EFFICACY AND TOLERANCE OF EXFOLIATING AGENTS 249 in cutaneous sites previously treated with AHAs. Both in vivo evaluations were monitored by refl ectance spectrophotometry. MATERIALS AND METHODS SUBJECTS In vivo experiments were performed on twenty healthy volunteers (females/males 14:6) of skin types II and III, aged 25–35 years. Between October 2006 and Septem- ber 2007, the volunteers were recruited after medical screening that included fi lling in a health questionnaire and physical examination of the application sites. Subjects exhibiting such features as sunburn, suntan, burn marks, or any other active lesions that might interfere with evaluation were excluded from the study. After they were fully informed of the nature of the study, substances, and procedures involved, the subjects gave their written consent. For the period of the studies, in vivo experiments were carried out on the volar forearms of each volunteer. Each subject rested for 15 minutes before the experiments, and room conditions were set at 22° ± 2°C and 40–50% relative humidity. Two research assistants were responsible for all recruit- ment and data collection. TEST MATERIAL Glycolic acid (70% cosmetic grade) and mandelic acid were supplied by A.C.E.F. (A.C.E.F. s.p.a., Fiorenzuola, Piacenza, Italy). Organic acids from white grape juice (a solution con- taining a blend of tartaric acid (18.5%), malic acid (12%), citric acid (3%), lactic acid (2.5%), gluconic acid (3.5%) and shikimic acid (0.5%)) were supplied by Bionap (Renegrape®, Bionap s.r.l., Italy). Exfoliating gel formulations were prepared by gelifi ca- tion with xanthan gum (1% w/w) of aqueous solutions containing three different concen- trations of glycolic acid (formulations labeled GLY), mandelic acid (formulations labeled MAN), and grape acids (formulations labeled GA). Standard samples of GLY, MAN, and GA were weighted and dissolved in water, obtaining solutions with a fi nal concentration of 10%, 30%, and 50% w/w for each acid. The pH of the test materials was adjusted to 3.5 by using sodium hydroxide. The test materials were alphabetically coded by the manufacturer, and neither the research assistants nor the volunteers knew the content of the blinded formulations. A sunless tanning formulation containing 5% w/w dihydroxy- acetone (A.C.E.F. s.p.a., Fiorenzuola, Piacenza, Italy) was prepared by mixing DHA in water and stirring for ten minutes. INSTRUMENTS Skin refl ectance spectra were recorded using a refl ectance visible spectrophotomer, X-Rite model 968 (XRite Inc. Grandville, MI), having 0° illumination and a 45° viewing angle, calibrated and controlled as previously reported (19). Refl ectance spectra were obtained over the wavelength range of 400–700 nm using illuminant C and a 2° standard ob- server.

JOURNAL OF COSMETIC SCIENCE 250 IN VIVO EVALUATION OF THE EXFOLIATING EFFECTS OF FORMULATIONS ON DHA-INDUCED SKIN PIGMENTATION For each subject, four sites on the ventral surface of each forearm were defi ned using a circular template (1 cm2) and demarcated with permanent ink. Baseline skin assessment was performed by refl ectance spectrophotometry on all sites. Each site was then treated with the 5% DHA formulation (200 μl) and kept under occlusive conditions with the use of Hill-Top Chambers (Hill Top Research Inc., Cincinnati, OH) for one hour, once daily for two consecutive days. After the removal of the chambers, the residual formulation was removed by gently wiping with cotton balls. One day after the second DHA application, the sites treated with DHA showed the development of a visually brownish coloration, and the skin refl ectance spectrum of each site was recorded by refl ectance spectrophotom- etry. Afterwards, three skin sites received a topical dose (200 μl) of 10% GLY, 10% MAN, or 10% GA formulation, applied by Hill-Top Chambers, once daily for 12 days. Application was completed within two minutes and terminated by cleaning the skin sites with cold water and neutralizing with 1% sodium bicarbonate solution. One site received no topical treatment (CONTR). For each site, skin refl ectance spectra were recorded over the monitoring period of two weeks that began at the conclusion of the 12 days. From the refl ectance spectral data, the melanin index (M.I.) was obtained using the following equation (equation 1) (20): § · = + ¨ ¸ © ¹ 650 700 1 1 M.I. log log 0.015 R R (1) where the log of inverse refl ectance values (log 1/R) is the apparent absorbance at a spe- cifi c wavelength (650 nm and 700 nm) and 0.015 is an adjusted instrumental factor. This index is calculated as the slope of the apparent absorbance levels from 650 nm to 700 nm and was used to measure both melanin and the melanogenic dose response. All the re- gions were measured in triplicate. After plotting M.I. values versus time, the time course of DHA-induced pigmentation disappearance was obtained for each site. The regenera- tion of the skin surface was obtained when the skin returned to the M.I. baseline, mea- sured before dihydroxyacetone treatment, and the DHA-induced pigmentation disappeared. The time (days) required to obtained MI baseline indicated the rate of skin exfoliation and was expressed as the “recovery time” value (RT) for each skin site. The RT was inversely related to the cell turnover acceleration induced by topical application of the exfoliating formulations. IN VIVO EVALUATION OF SKIN ERYTHEMA INDUCED BY TOPICAL APPLICATION OF THE FORMULATIONS In vivo evaluation of skin erythema by refl ectance spectophotometry was used to deter- mine the skin-irritant effect of the exfoliating agents after topical application. The experiments were performed on the same subjects as in the DHA-induced skin pigmenta- tion protocol after a rest period of three months. Nine skin sites (defi ned as described above and distinct from the sites used in the fi rst experiment) were treated with three