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%.
The in vitro mammalian cell gene mutation test can be used to detect gene mutations induced by chemical substances. This TG includes two distinct in vitro mammalian gene mutation assays requiring two specific tk heterozygous cells lines: L5178Y tk+/-3.7.2C cells for the mouse lymphoma assay (MLA) and TK6 tk+/- cells for the TK6 assay. Genetic events detected using the tk locus include both gene mutations and chromosomal events.
Cells in suspension or monolayer culture are exposed to, at least four analysable concentrations of the test substance, both with and without metabolic activation, for a suitable period of time. They are subcultured to determine cytotoxicity and to allow phenotypic expression prior to mutant selection. Cytotoxicity is usually determined by measuring the relative cloning efficiency (survival) or relative total growth of the cultures after the treatment period. The treated cultures are maintained in growth medium for a sufficient period of time, characteristic of each selected locus and cell type, to allow near-optimal phenotypic expression of induced mutations. Mutant frequency is determined by seeding known numbers of cells in medium containing the selective agent to detect mutant cells, and in medium without selective agent to determine the cloning efficiency (viability). After a suitable incubation time, colonies are counted.
The in vitro mammalian cell gene mutation test can be used to detect gene mutations induced by chemical substances. This TG includes two distinct in vitro mammalian gene mutation assays requiring two specific tk heterozygous cells lines: L5178Y tk+/-3.7.2C cells for the mouse lymphoma assay (MLA) and TK6 tk+/- cells for the TK6 assay. Genetic events detected using the tk locus include both gene mutations and chromosomal events.
Cells in suspension or monolayer culture are exposed to, at least four analysable concentrations of the test substance, both with and without metabolic activation, for a suitable period of time. They are subcultured to determine cytotoxicity and to allow phenotypic expression prior to mutant selection. Cytotoxicity is usually determined by measuring the relative cloning efficiency (survival) or relative total growth of the cultures after the treatment period. The treated cultures are maintained in growth medium for a sufficient period of time, characteristic of each selected locus and cell type, to allow near-optimal phenotypic expression of induced mutations. Mutant frequency is determined by seeding known numbers of cells in medium containing the selective agent to detect mutant cells, and in medium without selective agent to determine the cloning efficiency (viability). After a suitable incubation time, colonies are counted.
The in vivo alkaline single cell gel electrophoresis assay, also called alkaline Comet Assay is a method measuring DNA strand breaks in eukaryotic cells.
Each treated group is composed of a minimum of 5 animals of one sex (or of each sex as appropriate). A positive and a vehicle control group are also used. Administration of the treatment consists of daily doses over duration of 2 days or more, ensuring the test chemical reaches the target tissue which can be the liver, the kidney or other tissues if justified.
Tissues of interest are dissected and single cells/nuclei suspensions are prepared and embedded in agarose on slides. Cells/nuclei are treated with lysis buffer to remove cellular and/or nuclear membranes. The nuclear DNA in the agar is then subjected to electrophoresis at high pH. This results in structures resembling comets which by using suitable fluorescent stain, can be observed by fluorescent microscopy. Based on their size DNA fragments migrate away from the head to the tail, and the intensity of the comet tail relative to the total intensity (head plus tail) reflects the amount of DNA breakage.
The in vivo alkaline single cell gel electrophoresis assay, also called alkaline Comet Assay is a method measuring DNA strand breaks in eukaryotic cells.
Each treated group is composed of a minimum of 5 animals of one sex (or of each sex as appropriate). A positive and a vehicle control group are also used. Administration of the treatment consists of daily doses over duration of 2 days or more, ensuring the test chemical reaches the target tissue which can be the liver, the kidney or other tissues if justified.
Tissues of interest are dissected and single cells/nuclei suspensions are prepared and embedded in agarose on slides. Cells/nuclei are treated with lysis buffer to remove cellular and/or nuclear membranes. The nuclear DNA in the agar is then subjected to electrophoresis at high pH. This results in structures resembling comets which by using suitable fluorescent stain, can be observed by fluorescent microscopy. Based on their size DNA fragments migrate away from the head to the tail, and the intensity of the comet tail relative to the total intensity (head plus tail) reflects the amount of DNA breakage.
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.
The in vitro micronucleus test is a genotoxicity test for the detection of micronuclei in the cytoplasm of interphase cells. Micronuclei may originate from acentric chromosome fragments (i.e. lacking a centromere), or whole chromosomes that are unable to migrate to the poles during the anaphase stage of cell division. The assay detects the activity of clastogenic and aneugenic test substances in cells that have undergone cell division during or after exposure to the test substance. This Test Guideline allows the use of protocols with and without the actin polymerisation inhibitor cytochalasin B. Cytochalasin B allows for the identification and selective analysis of micronucleus frequency in cells that have completed one mitosis, because such cells are binucleate. This Test Guideline also allows the use of protocols without cytokinesis block provided there is evidence that the cell population analysed has undergone mitosis.
