IN VITRO SPF DETERMINATION ON HD6 PMMA 247 before and after application of the sunscreen. A PMMA plate covered with 10 mg of C.B. was used to obtain the blank transmittance spectrum from 290 to 400 nm in steps of 1 nm. Preliminary studies showed that the optimal amount of sunscreen per surface unit on these high-roughness plates is 1.3 mg/cm2 (5). Twelve milligrams of C.B. were spread with a saturated fi ngercot until a homogenous dis- tribution was achieved over the whole surface. A period of ten minutes is necessary for C.B. stabilization. Samples A and B were applied in parallel on the HD6 PMMA with and without C.B. pretreatment. The sunscreen product was spread over the whole surface by means of light strokes with a fi ngertip “presaturated” with the product. The different formulae studied were spread with the same protocol. The sample thus obtained was allowed to settle for 15 minutes in the dark at room temperature to ensure a leveling of the formula. A total of 9 UV trans- mission spectra (from 290 to 400 nm, 1-nm increment steps) were recorded on each plate at different locations. ROUGHNESS MEASUREMENT OF FILM PRODUCT SPREAD ONTO HD6 PLATES Roughness of the substrate. The roughness of the HD6 substrate was measured by non- contact surface topographic analysis using an “Altisurf 500” Lab-workstation from Altimet™, France. The system is composed of an optical sensor, a motion controller, an x-y translation stage, and a microtopography software Mountain Altimap module. The confocal optical sensor “Altiprobe” is based on a white light chromatic aberra- tion principle that allows a high resolution: 10 nm vertical and 1 μm horizontal. Five different areas of exactly 10×5 mm were analyzed according to the scheme illustrated in Figure 3. Surface topography parameters. As described in our previous publications (5,14), the control chart enables the roughness of an HD6 surface to be characterized. In the present publication, the control chart of the HD6 substrate with and without C.B. pretreatment was determined. For each covered PMMA plate, fi ve areas were ana- lyzed. We obtained the respective values for the ten parameters for every location, following which we determined the mean of fi ve values obtained for the fi ve different sites studied. Figure 3. Localisation of the fi ve areas measured with an “Altisurf 500” Lab-workstation.

JOURNAL OF COSMETIC SCIENCE 248 MICROSCOPIC ANALYSIS The behavior of samples A and B during spreading were followed by microscopy to understand more fully the role of pretreatment on product distribution. The microscopic observations were performed with a microscope (Axio Imager, Carl Zeiss S.A.S.) in con- junction with a camera (Axio Cam MRc.) The images were then analyzed with Axio Vision software. RESULTS AND DISCUSSION Different in vitro methodologies have already been proposed by the industry to assess the in vitro SPF of suncare products, but all the methods proposed showed poor correspon- dence between in vitro and in vivo values. The limitations of the existing in vitro methods were explored and showed the importance of the substrate used. A previous study, using the injected molded plates, revealed that results can be improved by controlling the physical characteristic (the roughness) of the plates. Nevertheless, this step seems to be necessary, but not suffi cient, to have complete control of the technique, as HD6 fails to reproduce the in vivo results in some cases. The question is why the HD6 sometimes fails to give reliable results. The present study investigates the chemical aspect of the spreading of different products on HD6 plates, offering either poor or good correlation with the in vivo results. The aim is to compare the interactions between product and substrate in both cases. First, the criteria of transparency and roughness were controlled on both types of plates (treated and untreated) in order to reproduce exactly the same experimental conditions and adhere to the Colipa guidelines concerning the transparency and the roughness con- trol chart (5). OPTICAL TRANSMISSION GUIDELINES According to Colipa guidelines, the minimum average optical transmission require- ment through a substrate treated with glycerin is 60% at 290 nm, 69% at 300 nm, and 81% at 320 nm. The present study complied with these recommendations. The optical characteristics of cocamidopropyl betain are compared with glycerin in Table II. The results of transmission obtained with the cocamidopropyl betain-treated HD6 plates showed that the transparency required is appropriate for UV analysis. This amphoteric pretreatement can be introduced in an in vitro UV spectroscopic method. Table II Percentage of Transmission at 290 nm, 300 nm, and 320 nm for the PMMA Plate HD6 Treated with C.B. Treatment PMMA HD6 + glycerin PMMA HD6 + TegoBetain F50 Wavelength (nm) 290 300 320 290 300 320 % Transmittance 66.7 71.8 83.1 68.5 73.6 84.9