568 JOURNAL OF I'HE SOCIETY OF COSMETIC CHEMISTS most widely employed assays are illustrated and described here. The first two assays are indicative of general corticoid activity. They may therefore be used as a preliminary screen for selecting those compounds which are to be further tested in order to determine if they also have topical anti-inflammatory activity. Thyroids Involutio•t Assay--This assay is run on adrenalectomized rats (3, 4). The sizes of the glands are measured, and the degree of in- volution caused by the steroid is determined as an indication of its potency. Antigranulo.ma Assay--Adrenalectomized rats are also utilized in this assay (3, 4). Granulomas are induced by subcutaneous implantation of cotton pellets on either side of the thorax. The degree of granuloma in- hibition achieved by a corticosteroid reflects its potency. Figure 3 shmvs inhibition of granuloma formati.on achieved with hydrocortisone and fluocinonide. This assay, as well as the thymus involution assay, mea- sures systemic rather than topical activity. Fibroblast Assay--The in vitro culture of fibroblasts can be used as an assay method for anti-inflammatory substances, and a direct correlation Figure 3. Inhibition of granulomas induced by subcutaneous implantation of cotton pellets A. Untreated controls B. Hydrocortisone, 1 mg C. Fluocinolone acetonide, 0.002 mg

CORTICOID, VEHICLE, AND SKIN INTERACTION 569 has been demonstrated between biological activity and corticoid bio- transformation at this target cell level (5). Further correlations can be obtained since similar structure activity relationships exist in vitro as are found in actual clinical use (6). For example, fiuocinolone acetonide and i, ts acetate, fiuocinonide, have been shown to be particularly resis- tant to biotransformati.on by fibroblasts and exhibit growth inhibitory activity at extremely low concentrations (7). These anti-inflammatory steroids also induce a morphological change consisting of a shortening ot• terminal processes resulting in a rounded fibroblast which appears to enable the fibroblast .to resist the chain reaction of cell destruction initiated by the inflammatory agent and the cytotoxic products elaborated during the inflammatory reaction. This permi½s the body's natural re- sources to clear up the inflamed area and repair the damaged tissue (6). Rat Ear Assay--The method employs 21-day old male rats. A mix- ture containing 20% pyridine, 5% water, 74% diethyl ether, and 1% croton oil, with or without the test compound, is inuncted to the ears once. Ears are removed 6 hours after administration and uniform areas of the ear are punched out with a No. 4 cork borer and weighed. The topical corticoids significantly inhibit the increase in weight of the treated ear (2). Human Vasoconstriction Assay--The human vasoconstriction assay, developed by McKenzie and Stoughton (8, 9), has become one of the most valuable assay methods for precise evaluation of topical cor'tico- steroid potency. The vasoconstri,ction assay measures the degree of visi- ble blanching caused by various dilutions of corticosteroids when ap- plied to the human skin (Fig. 4). A relationship has been demonstrated between the ability to induce vasoconstriction and the ability .to combat inflammation in therapeutic use (10, 11). Although many of these assays have been performed by different investiga,tors under different condi- tions, a compilation of the approximate relative biological potency ot• fluocinonide as compared to hydrocortisone is presented in Table I. Table I Biological Potency of Fluocinonide Relative to Hydrocortisone as 1 Potency Assay (approximate) Rat ear 350 Fibroblast 440 Vasoconstriction 400