BIOASSAY OF CONTACT ALLERGENS 155 milliliter of test compound is applied. This is covered with an occlusive dressing consisting of an outer layer of stretch adhesive (Elastoplast), a mid- die layer of wide-mesh gauze (K]ing) and an inner layer of Blenderm tape, but with the sticky side away from the skin. The adhesive part of the Elasto- plast overlaps the sides of the dressing. The dressing is fixed in place with adhesive tape (this may not be necessary). Twenty-four hours later, the dressing is removed and readings are made. The hair is clipped around the borders of the test site so as to enlarge it, precaution being taken to identify the test site by straddling marks made with a skin-marking pencil. Further readings are made at 48 hours, at 72 hours, and later, under special circumstances. Retestings at a different site for a second and third testing occasionally will bring out borderline earlier readings (5, 10). The appearance of the challenge sites is recorded as word descriptions xvhich are later scored (e.g., 0, trace, ñ, +, -4-+ . . . ) (9, 10). The scoring system rates the clinical intensity of the dermatitis induced by allergen as fol- lows: 0, normal skin ñ, very faint pink, nonconfluent +, faint pink ++, pale pink, usually slighfiy elevated +++, pale pink to pink, usually moder- ately elevated ++++, pink and thickened +++++, bright pink and markedly thickened. Intermediate scorings (trace, +ñ, etc.) are made as appropriate. Readings are best made by a senior person who has no knowl- edge of the test animals' prior history. Prospective Sensitizers Benzocaine, neomycin sulfate, hexachlorophene, Furacin, bithionol, and tri- bromosalicylanilide were obtained as powders.* Procaine crystals as Nova- caine©*, 5% Xy]ocaine©* ointment, 5% mafenide cream (Sulfamy]on©? cream), streptomycin sulfate, U.S.P.õ, picric acidll , and petrolatum, U.S.P., were purchased in small quantity. When a powder was incorporated in petro- latum, this was done directly, i.e., without prior solubilization. ]•ESULTS In a typical early experiment we compared two topical anesthetics, viz., benzocaine 5% and procaine 5%, each made up in U.S.P. grade petrolatum, as well as a commercial (lidocaine) topical anaesthetic, 5% Xylocaine oint- ment. Prospective sensitization was as outlined in Methods, except that the sensitization site xvas neither shaved nor treated with dry ice. The maximum * From Dr. F. N. Marzulli, Director, Dermal Toxicity Branch, Division of Toxicology, Food and Drug Administration, Washington, D.C. 20204. ? Winthrop Laboratories, New York, N.Y. Astra, Worchester, Mass. õ Eli Lilly, Pearl River, N.Y. If Allied Chemical, Morristown, N.J.

156 JOUBNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table I Comparative Trials: Benzocaine 5% vs. Procaine 5% Sensitization T-22 Challenge 1% Intraderma] 5% Topical 78 Benzocaine 5% 0 a tr 79 I 0 tr 80 0 0 81 0 0 82 0 + ñ 83 0 + ñ t.c .... 0 0 t.e. 0 0 84 Procaine 5% 13.0 mm q-q-+ ñ 85 ] 4.0 mm 86 9.0 mm 87 16.5 mm 88 12.5 ram 89 13.0 ram t.e .... 0 0 t.c .... 0 0 "1% suspension. reactions to intradermal and topical challenge tests made on Day 22 are shown in Table I. Clearly, procaine is the more efficient sensitizer 5% Xylo- caine did not sensitize any animals. In later experiments, the sensitization rate with 5% benzocaine was increased to 100% by shaving and freezing the sen- sit zation site. Under the latter circumstances, in different experiments, simuI- taneous prospective testing with 5% Xylocaine ointment, with 5% bithionol in peh'olatum (no ultraviolet light activation), with 5% Furacin in petrolatum, and with 5% tribromosalicy]ani]ide in petrolatum gave no sensitization. This accords with clinical and experimental results in man (5, 6). In another experiment, we tested the allergen •city of three different con- centrations of picric acid in petrolatum. The sensitization method was as out- lined in Methods. For challenge open patch tests were made on Day 16 with one drop of 1, %, and •A•% picric acid dissolved in dibutyl phthalate (10). The maximum readings and the arithmetic sum of the "pluses" of each group of testings are tabulated in Table II. As is generally, but not universally, true, the higher concentration of sensitizer elicited the stronger reaction and in- duced the higher incidence of sensitization. In another experiment, we prospectively sensitized groups of guinea pigs with 5% mafenide cream (Sulfamylon cream), 5% streptomycin sulfate sus- pended in petrolatum, and 5% neomycin sulfate suspended in petrolatum. In the respective groups (plus toxicity control groups) challenge was made with 5% mafenide cream, 5% streptomycin sulfate ointment, and 5% neomycin sul- fate ointment. Five per cent malehide cream and 5% streptomycin sulfate