A NEW BROAD-SPECTRUM SUNSCREEN 69 experimental animals (tortoiseshell guinea pigs) were treated with samples on five sepa- rate areas and, ten minutes later, were irradiated once with 0.26 J/cm 2 of UVB (1.4 mW/cm 2) or 22.32 J/cm • of UVA light (3.1 mW/cm2). This process was repeated daily for three (UVB) to eight (UVA) days. Test sites were followed over 22 days to evaluate pigmentation by means of a color difference meter (Nippon Denshyoku Co. Ltd.), and the intensity of the pigementation was expressed by Hunter's color difference formula (AL). In human volunteers, back skins were treated with samples and, 20 minutes later, were exposed to natural sunlight for 20-60 minutes in midsummer. Test sites were observed 14 days later to determine the minimal melanogenic dose (MMD). The MMD was determined as being the smallest dose required to produce minimally visible pigmenta- tion at 14 days. Pigmentation was assessed as follows: 0, no pigmentation 1 +, min- imal visible pigmentation 2 +, moderate pigmentation 3 +, intense deep pigmenta- tion. ASSAY OF ARACHIDONATE METABOLITES Shaved back skin of Hartley white guinea pigs was irradiated with UVB using SE lamps at an intensity of 0.6 mW/cm = at 305 nm (1 MED = 0.29 J/cm=). For PUVA treat- ment, the animals received 4.2 mg/kg body weight of 8-methoxypsoralen in 30% eth- anol intraperitoneally. Thirty minutes after administration, they were exposed to UVA of 3.5 mW/cm 2 at 365 nm (1 MED = 3.5 J/cm=), emitted from Toshiba FL20BLB lamps. Levels of arachidonate metabolites were determined in organ-cultured skin by radioim- munoassay (6). Thus, irradiated or non-irradiated skin was cut off at indicated hours postirradiation using a punch biopsy knife with a diameter of 6.5 ram, then placed on a 3.5-cm culture Petri dish supplemented with 0.5 ml Eagle's minimum essential me- dium (MEM) containing 20% FCS, 4 mM glutamine, 100 units/ml penicillin, and 100 }xg/ml streptomycin. Organ culture was carried out at 37 ø C with 5% CO•/95% air atmosphere for 24 hours. Radioimmunoassay for PGE2 was performed with 2000 g supernatant of whole culture medium after addition of 1.5 ml MEM into the culture dish according to the method of Jaffe (7). HISTOCHEMICAL PROCEDURE Melanocyte population was evaluated according to the method described previously (8). Breifly, skin specimens (2.5 X 2.5 cm) were removed from the flanks of guinea pigs. The tissues were rinsed in 0.1 M phosphate buffer (pH 6.8) and incubated in 1 M sodium bromide for 5 h at 37 ø C. The epidermal sheets separated from the dermis were fixed in 10% cold neutral formalin for 30 minutes, washed twice with 0.1 M phosphate buffer (pH 6.8), and incubated in 0.1% dihydroxyphenylalanin (dopa) in 0.1 M phos- phate buffer (pH 6.8) for 5 hours. The number of melanocytes (per square millimeter) was counted using an Olympus-BHA microscope at a magnification of X 200. In each specimen, the number of melanocytes was calculated by averaging the numbers found in 50-100 fields.

70 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS RESULTS ABSORPTION PROPERTIES MDC can easily interact with cationic metal compounds like boehmite to produce an MDC-boehmite complex (Figure 1A) that results in spectral changes with varying A O O COOH I (MDC) Boehmile 1,3 - dikelone sol r/-COO O O O + + D MD C •J, max = 341 nm + •(DOOC% oe: 30,300 c .- 4Q,ooo ao,ooo 20'0001:• ø ""•'ø o o o UV-B • Boehmite/VA-100 {wt/wt ) 3OO 4O0 Wavelength / nm • -- -- MDC.Boehmite(I/l) 496 .... MDC. Na /--"-•x, • Eusolex 8020 .• 2 / C / \ ----- Parsol 1789 ,..,.,"' •'"\...i I -f"•-'•---'"X , J I I I , 250 300 350 400 450 500 Wavelength ! nm Figure 1. Chemical structures of MDC and MDC-boehmite complex (A), spectrum changes of MDC (B), and changes in molecular absorbance coefficient (C) in the presence of boehmite, and comparison with other UVA sunscreens in the spectrum at 4% concentration of emulsion (D).