J. Soc. Cosmet. Chem., 41, 243-248 (July/August 1990) A quantitative method for assessing cosmetics for efficacy at reducing wrinkles A. M. THIELEMANN, H. CHAVEZ, M. A. SANDOVAL, and I. ORREGO, Departamento de Ciencias y Tecnologoea Farmacguticas, Facultad de Ciencias Quoemicas y Farmacguticas, Universidad de Chile, Casilla 233, Santiago, Chile. Received February 8, 1990. Synopsis This study postulates a quantitative method to assess cosmetics for wrinkle diminution. For this effect, the replica technique and scanning electron microscopy (SEM) were used. The technique used in this study consists essentially of three steps: obtaining a negative skin replica using a mixture of silicone plus a catalyst obtaining a positive skin replica from the negative replica and, finally, coating the positive replica with a gold-palladium mixture for further SEM observation and photography. The method was standardized and assessed using a base emulsion. When the effectiveness of the method was validated, a study was done of the potential effect of a sodium salt of 2-pyrollidone-5-carboxilic acid as a wrinkle diminisher. Two creams, only one of them containing the active principle, were applied to a female volunteer group for a certain period. A negative replica was taken before and after each cream application period. The assess- ment parameters used were length and width of a previously selected forearm wrinkle. The results showed that the proposed method can detect variations in the length and width of a previously selected wrinkle and that it can also be effectively used in the assessment of cosmetics used to diminish wrinkles. INTRODUCTION Skin dryness and wrinkle formation have increased the demand for cosmetic products. This has led to the development of methods for evaluating the various formulations used as wrinkle smoothers. There are various subjective methods to evaluate skin characteristics: the Kligman re- gression method (7), stereomicroscopy (8), magnified photography (3,4,9), and others. All these techniques require highly trained evaluators and a large number of volunteers. There are at present various in vivo and in vitro methods to assess quantitatively the hydrated state of the corneum stratum. The criteria vary widely, depending on the technique used. The methods may be either direct, such as transpirometry (1-3), or indirect, such as measurement of the skin elastic module (3,4) and in vivo conductance determination (5,6). A good review of these mechanical methods is that of Salter (3). Another direct method for evaluating the hydrated state of skin is one that uses electron 243

244 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS microscopy. Transmission electron microscopy assessments can be applied to biopsy samples, which provide information about the effects of cosmetics on skin (10). How- ever, because of the difficulties encountered in working with biopsies, skin replicas have been obtained by impressing silicone resins, which polymerize easily at ambient tem- perature. This technique has been widely used to determine general skin condition. A negative silicone skin replica is obtained, and from this a polyethylene positive skin replica is developed (10- 13). The positive replicas can be assessed by profilometry (14) or by scanning electron microscopy (15). The aim of our work is to develop a direct method that may provide quantitative information about the efficacy of wrinkle smoothers. The replica technique was used, and further analyses were performed by scanning electron microscopy. The measuring parameters were wrinkle length and width, since their values vary according to the hydrated state of the corneum stratum (16). MATERIALS AND METHODS Two emulsions were prepared: the base formulation (F•) and the same base plus 8% sodium salt of pyrollidone carboxylic acid (Nalidone •-vø) (F2). Both are oil-in-water emulsions. Both formulations were applied in summer to a group of five Caucasian female volun- teers whose ages ranged from 35 to 38 years. They did not suffer from any kind of allergy or cutaneous alteration and were not ingesting any kind of drug. The volunteers had normal skin according to the classification of Koyama et al. (15). A previously selected forearm wrinkle was determined. This comprised an area 1 cm 2, well marked with ink. This area was selected because it was little exposed and had a lower probability of being affected by the environment. Besides, it had low perspira- tion, which permits good skin replicas. All the wrinkles had the same orientation in relation to the arm axis 25 microliters of each formulation were applied over 1 cm 2 of skin. A cross-over design was used, with a 22-day-long non-application period between each treatment of two daily applications during 22 days. At the end of each period, the area under study was evaluated using the selected parameters. Both the wrinkle length, magnified 45 x, and the wrinkle width, magnified 1500 x, were photographed using a JOEL JMS-25 II R scanning electron microscope with a 6 x 7 Mamiya photographic camera adapted to it. A Helios Vernier was used for the determinations. The method used to obtain the replicas was that described by Ryan et al. (12). A Coltene silicone, Coltex Extrafine •!3•, and its catalyst, Coltex Extrafine Mix •!3• were used to obtain the negative skin replica. The positive replica was obtained by using low-den- sity polyethylene (Dow Chemical Co.). RESULTS AND DISCUSSION Before starting the experimental stage, the reproducibility of the technique was checked. For this purpose, two negative replicas of the same wrinkle were taken so as to generate the corresponding positive ones. Microphotographs obtained by scanning elec- tron microscopy were compared as to wrinkle length and width. Starting from one