localization of applied chemicals (Rothman, 1954). The present study is an attempt to resolve the problem by measuring the passage

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1 J. Physiol. (1961), 156, pp With 3 text-figures Printed in Great Britain RELATIVE PENETRABILITY OF HAIR FOLLICLES AND EPIDERMIS BY R. T. TREGEAR From the Chemical Defence Experimental Establishment, Porton Down, Wilts (Received 16 November 1960) The skin protects the body against foreign chemicals by restricting their passage through the epidermis; diffusion of common organic molecules through rabbit epidermis is two to three orders of magnitude slower than through dermis (Treherne, 1956). Such diffusion as does occur might be through the epidermis itself or through the defect structures, the hair shafts and sweat glands. Argument about this has hinged on the histological localization of applied chemicals (Rothman, 1954). The present study is an attempt to resolve the problem by measuring the passage through each route separately. METHODS A kg pig was anaesthetized with dial-urethane. The flank was washed and its hair clipped very short. Seven 1 in. (2.5 cm) circles were drawn on the skin, corresponding to the holes in a metal covering plate (Fig. 1). Tri-n-butyl phosphate (TBP) containing 32p of radioactivity 0-2 mc/mole and 2 % of a fluorescent dye (Uvitex W, Ciba) was placed on the skin within these circles with a fine entomological pin. Approximately 0d1 Aumole of TBP was placed in each circle in separate patches. In four of the circles the TBP was placed, as far as possible, so as to avoid the hair follicles and in the other three the hair follicles were deliberately filled with TBP. The number of hair follicles filled by the fluorescent dye was counted under ultra-violet light. A mica cover was sealed over each skin circle by surrounding adhesive tape and the metal plate already mentioned was pressed down over the entire set of circles to prevent movement during respiration. Each circle in turn was exposed to an end-window Geiger counter at 20 cm distance. These measurements continued for 4-6 hr, care being taken that the geometry of the system was not disturbed. At the end of this time the number of hair follicles covered by fluorescent dye was again measured. A dental X-ray film was pressed into contact with each mica cover and an autoradiograph of the 32p on the skin obtained. The mica covers were removed and the fluorescent dye on each skin circle was photographed. Finally, the animal was killed and each skin circle was dissected out, digested in nitric acid and its 82p content determined. In a few experiments the pig was killed 1 hr before the,b-emission measurements were concluded.

2 T. TREGEAR J;J-'--Counter Ij /i! i /Ij // ~~~2-5cm /.1, M Mica / cover Lead shields Skin circles Fig. 1. Diagram of the apparatus used; the mica cover is indicated. Note the small angle subtended by the TBP at the Geiger counter. x x x x = TBP. RESULTS Each tiny (approx. 5 nmole) drop of TBP placed on the skin spread to cover approx cm2, whether the drop involved a hair shaft or not. When a hair shaft was covered by TBP a bright spot of fluorescence could be seen at the entrance of the hair into the epidermis, indicating the presence of a pool of TBP. Many of these pools were still present at the end of the experiment. The hairs themselves fluoresced brightly, and by the end of the experiment fluorescence of the intra-epidermal part of the hair could be seen through the nearly transparent epidermis, showing that the marker dye had penetrated into the hair shaft. In four out of the seven skin circles used in each experiment an attempt was made to avoid the hair shafts during application of the TBP. This was not completely successful; the marker dye showed that the TBP reached some hair shafts immediately, and spread to a few more by the end of the experiment. The number was, however, small compared to those filled deliberately in the other three skin circles (Table 1). The,B emission from the 32p in each skin circle fell during the experiment; the rate of fall was constant after the first hour (Fig. 2). The fall of f emission reflects a movement of 32p out of the counting field of the Geiger tube. As the alkyl phosphates hydrolyse very slowly at 40 C (Cavalier, 1898) and do not oxidize at all (Kosolapoff, 1950) it is very

3 PENETRABILITY OF EPIDERMIS 309 likely that the 32p moved in association with TBP; thus the fall in P emission indicates a movement of TBP. The fall ceased immediately the animal was killed (Fig. 2). Since evaporation of TBP through hypothetical leaks in the mica-adhesive tape seal or diffusion of TBP deep into the dermis would not be immediately affected by the animal's death, these movements were assumed to be negligible. There was no apparent lateral movement of the TBP on the skin surface to be seen in the photographs TABLE 1. Average number of hair follicles covered by TBP in each skin circle when follicles were avoided (A) or deliberately filled (B) Time after application (min) A B (04) 25*0 (08) X3 (0.5) 23-7 (0 8) Values are derived from 40 (A) or 30 (B) skin circles: S.E. of means in parentheses. 30 -> ~~Ro 25 o 8 \* ~~~~~~~~Slope=d dx E 0~~~~~~ 0 0 0~~~~~~~~ I,~ 20I ~ ~ ~ ~ Slopilled \ Rf 1 ~ ~ ~ 1 Fig I Time after application (min) emission from a skin circle to which TBP has been applied. Symbols are those defined in the text.

