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

328 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS skin there should be allowed to elapse a period of time. This period varies with different observers and in searching the literature one finds that this period varies anywhere from five days to twenty-one days. It is this period during which time the individual is not in contact with the substance that is called the sen- sitizing period since the initial ap- plication of the tested substance to the skin has resulted in the probable deposition of small quantities of the test substance in the skin. The bio- logical effects of this become appar- ent on the second application. On second application the substances are again applied to the skin of the same subjects and in the same man- ner as done on the first application. This patch is kept in contact with the skin for a period ranging any- where from twenty-four to forty- eight hours. At the end of this time, these patches are removed and the reactions are noted. The second application of a substance tested, such as wearing apparel, would be re-wearing the same arti- cle of clothing so that it would be in contact in the same parts of the body, as on the first application. If on first application there has been no evidence of irritating effect on the skin and after the subsequent layoff period and a reapplication of the material to the skin there is a reaction, then the skin has been sensitized to that substance. The substance is therefore known as a sensitizing substance. Ira material gave very mild reactions on first application and the second appli- cation elicited much stronger re- actions in a significant number of subjects using the same concentra- tion of this test substance then the material once more can be consid- ered as being a sensitizer. If a material produced a marked reac- tion on first application, it is only proper to assume that the reaction would have been obtained in the second application if the same con- centration were used. Therefore where strong reactions are obtained on first application of the material, then on second application a much higher dilution is applied to the skin. If the reaction to the diluted mate- rial is as great as, or greater than, that obtained with a more concen- trated material on first application then this substance may also be said to be a sensitizing substance. Thus far we see that if a sub- stance is a primary irritant, we can control this irritating effect either by varying the time during which the material is kept in contact with the skin or by diluting it suffi- ciently with an innocuous substance and thereby reaching the point where the material is no longer irritating regardless of the time it is kept in contact with the skin. Therefore, with primary irritants we can regulate the action of the material or we can eliminate its irritating effects entirely. The situation is quite different with the substance which is a sen- sitizer. This substance on first ap- plication may give absolutely no reaction, or a very mild reaction, or in some cases it may also be a