j. Soc. Cosmet. Chem., 39, 107-119 (March/April 1988) A new in vitro method for transepidermal water loss: A possible method for moisturizer evaluation L. M. LIEB, R. A. NASH, J. R. MATIAS, and N. ORENTREICH, St. John's University, Jamaica, NY 11439 (L.M.L., R.A.N.) and Orentreich Foundation for the Advancement of Science, Inc., 910 Fifth Ave., New York, NY 10021 (J.R.M., N.O.) Received October 19, 1987 Synopsis Transepidermal water loss (TEWL) through the ventral skin of the hamster ear was determined using a modified flow-through diffusion apparatus. Tritium-labeled water was allowed to permeate through the dermal/epidermal layers. Water vapor was collected in a closed system by adsorption onto solid anhydrous calcium chloride in a separately attached receiver. The desiccant was removed, dissolved in water, and the radioactivity determined by liquid scintillation counting. The rate of TEWL was determined for various durations of exposure to the desiccated environment, in the temperature range between 5 and 40øC. TEWL rate reached a maximum one hour after exposure, and then decreased to a steady state with time. TEWL increased exponentially with increasing temperature in accordance with the Arrhenius relationship. An activation energy value of 13 Kcal/mole was obtained. At incubator temperature of 22øC, the rate of TEWL was found to be 152 _+ 14 p,g/cm2/hr, which agrees with previously reported values obtained by in vivo methods of testing. The present technique is proposed as a rapid in vitro method for measuring TEWL and as a possible pretest for assessing efficacy of potential skin-moisturizing agents. Four agents were studied for their effect on water permeability. The agents tested were mineral oil, castor oil, sesame oil, and 25% glycerin in water. Skin membranes treated with occlusive agents such as mineral oil, castor oil, and sesame oil showed a marked decrease in the TEWL rate, while those membranes treated with the humectant, 25% glycerin in water, showed a marked increase in the TEWL rate. Similar results using these same agents have been reported previously. INTRODUCTION Blank showed that proper hydration of the stratum corneum is a primary factor in maintaining skin softness and flexibility (1,2). Other constituents of skin, such as pro- teins and lipids, play a minor role by influencing the water-binding capacity of the stratum corneum. Appearance and barrier function of the skin is affected, if it is not optimally hydrated (3). This effect is exhibited in low relative humidity environments, where the skin becomes dry, flaky, and/or chapped. When skin hydration increases at higher relative humidity, the water content and the permeability of skin increases (1,2,4-10). It is considered supple, flexible, pliable, and less likely to chap, fissure, and crack (1,2, 11- 14). Some cosmetic chemists regard moisturization as any means to increase water content and maintain this level over a period of time (15). "A moisturizer," as defined by Kligman (16) "is a topically applied substance or product that overcomes the signs and 107

108 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS symptoms of dry skin." In simple terms, the alleviation of the dry skin state after "moisturizers" are applied externally may be partly due to the increased hydration or water content of the skin (1). One of the methods used to assess the hydrated state of the skin is to measure the percent decrease of transepidermal water loss (TEWL) on skin treated with a moisturizer versus untreated skin. TEWL is a passive diffusional process where water vapor diffuses from highly hydrated dermal layers via the stratum corneum to the exterior surface where it evaporates freely. Leveque et al. (17) recently made in vivo TEWL measurements (Servomed © evapor- imeter technique of Nilson) of dry skin patients and compared this method to the regres- sion method of Kligman (16). A statistically significant trend was observed with TEWL and dry skin, although the two did not correlate closely. It is noted that an increase in TEWL (from normal levels) during a dry skin state suggests a disturbance in the horny layer structure. However, during this state there's also a tendency of dry skin to form a thicker horny layer, counteracting this effect (17). This method of measuring moistur- izing efficacy via TEWL rate decrease has its limitations and is a subject of controversy. Other methods that have been used to hydrate the skin include (18): 1. applying ex- ternal water directly to the stratum corneum, and 2. increasing the rate of diffusion of water from the lower epidermal layers through the stratum corneum. According to Wu (19), transepidermal water loss is described by the term called flux and is expressed by Fick's second law: J = D(C) dC/dX where J equals flux, in Ftg/cm2/hr, C equals water concentration in the stratum cor- neum, •tg/cm 3, X equals the thickness of skin membrane, cm, and D(C) equals diffu- sivity of water in the stratum corneum, cm2/hr. D(C) is a constant developed by Wu (19) to describe the diffusivity of water and its relation to the barrier properties of the membrane utilized. FACTORS AFFECTING TEWL The environmental temperature, relative humidity, and skin source must be controlled during experimentation in order to determine TEWL. This requirement is evident by the large variation in TEWL values, reported previously: 100-1500 Ftg/cm2/hr (1,4,6,9,20-26). In vivo studies by Grice et al. (20,21) and in vitro experiments by Matias (22) have shown that TEWL will increase exponentially with increasing temperature. Reports of the effect of environmental relative humidity on TEWL, however, are in conflict (23-26). Using different experimental methods, Goodman and Wolf (23), Spruit and Malten (24), and Bettley and Grice (25) showed that the water loss through skin de- creases as relative humidity increases. More recent controlled in vivo studies by Grice (26) revealed a maximum TEWL rate between 50% and 75% relative humidity (RH). In vitro measurements with fetal hog periderm by Wu (19) have confirmed Grice's findings (26). The integrity and source of skin used in in vitro studies are also critical factors. It has been reported that diseased and damaged skin have increased TEWL (27-28). Dupuis (29) reported a TEWL value from forehead skin almost twice that from skin at other anatomic sites.