J. Cosmet. Sci., 61, 247–258 (May/June 2010) 247 Comparative in vivo study of the effi cacy and tolerance of exfoliating agents using refl ectance spectrophotometric methods LUISA RIZZA, GIUSEPPINA FRASCA, CLAUDIA BONINA, and CARMELO PUGLIA, Department of Pharmaceutical Science, School of Pharmacy, University of Catania, Viale A Doria 6, 95125 Catania, Italy. Accepted for publication November 16, 2009. Synopsis The aim of the present study was to compare the effectiveness and the safety of different topical agents (gly- colic acid, mandelic acid, and grape juice acid mixture) in skin exfoliation by objective instrumental meth- ods. To evaluate the exfoliating effects of these substances, a new experimental in vivo protocol based on DHA (dihydroxyacetone)-induced skin pigmentation was used. Skin acceptability towards acid application was investigated by the evaluation of skin erythema induced by topical application of these substances at in- creased concentrations. Furthermore, their photosensitizing effects were evaluated by determining the in- crease in sensitivity to UV-light exposure in cutaneous sites previously treated with acids. These in vivo evaluations were monitored by refl ectance spectophotometry. From the results obtained, we observed the differing capacities of the tested acids to increase the rate of skin regeneration, with a signifi cant reduction in the time required to obtain skin renewal. The study pointed out that glycolic acid (10% w/w) induced a faster skin exfoliation, a more intense erythema, and a higher photosensitizing effect in comparison with the mandelic acid and grape juice acid mixtures. Further evi- dence showed that the mandelic acid and grape juice acid mixtures were able to induce a slower and safer peeling action in comparison with glycolic acid. Finally, our results suggest that the methodologies and protocols used in this study may help in choosing the most appropriate topical agents for skin exfoliating treatments. INTRODUCTION Despite new and emerging modalities in the fi eld of dermatology, chemical peeling holds its own as an effi cacious technique available for treatment of cutaneous diseases and con- ditions and for aesthetic improvement (1,2). Chemical peeling involves the application of one or more chemical exfoliating agents to the skin, resulting in a wound-healing process that can regenerate the epidermis and restore photodamaged, wrinkled, blemished, acne- scarred, or blotchy skin to its original appearance (3,4). A variety of chemical peeling agents are available, such as glycolic acid, trichloroacetic acid, salicylic acid, pyruvic acid, resorcinol preparations, and solid carbon dioxide (5), and new agents are being researched to create new ways of peeling (1).

JOURNAL OF COSMETIC SCIENCE 248 Alpha-hydroxy acids (AHAs) are a class of compounds commonly used for chemical peel- ing, and glycolic acid is the most extensively studied of these acids (6). According to these studies, glycolic acid appears to induce an acid-dependent discohesion of corneo- cytes, and when used for a long period of time in high concentration, it is able to increase cell proliferation of the basal epithelial cells in the epidermis, elastic fi bers, and collagen (7,8). Many commercial skin-care products containing glycolic acid are proposed to coun- teract photoaging, decrease acne and pigmentary changes, or reduce stretch marks (8,9). As reported in several studies, better response is obtained only when glycolic acid is used at higher concentrations (50–70%) (10,11), increasing the risk of skin irritation. The use of glycolic acids in chemical peeling is strictly correlated with some undesirable side effects such as persistent erythema and pruritus, burning, post-infl ammatory hyper/ hypopigmentation, hypertrophic scarring, and infectious complications (3,11,12). More- over, recent experimental studies demonstrate that short-term application of glycolic acid sensitizes the skin to the damaging effects of UV light (13). To improve the safety of products, committees set up by associations of cosmetics manufacturers in Europe and in the USA recommend similar guidelines and, in particular, pH values higher than 3.5 and alpha-hydroxy acids contents lower than 7–10% (14). Recently, in order to achieve a balance between performance and risks, many common organic acids and combinations of them, such as mandelic acid, lactic acid, and natural acids from fruits (such as tomatoes, lemons, grapefruits, oranges, and limes) have been used in commercial products. The aim of the present study was to compare the effectiveness and the safety of different AHAs (glycolic acid, mandelic acid, and a blend of organic acids from grape juice) in skin exfoliation by objective instrumental methods. To evaluate the effi cacy of the exfoliating agents, a new experimental in vivo protocol based on DHA (dihydroxyacetone)-induced skin pigmentation and a non-invasive instrumental method was used. DHA is a three- carbon sugar, formally a derivate of glycerol, and it is the most common and safe cosmetic ingredient used in sunless tanning products (15). The pigmentation produced by DHA is the result of the chemical reactions (Maillard reaction) between the DHA and the amino acids of the corneocytes in the upper layers of the stratum corneum, forming poly- meric colored substances called melanoidins (15–17). Since DHA is bound in an irrevers- ible way to the free amino groups, the resultant color lasts for several days on the skin and is only removed by natural skin renewal. Skin regeneration occurs by the continuous generation of new cells in the basal layer that rise through the epidermal layers of the skin until they reach the stratum corneum, where the skin cells die and eventually fall or slough off (18). Therefore, the color intensity is directly related to the amount of DHA bound in the skin, and the durability of the staining by DHA is strictly dependent on the rate of skin cell renewal. On the basis of this assumption, in this study we tried to demonstrate that the use of DHA-induced pigmentation could be a valid method to estimate the activity of exfoliat- ing agents in promoting skin regeneration. For an objective evaluation of DHA-induced pigmentation, the study was carried out by non-invasive instrumental refl ectance spec- trophotometry, and spectral data were used to quantify skin color intensity. The safety profi le of the AHAs versus skin was studied by two different in vivo studies: the evaluation of skin erythema induced by topical application of acids at different con- centrations (10%, 30%, and 50% w/w) and the increase in sensitivity to UV light exposure