This value is based on internal experience and experiments to distinguish native and punched human skin samples. A lab-specific limit value Crenolanib nmr is necessary due to limited transferability: The measured resistance is dependent on the device, applied frequency, resulting current, ionic strength of the solution as well as the surface area of the skin sample (Fasano et al., 2002). The transepidermal water loss was measured after minimal 1 h of equilibration and drying of the skin surface. The moisture on the skin surface originating from rehydration of the frozen skin samples

or from TEER measurement needs to be evaporated to measure exclusively the water loss through the skin sample. With a VapoMeter (Delfin Technologies Ltd., Finland) the TEWL was determined under closed chamber conditions (Imhof et al., 2009). For this end the donor compartment of the diffusion cell was covered completely with the VapoMeter. The standard limit

of 10 g m−2 h−1 was used (Schäfer and Redelmeier, 1996b). To determine the absorption characteristics of tritiated, 3H-labeled, water, the receptor compartment was filled with physiological saline. An infinite dose (300 μl cm−2) with a specific radioactivity of 123 kBq ml−1 was applied to the surface of the skin. At distinct time points (0.5, 1, 2, 3, 4 and 5 h) receptor fluid was collected using a syringe. After the last sampling the skin was thoroughly washed with distilled water and cotton swabs. Receptor fluid was diluted with scintillation cocktail, measured by LSC and data were used to calculate the permeability constant (Kp) as described Selleck MK-2206 in Section 2.3. A generally accepted limit value of 2.5 ∗ 10−3 cm h−1 was used (Bronaugh et al., 1986). Using TWF as a pre-test, the radioactivity needs to be removed from the system before application of the test compound. Therefore, the receptor fluid was changed several times until the activity in a receptor fluid aliquot declined to 50 dpm (0.8 Bq). A 3H-labeled internal Celastrol reference standard was added to the 14C-labeled test compound formulation and applied to the skin (see Table 1 and Table 3). The concentration was determined by the specific radioactivity of the 3H-ISTD which was

chosen to be equal to the specific radioactivity of the 14C-labelled test compound (Table 1). In all samples 3H-activity was measured along with the 14C-activity by LSC. Absorption characteristics (AD and maxKp) were determined analogously, as described in Section 2.3. Following the final washing procedure at the end of the absorption experiment, 250 μl of methylene blue, 0.025% aqueous solution, was applied on top of the skin for 0.5 h and washed off with 0.7% aqueous Texapon® N70 solution. The receptor fluid was tested for permeated dye using a photometer operating at 661 nm. The concentration in the receptor fluid was determined via a calibration curve. Any staining of the epidermis was reported before digestion and processing for LSC measurements.