The in vitro macromolecular test method is a biochemical in vitro test method that can be used to identify chemicals (substances and mixtures) that have the potential to induce serious eye damage as well as chemicals not requiring classification for eye irritation or serious eye damage. The in vitro macromolecular test method contains a macromolecular reagent composed of a mixture of proteins, glycoproteins, carbohydrates, lipids and low molecular weight components, that when rehydrated forms a complex macromolecular matrix which mimics the highly ordered structure of the transparent cornea. Corneal opacity is described as the most important driver for classification of eye hazard. Test chemicals producing protein denaturation, unfolding and changes in conformation will lead to the disruption and disaggregation of the highly organised macromolecular reagent matrix, and produce turbidity of the macromolecular reagent. Such phenomena is quantified, by measuring the changes in light scattering (at a wavelength of 405 nm using a spectrometer), which is compared to the standard curve established in parallel by measuring the increase in OD produced by a set of calibration substances.

Phototoxicity is defined as a toxic response is elicited by topically or systemically administered photoreactive chemicals after the exposure of the body to environmental light. Several classes of photoreactive chemicals could cause phototoxic reactions when activated by light at otherwise non-toxic doses. Phototoxicity can be categorized as photoirritation, photoallergy, and photogenotoxicity. The purpose of this test is to measure the phototoxicity of a chemical. The main event in any phototoxic reaction is the absorption of photons of a wavelength that induces the excitation of the chromophore. Excitation energy is often transferred to oxygen molecules, followed by the generation of ROS (reactive Oxygen Species). Measurements are performed using a spectrometer. The determination of the ROS generation from irradiated chemicals with simulated sunlight is indicative of phototoxic potential.

The Vitrigel-Eye Irritancy Test (EIT) method is an in vitro test method that allows the identification of test chemicals not requiring classification and labelling for eye irritation or serious eye damage. This test measures the eye irritation potential of a test chemical based on its ability to induce damage to the barrier function of the human corneal epithelium (hCE) models used in the Vitrigel-EIT method. It is known that chemicals that are irritating to the eye first destroy tear film and epithelial barrier function of the eye, subsequently induce epithelial cell death, and finally produce stromal degeneration and endothelial cell death, resulting in corneal opacity. Therefore, the change of the epithelial barrier function is a relevant endpoint for detecting eye irritation. In the Vitrigel Eye Irritancy test method , time-dependent changes in the Transepithelial Electrical Resistance (TEER) values are indicative of damage to the barrier function of the corneal epithelium following exposure to a test chemical; this situation is similar to the observed damage of the rabbit cornea following exposure to a test chemical, which is an important mode of action leading to damage of the corneal epithelium and eye irritation. The Vitrigel-Eye Irritancy Test (EIT) method can be used within the limited applicability domain of test chemicals having pH > 5.0 (based on 2.5% weight/volume (w/v) preparation).

This Performance-Based Test Guideline (PBTG) describes in vitro assays, which provide the methodology for human recombinant in vitro assays to detect substances with estrogen receptor binding affinity (hrER binding assays). It comprises two mechanistically and functionally similar test methods for the identification of estrogen receptor (i.e. ERα) binders and should facilitate the development of new similar or modified test methods. The two reference test methods that provide the basis for this PBTG are: the Freyberger-Wilson (FW) In Vitro Estrogen Receptor (ER) Binding Assay Using a Full Length Human Recombinant ERα, and the Chemical Evaluation and Research Institute (CERI) In Vitro Estrogen Receptor Binding Assay Using a Human Recombinant Ligand Binding Domain Protein. This assay measures the ability of a radiolabeled ligand ([3H]17β-estradiol) to bind with the ER in the presence of increasing concentrations of a test chemical (i.e. competitor).  Test chemicals that possess a high affinity for the ER compete with the radiolabeled ligand at a lower concentration as compared with those chemicals with lower affinity for the receptor. This assay consists of two major components: a saturation binding experiment to characterise receptor-ligand interaction parameters and document ER specificity, followed by a competitive binding experiment that characterises the competition between a test chemical and a radiolabeled ligand for binding to the ER. These test methods are being proposed for screening and prioritisation purposes, but also provide mechanistic information that can be used in a weight of evidence approach.

