EFFICACY AND TOLERANCE OF EXFOLIATING AGENTS 251 different concentrations (10%, 30%, and 50% w/w) of GLY, MAN, and GA formula- tions (200 μl) under occlusion conditions by Hill-Top Chambers for 3–15 minutes depending on the subject’s sensitivity. After the chambers’ removal, the cutaneous sites were washed by means of cold water-soaked gauze pads. For each skin site the induced erythema was monitored for 50 hours by refl ectance spectrophotometry. Since erythema is due to an increase in blood count in the subpapillary plexus of the skin, erythema index (E.I.) values were calculated by subtracting the logarithm of inverse refl ectance (log 1/R) values at 510 nm and 610 nm (mainly due to melanin absorption) from the sum of log 1/R values at 540 nm, 560 nm, and 580 nm, which represent the wave- lengths of hemoglobin absorption peaks (equation 2) (19). All the regions were mea- sured in triplicate. ª § · § ·º = + + + ¨ ¸ ¨ ¸» « © ¹ © ¹¼ ¬ 1 1 1 1 1 E.I. 100 log 1.5 log log 2 log log R560 R540 R580 R510 R610 (2) To evaluate the time course of skin erythema, E.I. baseline values were subtracted from the E.I. values obtained after application of the formulations, to calculate Δ.E.I. values. For each site, plotting Δ.E.I. versus the time the area under the curve was computed us- ing the trapezoidal rule to obtain AUC (area under curve) dimensionless index values directly related to the degree of skin erythema. IN VIVO EVALUATION OF THE PHOTOSENSITIZING EFFECT INDUCED BY TOPICAL APPLICATION OF THE FORMULATIONS In order to determinate the photosensitizing effect of the exfoliating agents, the skin ery- thema induced after UVB irradiation was evaluated in the same group of subjects par- ticipating in the previous studies, after a rest period of three months. For each subject, four skin test sites were defi ned on the ventral surface of each forearm. Three sites received 200 μl of 10% GLY, 10% MAN, or 10% GA formulations, applied once daily for four consecutive weeks. As reported before, the acid applications were completed within two minutes and termination was performed by cleaning the skin sites with cold water and neutralizing with 1% sodium bicarbonate solution. One site was used as control (no topical treatment). At the end of the fourth week, all sites were exposed to a UVB irra- diation dose, corresponding to the minimal erythema dose (MED), by using an ultravio- let lamp, model UVM-57 (UVP, San Gabriel, CA) that emitted radiation in the range of 290–320 nm, with an output peak at 302 nm. The fl ux rate measured at the skin surface was 0.80 mW cm-2. For each skin site the in- duced erythema was measured by refl ectance spectrophotometry (equation 2) twenty-four hours after the irradiation exposure, and the photosensitivity was expressed as the per- centage calculated from erythema index values using equation 3: T E.I. E.I.C Photosensitivity% 100 E.I.C = × (3) where E.I.C is the erythema index of the no-treatment skin site and E.I.T is the erythema index of the sites treated with the formulations.

JOURNAL OF COSMETIC SCIENCE 252 DATA ANALYSIS All data obtained were submitted to a statistical analysis. All statistical comparisons in instrumental assessment were evaluated using repeat-measure analysis of variance (ANOVA) followed by the Bonferroni-Dunn post-hoc pair-wise comparison procedure. A p value of less than 0.05 was considered signifi cant. RESULTS IN VIVO EVALUATION OF THE EXFOLIATING EFFECTS OF THE FORMULATIONS ON DHA-INDUCED SKIN PIGMENTATION The effects of 10% GLY, 10% MAN, or 10% GA formulations on skin exfoliation rates were evaluated by objective instrumental observation of in vivo DHA-induced skin pigmentation disappearance. DHA-induced skin pigmentation was determined as a melanin index by refl ectance spectrophotometry. For each subject, skin refl ectance spectra were recorded and melanin indices were calculated over the monitoring period of the study. The trends of skin pigmentation disappearance are reported in Figure 1 by plotting M.I values versus time (days). As can be seen, after DHA-pigmentation, the formulations showed different trends in M.I. value reductions over time. Moreover, to better compare the skin exfoliation rate, the “recovery time” (RT) values are given in Table I for each subject admitted into the study. The results showed that 10% GLY, 10% MAN, and 10% GA formulations were effective in inducing skin M.I. reduction in comparison with the non-treated site (CONTR) (p 0.05). An RT value of 10% GLY was signifi cantly lower than an RT value of 10% GA (p 0.05). Furthermore, a signifi cant difference was found between the RT value of 10% MAN and that of the 10% GA formulation (p 0.05). Figure 1. Trends of the melanin index (M.I.) vs time (days) for subjects recruited in the CONTR (no topical treatment), 10% GLY (glycolic acid), 10% MAN (mandelic acid), and 10% GA (grape acids) groups.