The standard “OECD TG 435 In vitro membrane barrier test method for skin irritation”, published under the OECD guidelines on responsible business conduct for multinational enterprises (OECD guidelines), describes an in vitro membrane barrier test method used to identify corrosive chemicals. This test method uses an artificial membrane designed to respond to corrosive chemicals in a similar way to animal skin.
The in vitro membrane barrier test method can be used to test solids, liquids, and emulsions. However, aqueous substances with a pH in the range of 4,5 to 8,5 are generally not suitable for testing.
The test method described allows the identification of corrosive chemicals and mixtures and the sub-categorization of corrosive substances as permitted in the globally harmonized system of classification and labeling of chemicals. This classification is based on the time it takes for the substance to permeate through the membrane barrier.
This test system consists of two components:
When applying this test system, an appropriate number of replicates are prepared for each test substance and its corresponding controls. The times of application of the test substance to the membrane barrier are recorded and arranged in steps. The time (in minutes) between application of the test substance to the membrane barrier and barrier penetration is used to determine the corrosiveness of the test substance.
Skin corrosivity has traditionally been determined by applying the test chemical to the skin of live animals and assessing the degree of tissue damage after a specified period of time. Alongside the OECD TG 435 standard, a number of other in vitro test methods have been adopted as alternatives to the standard in vivo rabbit skin procedure (OECD TG 404) used to identify corrosive chemicals. OECD guidelines recommend the use of validated and accepted in vitro test methods.
In this modular approach, positive results from in vitro testing methods are used to classify a chemical as corrosive without the need for animal testing. In this way, the aim is to avoid the use of animals in testing and to prevent the pain and suffering that may occur if animals are used for this purpose.
This test method detects membrane barrier damage caused by corrosive test chemicals after application of the test chemical to the surface of a synthetic macromolecular membrane barrier, presumably through the same irritation mechanisms operating in living skin, via a “chemical detection system.”
Penetration or breaching of the membrane barrier can be measured by a number of procedures or chemical detection systems, including a change in the color of the pH indicator dye or other property of the indicator solution beneath the barrier.
The membrane barrier must be determined to be valid, i.e. relevant and reliable, for its intended use. This includes ensuring that different preparations are consistent in their barrier properties, e.g. being able to maintain a barrier to non-corrosive chemicals, being able to categorise the corrosive properties of chemicals within the corrosiveness subcategories of the Globally Harmonised System for Classification and Labelling of Chemicals. The classification assigned is based on the time it takes for a chemical to penetrate the membrane barrier into the indicator solution.
Before routine use of the in vitro membrane barrier test method is undertaken, laboratories must demonstrate their technical competence by correctly classifying the proposed proficiency substances in accordance with this standard. In cases where a listed substance is not available or is justified, another substance for which adequate in vivo and in vitro reference data are available may be used.
Tests must be carried out at ambient temperature (in the range of 17-25 degrees) and the components must comply with the conditions specified in the standard.
Before performing the membrane barrier test, a chemical compatibility test is performed to determine if the test chemical is detectable by the chemical detection system. If the chemical detection system does not detect the test chemical, the membrane barrier test method is not suitable for evaluating the potential corrosivity of that test chemical and a different test method should be used. The chemical detection system and exposure conditions used for the compatibility test should reflect the exposure in the subsequent membrane barrier test.
If appropriate for the test method, a test chemical that has been qualified by a compatibility test should be subjected to a time scale category test, which is a screening test to distinguish between weak and strong acids or bases. For example, a time scale categorization test is used in the validated reference test method to indicate which of the two time scales should be used depending on whether significant acid or alkali reserve is detected. Depending on the acid or alkali reserve of the test chemical, two different breakthrough time scales should be used to determine its corrosivity and the Globally Harmonized System of Classification and Labeling of Chemicals skin corrosivity subcategory.
The membrane barrier consists of two components: a proteinaceous macromolecular aqueous gel and a permeable supporting membrane. The proteinaceous gel should be impermeable to liquids and solids but can be eroded and made permeable. The fully constructed membrane barrier should be stored under prescribed conditions that have been shown to prevent gel degradation, such as drying, microbial growth, shearing or cracking, which would reduce its performance. Acceptable storage times should be determined and membrane barrier preparations should not be used after this period. The permeable supporting membrane provides mechanical support to the proteinaceous gel during the gelation process and exposure to the test chemical. The supporting membrane should prevent the gel from sagging or shearing and should be readily permeable to all test chemicals.
For the chemical detection system, the indicator solution, which is the same solution used for compatibility testing, should respond to the presence of a test chemical. A pH indicator dye or combination of dyes, such as cresol red and methyl orange, should be used that will show a color change in response to the presence of the test chemical.
Among the numerous tests, measurements, analyses and evaluation studies that our organization has carried out for businesses in various sectors, it also provides testing services defined in the “OECD TG 435 In vitro membrane barrier test method for skin irritation” standard with its trained and expert staff and advanced technological equipment.
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