4 310 R.T.TREGEAR or autoradiographs taken at the end of the experiment, so that screening by the metal covering plate may also be discounted. Changes in thickness of the TBP on the skin surface, or movement within the epidermis, are unlikely to have much affected the emission, for the distances involved are of the order of 10-2 mm and the half-thickness for 32p is approx. 1 mm (Faires & Parkes, 1958). It therefore follows that the reduction in / emission was due to the only remaining route, the passage of TBP through the epidermis or hair shafts and into the systemic circulation, and it is on this assumption that the following results are construed. 32p distributed throughout the body would be virtually completely screened from the Geiger tube so that the rate of fall of / emission should be proportional to the rate of penetration of TBP into the systemic circulation: m dr P = X Rf dt where p = penetration rate of TBP into the system (nmole/min), Rf = / emission at the end of the experiment (counts/sec), m = TBP in skin at the end of the experiment (nmole), dr/dt = rate of change of,b emission (counts/sec/min). During dynamic equilibrium (straight line in Fig. 2) this penetration rate was calculated for the TBP on each skin circle. It was not significantly greater when the hair follicles were covered than when they were avoided; in fact it was slightly less (Fig. 3). This small difference was investigated further by calculating the penetration rate per unit mass of TBP (pm) and per unit area covered (Pa): pm = prf/mr., and pa p/a, where Ro = / emission at the start of the experiment (counts/sec), A = area of skin covered by TBP (cm2; calculated from the average of the darkened area in the autoradiograph and the fluorescent area in the photograph). Both these measures of penetration rate indicated that it was slightly slower when the hair follicles were covered. The difference was, however, barely significant (Table 2). The maximum contribution of the hair follicles to the penetration rate statistically consistent with these results was approximately calculated by taking the value of B-A in Table 2 at 2-5 x the S.E. above the mean; this gave values for the difference of 2 and 13 % of the mean value of A or B, i.e. the addition of the extra hair follicles in group B did not increase the penetration rate by more than

5 PENETRABILITY OF EPIDERMIS %, or approx. 20 pmole/min (cf. Fig. 3). The maximum follicular transport possible was therefore 1 pmole/min/follicle; it is more probable that involvement of the hair follicles actually reduced the penetration of the TBP. 500 _ 400 o 300 E 0~. G) 2001 ~~ Number of hair follicles Fig. 3. The effect of covering hair follieles with TBP on its penetration rate. Each point is the mean from 3-4 skin areas; the lines join points obtained on the same animal. TABLE 2. Penetration of TBP through skin in which the hair follicles were either avoided (A) or deliberately covered (B) Pm (kmin-1) pa (pmolefmin/cm2) A B B-A A B B-A 2* F * Mean (s.e. 0-09) (s.e. 16) Each value is the mean of four (A) or three (B) skin areas in a single experiment.

