CYTOLOGY OF STRATUM CORNEUM 5 5 site when tested within a time period of six weeks (10). The following compounds, freshly made and used for not more than seven days, stored in a cool place and kept light protected, were applied: (1) 1.5% isotretinoin (dissolved in acetone) on the distal volar side of the right forearm (2) 0.05% tretinoin (dissolved in equal parts of ethanol and propyleneglycol) on the proximal volar side of the right forearm (3) ethanol and propyleneglycol on the distal upper part of the right arm (4) 0.5% motretinid on the distal volar side of the left forearm and (5) proximal to it, the gel base. Formulations were not randomized among application sites, as treatment response is negligible for small areas such as used in this study (unpublished data). The chemical structure of the retinoids are shown in Figure 1. Application was made daily for four weeks (five days per week). The compounds were applied to the skin with a cotton swab, so that a thin film covered the test area. Corneocytes were sampled once a week as described below. Two weeks after applications had been discontinued the last sample was obtained. In the second experiment another five volunteers (two female, three male, ages 26-32 years) took part. In this study two different concentrations of isotretinoin (1.5% and 0.5 %), dissolved in acetone, were applied on the right forearm. The solvent acetone was applied on the right upper arm. Two different concentrations of motretinid (0.5 % and 0.25%) and the gel base were tested on the left arm. Corneocytes were collected using the detergent scrub technique (1,9, 11). In this procedure a glass cylinder with an area of 3.8 cm 2 is pressed firmly onto the skin and 0.5 ml of phosphate-buffered 0.05% Triton-X-100 solution is added. The skin within the glass cylinder is rubbed gently with constant pressure with the blunt end of a teflon rod for 30 seconds. The suspension of corneocytes is obtained with an Eppendorf pipette. The corneocyte count per cm 2 skin surface increases with time of rubbing and pressure. Therefore, for all samples 30 seconds were kept constant (1,2). After staining with methylene blue and rhodamine B (2,9), some drops of the suspension were applied on a microscopic glass slide and cov- ered with a cover glass. The samples were evaluated 24 to 48 hours after they had been dried at room temperature. To count corneocytes the stained cell suspension was filled into a hemocytometer (Fuchs-Rosenthal) (1,2,9). The surface area of the corneocytes was analyzed using a projection microscope with a projection mirror (Carl Zeiss), a ) 100 oil immersion lens, and a semiautomatic analyzer system (Videoplan © Kontron, FRG). Fifty cells from each test site, randomly chosen, were evaluated and the mean value and standard deviation determined. The surface area was expressed in square mi- crometers (•m2). The error of this technique is less than 3% (12). RESULTS EXPERIMENT ! Clinical efficts. The visible effects caused by the retinoids on the skin were quite dif- ferent. To illustrate this we chose a classification system: (I) No visible irritation (II) slight irritation, i.e., some scaling (III)weak irritation, i.e., some erythema, scaling (IV) moderate irritation, i.e., erythema, scaling, some exudation and (V) strong irrita- tion with erythema, scaling, exudation, crusts. In the first experiment 1.5 % isotretinoin led to dermatitis in each test person (Table I). The volunteers reacted in a similar way to tretinoin. The vehicle (ethanol and propyl- eneglycol at equal parts) also caused some reactions probably due to the relatively high

56 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table I Experiment I: Irritation of Three Retinoids and Their Vehicles on Normal Forearm Skin (N = 5) After Application Once Daily 5/7 d for 28 d Irritation None Slight Weak Moderate Strong Isotretinoin 1.5% Tretinoin 0.05 % Ethanol and propyleneglycol aa Motretinid 0.5 % Gel base 1 3 1 1 2 2 2 1 concentration of propyleneglycol. Marked reactions to all three substances were seen in a single person isotretinoin, tretinoin and ethanol-propyleneglycol had to be discon- tinued after three weeks. At the sites where motretinid was applied only slight desqua- mation was observed, and no alteration at all in two persons. No reaction occurred to the gel base. Cytological efficts. The changes in the exfoliative cytology during the trial and after discontinuing are summarized in Figure 2. There was an immediate almost twofold increase of cell counts in the first week following the application of tretinoin. Also counts in areas treated with isotretinoin and motretinid increased. The maximum was •c 400 +50- -50• CORNEOCYTE COUNTS TREATMENT Figure 0-0 ISOTRETINOIN 1.5Y. ,--, TRETINOIN O.05Z o--o ETHANOL+ PROPYLENEGLYCOLWa &--& MOTRETINID 0.5Y. /•--/• GEL BASE 2. Experiment I: Changes in corneocyte counts.