612.793.5 s O 4i.7et 1..-.s FACTORS INFLUENCING THE PENETRATION OF THE SKIN BY CHEMICAL AGENTS. By H. CULLIUMBINE, M.D., Ch.B., M.Sc. (Chemical Defence Experimental Station, Porton, Wilts.) (Received for publication 22nd January 1947.) EXPERIENCE during the war with the common vesicants, mustard gas and lewisite, suggested that these compounds would be suitable agents for a study of the factors influencing the penetration of chemical compounds through the skin. After penetration they produce skin reactions which are easily recognisable and which are capable of a quantitative assessment so that the experimental findings can be subjected to statistical control. We have, therefore, used mustard gas-in the liquid and vapour states-and lewisite (in the vapour state) as the vehicles for studying some of the problems of penetration through the skin. METHODS. The skin of the human forearm and the depilated skin of the rabbit's abdomen were the sites investigated. In the human experiments the under side of the forearm was placed over the mouths of test tubes containing mustard gas, care being taken to cover the tubes completely, thus allowing no leakage of vapour. The tubes were contained in a water-bath thermastatically controlled at 30 C. Hence the forearm was exposed to a constant concentration of mustard-gas vapour. Six such exposures were made on each subject, i.e. three on each forearm. Different conditions of the skin were produced at each of these six exposure sites and the site of each different condition was varied from subject to subject. The time of exposure of the bare skin to mustard-gas vapour was four minutes and the skin reactions caused were noted 24 and 48 hours after exposure. For the purpose of statistical analysis the noted skin reactions were given the following numerical value No reaction........ O Faint redness (E-)....... 1 Definite redness (E)....... 2 Redness and swelling (E +)..... 3.5 Pinhead vesication (V-)...... 5 Vesication....... 6 Vesication with spreading subcutaneous cedema (V +). 8 These numerical values were arrived at by exposing for varying periods of time the normal untreated forearms of a large group of subjects to an atmosphere saturated with mustard-gas vapour at 30 C. These " normal " exposures showed that- A 1 minute exposure caused an E- reaction,,2.,,,,, E,,,3-5,,,,,,E+,,5,,,,,,V-,,,,, V,, VOL. XXXIV, NO. 2.-1947. 83 6
84 Cullumbine That is, the values we have assigned to the reactions occurring in the treated skin should be roughly proportional to the amount of effective mustard-gas vapour entering that treated skin. The experiments with lewisite vapour were performed in a similar manner to those for mustard-gas vapour and a similar numerical assessment of the severity of the skin reactions produced was used. In the experiments with liquid mustard gas 0-32 mm. diameter drops were placed on the skin-either of the human forearm or of the depilated rabbit abdomen-and the skin reaction noted. The method of statistical analysis used for the results of these experiments was Fisher's method of analysis of variance for Latin squares. In the preliminary experiments some of the physiological properties of the skin were modified in turn and the effect of this modification on the reaction of the skin to the vesicants then determined. In the later experiments the forearms of the subject were placed in a small asbestos-lined chamber in which the temperature and relative humidity could be varied between 600 F. and 1000 F., and between 40 per cent. and 100 per cent. respectively. The humidities were estimated by means of dry and wet bulb thermometers and a hair hygrometer. Superficial skin temperatures were measured by means of a standardised thermocouple galvanometer. The relative accuracy of the readings was about 0.10 C. and the absolute accuracy was about 0.25 C. The moisture on the surface of the skin was assessed roughly by drying with weighed dry cotton-wool swabs. As judged by skin temperature and sweat estimations it was found that it took about half an hour for the skin to come into equilibrium with the atmosphere in the chamber. RESULTS. Table I summarises the effects obtained when some of the physiological properties of the skin were modified in turn. Similar results were obtained with liquid-mustard gas, mustard-gas vapour and lewisite vapour, and on human and rabbit skin. In general, we can say that the skin reaction is increased by: (1) capillary constriction, (2) defatting with xylol (defatting with ether did not significantly alter the reaction), (3) water on the skin. The skin reaction is reduced by: (1) applying fat to the skin, (2) producing sweating by the injection of pilocarpine. In the "atmospheric " experiments the following local skin conditions were produced: (1) Hot moist skin-a hot, sweating skin, the sweat not evaporating from the skin since the atmosphere was saturated with watervapour. (2) Hot normal (moisture) skin-a hot sweating skin with normal sweat evaporation occurring.
