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.

METHOD FOR TRANSEPIDERMAL WATER LOSS 109 Application of topical vehicles can either increase or decrease the hydration of the stratum corneum depending on how the vehicle alters water activity in the barrier. Oily, occlusive materials, such as petrolatum, mineral oil, lanolin, and isopropyl my- ristate, significantly decrease the rate of water loss from the skin. These agents can be considered moisturizing agents since they are able to increase skin hydration. Humec- tants, agents that act to bind water to skin, such as glycerin and propylene glycol, can increase skin moisture by interacting with the atmospheric moisture in conditions of moderate to high humidity. When the relative humidity of air decreases, humectants on the skin, however, will extract moisture from the deeper layers. Rieger and Deem have shown that humectants increase the in vitro TEWL at conditions of low humidity (30). Humectants alone, therefore, only function as a moisturizer in proper atmospheric conditions. It is conceivable that the moisturizing emulsions can still decrease TEWL, at various humidities, even with a humectant present in the formula. Decreasing TEWL presumably increases the pool of water available to hydrate the skin. It is pos- sible to determine the influence of TEWL on moisturizer effectiveness by comparing a TEWL value on untreated skin to that value obtained from moisturizer- or occlusive agent-treated skin (30). In vitro methods for TEWL measurement can be utilized to pre-evaluate the potential efficacy of moisturizers in human skin. Present in vitro methods (1,2,4,7,8, 19,30- 33) are time-consuming to use for this purpose. Another limitation is that water loss from the skin can conflict with water loss from the moisturizer. The objective of the present research was to develop a simple and accurate method that permits rapid evaluation of TEWL of potential moisturizers by a quantitative measurement of moisture loss through an animal skin membrane. This method uses a tritiated-water tracer technique which can eliminate erroneous TEWL values that may result from the water evaporation from a moisturizer. EXPERIMENTAL PROCEDURE APPARATUS DESIGN A modification of a flow-through diffusion cell originally designed by Bronaugh (34) was utilized. The membrane was full-thickness, cartilage-stripped skin taken from the ventral ear of the male Syrian golden hamster (Harlan-Sprague Dawley, Indianapolis, IN) as described by Matias (35). Tritiated water (HTO) permeated through the mem- brane into the receptor compartment of the diffusion cell. Flux (TEWL) was deter- mined, via scintillation counting, by the measurement of HTO that adsorbed on anhy- drous calcium chloride in the receptor cell. A TEWL rate-vs-time study was conducted to determine the time to steady state or equilibrium conditions at 32øC. The prepared cells were then exposed to temperature increments within the range between 5 and 37øC, and then TEWL measured at steady state. A plot of the logarithm of TEWL vs the reciprocal of absolute temperature was used to determine the energy of activation (by slope analysis) in accordance with the Arrhenius relationship. An evaluation of TEWL was conducted for skin treated with three occlusive agents: mineral oil, castor oil, and sesame oil, and skin treated with a humectant (25% glycerin