CHALLENGES TO SUNSCREEN EVALUATION 589 at 420 nm, it provides exposure conditions closely matching Miami Peak Sun 26 S Direct and COLIPA sun. In our study the irradiation fl ux was normalized to 1.57 W/m2 at 420 nm in order to expedite the pre-irradiation step. SUBSTRATES The FDA’s proposed rules use optical grade roughened quartz plates as substrates with an application dose of 2 mg/cm2, which is applied with a gloved fi nger (pre-saturated with test article) within approximately ten seconds with a very light spreading action. Optical grade roughened quartz substrates with dimensions of 5 cm × 5 cm × 2 mm (Q-Glass) were obtained from the Q-Glass Company (3). According to the manufacturer, Q-Glass plates are roughened on one side in a reproducible manner. It should be noted that the roughness of quartz plates is not specifi ed by the FDA however, L. Ferrero et al. showed that this parameter is important for uniform application and reproducible measurements on roughened substrate (4). In the proposed rules, the FDA requested comment regard- ing the suitability of other possible substrates, which prompted the evaluation of an al- ternative substrate, Vitro Skin® N-19 in this study. It was pre-cut in 6.2 cm × 6.2 cm pieces to provide a suffi cient area for transmittance measurements at the required 12 loca- tions and pre-hydrated according to reference 5. References (blanks) and substrates with applied test articles were placed on slide mounts, positioned inside the Ci65A, and pre- irradiated simultaneously in order to reach the required PIDs. TRANSMITTANCE ANALYZERS Labsphere UV 2000S and Optometrics 290S transmittance analyzers were employed in this study: • The Labsphere UV 2000S (Figure 2) utilizes a Xenon UV source, 10 watts, with fl ash radiation. It has an integrating sphere and a photodetector, and it provides a continu- ous emission spectrum from 290 nm to 400 nm, with suffi cient illumination at each wavelength, but not in excess of 0.2 J/cm2 the dynamic range from 290 nm to 400 nm is at least 2.7 absorbance units. • The Optometrics SPF-290S transmittance analyzer has a Xenon UV source, 125 watts, with an integrating sphere, a monochromator, and a photomultiplier the dynamic range from 290 nm to 400 nm is 2.5 absorbance units. MEASUREMENTS AND CALCULATIONS The determination of the UVAI/UV ratio according to the FDA’s proposed methodology consists of two consecutive parts. The fi rst part requires conducting spectral transmit- tance measurements in order to calculate the value of mean transmittance and its stan- dard deviation from 290 nm to 400 nm at 5-nm increments, and the second part requires determining the coeffi cient of variation. At least 12 measurements of transmitted spectral irradiance are required for reference without sunscreen: [C(λ)1,C(λ)2…C(λ) 12], and for the substrate with applied sunscreen: [P(λ)1,P(λ)2…P(λ) 12]. These calculations gener- ate 23 transmittance values with associated standard deviations, one for each 5-nm

JOURNAL OF COSMETIC SCIENCE 590 increment from 290 nm to 400 nm. The value of mean transmittance and its standard deviation are calculated according to the following equations: O)/n O O Oº)) O O)/n ª º u ª « » « » ¬ ¬ ¼ ¦ ¦ 2 2 1 2 1 ( ( ) ( ( )) ( and ( ( )) ( n n P P s C s P( T(O) s C C(O¼) C Standard deviations of reference s(C(λ)) and sample s(P(λ)) are also calculated accord- ingly: O O O O ¦ ¦ 2 2 1 ( ) ( ) ( ) ( ( )) and ( ( 1) ( 1) n n C C P P(O) s C s P(O1)) n n The coeffi cient of variation (CV) is determined by dividing the standard deviation by the mean and expressing the resulting value as a percentage. The CV provides an indication of the uniformity of the sunscreen layer, and this value should be less than 10%. At least fi ve repetitions are used for each test article. It should be noted that when measurements are taken on the Labsphere UV 2000S, they can be extracted as transmittance, absorbance, or mPF values. The Labsphere UV 2000S measures transmittance values in the wider range of 250-450 nm, and the special calcu- lating template was created to extract only the required transmittance values from 290 nm to 400 nm at intervals of 5 nm. The Optometrics SPF 200S provides an option to measure transmittance values from 290 nm to 400 nm at intervals of 5 nm, but the values can only Figure 2. Optical design of Labsphere UV 2000S transmittance analyzer (courtesy of Labsphere).