Penetration of the Skin by Chemical Agents 85 TABLE I. Skin reaction Physiological Modification Method of compared with factor. modification. reaction of normal skin. NORMAL SKIN Circulation. Capillary Application of mustard No change, though dilatation. plaster to skin for li constant tendency hours. to be reduced. Reactive hyperiemia. Capillary H.I. 5m. liq. adrenalin. Increased (p=0.01). constriction. hydrochlor. Stasis. Sphygmo manometer No change. applied to upper arm and maintained at 200 mm. Hg. during exposure. Sweating. Increased. H.I. 0 4 c.c. 2 per cent. Reduced (p = 0.01). pilocarpine. Fat content of Reduced. Xylol applied. Increased (p =0.05). skin surface. Ether applied. No change. Increased. Adeps Lanae applied. Reduced (p = 0.05). Water content Increased in H.I. 0 4 c.c. normal No change. of skin. skin surface. saline. Increased on Water applied. Increased (p = 0.01). skin surface. H.I. =Hypodermic injection. TABLE II. ha Mean increase Local condition Mean cange in moisture on Effect on reaction of skin to of the skin. temperature. of sq. vesicants. of skin. Hot moist. +2.520 C. 1X5 mg. Increased markedly (p =0 001). Hot dry. +2.750 C. Nil. Reduced (p=0 01). Hot normal. + 2.260 C. Nil. No significant change though constant tendency to reduce. Normal moist Nil. 2X2 mg. Increased (p = 0.01). Normal dry. Nil. Nil. No change.
86 Cullumbine (3) Hot dry skin. (4) Normal (temperature and moisture) skin. (5) Normal (temperature) dry skin. (6) Normal (temperature) moist skin. In Table II are given the changes produced by those local skin conditions on the reaction of the skin to the vesicants. It will be seen that a hot moist skin shows the greatest increase in its reaction and a hot dry skin the greatest reduction. The presence or absence of moisture on the surface of the skin would seem, therefore, to be the determining factor. Confirmation of this is obtained from the following experimental facts: (1) hot dry skin when moistened with water has its reaction to mustard-gas vapour increased; (2) normal skin when moistened has its reaction increased; (3) a hot moist skin when dried has its reaction decreased; (4) there is no statistical correlation between skin temperature or rise in skin temperature and the reaction to mustard gas or lewisite. Moisture is not the sole factor though, since hot moist skin gives a greater reaction than cool moist skin, and cold moist skin and cold dry skin give the same reaction (see Table III). TABLE III.-EFFECT OF COLD ON THE SKIN REACTIONS TO VESICANTS. Condition of skin. Effect on reaction of skin to vesicants. Cold dry. No change, though constant tendency to be reduced. Cold wet. No change, though constant tendency to be reduced. Normal moist. Increased (p = 0 01). (The cold skin was produced by applying rubber bags of ice to the forearm for half an hour before exposure to vesicants.) To be sure that we were not under-assessing the effect of heat alone, arms were exposed to a hot dry atmosphere with a temperature of 1200 F. The skin reaction was even further reduced, although the skin temperature was raised by 40 to 70 C. Perhaps sweat on the skin behaves differently from water on the skin? The ph of sweat is about 4-5 and is mainly due to lactic acid. Experiments with aqueous solutions of lactic acid and of hydrochloric acid at ph 4.5 showed that these behave quantitatively like water in
Penetration of the Skin by Chemical Agents increasing the reaction of the skin to the vesicants, so that neither the lactic acid content nor the acidity of the sweat can be important factors. Again, it can hardly be the fat content of the sweat which enhances its effects, since fat alone reduces the skin reaction. DIsCuSSION. In a study of the action of mustard-gas vapour, liquid mustard gas and lewisite vapour upon human skin and rabbit skin we have concluded that the reaction of skin to these vesicants is increased by cutaneous capillary constriction during exposure, defatting the surface with xylol before exposure and the presence of water on the skin during exposure, while the skin reaction is reduced by applying anhydrous fat to the skin before exposure and by producing sweating by the injection ofpilocarpine. Can we say, therefore, that moisture on the skin or local capillary constriction increases the amount of vesicant penetrating the skin and that fat on the skin decreases the amount penetrating? The problem is complicated by the fact that, after penetration, some of the vesicant remains in the skin to produce the local reaction while the rest is absorbed into the general circulation. Hence variations in the local circulation, by altering the amount of vesicant remaining in the skin, should alter the local skin reaction. However, the mere application of water to the surface of the skin does increase the skin reactions obtained, and here no alteration in the local circulation is likely to have occurred. Again, a hot moist skin produces a greater skin reaction than does a hot dry skin where the skin temperatures, and presumably the local blood flows, are