The in vitro micronucleus test is a genotoxicity test for the detection of micronuclei in the cytoplasm of interphase cells. Micronuclei may originate from acentric chromosome fragments (i.e. lacking a centromere), or whole chromosomes that are unable to migrate to the poles during the anaphase stage of cell division. The assay detects the activity of clastogenic and aneugenic test substances in cells that have undergone cell division during or after exposure to the test substance. This Test Guideline allows the use of protocols with and without the actin polymerisation inhibitor cytochalasin B. Cytochalasin B allows for the identification and selective analysis of micronucleus frequency in cells that have completed one mitosis, because such cells are binucleate. This Test Guideline also allows the use of protocols without cytokinesis block provided there is evidence that the cell population analysed has undergone mitosis.
The in vitro micronucleus test is a genotoxicity test for the detection of micronuclei in the cytoplasm of interphase cells. Micronuclei may originate from acentric chromosome fragments (i.e. lacking a centromere), or whole chromosomes that are unable to migrate to the poles during the anaphase stage of cell division. The assay detects the activity of clastogenic and aneugenic test substances in cells that have undergone cell division during or after exposure to the test substance. This Test Guideline allows the use of protocols with and without the actin polymerisation inhibitor cytochalasin B. Cytochalasin B allows for the identification and selective analysis of micronucleus frequency in cells that have completed one mitosis, because such cells are binucleate. This Test Guideline also allows the use of protocols without cytokinesis block provided there is evidence that the cell population analysed has undergone mitosis.
The purpose of the unscheduled DNA synthesis (UDS) test with mammalian liver cells in vivo is to identify substances that induce DNA repair after excision and removal of a stretch of DNA containing a region of damage induced by chemical substances (solid or liquid) or physical agents in the liver.
The test is usually based on the incorporation of tritium-labelled thymidine, 3H-TdR, (during 3-8 hours) into the DNA of liver cells which have a low frequency of cells in the S-phase of the cell cycle. The uptake of 3H-TdR is usually determined by autoradiography. Rats are commonly used, and the number of animals should be at least three analysable animals per group. Normally, at least two dose levels are used. A limit test may be performed if no effects would be expected at a dose of 2000 mg/kg bw/d. Test substances are generally administered as a single treatment by gavage using a stomach tube or a suitable intubation cannula. Liver cells are prepared from treated animals 12-16 hours after dosing of animal. After autoradiography, normally 100 cells are scored from each animal from at least two slides. A positive result from the UDS test with mammalian liver cells in vivo indicates that a substance induces DNA damage in mammalian liver cells in vivo that can be repaired by unscheduled DNA synthesis in vitro. A negative result indicates that, under the test conditions, the test substance does not induce DNA damage that is detectable by this test.
The mouse heritable translocation test detects structural and numerical chromosome changes in mammalian germ cells as recovered in first generation progeny.
The types of chromosome changes detected in this test system are reciprocal translocations. Carriers of translocations and XO-females show reduced fertility which is used to select first generation progeny for cytogenetic analysis. Translocations are cytogenetically observed in meiotic cells at diakinesis metaphase I of male individuals. The test is usually performed by analysis of male first generation progeny. About 500 first generation males per dose level are required. One dose level is tested, usually the highest dose associated with the production of minimal toxic effects, and administered by oral intubation or intraperitoneal injection. A single administration of the test substance or the administration of the test substance on 7 days/week for 35 days, are possible. The test substance can be solid, liquid, vapour or gaseous. For translocation heterozygosity one of two possible methods is used: fertility testing of first generation progeny; or cytogenetic analysis of all male first generation progeny are possible. A test substance producing neither a statistically significant increase in the number of translocations observed for at least one test point, nor a statistically significant, dose-related, increase in the number of translocations observed, is considered non-mutagenic in this system.
This test measures chromosome events in spermatogonial germ cells and is, therefore, expected to be predictive of induction of inheritable mutations in germ cells.
Male Chinese hamsters and mice are commonly used. Animals are exposed to the test substance (liquid or solid) by an appropriate route of exposure, usually by gavage or by intraperitoneal injection. Then, they are sacrificed at appropriate times after treatment. Each treated and control group must include at least five analysable males. Test substances are preferably administered once or twice but they may also be administered as a split dose to facilitate administering a large volume of material. Prior to sacrifice, animals are treated with a metaphase-arresting agent. Chromosome preparations are then made from germ cells and stained, and metaphase cells are analyzed for chromosome aberrations. A limit test may be performed if no effects would be expected at a dose of 2000 mg/kg bw/d. Positive results from the in vivo spermatogonial chromosome aberration test indicate that a substance induces chromosome aberrations in the germ cells of the species tested.