160 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS (b) allergen, and (c) allergen emulsified in CFA. A week later, allergen is applied under a closed dressing to the area, and the dressing left in place for 48 hours. Challenge is made after a further 9, weeks. The maximization meth- od of Magnusson and Kligman virtually eliminates the false negative results of the Landsteiner-Draize technique (Ref. 6, Table 8:6). However, there are several drawbacks to the maximization test. Materials for testing are admin- istered to the skin both topically and parenterally. Thereby, the variable of the efficiency of penetration into the skin of prospective allergen is eliminated. Passage through the barrier layer (stratum corneum) is a prerequisite sensitization and for challenge. Materials, such as nickel, that penetrate poorly are poorer sensitizers than they would be otherwise. It seems wise to avoid the artifice of an unnatural route of administration of a contact allergen. Mate- rials designed for topical use are best tested for by the topical route alone. Conversely, materials to be assayed for parenteral allergenicity should be given by the particular parenteral route alone, at least in final testing. The Magnusson-Kligman maximization test is designed to optimize the conditions for inducing sensitivity with a particular substance. Accordingly, very high concentrations of prospective sensitizer are used and there is a standard sol- vent, usually petrolatum. It is the purpose of our test to assay the allergenicity of final materials, e.g., topical antibiotics, perfumes, etc., in their ultimate con- centrations and vehicles. Clearly, the interaction of vehicle, active material (s) and preservatives as well as the particular concentrations of each will signifi- cantly affect the sensitization potential of a given preparation. It is the likeli- hood for inducing sensitization with proposed final products that our test is designed to measure. Of course, preliminary formulations can be assayed as well. What is the mechanism of action of Freund's complete adjuvant in its inten- sification of acquisition of delayed hypersensitivity in the guinea pig? On the basis of studies utilizing a variety of cellular and soluble allergens, we have proposed the following scheme (12). Allergen administered to the skin inter- acts with circulating immunocompetent lymphocytes ("T" cells) thereby be- coming immunocommitted. These lymphocytes pass to regional lymph nodes and clonalize. Freund's complete adjuvant acts in the regional node by mak- ing more efficient this clonalization. An important practical question is wheth- er intensification by Freund's complete adjuvant changes the relative ordering of the sensitization potential of different materials or instead more or less reg- ularly magnifies a given level of sensitivity. Our experience, and that of Mag- nusson and Kligman, and, additionally, a large body of information utilizing CFA to intensify the acquisition of sensitivity to macromolecular antigens sug- gests that there is no reordering of allergens. It is to experiments from Landsteiner's laboratory that we owe observations that (a) Freund's complete adjuvant enhances the acquisition of allergic con- tact dermatitis in the guinea pig, (b) multiple exposures to a simple chemical

BIOASSAY OF CONTACT ALLERGENS 161 allergen increase the sensitization rate, and (c) previous irritation makes a skin site more agreeable for the induction of sensitivity (13-15). The tech- nique of administering allergen under occlusion to the guinea pig was intro- duced by Buehler (16). Numerous studies in man and experimental animals have demonstrated that occlusion renders the barrier layer of the skin more permeable. Clearly, the test method for the evaluation of putative contact allergens that is proposed here requires extensive evaluation to determine its useful- ness. In a limited experience we have found it reliable. It should be empha- sized that it is particularly suited for rating the allergic potential of unknown against known materials, especially where all are designed for the same final use. For example, a proposed topical antibiotic for a given purpose would be compared with two or more topical antibiotic preparations that are currently used for that purpose. Of course, all preparations would be studied, in differ- ent guinea pigs, at the same time. The results of such studies would then not give absolute ratings, but would imply a greater or lesser risk of sensitization by the unknown as compared to the known. With experience, certain likely extrapolations could be made between results of individual experiments. Experience in other laboratories will doubtless improve our method. For instance, it may be that extensive shaving of the sensitization site is unneces- sary and, in fact, that only clipping of the site is necessary. We have no ex- periments directly to that point. (Received October 16, 1972) PREFERENCES (1) Rostenberg, A., and Haeberlin, J. B., Studies in eczematous sensitizations. III. The development in species other than man or the guinea pig, J. Invest. Dermatol., 15, 233-47 (1950). (2) Asherson, G. L., and Ptak, W., Contact and delayed hypersensitivity in the mouse. I. Active sensitization and passive transfer, Immunolog!t, 15, 405-16 (1968). (3) Simon, F., Observations on poison ivy hypersensitiveness in guinea pigs, J. Immunol., 30, 275-86 (1936). (4) Chase, M. W., H!lpersensitivity to Simple Chemicals, Harvey Lecture Series 61, 1965-1966, Academic Press, New York, 1967, pp. 169-203. (5) Marzulli, F. N., Carson, T. R., and Maibach, H. I., Delayed contact hypersensitivity studies in man and animals, Proc. Joint Conf. Cosmet. Sci., Washington, D.C., 107--22 (1968). (6) Magnusson, B., and Kligman, A.M., Allergic Contact Dermatitis in the Guinea Pig, Charles C. Thomas, Springfield, Illinois, 1970. (7) Draize, J. H., Woodward, G., and Calvery, H. O., Methods for the study of irritation and toxicity of substances applied to the skin and mucous membranes, J. Pharmacol. Exp. Ther., 82, 377-90 (1944). (8) Epstein, E., Allergy to dermatologic agents, J. Amer. Med. Ass., 198, 517-20 (1966). (9) Maguire, H. C., and Chase, M. W., Exaggerated delayed-type hypersensitivity to simple chemical a]lergens in the guinea pig, J. Invest. Dermatol., 49, 460-8 (1967). (10) Maguire, H. C., and Chase, M. W., Studies on the sensitization of animals with simple chemical compounds. XIII. Sensitization of guinea pigs with picric acid, J. Exp. Med., 135, 357-75 (1972).