performed in Bronaugh flow-through diffusion cells •. Dermatomed skin sections were placed in diffusion cells stratum comeurn-side up and skin surface temperature was maintained at 32 0 C. Receptor fluid was collected at a flow rate of 1.5 ml/hr in 6-hr fractions for 24 hr. Perfusion was ceased at various time intervals and skin viability was determined by the lactate assay or the MTr assay. Lactate Assay. Anaerobic glucose utilization in skin was determined by measuring lactate in the collected receptor fluid fractions with a Sigma © diagnostic kit. Briefly, a 0.20 ml aliquot from each 6-hr fraction was mixed with 2.8 ml of assay reagent. Samples were incubated in a water shaker bath at 37 øC for 45 min. The UV absorbance of the reaction mixture at 340 nm was measured spectrophotometrically and --'as proportional to the amount of lactate originally present. MTr Assay. MTr was dissolved in HHBSS at 2 mg/ml and warmed to 37 øC. At specified time intervals, the area of skin perfused was cut and placed into a 6-well tissue culture plate. To each well 2 ml of MTr solution was added and the plate incubated for 2 hr at 37 øC and 90% humidity on a rotating platform. Fresh control skin was dermatomed and also assayed after equilibrating in I-IHBSS for 20 min. After incubation the MTr solution was removed and each skin section was washed with phosphate-buffered saline. Isopropanol was added to extract the blue MTr-formazan crystals from skin by agitating plates on rotating platform at room temperature for 1 hr. Two ml of extract was diluted with 2 ml of isopropanol and the absorbance was measured at 540 nm. Results and Discussion The rates of anaerobic glucose utiliza, tion from perfusion of HGP skin with HHBSS and HHF• •' + ß 4% BSA are illustrated in Fig. 1 by plotting the micromoles oflactate produced per hour in each 6-hr