This Test Guideline describes an in vitro procedure the identification on its own of chemicals (substances and mixtures) not requiring classification (No Cat), requiring classification for eye irritation (Cat 2) and requiring classification for serious eye damage (Cat 1) according to the UN GHS ocular hazard categories. 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. The test evaluates the ability of a test chemical to induce cytotoxicity in a RhCE tissue construct, as measured by the MTT assay. RhCE tissue viability following exposure to a test chemical is measured by enzymatic conversion of the vital dye MTT by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues. Cytotoxicity is measured at different time points of exposure; this is one of the methodological differences with the original TG 492.

This Test Guideline describes 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. The test evaluates the ability of a test chemical to induce cytotoxicity in a RhCE tissue construct, as measured by the MTT assay. Coloured chemicals can also be tested by used of an HPLC procedure. RhCE tissue viability following exposure to a test chemical is measured by enzymatic conversion of the vital dye MTT by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues. The viability of the RhCE tissue is determined in comparison to tissues treated with the negative control substance (% viability), and is then used to predict the eye hazard potential of the test chemical. Chemicals not requiring classification and labelling according to UN GHS are identified as those that do not decrease tissue viability below a defined threshold (i.e., tissue viability > 60%, for UN GHS No Category).

This Test Guideline describes a cytotoxicity-based in vitro assay that is performed on a confluent monolayer of Statens Seruminstitut Rabbit Cornea (SIRC) cells, cultured on a 96-well polycarbonate microplate. After five-minute exposure to a test chemical, the cytotoxicity is quantitatively measured as the relative viability of SIRC cells using the MTT assay. Decreased cell viability is used to predict potential adverse effects leading to ocular damage. Cell viability is assessed by the quantitative measurement, after extraction from the cells, of blue formazan salt produced by the living cells by enzymatic conversion of the vital dye MTT, also known as Thiazolyl Blue Tetrazolium Bromide. The obtained cell viability is compared to the solvent control (relative viability) and used to estimate the potential eye hazard of the test chemical. A test chemical is classified as UN GHS Category 1 when both the 5% and 0.05% concentrations result in a cell viability smaller than or equal to (≤) 70%. Conversely, a chemical is predicted as UN GHS No Category when both 5% and 0.05% concentrations result in a cell viability higher than (>) 70%.

This Test Guideline describes an in vivo assay that detects chemicals that may induce gene mutations in somatic and germ cells. In this assay, transgenic rats or mice that contain multiple copies of chromosomally integrated plasmid or phage shuttle vectors are used. The transgenes contain reporter genes for the detection of various types of mutations induced by test chemicals. A negative control group and a minimum of 3 treatment groups of transgenic animals are treated for 28 consecutive days. Administration is followed by a period of time, prior to sacrifice, during which the agent is not administered and during which unrepaired DNA lesions are fixed into stable mutations. At the end of this period (usually 3 or 28 days depending on the cell type and requirements), the animals are sacrificed, genomic DNA is isolated from the tissue(s) of interest and purified. Mutations that have arisen during treatment are scored by recovering the transgene and analysing the phenotype of the reporter gene in a bacterial host deficient for the reporter gene. Mutant frequency, the reported parameter in these assays, is calculated by dividing the number of plaques/plasmids containing mutations in the transgene by the total number of plaques/plasmids recovered from the same DNA sample.

This Test Guideline describes an in vivo assay that detects chemicals that may induce gene mutations. In this assay, transgenic rats or mice that contain multiple copies of chromosomally integrated plasmid or phage shuttle vectors are used. The transgenes contain reporter genes for the detection of various types of mutations induced by test substances. A negative control group and a minimum of 3 treatment groups of transgenic animals are treated for 28 consecutive days. Administration is usually followed by a 3-day period of time, prior to sacrifice, during which the agent is not administered and during which unrepaired DNA lesions are fixed into stable mutations. At the end of this 3-day period, the animals are sacrificed, genomic DNA is isolated from the tissue(s) of interest and purified. Mutations that have arisen during treatment are scored by recovering the transgene and analysing the phenotype of the reporter gene in a bacterial host deficient for the reporter gene. Mutant frequency, the reported parameter in these assays,is calculated by dividing the number of plaques/plasmids containing mutations in the transgene by the total number of plaques/plasmids recovered from the same DNA sample.