Alternative Approaches For Skin Integrity Testing
The in vitro percutaneous absorption method has been described in detail in several publications and previously in our blog posts and whitepapers. One interesting question that has arisen recently is how to provide data for skin integrity testing.
Assessment of in vitro absorption provides valuable information when predicting dermal absorption in vivo. Skin membrane integrity is an essential component of any in vitro method. This is further described in the test guideline OECD 428. If skin membrane integrity is compromised during any preliminary handling of the tissue, this will affect the permeability of the membrane with test articles. For many years, the conventional means of evaluating membrane integrity has been by measurement of the flux of a standard. Historically, assessment of the membrane’s permeability to tritiated water (T2O) and the generation of a permeability coefficient (Kp) have been used to confirm that the skin membrane was intact prior to application of the test penetrant.
The most common approach used is tritiated water 3H2O (or T2O), where the permeability of the membrane to T2O is determined and the permeability coefficient (Kp) for T2O is calculated over a number of hours. However, this method is time consuming and the use of radioactivity is costly and has safety implications, which in a lot of jurisdictions, is becoming more difficult to maintain in a laboratory setting.
Here is the basic procedure for using tritiated water:
To assure barrier integrity of each skin section, its permeability to tritiated water should be determined before application of the test products. Following a brief (0.5–1 hour) equilibrium period, 3H2O is layered across the top of the skin by dropper so that the entire exposed surface is covered (approximately 100–150 μ l). After 5 minutes, the 3H2O aqueous layer is removed and the skin surface blotted dry. At 30 minutes after application, the receptor solution is collected and analyzed for radioactive content by liquid scintillation spectrometry. Skin specimens in which absorption of 3H2O is less than 1.25 μ l-equ are considered acceptable. In case of failure, the criterion should be either be discarded or used as a non-dosed, blank analytical control chambers.
From the other sources, the skin barrier test results demonstrated that after 20 minutes application of 3H2O onto human skin, the passage (of 3H2O) was 0.3-0.5% ±0.1 % of applied dose.
For more information on this technique, please see TJ Franz and PA Lehman's work on "The use of water permeability as a means of validation of skin integrity in In Vitro percutaneous absorption studies." (J Invest Dermatol 1990; 94: 525, The journal of pharmaceutical sciences)
Liquid scintillation counting is a standard laboratory method in the life-sciences for measuring radiation from beta-emitting nuclides. The only alternative method is skin Electrical Resistance (ER) measurement.
The integrity of the membranes was determined by measurement of the electrical resistance across the skin membrane, concurrently and in the same cells used for the T2O method. Resistance across each membrane was measured by passing a fixed current across the skin preparation using a PRISM Electronics AIM6401 LCR data bridge connected to two stainless steel electrodes, using a setting of 100 kHz. This was undertaken at least 30 min after T2O contact to ensure temperature and humidity equilibration. One electrode probe, at least 50 mm long, was inserted to the bottom of the receptor chamber arm, below the saline level. The donor chamber probe was positioned in the saline above the skin preparation, taking care not to touch the skin membrane. Following a few seconds of stabilization, the reading was recorded.
This study was performed by D.J. Davies et al. "Multi-species assessment of electrical resistance as a skin integrity marker for in vitro percutaneous absorption studies" (Toxicology in Vitro 18 (2004) 351–358)
This study has shown that electrical resistance is a quick, valuable tool for measuring the integrity of a variety of skin membranes without the need for the use of radiolabeled material. When compared with the standard T2O technique, it has been demonstrated that the measurement of resistance across the membrane provides a robust measure of skin barrier integrity and is therefore a suitable method for the acceptance/rejection of membranes for in vitro percutaneous absorption measurements. ER cut-off values were provided for these studies that were proposed should be used to qualify skin integrity of specimens from different species. However, it was recognized that this validation related to their own static cell equipment and, importantly, the actual databridge settings used to measure the ER. Other laboratories using different equipment or methodology would need to perform their own validation in house before utilizing ER as their measure of skin integrity.
Another interesting technique that can be evaluates is an FTIR technique. An adapted FTIR technique might provide sensitive, accurate and fast measurement of stable isotopes in water D2O instead of radioactive T2O. Classical measuring cells with water-resistant CaF2 windows can be used in this applications. 0.2 mm pathlength can be used for the –OD determination within the range of the detection limits. The measurements can be carried out on certain FTIR instruments. The samples can be directly transferred to the IR measuring cell. These minimal sample preparation steps might be sufficient to meet the requirements for fast and relatively interference-free sample preparation and measuring techniques.
The high sensitivity of this method is based on high baseline stability, high signal-to-noise ratio and data accuracy of the photometric method. For the water sample, a detection limit of 0.003% deuterium can be obtained. Furthermore, FTIR is considered to be a fast technique, as only 10 minutes is needed from sample preparation to data evaluation. FTIR measurements with respect to water kinetics are all comparable with a reference MS method.
It must be cautioned that this is a novel concept that requires further investigation. For those labs that need to perform skin integrity testing and cannot acquire a radioactive license, this technique may be worth further examination.