the same. Hence, moisture on the surface of the skin increases the amount of vesicant that can penetrate the skin and, by a similar reasoning, we conclude that anhydrous fat will produce the reverse effect. Our experiments give no definite indication as to the influence of the local blood flow on the ability of substances to penetrate the skin. Hyperaemia, produced in a variety of ways (cold, reactive hypermemia, heat), had no significant effect, though the tendency in most cases was to reduce the skin reaction. In these cases the dilatation of the local skin vessels presents a larger blood-absorbing area so that presumably more of the vesicant penetrating is absorbed into the blood stream and carried away in the skin. [Moritz and Henriques (by communication, 1942, unpublished) have demonstrated, with mustard gas containing radio-active sulphur, that about 90 per cent. of the penetrating mustard gas is conveyed away by the circulation; the absorbing of merely another 5 per cent. would reduce the amount available for producing local effects in the skin by half.] Conversely, the injection of adrenaline, to produce local capillary constriction and so reduce the vascular absorption, should and did increase the skin reaction. Moreover, as 87
88 Cullumbine presumably most substances penetrating the skin are absorbed to some extent into the general circulation, we can say that local anaemia will tend to increase and local hyperaemia will probably reduce their effect on the skin. The influence of the activity of the sweat glands would seem to depend chiefly on whether the moisture they produce is allowed to evaporate away or remain on the surface of the skin. With the hot, moist skins the sweat accumulated on the skin, more vesicant penetrated and so a greater skin reaction occurred. The sweat glands of the hot, dry skins were similarly active but yet the skin reactions to the vesicants were much reduced. Moreover, if gross sweating were produced artificially by the injection of pilocarpine then the skin reaction was again reduced. Why should surface moisture facilitate skin penetration? To answer this question let us consider mustard gas as an example. Ogston in a review (unpublished) of the chemical reactions of mustard gas has pointed out that "there is no evidence that mustard gas reacts in vivo by any mechanism other than the substitution of the chlorine atom in an aqueous phase." Hence, it is reasonable to postulate that mustard gas will penetrate and react more efficiently in the presence of moisture than in its absence. The low solubility of mustard gas in water would be no hindrance to penetration, since the ready reaction of the mustard gas in the epithelium would necessitate more of the mustard dissolving in the aqueous phase to keep the latter saturated, and so on. The chemical reactions involving mustard gas in the skin would no doubt be accelerated by temperature in a manner similar to other chemical processes; in addition, the solubility in water will increase with rising temperature. Therefore, we should expect moist skin to give a bigger reaction than dry skin and hot moist skin a bigger reaction still. As mustard gas is very soluble but relatively inert in fat, a layer of fat on the skin merely interposes a good solvent barrier between the mustard atmosphere and the skin, thus trapping the vesicant in an unreactive phase from which it may later evaporate away. (This argument applies only to anhydrous fat.) Extreme cold, e.g. the application of ice to the skin, by producing hyperawmia and also reducing the rate of reaction of mustard in the skin, we should expect to reduce the skin reaction. A similar train of reasoning could be applied to any substance whose biochemical reactions chiefly occur in an aqueous phase. The above experimental findings would form the basis of a logical explanation for the well-established fact that hot and sweaty skin is more sensitive to mustard gas than is cool and dry skin. Again, we should expect rubefacients, by producing a hyperaemia and therefore increased vascular removal of mustard gas, to assist the decontamination of the skin from mustard gas. This indeed has proved to be so; we
Penetration of the Skin by Chemical Agents 89 have found that methyl salicylate, camphor and allied substances, when added to anti-gas ointments, increase the decontaminating efficiency of those ointments. SIUMMARY. Using the common vesicants, mustard gas and lewisite, a study has been made of the factors influencing penetration of the skin. It has been concluded that penetration is facilitated by the presence of moisture on the skin and is hindered by the presence of fat. Defatting the surface of the skin will increase penetration, while active sweating with evaporation reduces penetration. The influence of a rise in external temperature is more complex, depending on whether the sweat is allowed to evaporate away and the degree of hyperawmia produced. ACKNOWLEDGMENT. I am indebted to the Chief Scientific Officer, Ministry of Supply, for permission to publish this paper.