PERCUTANEOUS ABSORPTION 493 absorption in vivo (54, 91). This technique is useful in determining the presence of the compound in the various anatomical layers of the skin and in gaining some idea of the relative concentration. However, it is of limited value for quantitative investigations. Enhancement of penetration It is sometimes necessary to simulate 'conditions of use' where the skin surface may be exposed to injury and loss of its protective keratin expected. One method frequently employed is to remove keratin by the successive application and stripping of adhesive cellulose tape to the same cutaneous site. The number of consecutive strippings is usually about 25 (53, 92). The depth of epidermis removed by the adhesive is not uniform. Histological studies of tape removed after firm application revealed that in some areas the complete epidermal barrier is removed but in other areas only the merest trace of keratin (93). Other authors claim that a uniform separation of the keratin barrier is achieved by this method (29) so that the barrier is uniformly weakened. The skin barrier can be removed also by other methods. Recently Parekh et al (57) scarified the skin with a 'dull' razor to the point of localized bleeding in order to assess the absorption of sodium pyridinethione from damaged skin. Percutaneous absorption 'in vitro' methods In vitro measurements are found particularly useful in comparing the rates of diffusion of different compounds (74, 94, 95) and in obtaining some idea of the rate of the transepidermal passage of highly toxic substance prior to in vivo tests (42). They also provide a means of obtaining a better understanding of the factors that influence percutaneous absorption in vivo (9, 96) Both human and animal skin have been used for in vitro studies. Human skin is obtained at necropsy generally from the abdomen (42, 97) and from the abdomen, flanks or back in the case of animals (98, 99). Although the methods employed for the in vitro measurement of percutaneous absorption vary considerably in details, they follow a general pattern. The specimen of skin is trimmed to a suitable size and is mounted in a hollow chamber so that it divides the chamber into two compartments. The two surfaces of the skin are bathed in a suitable fluid. One of the fluids contains the test sub- stance. Passage through the skin is then measured either by the 'dis- appearance' of the test substance from one chamber, by its appearance in the other or by both methods combined. Full details of the construction of

494 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS a chamber suitable for such experiments and of the techniques employed are found in other publications (42, 94-98, 100, 101). The applicability of in vitro techniques is largely due to the non-cellular nature of the main epidermal barrier, the keratin layer, so that vital pro- cesses dependent on the integrity of epidermal cells are not involved: these cells lose their viability soon after death or removal from live animals. Knowledge of the rate of transepidermal passage of chemicals is im- portant in an assessment of the hazard of systemic toxicity from chemicals applied percutaneously. The in vivo method of 'analysis by difference' using a suitable radio-active technique is the most appropriate for assessing percutaneous absorption of cosmetic compounds. If such a study is first carried out in animals it may need to be supplemented by in vitro studies on excised human and animal skin in order to obtain some idea of the expected rate of penetration in humans. In vitro studies may also assist in studying factors which influence absorption of specific chemicals under defined conditions, such as temperature and pH. FACTORS INFLUENCING PERCUTANEOUS ABSORPTION Physico-chemical factors Temperature In most experiments on percutaneous absorption the environmental temperature is kept at about 37øC. Under the ordinary conditions of life, however, the skin .[emperature fluctuates considerably, especially in the exposed parts. Fluctuations in skin temperature are known to influence percutaneous absorption. Whitehouse, Hancock and Haldane (102) in their study of the passage of water and gases through the human skin, demon- strated an increase in the rate of percutaneous absorption of oxygen on raising the environmental temperature of man. Brown and Scott (103) later showed an increase in the absorption of methyl salicylate due to an increase in the skin temperature. Fritsch and Stoughton (104) investigated the effect of temperature on the in vitro percutaneous absorption of acetylsalicylic acid on human skin. They found that at 40øC and 88•o rh, the transepidermal passage of salicylate was about eight times times greater than it was at 10øC. An increased rate of percutaneous penetration of alcohols (C•.-C8) over the range of temperature 5ø-50øC was observed by Blank, Scheuplein and MacFarlane (105) using in vitro