4 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS solubilized in Solvene 350 prior to addition to the Hionic Fluor solution and scintil- lation counting. IN VIVO MODEL Hairless male mice (n = 4) were sedated with chloral hydrate, and their flanks were then wiped with acetone until TEWL rates exceeded 2.0 mg/cm2/hr. Four 0.5-cm 2 circles were delineated on each flank with a black felt tip marker. Approximately 3 •1 of each of the following two test solutions was then applied to each circled area and gently rubbed in for 1 minute: 1) Petrolatum "neat" was prepared by drying down the appropriate amount of 3H- docosane in a tube, and then adding warmed petrolatum (in liquid state) with vigorous vortexing to a final concentration of 5.4 p•ci/ml (0.06 •ci/10 •g petrola- tum). Each application of the test mix was preceded by re-warming, then quickly applying the test mix before the petrolatum solidified. 2) 1% petrolatum was made by drying down 3H-docosane in a vial, adding petrolatum, and solubilizing both together in propylene glycol:ethanol (7:3 vols) to a final concentration of 0.08 •ci/•l (0.08 •ci/10 •g petrolatum). Mice remained asleep under a heat lamp for 2V2 hours, and then were bled and sacri- ficed. The surface of each test site was then wiped extensively with a cotton pellet to collect all excess surface test solution. An area 1.25 cm 2, with the 0.5 cm 2 circle within it, was sequentially tape stripped until tape no longer was adherent to the underlying skin layer (usually three strips). Each tape was collected and counted separately. The tape-stripped area of skin was excised and heat split at 60øC for 1 minute, yielding epidermis, dermis, and fat (scraped from the underside of the dermis). All samples (cotton balls, tapes, epidermis, dermis, fat, and blood) were saponified overnight at 50øC in 45% KOH in methanol, cooled, and extracted three times with petroleum ether. The extracts were dried down, redissolved in Scintisafe, kept in the dark for 18 hours, and then counted. RESULTS PENETRATION OF HEXADECANE THROUGH INTACT PORCINE SKIN In the first set of experiments we determined •4C-hexadecane absorption across intact skin. Regardless of the vehicle employed, most of the applied label remained unabsorbed on the outer surface of the skin samples, with the greatest levels of unabsorbed radio- activity occurring with the "neat" petrolatum vehicle (representative experiments shown in Table I). After removal of the non-absorbed label, we then determined the levels of •4C-hexadecane in subjacent skin layers. As seen in Figure 2, regardless of the vehicle, 95-98% of the absorbed label remained in the stratum corneum, with very low levels of radioactivity reaching either the nucleated layers of the epidermis or dermis. More- over, no label reached the receptor fluid, demonstrating that the label did not traverse the dermis under the conditions of these studies. These studies show that the relatively short-chain alkane, hexadecane, remains restricted to the stratum corneum, with little label reaching lower layers and none reaching deeper tissue levels. Moreover, whereas the

TOPICAL HYDROCARBONS 5 Table I Influence of Vehicle on Hexadecane Absorption and Transdermal Delivery (% Total Recovered Label) Vehicle Layer Petrolatum Polydecene Mineral oil Soya oil Cream (w/o) Surface 83 64.2 58.3 56.4 56 Horny layer 16.3 34.8 40.4 41.8 41.6 Epidermis 0.4 0.4 0.5 1.0 1.2 Dermis 0.3 0.6 0.8 0.8 1.1 Receptor fluid 0 0 0 0 0 Total 100.0 100.0 100.0 100.0 99.9 Distribution of absorbed C1 4-hexadecane lOO% 75% 50% 25% 0% percentage of absorbed C14-activity petrolatum polydec. miner. oil soya oil cream horny layer epidermis ß 2,2 dermis F• 1 receptor fl. I=1=1 o 97,2 1,1 1,7 o g•i,8 1,3 1,9 o 95,9 2,4 1,7 o 94,7 2,7 2,•I o Figure 2. Absorption of •4C-hexadecane across intact, freshly excised porcine skin. Regardless of the type of vehicle employed, almost all of the label remains restricted to the stratum corneum. Although small quantities of label reached the nucleated layers of the epidermis and dermis, none appeared in the receptor fluid, which corresponds to the vascular compartment in this model. different types of vehicles influenced the extent of hexadecane absorption, they did not influence the delivery of the label to deeper layers. PENETRATION OF DOCOSANE THROUGH INTACT PORCINE SKIN We next assessed the effect of increasing hydrocarbon chain length on penetration characteristics across porcine skin. Utilizing 3H-docosane (C 24:0) we again found that most of the alkane remained on the skin surface regardless of the vehicle (representative experiments shown in Table II). However, much less radioactivity was absorbed from petrolatum than from the other vehicles. Surprisingly, despite the tendency of docosane to remain on the skin when dissolved in petrolatum, this was not reflected in the ability of the absorbed label to penetrate to deeper skin layers (Figure 3). Although a slightly