488 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS water content. However, because of the limited knowledge of the factors influencing skin conductivity, this test cannot be recommended to replace the more orthodox methods. Percutaneous absorption in vivo 'Disappearance' technique or 'analysis by difference' The techniques employed for the application of the test material on the skin surface and for measuring its percutaneous absorption vary consider- ably in detail but conform to a few general principles. A measured volume of the compound is applied to the skin if the com- pound is a liquid in its natural state. Parathion and $arin are examples of compounds applied to the skin in undiluted form in order to estimate their rates of absorption in experimental animals. If the test compound is a solid, a known amount is usually dissolved in a specified volume of a liquid vehicle and a measured volume of this solution is applied (19, 54, 64-66). Occasionally it may be desired to deposit a very small quantity of test material in solid form on the skin. It is often more practicable to apply the material in a volatile solvent and to allow the solvent to evaporate quickly rather than to apply the material directly. Thus, Feldmann and Maibach (67) in their studies on percutaneous penetration of steroids in man applied the test steroid dissolved in acetone. Evaporation of the acetone was assisted by blowing a stream of air over it. It would appear that this pro- cedure assists percutaneous absorption by increasing the local concentra- tion (68). On occasions it may be essential to estimate the percutaneous absorption of a test substance contained in an ointment base. A weighed amount of the ointment, containing a known concentration of the test material, is usually rubbed on the skin in a defined manner and protected with a non-absorbable covering. Radio-active emission may be measured through this covering (69-71). An accurate (4-10•o) and simple method has been developed by Hadgraft, Barrett and Sarkany (72). It consists of producing a disc of the preparation on a small square of polyethylene by means of a thin tin sheet into which a circular hole has been cut. The tin sheet is placed over the polyethylene and a quantity of the test preparation is drawn across the hole by means of a spatula. The polyethylene square containing the disc of ointment is then removed and applied to the skin surface. One of the first methods employed in the investigation of absorption through intact skin in living animals was devised by Hediger (73) and called

PERCUTANEOUS ABSORPTION 489 the method of 'analysis by difference'. This method involves the application to the skin of an accurately weighed amount of the penetrant and the estimation of how much remains in the deposit after different periods of time. In the original method, used extensively with little modification until recently, Hediger (73) used a bell-shaped glass vessel cemented to the skin. Samples were taken periodically from the skin enclosed within the vessel and removed for chemical analysis. This method often needed elaborate analytical procedures and was limited by the sensitivity of the analytical method employed (21, 74). The advent of isotope labelling has considerably simplified the method of 'analysis by difference' since the amount remaining on the skin could be estimated relatively easily from its radio-active emission. These radio-active techniques have introduced difficulties of another sort, since the type of radio-active emission could have a considerable influence on the sensitivity and accuracy of the measurements. Gamma emission traverses readily the thickness of the corneum and epithelium so that compounds labelled with such isotopes do not offer any major difficulties in detection but the true passage of such isotopes through the cutaneous barrier may not always be readily assessed since the high penetrating properties of the gamma emission may give a positive result from regions beyond the cutis vera, e.g. sub- cutaneous tissue. On the other hand, [I-emission does not possess such great penetrating properties so that failure to detect radio-active emission need not necessarily imply complete absorption: substances labelled with [I-emitting isotopes may, for example, lodge in the skin appendages without detection (75-77). These complicating factors may affect materially the results, and it is essential to take these factors into account. For this purpose some workers excise the skin and subcutaneous tissue after completing a series of counts in vivo and measure separately the residual radio-activity in those two tissues (57, 76). The type of correction applied then depends on the type of radio-active emission. In the case of T-radiation, any residual radio-activity in the subcutaneous tissue is subtracted from the readings obtained in vivo since the fraction of the compound responsible for this emission has passed through the skin. Residual activity in the subcutaneous tissue from [I-emission need not be taken into account since it is unlikely that it would affect readings taken at the skin surface. On the other hand, residual •-emission in the skin itself needs to be taken into account since it represents radio-active material deep in the skin (usually pilosebaceous apparatus) which may have escaped detection (74). A few selected examples may serve to illustrate the way in which per-