STATEMENT OF PROBLEM AND CRITICAL REVIEW OF PAST METHODS 61 penetration and cite one or more examples of investigative work in which each of the methods has been used. I believe it is possible to fit most of the methods into the following categories: 1. Penetration into"models" 2. Study of changes in the histology of the skin 3. Use of tracers a. Dyes b. Fluorescent compounds c. Radioactive elements 4. Measurement ofphysiologicalreactions 5. Analysis of tissues: sk. in, blood, urine, etc. 6. Loss ofpenetrant from the cutaneous surface PENETRATION INTO "MODELS" One of the "models" which has been used to simulate skin is agar gel. It has been commonly used in studying the release of antibacterial agents from various ointment bases (4). It is often assumed that those ointment bases which best release antibacterial agents to an agar gel will similarly release ' these substances to the skin when the ointments are placed on the skin. The continuous phase in an agar gel is water. If one observes the transfer of a chemical from some vehicle, petrolatum for example, into an agar gel and the subsequent movement of that chemical in the gel, it is equivalent to observing the transfer from the vehicle into water and the ensuing diffusion through water. In subsequent papers in this symposium, the chemical and physical structure of the skin will be discussed. It is much more complex than a single aqueous phase. It seems unsatisfactory, therefore, to assume that data obtained from a study of penetration into agar gel are applicable to penetration into skin. STUDY OF CHANGES IN THE HISTOLOGY OF THE SKIN When salicylic acid in various vehicles is placed on the skin it produces keratolysis. Strakosch (5) has studied penetration of this substance into the skin by taking biopsies of the skin and examins histologic sections for evidence of kerato]ysis. It is difficult to measure keratolysis quantita- tively under these conditions, and certainly this investigator made no claims that this method would permit quantitative measurement of the amount of salicylic acid which had penetrated the skin. Cotty, in a subsequent paper in this symposium, will discuss another method by which penetration of salicylates may be studied. Goldzieher and his associates (6) also have used histologic sections of skin in their study of the penetration of estrogen. We should delay any discus- sion of this work until after Dr. M. A. Goldzieher has told us about it in

JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS his own words. We are fortunate that he is here in person to present his observations. Special histochemical methods can be used on a histologic section for detecting the presence of a substance which had been placed on the cu- taneous surface before the section was prepared (7). If care is used, this method can show whether or not penetration has occurred, but it will not give quantitative data. UsF. oF T•t^cF. Rs One of the early methods used for studying penetration of fats into skin employed an oil-soluble dye to color the fat globules. Histologic sections of the treated skin were then examined for the presence of dyed globules (8). When using any method in which a tracer is employed, one must always be suspicious that the tracer may become separated from the substance to which it was originally added and attach itself to some other substance. When a dyed fat globule is observed in a histologic section of skin, one must determine whether it is the globule to which the dye was originally attached or whether the dye has become dissolved in one of the fats previously present in the skin. If the dye has shifted from one fat to another, the presence of colored globules in the histological preparation is positive evidence only of penetration of the dye and provides no information about the penetration of the original fat. In a modification of the dye method, a fluorescent material rather than a dye is used, and the histologic sections are examined under a micro- scope, the condenser system of which can transmit ultraviolet radiation. This method is open to the same criticism as the one in which a dye is used as a tracer. One cannot be sure that the fluorescent material remains with the substance whose penetration is being studied. This objection is not valid if the penetrating substance is itself fluorescent. The penetration of vitamin A (9) and some hydrocarbons, e.g., benzyprene (10), which are fluorescent, has been studied by means of fluorescent microscopy. One pitfall in this method is the fact that skin naturally contains substances which fluoresce at a wavelength quite similar to that of the fluorescence from vitamin A and benzpyrene. Fluorescence from normal constituents of the skin must be differentiated from that emanating from absorbed substances. In recent years, the use of radioisotopes for tracing penetration (11) has become increasingly popular. As a rule, an attempt is made actuall.y to synthesize the substance being studied so that it includes a radioisotope. At times, the molecular structure of the penetrating substance is modified somewhat by incorporation of the radioactive element, as for instance in the iodination of an unsaturated fat with I TM (12). In working with radioactive compounds, one must always make certain that the radioactive