| We are particularly grateful to Krys Bottrill, who is the editor of the reports. |
INVITTOX - LIST OF AVAILABLE PROTOCOLS
This test is designed to detect damage to the eye after application to the conjunctiva. This test is designed to detect damage to the eye after application of test substance to the conjunctiva. The cytotoxic effect of chemicals upon cells in culture is measured by cell viability (neutral red uptake) method. This method enables the in vitro cytotoxicity testing of dental restorative materials which may then be related to dental toxicity likely to occur in vivo. Specific cell types are isolated from the kidney and the cytotoxic effect of chemicals assessed by examining cell glucose and/or fatty acid oxidation and de novo protein synthesis. This method measures the leakage of DNA and lactate dehydrogenase (LDH, EC. 1.1.1 27) from lymphocytes into the surrounding medium as an indicator of cytotoxicity. This method also includes an assay of intracellular (mitochondrial) diaphorase as a measure of cellular activity (MTT assay). This method enables the isolation of different populations of liver cells and describes their subsequent culture. The Allium test provides a rapid screening procedure for chemicals, pollutants contaminants, etc. which may represent environmental hazards. Root growth inhibition and adverse effects upon chromosomes provide an indication of likely toxicity. Membrane permeability of perfused cell cultures, as determined by the efflux of [3H]-2-deoxy-D-glucose-6-phosphate, is used as an indicator of the cytotoxic effect of chemicals. This test is designed to detect irreversible toxic effects on both cell growth and survival, by the evaluation of colony-forming efficiency, in rat hepatoma cell lines MH1C1 and HTC. The cytotoxic effect of chemicals upon cells in culture is measured by the change in total cell protein arising from the inhibition of cell proliferation (Kenacid Blue R dye binding method). Collagenase perfusion of rat liver yields a hepatocyte suspension which may be exposed to test compounds in order to assess their effects on cell viability and enzyme leakage. The cytotoxic effect of test compounds on bovine spermatozoa is determined by the measurement of spermatozoa motility and velocity using videomicrography and automatic computer analysis, and ATP contents. The cytotoxic effect of chemicals upon the protozoan, Tetrahymena thermophila, in culture is assessed by studying their effect upon the normal motility of the organism. It is suggested that the Tetrahymena thermophila test may be useful as part of a small battery of in vitro tests in the evaluation of the ocular irritancy potential of chemicals. Flow cytometry is used to monitor drug-induced changes in DNA and protein contents of hepatocytes cultured at physiological oxygen concentrations. Changes in the balance of cytoskeletal proteins after exposure to test compounds can be detected by indirect immunofluorescence microscopy and quantitative biochemical methods. Tumour cell lines cultured as aggregates can be utilised for in vitro radiosensitivity and/or chemosensitivity tests. Chemical effects are monitored by studying the changes in spheroid diameter measured by laser diffraction. Skin fibroblasts are incorporated into 3-D collagen lattices containing the test compounds. An inhibition of lattice contraction indicates a possible toxic effect which is verified by trypan blue exclusion for cell viability. This procedure describes a method for establishing short-term explant cultures of oesophageal mucosa. Adverse effects produced by exposure to radiation or test compounds can be detected as an inhibition of cell outgrowth. This method enables the culturing of thyroid cells without loss of differentiation and medium change. It is potentially useful for the long-term study of drug effects on the thyroid gland. Reverse Mutation in Histidine-requiring Strains of Salmonella typhimurium (complies with OECD Guideline 471) This procedure evaluates the mutagenic potential of test chemicals by their effect on five histidine requiring strains of the bacterium, Salmonella typhimurium in the absence and presence of a rat liver metabolising system. This procedure describes the use of L929 mouse fibroblast cells cultured in vitro in an agarose overlay assay to assess the toxicity of test substances. The assay may be useful in assessing the irritation potential of test substances (e.g. surfactant-based products) as an alternative to the Draize rabbit eye test. Macrophage cells in culture may be exposed to particulate matter, and resultant effects upon cell viability determined by vital dye exclusion and enzyme leakage assays. The cytotoxic effect of chemicals upon yeast (Saccharomyces cerevisiae) cells in culture is determined by inhibition of cell proliferation, as measured by cell density. The effect of chemicals on the activity of the plasma membrane-bound H+-ATPase, isolated from yeast (Saccharomyces cerevisiae) cells, is used as a measure of their toxicity. The effect of chemicals on the activity of the plasma membrane-bound Na+/K+ -ATPase isolated from Chinese Hamster Ovary (CHO) cells is used as a measure of their toxicity. The inhibition of CHO cell proliferation provides an overall assessment of the toxicity of the test substance. An in vitro red blood cell assay is presented which allows the estimation of the irritation potential of tensides and tenside containing materials such as shampoos, shower gels, cleaning products, etc. The estimation is based on the fact that surfactants interact strongly with cellular membranes and proteins. Both effects are measured photometrically by use of the inherent native dye, oxyhemoglobin. Unlike other cell-based systems, the RBC assay is able to differentiate between membrane damage (haemolysis) and protein damage (denaturation). This simple cell culture-based cytotoxicity test (in which cell viability is determined by uptake of the dyes ethidium bromide and fluorescein acetate) has been developed as a general test for acute toxicity. The cytotoxic effect of test chemicals in V79 cell culture can be determined by assessing damage to the plasma membrane as determined by a nucleic acid leakage assay. In this test, rabbit-derived corneal cells are cultured in the presence of test compounds, the toxicity of which are determined by their effect upon cell viability. A decrease in cell number, as measured by uptake of the dye Neutral Red, serves as an indicator of