PERCUTANEOUS ABSORPTION OF VITAMINS 161 is effective not only in proving percutaneous absorption but also in deter- mining rate of penetration. VITAMIN A There is no doubt that vitamin A is absorbed well through the skin of the rat, mouse and probably the guinea pig. Demonstration of percu- taneous absorption in man has required very sensitive methods. Tests for cure of night blindness and more recent tracer studies with labeled vitamin A show small but significant absorption. ANIMALS Studer and Frey (10) appear to be the first to report that topical appli- cation of pure vitamin A caused transient thickening of the skin of rats. Further studies by Studer (11) showed that local application of large doses of vitamin A to the skin of rats produced both an increase of cell division and an increase in the thickness of the epidermis. However, both the rate of cell division and the thickness of the epidermis returned to normal despite continued application of vitamin A. Topical vitamin A was shown to have an inhibiting effect on keratinization in the rat by Sabella, Bern and Kahn (12). A thickening of the stratum granulosum was observed with a resultant decrease in keratin formation and a thinner horny layer. The effect is entirely local untreated skin areas showed no significant altera- tion (12, 13). Lawrence and Bern (14) found that the epidermal response of mice to small quantities of vitamin A appeared to follow a dose-response relationship in which the degree of epidermal hypertrophy was roughly proportional to the log of the dose. Epidermal hypertrophy was initiated by a concentration of about 1 unit of vitamin A per day. •-Carotene, applied in the mole-equivalent of 100 units of vitamin A per day, failed to induce a significant change in epidermal thickness. Mont•gna (15), using the fluorescence of vitamin A in near ultraviolet light, showed that vitamin A dissolved in alcohol or chloroform penetrates the intact skin of guinea pigs quickly by way of the sebaceous glands when dissolved in ]inoleic acid, oleic acid or in a viscous petrolatum paste, it penetrates more slowly. The absorption of vitamin A through the skin was demonstrated by Helmet and Jansen (16) and Eddy and Howell (17) by the weight gain shown by vitamin A-deficient rats in response to topical application of fish liver oil. Sobel (18), in similar experiments found local application of vitamin A to be about one-seventh as effective as oral dosage in promoting growth of deficient rats. Studies by Sobel et al. (13) indicate that the amount of vitamin A stored in the liver and kidney, the amount circulating in the blood stream after twenty-eight days of topical application and the utilization measured by the increase in body weight after depletion vary with the concentration of the vitamin applied, the medium of application

162 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS and the physical condition of the animal at the time of depletion. The curative effect of topically applied vitamin A on the progressive histological changes in rat skin during vitamin A deficiency was demonstrated. This group (19) further found that very large doses of topically applied vitamin A depressed the weight gain of rats. A small but significant amount of absorption through the skin was found by St•ttgen (20) who determined the penetration of tritium-labeled vita- min A both in vitro and in vivo skin tests. After application of labeled vitamin A with the usual precautions in vivo to prevent licking of the site, the area of application was excised and sectioned in a freezing microtome. The individual skin layers were analyzed for radioactivity in a flow counter. Even the lower layers showed radioactivity with some indication of a saturation effect. A slow rate of systemic penetration was indicated by the small difference between the in vivo and in vitro relationships. Because of the difficulty of measurement of the weak energy emission of the tritium and the possible lack of stability of the labeling, these experiments were re- peated and the results confirmed by Stiittgen (21) with carbon-labeled vitamin A. MAN The absorption of topic'ally applied vitamin A and its effect on the skin of man are not as easily demonstrated as in animals. Fisher and Herr- mann (22), using planimetric measurements, found no increase in the thickness of adult skin after daily application of about 9000 I.U. of vitamin A palmirate for a period of three to five weeks. On the other hand, Reiss and Campbell (23) found by histological examination that local application of about 500 I.U. of vitamin A palmirate twice daily for three weeks resulted i'n thickening and other changes of the stratum granulosum of senile skin. The difference in results may be due to the age difference of the test subject. Histological studies by Puccinelli and Bellone (24) indicated an acceleration of normal cornification after topical application of vitamin A to human skin. The effects of locally applied vitamin A on serum levels of vitamin A and on hair are not the same in humans as in animals. Thus, Flesch (25) and Sttittgen and Wrist (26) found no rise in the vitamin A serum levels of humans after the application of large doses. Hair loss after topical vita- min A was found in animals only (27). Evidence for the penetration of vitamin A through the skin is found in the studies of Mandelbaum and Schlessinger (28) who made dark adapta- tion measurements on human subjects on a vitamin A-deficient diet. Topi- cal vitamin A was found to exert both preventive and therapeutic effects. They concluded that vitamin A is absorbed through the skin in physiologi-