UV SCREENING EFFECT OF SUNSCREENS 293 from El-Nasr Pharmaceutical Co., Egypt. Methylparaben and propylparaben were cour­ tesy of Alexandria Pharmaceutical Co., Egypt. Nutrient agar was purchased from OXOID Ltd, Basingstoke, Hampshire, U.K. Sodium chloride was obtained from Arabic Laboratory Equipment Company, Egypt. METHODS Microbiolgical procedures and calculation of DRY. Sunscreen preparations were applied as a continuous film on a UV-transparent membrane (Deema Packing and Packaging Ma­ terials, Egypt) covering nutrient agar plates on which E. coli was spread (7). The initial count/plate was adjusted at 200 colonies using optical density measurements (14). Plates were transferred to a laminar biological safety cabinet (NuAire, Inc., Plymouth, Minn., U.S.A.), with a UV lamp providing an average intensity of 10 mW/cm2 at the horizontal plane defined as the bottom of the work surface of the cabinet (7 6 cm away from the lamp). The plates were placed on a platform, 35 cm away from the UV lamp unless otherwise stated. The UV exposure time ranged from 20 seconds to two hours, depend­ ing on the SPF label claim of the product or the composition of the test formulation. Five plates were prepared for each exposure time. Non-irradiated plates were used as a control for the initial count per plate. After overnight incubation at 3 7°C, colonies were counted and the DRT determined. The DRT was estimated either mathematically or graphically from the regression equation obtained or the plot relating the average log survivors to the UV exposure time, respectively. Only data obtained with a correlation coefficient higher than 0.9, for the relation between log survivors and exposure time, were taken into account. Effect of experimental variables on the DRY. The influence of some experimental variables on the DR T of some market sunscreen products was investigated. These variables included the distance between the sunscreen/£. coli system and the UV lamp (35 and 76 cm), the sunscreen film thickness (1 and 2 mg/cm2), and the initial bacterial count/plate (100, 150, 200, and 300 colonies per plate). Reproducibility of the results was assessed by repeated testing of the product Luna® SPF 12. The sensitivity of the method was checked by testing market sunscreen products with SPF values ranging from 12 to 100. Testing potential applications of the method. The suitability of the method as a quality control tool was challenged by testing the effect of some formulation variables on the DRT. The effect of increasing the concentration of an oil-soluble sunscreen agent, benzophenone-3 (1 %, 2%, 3%, and 6%) and inclusion of additional sunscreen agents (phenylbenzimidazole sulphonic acid [2%} and titanium dioxide [5%}) on the DRT of a 2% benzophenone-3 o/w test lotion (Formulation 1) was investigated. DRTs of indi­ vidual o/w lotion formulations containing benzophenone-3, BZ-3 (2%), phenylbenz­ imidazole sulphonic acid, PBSA (2%) and titanium dioxide, TiO2 (5%) were also determined and compared to their corresponding blends. In addition, the suitability of the method for prediction of SPF was tested by correlating DR T data obtained for a series of market sunscreen products with their corresponding SPF label claim. RES UL TS AND DISCUSSION EFFECT OF EXPERIMENTAL VARIABLES ON THE ORT The effect of three experimental variables, namely the distance between the sunscreen/£. coli system and the UV lamp, the sunscreen film thickness, and the initial bacterial

294 JOURNAL OF COSMETIC SCIENCE Phase A (oil phase) Lanolin Benzophenone-3 (BZ-3) Titanium dioxide (TiO2) White petrolatum Stearic acid Propy1paraben Formulation 1 Phase B (aqueous phase) Phenylbenzimidazole sulphonic acid (PBSA) Methylparaben Edetate disodium Propylene glycol Triethanolamine Purified water to % (w/w) 5.00 X.00 Y.00 2.50 4.00 0.05 % (w/w) Z.00 0.10 0.05 5.00 Q.S. 100 count/plate on the DR T as a photoprotection assessment parameter was investigated using sunscreen lotion of a commercial brand (Luna®). Survival graphs for products Luna® SPF 12, 27, and 35 were obtained by plotting log survivors versus exposure time upon irradiation of the sunscreen/£. coli systems under study by a UV lamp placed at a distance of either 35 cm or 76 cm. Straight-line relationships were obtained (Figure 1). The decimal reduction time, DR T, was calculated from the graphs as the exposure time required for 90% reduction in the viable E. coli count (Table I). Greater DR T values indicate a higher degree of photoprotection. Expectedly, log survivor values were gen­ erally larger for test products with a higher SPF label claim. Decreasing the distance from the UV source from 7 6 cm to 3 5 cm resulted in a decrease in log survivors in all cases. It is worth noting that the test products with high SPF values (Luna® SPF 27 and 35) provided photoprotection with almost no change in bacterial count throughout the UV exposure period at 7 6 cm, precluding the calculation of DR T values for both products under the specified conditions. This could be attributed to a decrease in the killing effect of UV radiation at a longer distance from the UV source, as the intensity of radiation falling on a given area is governed by the inverse law (15 ). Sensitivity of photoprotection data obtained due to the change in the UV dose in terms of distance from the UV source allows the manipulation of this variable to enable testing sunscreen products with different SPF values. This can be achieved by reducing the distance or increasing the UV dose for products or test formulations, providing greater photoprotection. The effect of the sunscreen film thickness on the DR T was examined at two levels, 1 2 2 mg/cm and 2 mg/cm , using the test product Luna® SPF 12. The DRT calculated at 2 mg/cm2, the sunscreen film thickness approved for in vivo SPF determination (16), was 3.63 min. Decreasing the film thickness to 1 mg/cm2 reduced the DR T to 1.25 min (:::::65% reduction). Results indicate sensitivity of the method to the variation in the photoprotective effect of sunscreen products as a result of a change in the quantity applied per unit area. This shows the reduction in photoprotective effect upon reducing the applied amount of sunscreen, which is the case with all consumers who apply only 25-50% of the recommended amount of sunscreen product (17). The effect of the initial count of E. coli per plate on the DRT is shown in Figure 2. The DRT increased with increasing initial bacterial count per plate from 100 to 150. A