The ability of cells to reduce tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) provides an indication of mitochondrial integrity and activity which, in turn, may be interpreted as a measure of viability and/or cell number. The assay has therefore been adapted for use with cultures of exponentially growing cells. Determination of their ability to reduce MTT to the formazan product after exposure to test compounds, compared to the untreated control, enables the relative toxicity of test chemicals to be assessed.
In Dermaclaim, we have adapted this ECVAM protocol to yield a cytotoxic potential (number), through which you can conduct screenings of different compounds at the same time and determine the cytotoxic and irritating potential of finished products and ingredients in the initial phases of product development.
This OECD 439 (July, 2015) protocol may be used to determine the skin irritancy of chemicals either as a stand-alone replacement test for in vivo skin irritation testing or as a partial replacement test within a testing strategy.
It is based on the in vitro test system of reconstructed human epidermis (RhE), which closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis. The RhE test system uses human derived non-transformed keratinocytes as cell source to reconstruct an epidermal model with representative histology and cytoarchitecture.
This Test Guideline describes a method to evaluate photo-cytotoxicity by the relative reduction in viability of cells exposed to the chemical in the presence versus absence of light. Cytotoxicity in this test is expressed as a concentration-dependent reduction of the uptake of the Vital dye Neutral Red (NR) when measured 24 hours after treatment with the test chemical and irradiation.
The in vitro 3T3 NRU phototoxicity test is used to identify the phototoxic potential of a test substance induced by the excited chemical after exposure to light. Substances identified by this test are likely to be phototoxic in vivo following systemic application and distribution to the skin, or after topical application.
This test allows the identification of corrosive chemical substances and mixtures and it enables the identification of non-corrosive substances and mixtures when supported by a weight of evidence determination using other existing information. The test protocol may also provide an indication of the distinction between severe and less severe skin corrosives.
The test material (solid or liquid) is applied uniformly and topically to a three-dimensional human skin model, comprising at least a reconstructed epidermis with a functional stratum corneum. The principle of the human skin model assay is based on the hypothesis that corrosive chemicals are able to penetrate the stratum corneum by diffusion or erosion, and are cytotoxic to the underlying cell layers.
This protocol OECD 492 provides an in vitro procedure allowing the identification of chemicals (substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage in accordance with UN GHS.
It makes use of reconstructed human cornea–like epithelium (RhCE) which closely mimics the histological, morphological, biochemical and physiological properties of the human corneal epithelium.
This guideline presents general principles for measuring dermal absorption and delivery of a test substance using excised skin. Skin from many mammalian species, including 3D reconstructed skin, humans explants, or pigskin, can be used. The permeability properties of skin are maintained after excision from the body because the principal diffusion barrier is the stratum corneum.
The test substance, which may be radiolabelled, is applied to the surface of a skin sample separating the two chambers of a diffusion cell. The chemical remains on the skin for a specified time under specified conditions, before removal by an appropriate cleansing procedure. The receptor fluid is sampled at time points throughout the experiment and analysed for the test chemical and/or metabolites using HPLC-MS/MS, ELISA, etc.
The bacterial reverse mutation test uses amino-acid requiring strains of Salmonella typhimurium and Escherichia coli to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base pairs. The principle of this bacterial reverse mutation test is that it detects mutations which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesize an essential amino acid. The revertant bacteria are detected by their ability to grow in the absence of the amino acid required by the parent test strain.
Suspensions of bacterial cells are exposed to the test substance in the presence and in the absence of an exogenous metabolic activation system. In the plate incorporation method, these suspensions are mixed with an overlay agar and plated immediately onto minimal medium. After two or three days of incubation, revertant colonies are counted and compared to the number of spontaneous revertant colonies on solvent control plates.