j. Cosmet. Sci., 53, 269-282 (September/October 2002) Comparative study of the effects of cosmetic formulations with or without hydroxy acids on hairless mouse epidermis by histopathologic, morphometric, and stereologic evaluation LOCIA HELENA TERENCIANI RODRIGUES and PATRICIA M. B. G. MAIA CAMPOS, Faculty of Pharmaceutical Sciences of Ribeiraio Preto, University of S•o Paulo, Av. do Caf s/n, 14040-903, Ribeiraio Preto, SP, Brazil. Accepted for publication March 15, 2002. Synopsis Hydroxy acids have been extensively used in cosmetic and dermatologic formulations because of their satisfactory results in terms of maintaining a young and healthy skin and in recovering aged skin. The purpose of the present study was to examine whether treatment with glycolic acid, lactic acid, a fruit acids mixture, malic acid ester, or salicylic esters of lipophilic acid produces different cosmetic results and produces changes in hairless mice epidermis. Histopathologic, morphometric, and stereologic studies were carried out to investigate the possible alter- ations caused by formulations containing hydroxy acids or derivatives. Statistical analysis from the results indicate that, under present experimental conditions, formulations containing glycolic acid or lactic acid or a fruit acids mixture or malic acid ester acted on the epidermis, causing a thickening of the epithelium where the basal and spinous layers also showed increased thickness. The cells reached the largest volume, and the nuclei were also more voluminous. The results demonstrate that cosmetic benefits from the use of hydroxy acids are caused by modification of the skin surface, the epidermis. The hydroxy acids were more effective when compared with the control and their derivatives in a mouse model. INTRODUCTION Hydroxy acids (HAs) constitute a class of compounds that exert specific and unique effects on skin structures. They have been shown to have profound effects on keratini- zation and corneocyte cohesion (1,2). The cosmetic effects of HAs on the stratum comeurn include normalization of stratum corneum exfoliation, resulting in an increased plasticization and a decreased formation of dry flaky scales on the surface of the skin (3-6). A thinner stratum corneum is more flexible and compact. Address all correspondence to Dr. Patrfcia M. B. G. Maia Campos. 269

270 JOURNAL OF COSMETIC SCIENCE Though their mechanism of action is not yet fully understood, HAs are also thought to stimulate synthesis of collagen and mucopolysaccharides in the dermis (6). At use levels under 10 percent, skin care benefits are derived through a continued pattern of product usage. Continued use of products with hydroxy acid levels below 10 percent has been shown to result in a gradual reduction of fine lines and an improvement in skin texture through accelerated desquamation. At higher levels (greater than 30 percent), HAs function as chemical peeling agents that rapidly thin the stratum corneum. At these higher concentrations they achieve the same perceptible reduction in fine lines and an improvement in skin texture, though these effects are manifested more rapidly (7). One important question in the HA formulation is the relationship between the pH and topical efficacy of the formulation. The efficacy of HAs is related to the free acid concentration of the formulation--the lower pH, the higher the free acid concentration of the formulations and the more efficient the formulation (8,9). However, there is a clear inverse relationship between the pH of HA preparations and their potential for skin irritation. The lower the pH, the higher the irritation the higher the pH, going towards neutrality at 4.4, the more the irritation drops off (10). Recently emerged in the cosmetics market have been HA derivatives as substitutes for HAs in formulations. These products did not interfere with normal skin pH values, and they claim to maintain the HAs' activities (11,12). Identifying other compounds with similar effect and improved tolerance would be greatly valued. The fruit acids mixture has also been suggested as effective and safe to use in cosmetic products (11). The objective of this study was to investigate the histopathologic, morphometric, and stereologic alterations caused by the application of the cosmetic formulations containing glycolic acid, lactic acid, a fruit acids mixture, malic acid ester, or salicylic esters of lipophilic acid on the hairless mice epidermis. MATERIAL AND METHODS FORMULATIONS STUDIED The formulation consisted of 20% cetearyl alcohol/ceteareth-20, 1% squalane, 3% pro- pylene glycol, 0.15% methyl dibrome glutaronitrile, phenoxy-ethanol, and distilled water. The cream formulation was supplemented or not with 8% glycolic acid 70%, 5% lactic acid 85%, 3% fruit acids mixture, 5% malic acid ester, or 1% salicylic esters of lipophilic acid. BIOLOGICAL ASSAY Animals. Adult male hairless mouse, weight on average 30 g, were used. The animals where kept in individual cages and received a commercial ration and green food (rami), as well as water ad libitum. Treatment. One group of five animals was used as a control and the other six groups of five animals were used for application on the back area of 1 cm 2 of the cream formulation or the cream formulation containing HAs or their derivatives. The formulations were applied once a day for 15 days.