BIOASSAY OF CONTACT ALLERGENS 159 DISCUSSION The success of experiments dealing with the acquisition and expression of allergic contact dermatitis in the guinea pig requires the use of healthy ani- mals. Vitamin C deficiency, to which the guinea pig is uniquely susceptible, and bacterial infections (particularly with Streptococcus C ) must be carefully avoided. The albino Hartley guinea pig is widely available and is well suited for ordinary experiments in allergic contact dermatitis. If, for special experi- ments, histocompatible animals are required, the inbred Family II or Family XIII can be used. However, these inbred strains are less fertile than the Hart- leys and on the average are somewhat more difficult to sensitize. Which sex is to be preferred? We have not been impressed with significant differences of skin reactivity between males and nonpregnant females. However, the female has the advantage of being more placid, and thereby easier to handle and less apt to have damaged skin from fighting. Pregnant guinea pigs give poorer and less reliable skin reactions and should not be used. Guinea pigs do establish a social order and should not be rehoused after the start of an experiment. Verv young guinea pigs, less than 2 weeks of age, sensitized poorly the defect ap- pears to be peripheral h• that their skin does not express the reaction of aller- gic contact dermatitis even when the animals are passive sensitized (with viable cells from adult sensitized guinea pigs ). According to Magnusson and Kligman, guinea pigs older than I year do not sensitize well (6). The cause for this is not known. In practice, we select young adult animals weighing close to 400-500 g. The weight and sex of the guinea pig should be controlled between groups where comparisons are to be made. Warm temperatures in the animal room, as during the summer in the absence of air conditioning, tend to impair the delayed-type immunological responsiveness of the animal. The requirement for a healthy, well-cared for, suitably chosen guinea pig in experiments dealing with allergic contact der- matiris cannot be overstressed. The problem set in the predictive testing of possible contact allergens is to identify those materials that will cause significant difficulty in man. In the Landsteiner-Draize assay, the test material is dissolved, or suspended, in sa- line, and injected intradermally into the clipped dorsal skin of the guinea pig (7). The sensitizing schedule consists of a total of 10 injections given thrice weekly the volume of the first injection is 0.05 ml, that of the others 0.1 ml. Two weeks after the last sensitizing injection, the animal is challenged with an intraderma! injection of 0.05 ml delivered to normal skin. A significant dif- ference in the reactions to the first sensitizing injection and the challenge in- jection is interpreted as sensitivity. Strong and moderately strong sensitizers can be identified by this technique. Magnusson and Kligman developed a maximization method in the guinea pig for identifying contact allergens (6, 11 ). Their sensitizing protocol involves the following: simultaneous but sepa- rate paired intradermal injections of (a) complete Freund's adjuvant (CFA),

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