336 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS from a single donor, yielded specimens for four penetration chambers run simulta- neously, that is three samples and one blank test. BHA (Fa. Merck, Darmstadt, FRG) and [ • C]BHA (Amersham Buchler, Braunschweig, FRG) were added to a water-in-oil ointment (Beiersdorf AG, Hamburg, FRG) to prepare ointments containing 1% or 0.07% BHA. The final specific activity of the ointment applied to the skin was 5.47 X 10- 4p, Ci/mg. METHODOLOGY The experimental procedure of the permeation study was as described by Schaefer et al. (3). Using a small glass spatula, about 10 mg of the ointment were applied to the skin surface to cover an available diffusion area of 1.77 cm 2. Exact amounts were determined by differential weighing. The skin specimen was then inserted into a penetration cham- ber (4) kept at a constant temperature of 32øC. The acceptor medium was physiological saline (6 ml). The ointment was left on the skin for either four hours (1% BHA) or 16 hours (0.07% BHA), and then carefully wiped off. The horny layer was removed by 10-20 strippings with adhesive tape, depending on the individual skin tissue. Samples of 0.28 cm 2 diameter were punched out of the skin, frozen, and sectioned into layers of 100-}xm thickness with a microtome (Mikrotom- Kryostat II, Lab-Tek Instruments, Miles GmbH, Frankfurt, FRG). These skin sections were dissolved in, and the tapes were treated with, Soluene 350 (Packard Instruments, Frankfurt, FRG), whereas whole-skin samples were treated with 10 N KOH for four hours at 55-60øC. The residues, the tape solutions, the dissolved skin layers, and the receptor fluids were thoroughly mixed with scintillator cocktail and analyzed for [•4C] content (Minaxi Tricarb Liquid Scintillation Counter, Packard Instruments). The amounts of [•4C] detected in the analyses were set in relation to the amounts of [•4C] in the applied dose, and thus used to calculate the absorption and recovery rates. RESULTS Following application of ointment to the skin, no measurable amounts of radioactivity were detected in the receptor phase after four hours, even if the concentration of the applied dose was 1% BHA and the skin had been stripped before application to remove the stratum corneum. However, after 16 h on excised whole-thickness skin, measurable amounts were found in the chamber solution. Samples from five persons (2 male, 3 female) were examined (Table I). The overall mean rate of 0.18 }xg BHA/1.77 cm2/16 h (0.05-0.44 }xg BHA) had penetrated into the receptor fluid, i.e., 2.68% of the applied dose. Three samples from a person were tested to localize and determine the amounts of BHA in and on the skin and to obtain the recovery rate (Table II): 30.5% remained on the skin surface, whereas 6.7% was found in the horny layer and 50.8% in the whole-skin specimen after stripping. The concentration of BHA measured in the receptor solution was low (less than 10% of its solubility of 15.4 mg/1) and could thus sustain its driving force for permeation (5). The amounts found in the skin samples in relation to the skin depth are shown in Figure 1. In Figure 2 the radioactivity of the penetrating substance in each

ABSORPTION OF BHA 337 Table I In Vitro Permeation of 0.07% BHA in a W/O Ointment (10 mg) Through Whole-Thickness Excised Human Skin (surface area 1.77 cm 2) After 16 h (6 ml physiological saline as acceptor medium) BHA equivalents in Ftg, (mean -+ standard deviation, n = 3) % Applied dose d, 75 yr 0.17 -+ 0.044 2.2-3.0 d', 90 yr 0.33 ñ 0.120 3.2-7.1 •?, 58 yr 0.1! ñ 0.055 0.7-2.0 9, 81 yr 0.11 ñ 0.012 1.5-1.8 9, 24 yr O. 18 ñ O. 140 1.6-5.3 successive tape strip is plotted. Their sum represents the BHA concentration in the horny layer. DISCUSSION The use of in vitro preparations of human skin to study percutaneous absorption has been evaluated and compared to results obtained in living humans by Franz (6). He demon- strated that the in vitro data can be used to indicate the extent of in vivo dermal absorption. The combined in vitro results of the acceptor phases (Table I) give an overall mean permeation rate of 0. 179 Ftg/1.77 cm2/16 h (SD - 0.125 n -- 15). Presuming this experimental permeation rate and a daily application of a body lotion containing 0.07% BHA on a total skin surface of 1.8 m 2 (7) of a person with 60 kg body weight, the mean absorption rate would be 0.03 mg/kg bw/d, that is 6% of the acceptable daily intake through food. If one presumes a daily consumption of only 10 g of a body lotion Table II BHA Equivalents in Three Parallel Samples of a Skin Specimen (•?, 24 yr) BHA equivalents (Ftg) % Applied dose Residue on the skin surface Horny layer (10 strippings) Whole-skin sample after stripping (solubilized in 10 N KOH, 4 h at 5540øC) Amount in the chamber fluid Total recovery 2.O2 31.60 1.75 29.3O 1.96 3O.7O O.38 5.89 0.41 6.89 0.53 7.52 3.28 3.23 3.27 O. 10 0.06 0.37 51.30 54.2O 46.9O 1.56 1.02 5.34 9O.4O 91.40 9O.5O