76 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS '• .15 E ,. • .10 c). m ..., .0,5 o E (b) I I I I I 2 4 6 Time (hours) Figure 2.--Rates of penetration of perfused pig skin by tri-butyl phosphate as measured by (a) disappearance from the skin surface and (b) appearance in the perfusate. At the times indicated the perfusion flow rate was reduced. valid conclusion only when bland or unreactive substances are concerned, and the characteristics of a test-substance must be carefully considered before reliance is placed on any particular method of' measuring skin penetration. A very important advantage offered by the surface clearance method is that results can be obtained with minute quantities of test material so long as the activity of the trace element is high enough to operate a Geiger counter the toxic effects of such small quantities would be negligible. This means that a number of experiments can be carried out on the same subject and if arrangements are made to screen the test applications from the Geiger counter, but allow each to be exposed in turn, then the experi- ments can run concurrently. This technique has allowed us to study the possible significance of the hair follicle as an easy port of entry into the skin. To do this very small drops of labeled material were applied either to hairless regions of the skin or directly to the hair follicles. The liquid was transferred on the end of a fine wire in which a knot had been tied to provide a capillary retentive system. The wire was dipped in the test liquid and touched on the skin several times. The amount transferred each time was known to be within the range 0.5-5 microgram but the exact

METHODS FOR MEASURING PERCUTANEOUS ABSORPTION 77 amount was determined from its radioactivity at the GM tube after it had been applied. The operation was carried out under a low power microscope. As a check on the disposition of the droplets a photograph of the skin was taken to show the TABLE 2.--RATES OF PENETRATION OF PIG SKIN BY TRI-BUTYL PHOSPHATE (t•g./cm.2/-M•.) Application direct to 0.33 (10 results, hair follicles range 0.15-0.65) Application to inter- 0.28 (10 results, fo!licular epider- range 0.11-0.40) mis hairs and an autoradiograph to show the location of the droplets. Super- imposition of these showed very ac- curately whether a drop was quite clear of follicles or coincided with a follicle. The results on pig skin showed that the rate of absorption into the skin was nearly the same whether the follicles were involved or not. All .reliable methods of measuring chemical penetration of the skin should lead to the same result, although much depends on the type of information required. We may need to know how much enters the skin to produce a reaction, or how much enters the circulation to produce systemic effects. If we are considering systemic absorption from an application to the skin, then these methods should tell us the amount absorbed into the body, and a direct measure of this provides a standard by which a method can be evaluated. Direct measurements of systemic absorption have therefore been carried out on rats and rabbits, using tri-butyl phosphate. This is a convenient liquid because of its low volatility, inappreciable toxicity, stability and it is readily labeled with P32. The procedure was to take a group of about ten animals each with the same region of skin clipped free of hair and to apply to each animal an accurate dose of the liquid. At various times after the application, the skin in the region of the application was removed, and the animal sacrificed. The body of the animal then completely dis- solved in hot nitric acid and the resulting solution assayed for total content of the trace element. From this was calculated the total quantity of the test substance absorbed by the animal. The results again demonstrated that the absorption had proceeded slowly at first, but then accelerated and TABLE 3.--A COMPARISON OF THE RATES OF PENETRATION OF SKIN BY TRI-BUTYL PHOSPHATE MEASURED BY VARIOUS TECHNIQUES Rates are expressed as (ug./min./cm. 2) ß Species- , Method Rat Rabbit Pig Resected skin ... 1.6 (0.6-2.5 Perfused skin 0.80 1.7 (0.5-1.2) (0.8-2.3) Intact skin (disappearance from surface) ... 2.3 (1.0-3ß 7) Intact skin (systemic absorption) 0.70 9.. 0 (0.4-1.2) (1.7-2.3) 0.16 (0.08-0.35) 0.15 (0.07-0.23) 0.13 (0.07-0.20) ß . .