COSMETIC FILMS ON THE SKIN 153 reimpregnated and extracted) were collected in weighing bottles and dried in a vacuum desiccator over P2Oa to constant weight. Determination of Moisture Uptake--The ability of the three types of skin scales to absorb moisture was determined by placing scrapings in a moisture chamber at 90% R.H. for 48 hours. The difference between the initial and final weight represents the moisture absorption. Penetration---The question of penetration of Aqualizer E-J remains still unanswered. In other words, when Aqualizer E-J is applied to the skin does it only cling to the surface, or does it penetrate the stratum corneum? To answer this question, a microscopic method was devel- oped which permits determination of the presence of Aqualizer E-J and of natural NMF by two different staining reactions. These staining reactions are called Reaction N and Reaction R. Reaction N is a nin- hydrin reaction and is performed as follows: With the aid of a needle some cell material is placed on a dry microscope slide. The stratum corneum cells are covered with a drop of ninhydrin reagent and immediately covered with a cover glass. The slide is heated for 30 seconds at 60øC and immediately observed under the microscope for blue to violet coloration of the cells. The reagent is prepared by dissolving 0.3 g of ninhydrin in 100 ml of butanol and adding 3 ml of glacial acetic acid. Reaction R is a TTC (triphenyltetrazolium chloride) reaction and is performed as follows: A small amount of cell material is placed on a dry •nicroscopic slide, and two drops of the reagent are added. A cover glass is placed on top, and the slide is exposed to a tempera- ture of 55-60øC for 3 minutes. The red coloration of the cells is observed under a micro- scope. The reagent is prepared by mixing one part of a 0.5% solution of TTC in distilled water with two parts of a 0.5 N NaOH solution. To this mixture one part of a 1 2 sodium lauryl sulfate solution in water is added. The solution is cloudy at first but clears on shaking. A very small p•ecipitate settles out the supernatant clear solution is used. For these tests, the skin was treated in the same way as described above. The cell layers of the stratum corneum of the epidermis were scraped off, layer by layer, with a sharp scalpel. Each layer of cells was examined separately under the microscope for the presence of Aqualizer E-J or NMF to determine the penetration of Aqualizer E-J. For the evalua- tion of staining reactions the following scores were used: No reaction ............................................. 0 Slight reaction ........................................... 1 Definite reaction ......................................... 2 Strong reaction .......................................... 3 Very strong reaction ...................................... 4 The stronger the reaction the more Aqualizer E-J or NMF is present.

154 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS E•ect of Sebum on Water Vapor Transmission After the efficacy of the synthetic NMF was established, the effect of human sebum on the breathing of the skin was studied by measuring the insensible perspiration. For these studies the method of Gregory (18), which is basically an electrical hygrometer, was used. For the investigations described in this paper the following instrument was con- structed. Two copper electrodes were melted into the bottom of a glass beaker having a diameter of 3 cm. A cotton thread, 5 cm long, saturated with K C1, is wound around these electrodes in the form of a figure eight. This saturated thread serves as a semiconductor. The open end of this glass cell is padded with rubber so that the edge of the cell may be pressed air tight against the skin. The cell is connected to a LF 39 WTM* conductance meter. Absorption of moisture changes the resistance of the cotton thread, which is measured on the instrument. 0 2 4 6 B 10 12 14min Figure 2. Sum of five measurements with a cell on a phantom during 15 minutes temperature variations of water -- q-0.2øC Each cell is calibrated over a water surface serving as a model. As Fig. 2 shows, the deviations of one cell for five measuremen ts during 15 minutes and at temperature changes of the water of +0.2øC are relatively small. During these measurements it was proven that the galvanometer reading/time interval is the best usable measurement for the insensible perspiration. The resistance of the KC1 saturated cotton thread changes, depending on the presence of humidity in the cell, and is related to moisture uptake or moisture loss. The instrument also allows absolute measurements of the insensible perspiration. In this case the cell is calibrated as follows: The cell is connected with a flat cylindrical beaker having the same diameter as the cell. The beaker contains a weighed amount of water. After the galvanometer shows a certain reading, the beaker with water is removed * Wissenschaftlich-technische Werkstatte GMBH, Weilheim/Oberbayern, Germany.