64 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS emulsion and produce a shadow even when no radioactivity is present. Such shadows in the epidermis, follicular lining and around the sebaceous gland can be seen in Fig. 1, an "autogram" which was prepared from normal human skin containing no radioisotope. Third, if the tissue and photosensitive emulsion are on separate slides which, during exposure, are clamped tightly together, a pressure or stress artifact can be produced from hard tissues when no radioactivity is present. Figure 2 is an "autogram" in which the shadow was produced by pressure of the hair against the emulsion no radioisotope was present. MEASUREMENT or PHYSIOLOGICAL REACTIONS Development of a wheal following application of histamine to the skin (16), vasodilatation following application of nicotinic acid esters or other vasodilators (17), and anesthesia following topical application of local anesthetics (18) are examples of physiological reactions which have been used in studying the penetration of pharmacologically active substances. Reactions of this sort give a clear yes-or-no answer to the question of penetration: if a reaction is produced, the penetrating substance must have reached the deeper layers of the skin where lie the blood vessels and nerves. Such reactions, however, may not be directly proportional to the amount of material which has penetrated and seldom have methods of this kind yielded accurate, quantitative measurements of material which has penetrated. ANALYSIS or TISSUES The amount of a substance which penetrates into and through the skin after being applied to the cutaneous surface can be determined by analysis either of the skin itself, or, if there has been systemic dissemination, of some distant tissue. Before analyzing any specimen of skin, it is of course essential that the cutaneous surface be thoroughly cleansed of any residue of the applied material which has not penetrated into the actual tissue of the skin. With some materials, this is difficult to accomplish. If one is observing penetration into the skin only, then this method is applicable to either excised skin or skin on the living animal. Analysis of full-thickness skin provides no indication of how far into the skin the applied substance has penetrated. The results of analysis would be the same, whether the applied substance was only in the stratum corneum or had penetrated below the major barrier. If a liquid receptor is held in contact with the derreal side of a specimen of thin, excised skin, it is sometimes possible, by analyzing the receptor fluid, to determine the amount of applied material which has passed entirely through the skin (19). By analysing the receptor fluid at successive intervals, the rate of penetration may be determined by a test of this type.

STATEMENT OF PROBLEM AND CRITICAL REVIEW OF PAST METHODS 65 -, I•'igure 1.--"Autogram" produced by 16-day contact of a section of normal skin with a radiosensitive emulsion. Note the shadows in the hair-root sheaths, around the sebaceous gland and in the epidermis. These shadows are artifacts produced by chemical reactions. The section was in contact with the radiosensitive emulsion during photographic development. .. __ •:?•'.. .. ,• ...... .•,,• ..• -: -• . :: • • '-.{ ,. ,. ,•-¾ ?• • .:..-.•,.•:•.' '.•::' ...... . ...... • :* ..... .:.. '• X-:'&':%•:' :z,...t".' 7 ..... ß ..:. '...**•,-,: ,,.:*-•':. '5: , ..... :•,•. • :L'7 : ... . --,--:- .. . ............. .: .: , ......... ?•: ?• ............. • .• _ ......- .......... . . . .... : . ,& ,• -" .... .--.: - ...,,•_.' ,.. ......,•..,,,,a,•. ' ....-"•v•. :'-•'•. •. . •, B w ,v --. FigUre 2.--z/. Photomicrogram of normal skin. Hemotoxylin-eosin stain. B. "Autogram" produced by 76-day contact, with pressure, of a section of normal skin with a radiosensitive emulsion. Note the shadow which corresponds to the hair. This shadow is an artifact pro- duced by pressure of the hair against the emulsion. The section was not in contact with the radiosensitive emulsion during photographic development.