potential cytotoxicity. This test has been proposed as a potential replacement alternative for the Draize Eye Irritation test. In this test rabbit articular chondrocytes are cultured in the presence of test compound, the toxicity of which is then determined by its effect on the production of proteoglycan by the cells, as detected by the dye Alcian Blue. Leakage of lactate dehydrogenase and alanine aminotransferase from rat and mouse liver slices exposed to the test compound is used as a measure of hepatotoxicity. The potential cytotoxicity of compounds under hypoxic conditions is determined by exposing cell cultures to test compounds in a low oxygen atmosphere. Subsequent cell survival is determined by the MTT and methylene blue colorimetric assays. Human A431 cells and mouse 3T3 cells are exposed in culture to UV light both in the presence and absence of test compound. Phototoxicity is expressed as a decrease in cell viability as determined by the MTT assay. This test examines the effect of contaminated sea-water on the growth of the marine alga, Dunaliella tertiolecta. Inhibition of growth provides an indication of likely toxicity. The cytotoxic effect of chemicals upon Balb/c 3T3 cells in culture is measured by cell viability (Neutral Red Uptake) and total cell protein (Kenacid Blue R dye binding method). The potential irritancy of compounds may be detected by observing adverse changes which occur in the chorionallantoic membrane of the egg after exposure to test chemicals. Potential embryotoxicity is assessed by monitoring the effect of the test compound on total protein synthesis, and DNA synthesis in cultured human foetal lung fibroblasts. Rat lung epithelial cells can be used to determine cytotoxicity of select compounds because of their ability to metabolise xenobiotics. This bioassay utilises cultured H-4-II-E rat hepatoma cells to assess the aryl hydrocarbon hydroxylase (AHH) inducing potencies of planar aromatic hydrocarbons and/or contaminated environmental samples. The response of the cells to pure test chemicals or extracts of mixtures is compared with their response to the standard 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Two cytotoxicity tests are used in parallel to investigate the toxicity of implant materials used in medicine and dentistry. In this test kidney-derived cells are cultured in the presence of test compounds whose cytotoxicity is then determined by the Neutral Red method, and serves as an indicator of potential nephrotoxicity. AVEC-DIC microscopy in combination with mitochondria-specific fluorescence allows a quantitative analysis of cell organelle dynamics and fine structure in cell cultures exposed to test compounds. The AIDA system can be used, under precisely controlled environmental conditions, to predict the dermal absorption of the test substance in vivo. This procedure describes a method for routinely establishing finite but abundant primary explant cultures of human (and bovine) lens epithelial cells. Long-term cultures of bovine whole lenses are used to assess the effect of drugs and chemicals on the refractive index (focal length) and transparency of the lens tissue. These endpoints are measured simultaneously by a computer-driven scanning laser system. This protocol describes the use of SEMV cells (a cell line derived from ram seminal vesicles) in studies into prostaglandin H synthase-mediated metabolism of xenobiotics in intact cells. Tumour promoters can be detected by their ability to cause the initiated Bhas 42 cells to lose contact inhibition. The inhibition by a test compound of the uptake of the glucose analogue, a-methyl glucose by freshly isolated proximal tubular cells from rat kidney is used as a measure of acute early-stage nephrotoxicity. The cytotoxic effect of chemicals upon mammalian cells, such as BALB/c 3T3 and HepG2, in culture is measured by highest tolerated dose (HTD), cell viability (Neutral Red) and total cell protein (coomassie blue). The effect of the test substance on the transfer of the dye lucifer yellow between SV-40-transformed hamster fibroblasts is an indication of potential tumour-promoting activity. The results of cytotoxicity tests in primary cultures of rat hepatocytes and in MDBK and McCoy cells can be used to predict the in vivo 4-wk maximum tolerated dose in rats and dogs. A correlation between in vitro cytotoxicity, as measured in this system, and LD50 values in rats and mice has also been established. Complement activation that takes place when human plasma and the test compound are incubated in the presence of light is taken as an indication of potential phototoxicity of the test compound. Primary cultures of newborn rat skeletal muscle cells can be used to evaluate the myotoxicity of various drugs. Reaggregates of isolated chick embryo myocardial myocytes are used in a simple tissue culture based test as a model for the cardiotoxicity of compounds. The cDNA of different members of the cytochromes CYP family can be inserted into cell lines such as V79 Chinese Hamster cells, which are used for in vitro toxicity testing. This means that the metabolites of xenobiotics are produced in the same cells in which any toxic effect will be observed, thus negating the problems associated with co-cultures and the use of subcellular fractions. In this modification of the HET-CAM test, trypan blue staining provides an objective and quantitative assessment of the degree of damage to the chicken chorioallantoic membrane (CAM). The degree to which a surfactant causes denaturation of haemoglobin can be used to predict its potential to cause ocular irritation. This bioassay utilizes cultured Hepa-lclc7 (Hepa-1) mouse hepatoma cells to assess the CYPlA1-inducing potency or cytotoxicity of pure test chemicals or environmental samples. In the Hepa-l induction test , the CYPlA1-inducing potency of the test sample is detected as increased aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin O-deethylase (EROD) activities. In the Hepa-l cytotoxicity test , the effect of the sample on cell viability is measured. The effect of chemicals on 3T3 cells and on ES cells, a permanent cell line derived from mouse embryonic stem cells, can be used to predict teratogenic potential. The effect of a test compound, in the presence and absence of S-9 mix, on the differentiation and growth of rat limb bud and CNS cells in vitro indicates whether it is potentially a teratogen in vivo. |
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