200 JOURNAL OF COSMETIC SCIENCE The Clinical and Laboratory Assessment of Skin Whitening Walter Smith, Ph.D. Dermac Laboratory, Stamford, CT and Scott Norton, Ph.D., University of Texas, Denton Skin whitening can be evaluated with a variety of in vitro and in vivo test methods with duel goals of optimizing product performance and providing substantiation for product claims. Test tube assays monitoring the tyrosinase dependent conversion of tyrosine to L-Dopa have been useful in comparing the activity of tyrosinase inhibitors. Our tests indicate that kojic acid, ascorbyl magnesium phosphate, Mulberry extract, and numerous herbal extracts are effective enzyme inhibitors. This in vitro activity however does not always translate into clinical effectiveness. In a different test system we have evaluated the inhibition of delayed hyper-pigmentation resulting from complete removal of the stratum corneum via tape stripping. This in vivo test has confirmed activity of kojic acid, and some Mulburry extracts, however vehicle differences could markedly influence activity, and correlation with the in vitro assay was poor. Finally we have evaluated the skin whitening effects of several cosmetic prototypes over a three to four month period. Clinical grading, photography, and the Minolta Meter have proved useful in assessing skin color. Good correlation is observed with the three methods, with clinical grading being most sensitive. We have observed that herbal extracts, hydroquinone, and combinations of AHA's can induce measurable and consumer relevant skin lightening after two to three months of product use. In most cases a general skin lightening is observed without preferential lightening of "age spots". AN IN VITRO METHOD FOR SCREENING WHITENING AGENTS Gopa Majmudar, Ph.D., George Jacob, Yolanda Laboy and Lou Fisher, Ph.D. Mary Kay HoMing Corporation, Dallas, TX 75247 INTRODUCTION: The development of successful whitening skin care products depends on the use effective whitening or depigmenting ingredients that reduce melanin production in melanocytes. We utilized a three dimensional human skin model, Melanoderm, to screen whitening agents before testing the product on humans. Melanoderm is a living skin equivalent in vitro model of the human epidermis consists of well differentiated human keratinocytes and melanocytes. Biochemical, histological and ultrastructural properties of Melanoderm are sim- ilar to human epidermis. The cross section of the Melanoderm shows the presence of stratum corneum, keratinocytes and dendritic melanocytes localized in the basal cell layer. Melanocytes stain positive when exposed to L-dihydroxyphenyl alanine (L-DOPA), a precursor of melanin. A relative activity of whitening agents such as kojic acid, lactic acid and magnesium ascorbyl phosphate (MAP) was measured on Melanoderm. Total kojic acid in the product was measured by high performance liquid chromatography (HPLC). A combination of in vitro test on human skin equivalent culture and HPLC method is useful to evaluate the efficacy, stability and toxicity of whitening ingredients and products before clinical testing. Finally, the product was tested on human subjects to obtain a better correlation with in vitro test results. MATERIALS AND METHODS: In Vitro Testing: Melanoderm was purchased from Mat Tek Co, Ashland, MA. Kojic acid, lactic acid and MAP in aqueous or anhydrous base and the base alone were applied to Melanoderm and incubated for two to three days. At the end of incubation, the cream was removed. Melanoderm was washed in phosphate buffer saline (PBS) and incubated in 0.1% L-DOPA for one hour at room temperature. After one hour Melanoderm was placed in 0.1% fresh L-DOPA and incubated 4 to 16 hours. Optical density was measured at 490rim using a microplate reader. HPLC: Kojic acid assay was performed using a HP laser jet system with a four plus integrator. A C s, ODS -5, 150 X 4.5 mm column (Metachem//0297) and a mobile phase composed of water acetonitrile (20:80 v/v) were used. The wavelength of detection was 254nm. Samples were diluted with water:acetonitrile (50:50 v/v).

PREPRINTS OF THE 1998 ANNUAL SCIENTIFIC SEMINAR 201 Clinical Testing: Ten healthy adult subjects, who gave informed consent, participated in a 12 week study. All subjects applied anhydrous and aqueous base products containing kojic acid and a retail product containing hydro- quinone on their forearms twice daily. Baseline and 24 hours later, chromometer measurements were taken at each of the test sites. RESULTS AND DISCUSSION: Kojic acid (1%), lactic acid (3%) and 1% MAP in nonionic aqueous base were applied for three days on Mclanoderm. Melanoderm was treated with L -DOPA as described in Materials and Methods and examined under the microscope. The treated Melanoderm looked dendritic and healthy, suggesting that whitening agents and a base were not toxic. A quantitative analysis (Table-1) showed 48% and 46% and 33% inhibition by kojic acid, lactic acid and MAP respectively. To confirm our results and check the stability of kojic acid in different bases, we developed a new HPLC method to measure kojic acid in different products. Surprisingly, HPLC analysis showed that kojic acid in aqueous base was not stable (Table-2). Based on these data, we developed a new anhydrous base and tested on Melanoderm with kojic and lactic acids (Table -1). HPLC data suggested that kojic acid was stable in the anhydrous base (Table -2). Table 1 Effect of whitening ingredients on Melanoderm Ingredients Concentrations Tyrosinase Inhibition (%) (%) Aqueous Base Anhydrous Base None 0.00 0.00 0.00 Kojic acid 1.00 47.98 41.00 Lactic acid 3.00 46.20 22.00 MAP 1.00 33.55 ND Table 2 HPLC analysis of kojic acid Base/Vehicle I Time (weeks) Kojic acid concentration (%) 25 ø C Lost 37 ø C Lost (%) (%) (%) (%) Aqueous 0.00 1.15 0.00 ND ND 2.00 1.23 0.00 0.98 14.79 5.00 0.14 87.83 0.18 86.35 Anhydrous 0.00 0.99 0.00 ND ND 8.00 ND ND 0.78 21.22 12.00 ND ND 0.80 19.20 26.0 0.97 2.03 ND ND To confirm our in vitro data, clinical testing was conducted with anhydrous and aqueous nonionic bases containing kojic acid. There was a gradual increase in lightening of the skin over a period of three months with the anhydrous base containing kojic acid (Table -3). Non-ionic aqueous base containing kojic acid was less effective (Table -3), probably due to loss of kojic acid over a period of three months. Thus, we obtained a correlation among in vitro, analytical (HPLC) and clinical data on whitening/lightening agents.