6 312 R. T. TREGEAR DISCUSSION There was no indication in these experiments that any TBP passed through the hair follicles to the circulation. The hair follicles appeared to be no more penetrable than the same area of epidermis. Since there is vastly more epidermal than follicular area on the surface of pig skin, the transport of TBP applied generally to the skin would be nearly all through the epidermis. This can be put quantitatively: the maximum possible contribution of the follicles was 1 pmole/min/follicle and the number of hairs on pig skin is about 20/cm2, so the follicular transport could not be greater than 20 pmole/min/cm2, a small fraction of the observed total penetration rate of generally-applied TBP, 1300 pmole/min/cm2 (Tregear, 1960). How far may this result be generalized? It seems likely that in man hair follicles are also unimportant, for their histological structure is similar to those of the pig, and they are no more frequent, except on the hairy areas. Nearly all other mammals have a much greater hair density than the pig or man, so that the hair follicles may be more important; indeed, in species such as the rabbit the area of epidermis within the hair follicles, calculated from histological sections, exceeds the exposed surface of the skin, so that if the penetrability of the epidermis is similar in the two sites the hair follicles should represent the dominant site of entry. Treherne (1956) showed that a variety of organic solutes in water penetrated rabbit skin at speeds related to their molecular diameter and their oil:water solubility ratio, and he suggested that they all passed through the same structure. If this is also true of pig and human skin, the results obtained with TBP, a stable aliphatic ester of no known special properties, should apply to other chemicals. The evidence on which it has been suggested that foreign chemicals pass through the skin predominantly via the hair follicles is histological. It has been observed that many materials placed on the skin surface reach the lumen of the follicles (MacKee, Herrmann, Baer & Sulzberger, 1945; Axelrod & Hamilton, 1947; Sulzberger, 1948; Borelli & Metzger, 1957), although this is not always the case (Blank, Griesemer & Gould, 1958). This is a necessary but not a sufficient condition for penetration via the hair follicles to occur. Concentrations of chemical in the dermis around the hair follicles have also once been observed (MacKee et al. 1945); this showed that the chemicals used (water solutes of various types) had probably passed through the lining epithelium or sebaceous glands. It did not, however, show how much had passed through. In general, histology, unless combined with a rate study, cannot demonstrate the relative importance of competitive routes of penetration. In man, though not in the pig, a further defect in the epidermis is

7 PENETRABILITY OF EPIDERMIS 313 provided by the sweat glands. Since these glands are several times more numerous on the palms than on the body surface in general (Kuno, 1956), and the palms are stated to be less penetrable than other parts of the skin (Herrmann, 1945; Shelley & Melton, 1947), it is unlikely that the glands represent an important route of entry of foreign chemicals on sites other than the palms. In conclusion, it seems probable that the protective impenetrability of human skin is limited by the structure of the epidermis itself, and not by sweat glands or hair follicles acting as holes punched in it. SUMMARY 1. Tri-n-butyl phosphate (TBP) labelled with 32P was applied in small drops to the flank skin of an anaesthetized pig in seven circular areas. In four of these the hair follicles were avoided and in three they were deliberately covered with TBP. 2. The penetration of the TBP to the circulation was estimated from the steady reduction in f emission from the 32P on the skin. 3. This penetration was slightly faster when the hair follicles were avoided than when they were covered. 4. It was concluded that the hair follicles of the pig are no more penetrable than the epidermis. REFERENCES AXELROD, D. J. & HAMILTON, J. G. (1947). Radio-autographic studies of the distribution of lewisite and mustard gas in skin and eye tissues. Amer. J. Path. 23, BLANK, I. H., GRIESEMER, R. D. & GOULD, E. (1958). Penetration of an anticholinesterase agent (sarin) into the skin. II. Autoradiographic studies. J. invest. Dermat. 30, BORELLI, S. & METZGER, M. (1957). Fluorescenzmilaroskopische Untersuchungen uber die percutaner Penetration fluorescierender Stoffe. Der Hautarzt, 8, CAVALIER, J. (1898). Vitesse de saponification des 6thers phosphoriques. C.R. Soc. Biol., Paris, 127, FAIRES, R. A. & PARKES, R. H. (1958). Radio-isotope Laboratory Technique, p. 19. London: George Newnes. HERRMANN, F. (1945). Introduction of allergens into the skin by inunction with intraderm. Ann. Allergy, 3, KOSOLAPOFF, G. M. (1950). Organophosphorus Compounds, p New York: Wiley. KuNo, Y. (1956). Human Perspiration, p. 64. Illinois: Charles C. Thomas. MAcKEE, G. M., HERRMANN, F., BAER, R. N. & SULZBERGER, M. B. (1945). Histologic studies on percutaneous penetration with special reference to the effect of vehicles. J. invest. Dermat. 6, ROTHMAN, S. (1954). Physiology and Biochemistry of the Skin, pp Illinois: Chicago University Press. SHELLEY, W. B. & MELTON, F. M. (1947). Studies on absorption through normal human skin. Fed. Proc. 6, SULzEBERGER, M. B. (1948). Demonstrating the presence of sulfonamides in the tissues. Science, 98, TREGEAR, R. T. (1960). The epidermis as a thick membrane. J. Physiol. 153, 54-55P. TREHERNE, J. E. (1956). Permeability of skin to some non-electrolytes. J. Physiol. 133,