326 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS even mechanical irritation. Second- ary irritation is a specific reaction of the body as a whole to a sub- stance. It is an allergic response of the body based upon an allergen- antibody reaction. A secondary ir- ritant shows no irritation at the end of the first application. How- ever, on reapplication skin irritation results. In the interim between the first application and second application, the body's defensive mechanism begins to form anti- bodies as a reaction to contact. When the substance is reapplied to the skin a second time anti- bodies are already present and the antagonism between these anti- bodies and the substance produces a reaction. This reaction may be local, that is, confined only to the site of the reapplication, or it may be general, that is, over the entire skin surface as well as mucous membranes and the viscera. Thus step 3 of the patch test procedure, the recuperation period, must be of duration sufficiently long to enable the body to produce the antibodies necessary to react with the sub- stance when it is reapplied. There are many properties of primary irritants which are factors in the testing of these irritants. These are concentration, volatility, duration of contact, area of contact, pH, and osmotic pressure. The concentration of a substance which is placed on the skin is an important factor, since, if a substance is known to be irritating in high concentra- tions it may not be in lower con- centrations. In studying a sub- stance which shows a tendency to- ward being a primary irritant, it is wise to work with high dilutions so that reactions obtained are not violent. Concentrations of the sub- stance may gradually increase as the solvent vaporizes or evaporates and produce a misleading result. Just as much, can be learned about irritating properties of a primary irritant from a mild reaction in higher di- lution as can be learned from a severe reaction in low dilutions. The boiling point and vapor pres- sure of the solvent are also very im- portant since highly volatile sub- stances are irritating by virtue of their burn-producing capacity for example kerosene is a widely-used solvent yet the number of cases of dermatitis produced by kerosene is relatively small. However, im- proper use of kerosene produces an irritation of the skin. Thus if a substance such as kerosene is used as a solvent, the patch would be removed within an hour after application. If we permit it to stay on the skin for twenty-four hours, such as is done with water- solvent patches, a large blister similar to a first degree burn would result. However, the reaction which is produced on the skin with kero- sene in an hour disappears within a very short time. The irritating qualities of a highly volatile sub- stance such as kerosene can be mitigated to a great extent by dilut- ing it with an innocuous substance such as heavy mineral oil and there- by permitting a longer period of contact with the skin. Eventually

EXPERIENCES WITH HUMAN PATCH TEST METHOD 327 one may reach a dilution of a mix- ture of kerosene with mineral oil which could be kept in contact with the skin indefinitely without pro- ducing any irritation. The length of time which a mate- rial is kept in contact with the skin is determined by the end use of that material. Therefore, if the end use of a substance under investi- gation calls for only a few minutes' contact before it is washed off, step 1 is relatively short in duration, a perio•t of twenty-four to forty-eight hours should be a long enough dura- tion of contact. However, many substances in their end use are to re- main in contact with the skin for long periods of time, such as the dyes and the sizing chemicals used in textiles which go into clothing and also the chemicals which go into the manufacture of cosmetics. These substances should be placed in intimate contact with the skin for longer periods of time, for at least five days. Another factor in determining the irritating properties of a substance as determined by the end use is the area of the skin exposed to that substance. For example, lipstick in its end use is confined to a small area of the body. Stockings or leg paint cover a larger area of the body. After-bath powders and cloth- ing cover an even larger area of the body. Soaps come in contact with almost the entire surface of the body and if one is unfortunate enough, soaps may even come in contact with the conjunctiva of the eye. It follows that the area of skin exposed to the substance in the patch test should be in propor- tion to the area of the skin exposed to the substance in its ultimate use. Thus, in order to test lipstick, a patch test covering a small area suffices. Under ideal conditions of testing, if a textile dye were to be studied for irritating properties, it would be exceedingly advanta- geous to have an article of apparel, which. could be colored with this dye, worn by the subjects. Where a cosmetic is to be placed over a large area of the body, the size of the patch which is used in the patch test should be increased consider- ably so that it covers a minimum of at least four to six square inches, where indicated, and to test it as closely as possible to actual-use conditions. Thus far, we have spoken of the first application of a substance to the skin. This first application will determine whether the sub- stance studied is a primary irritant or not. It will also determine the extent of, or what measure of, primary irritation the substance causes. We also try to determine the dilution at which the irritating properties are eliminated. We have considered the concentration of the material studied, the end use to which the material is to be placed, the length of time that the material is to be in contact with the skin, and the size of the patch which is to be used, and whether or not the patch should be a sealed one. When this first application of the substance is removed from the