A NEW IN VITRO METHOD TO DETERMINE SPF 103 dose for each EBT3 fi lm is calculated as follows: 41 w/m2 × time × area. For example, 30 min irradiation will give 237.12 J dose on the fi lm. The calibration curve was obtained by the four points with FilmQA pro software [the function of irradiation energy and red, green, and blue (RGB) color, respectively]. The average relationship between irradiation dose and fi lm color change (RGB) can be established through the calibration curve. A total of 2.0 mg/cm2 sunscreen was applied on skin for the in vivo SPF test. To be con- sistent, 64.3 mg sample (2.0 mg/cm2) was also applied on EBT3 fi lm and evenly distrib- uted with gloved fi nger, from top to bottom and side-to-side with minimal pressure, avoiding spotting on the edges. After 30 min of UV irradiation, the irradiation dose of EBT3 fi lms with and without application of sunscreens was calculated by the calibration curve. The in vitro SPF value was calculated by the following: Dosage without sunscreen SPF= Dosage with sunscreen RECEIVED RECEIVED (3) RESULTS EBT3 FILM COLOR CALIBRATION EBT3 fi lm is composed of an active layer, nominally 27 μm thick, containing a radiation- sensitive active component, marker dye, stabilizers, and other additives. The active layer is between two, 120 μm transparent polyester substrates. After exposure to UV light, the color of active layer changes from green to dark. The color change (RGB) will have a de- fi ned relationship with cumulative UV irradiation dose. The calibration curves of EBT3 fi lm after 0, 10, 20, and 30 min UV irradiation are shown in Figure 1. All color param- eters, RGB, may be used for calculation, but red parameter was chosen due to the highest sensitivity in our data range. IN VITRO SPF TEST BY EBT3 FILM Applying this in vitro SPF test method, a commercial sunscreen lotion (SPF 30, PA++) was tested by EBT3 fi lm (Figure 2). Film with and without sunscreen was irradiated by Q-SUN chamber for 30 min. The absorbed dose of fi lm without sunscreen is 237.12 J, whereas the average calculated absorbed dose of fi lm with sunscreen is 7.96 J. Accord- ingly, in equation (3), the calculated SPF of sunscreen lotion is 29.8 ± 3.2. The SPF result is very close to the labeled SPF value of 30. Like the in vivo SPF test (equation 1), the new in vitro SPF test (equation 3) is also based on UV dose cumulate response, taking into ac- count sunscreen photostability. INFLUENCE FACTORS ON EBT3 FILM Different irradiation time and dose will affect the test results. Figure 3 shows the in vitro SPF results of two commercial sunscreen lotions based on EBT3 fi lm with different Q-SUN irradiation time. For SPF 20 sunscreen, the measured SPF value decreases from

JOURNAL OF COSMETIC SCIENCE 104 Figure 1. Calibration curve to set up the function between irradiation dose and response. 35 after 10 min irradiation time to 11 after 40 min irradiation time, whereas SPF 30 sun- screen maintains approximately 25–30 over 40-min period. In theory, different irradiation time or dose should not affect the fi nal SPF results, but the exception exists if the sunscreens are not UV stable. For example, SPF 20 sunscreen de- grades under extended UV irradiation. Therefore, the measured SPF value is much lower after 40 min irradiation. The fi lm color change, with and without sunscreen, need to be within the range of the calibration curve to ensure accuracy. Too dark or too light will affect the accuracy of the results. In this study, 30 min irradiation time and 41 w/m2 intensity was utilized for the remainder of the experiments. IN VITRO SPF TEST ON COMMERCIAL PRODUCTS To validate the new in vitro SPF test, the process was repeated on 15 commercial sun- screen products purchased in the market. The UV fi lters included in the products, labeled SPF value on the bottle, and in vitro SPF values measured by EBT3 fi lm are shown in Table I. Some products contain organic UV fi lters only, while others contain inorganic UV fi lters only, or together with organic UV fi lters. The SPF values labeled on the bottles vary from 15 to 50+ and PA to PA++++. When inexact SPF values were given, an addi- tional 5 were added onto the labeled value. For example, for SPF 30+, SPF 35 will be used in the calculations. For commercial products with organic UV fi lters only (samples 1–8), the correlation of in vitro results and labeled SPF value is illustrated in Figure 4, the trend line of EBT3 fi lm data is R2 of 0.9266. In the best situation, the interception of the equation should be close