', TRANSCUTANEOUS ABSORPTION OF VITAMIN A 171 ' long as the chemical method of assay showed that the vitamin po- tency was within a -•10 per cent range of the theoretical. In the majority of the experi- ments, topical applications of vita- min A were made by spreading approximately 0.3 gm. of the petro- latum, fortifiedwith the vitamin con- centrate, over the defined, cutane- ous areas and allowing it to remain in contact with the skin for two hours. At the end of this period, the preparation was removed, as com- pletely as possible, with cleansing tissue, and the entire ventral surface of the chest and abdomen was scrubbed thoroughly with a sponge, which had been saturated with a soap solution. Then the skin sur- face was rinsed with warm water and dried, by wiping with a towel, before the rat was returned to its cage. Throughout the test periods, all }ats were maintained in individual cages and fed the vitamin A-defi- cient diet which has been prescribed for oflScial use in biologic assays for vitamin A (5). Topical applications of vitamin A in the petrolatum base were made to the same areas of skin, once daily, except Sundays, of each week of the test period. All test areas of skin were clipped free of fur at intervals of one week. Judgments of systemic effects attributable to vitamin A applied to rats' intact skins have been based upon three criteria: 1. Gains in weights of young rats maintained on a vitamin A- o deficient diet these were re- corded at intervals of one week during test periods. Storage of vitamin A in the animals' tissues determined at the time of sacrifice. Correction of the abnormal, morphological changes in the rats' tissues which are indica- tive of vitamin A deficiency. Both preventive and curative types of experiment have been utilized in these studies of absorp- tion of vitamin A through skin. In the preventive experiment, rats at weaning age were placed upon the vitamin A-deficient diet. Applications on the base containing vitamin A were also begun at this time with the objective of deter- mining whether sufiScient vitamin A could be absorbed through the skin to influence either the rate of growth of the animals or the time of appear- ance of the characteristic gross signs of vitamin A deficiency. These animals formed the test group. At the same time, litter mates were placed upon the vitamin A-free diet and given applications of the base lacking vitamin A. These animals constituted a positive control group. Since this latter group received no source of vitamin A but otherwise were maintained under identical exprimental conditions, any differ- ence in growth records between the test group and the control group could be attributed only to the effect of vitamin A absorbed through the skin. The method employed in the cura-

ß 172 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS tive experiments differed from that specified in the Pharmacopoeia of the U.S. (5) for the biologic assay of vitamin A only in the fact that, in these experiments, the material con- taining vitamin A was applied to the intact skin instead of being fed by mouth. a typical preventive experiment. Ten rats, at weaning age, were divided into two groups of litter mates. Each group of five rats in- cluded equal numbers of males and females. During an experimental period of eight weeks, the rats in one group (test group) received daily, IOO i.-' 80 -- •o o rn _• 4.o •o • 0 I 2 3 4 5 6 7 8 WEEKS OF MAINTENANCE ON A VIT•AMIN A-FREE DIET Chart I.--Comparative average gains in weight of rats maintained on a vitamin A-free diet and receiving daily topical applications of either petrolatum or petrolatum containing 2100 units of vitamin A per gram. O, 5 rats receiving applications of base containing vita- min A O, 5 rats receiving applications of base without vitamin A. At weaning age, the rats were placed on the vitamin A-free diet and allowed to deplete their bodies of vitamin to such an extent that their weights were either stationary or declining for a period of one week. At this time, the animals were con- sidered vitamin A-deficient and topical applications of the vitamin were begun. In each case, one or more litter mate animals were main- tained until death on the vitamin A- free diet and without applications of any kind, thus serving as negative controls. EXPERIMENTAL RESULTS Chart I summarizes the results of topical applications of petrolatum containing 2100 units of vitamin A per gram, but their litter mates in the other group (control group) received comparable applications of the pet- rolatum without the vitamin con- centrate. Both groups grew at essentially the same rate for the first four weeks, but, after that period, the growth curve for the controls, receiving the plain petrolatum, lev- eled off and finally declined. How- ever, the growth curve for the test rats, receiving-the petrolatum con- taining vitamin A, continued to show practically constant weekly gains in weight. The slight change