By Date


  • 28-July-2015

    English

    Test No. 404: Acute Dermal Irritation/Corrosion

    This method provides information on health hazard likely to arise from exposure to liquid or solid test substance by dermal application. This Test Guideline recommends sequential testing strategies, which include the performance of validated and accepted in vitro or ex vivo tests for corrosion/irritation.

    The albino rabbit is the preferable laboratory animal. The substance to be tested is applied in a single dose to a small area of skin (approximately 6cm²) of an experimental animal; untreated skin areas of the test animal serve as the control. The exposure period is 4 hours. Residual test substance should then be removed. The dose is 0.5ml (liquid) or 0.5g (solid) applied to the test site. The method consists of two tests: the initial test and the confirmatory test (used only if a corrosive effect is not observed in the initial test). All animals should be examined for signs of erythema and oedema during 14 days. The dermal irritation scores should be evaluated in conjunction with the nature and severity of lesions, and their reversibility or lack of reversibility. When responses persist to the end of the 14-day observation period, the test substance should be considered an irritant.

  • 28-July-2015

    English

    Test No. 493: Performance-Based Test Guideline for Human Recombinant Estrogen Receptor (hrER) In Vitro Assays to Detect Chemicals with ER Binding Affinity

    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.

  • 28-July-2015

    English

    Test No. 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage

    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).

  • 28-July-2015

    English

    Test No. 435: In Vitro Membrane Barrier Test Method for Skin Corrosion

    This updated Test Guideline 435 provides an in vitro membrane barrier test method that can be used to identify corrosive chemicals. The test method utilizes an artificial membrane designed to respond to corrosive chemicals in a manner similar to animal skin in situ.

  • 28-July-2015

    English

    Test No. 491: Short Time Exposure In Vitro Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage

    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%.

  • 28-July-2015

    English

    Test No. 240: Medaka Extended One Generation Reproduction Test (MEOGRT)

    This Test Guideline describes the Medaka Extended One Generation Test (MEOGRT), which exposes fish over multiple generations to give data relevant to ecological hazard and risk assessment of chemicals, including suspected endocrine disrupting chemicals (EDCs).  Exposure in the MEOGRT starts with spawning fish (P or F0 generation) and continues until hatching (until two weeks post fertilization, wpf) in the second (F2) generation. This Test Guideline measures several biological endpoints.  Primary emphasis is given to potential adverse effects on population relevant parameters including survival, gross development, growth and reproduction (fecundity).  Secondarily, in order to provide mechanistic information and provide linkage between results from other kinds of field and laboratory studies, where there is a posteriori evidence for a chemical having potential endocrine disrupter activity (e.g. androgenic or oestrogenic activity in other tests and assays) then other useful information is obtained by measuring vitellogenin (vtg) mRNA (or vitellogenin protein, VTG), phenotypic secondary sex characteristics (SSC) as related to genetic sex, and evaluating histopathology.

  • 28-July-2015

    English

    Test No. 476: In Vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes

    The in vitro mammalian cell gene mutation test can be used to detect gene mutations induced by chemical substances. In this test, the used genetic endpoints measure mutation at hypoxanthine-guanine phosphoribosyl transferase (HPRT), and at a transgene of xanthineguanine phosphoribosyl transferase (XPRT). The HPRT and XPRT mutation tests detect different spectra of genetic 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.

  • 28-July-2015

    English

    Test No. 478: Rodent Dominant Lethal Test

    The purpose of the Dominant lethal (DL) test is to investigate whether chemical agents produce mutations resulting from chromosomal aberrations in germ cells. In addition, the dominant lethal test is relevant to assessing genotoxicity because, although they may vary among species, factors of in vivo metabolism, pharmacokinetics and DNA-repair processes are active and contribute to the response. Induction of a DL mutation after exposure to a test chemical indicates that the chemical has affected germinal tissue of the test animal.

    This modified version of the Test Guideline reflects more than thirty years of experience with this test and the potential for integrating or combining this test with other toxicity tests such as developmental, reproductive toxicity, or genotoxicity studies; however due to its limitations and the use of a large number of animals this assay is not intended for use as a primary method, but rather as a supplemental test method which can only be used when there is no alternative for regulatory requirements.

  • 28-July-2015

    English

    Test No. 490: In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene

    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.

  • 28-July-2015

    English

    Test No. 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

    This screening Test Guideline describes the effects of a test chemical on male and female reproductive performance. It has been updated with endocrine disruptor endpoints, in particular measure of anogenital distance and male nipple retention in pups and thyroid examination.

    The test substance is administered in graduated doses to several groups of males and females. Males should be dosed for a minimum of four weeks. Females should be dosed throughout the study, so approximately 63 days. Matings "one male to one female" should normally be used in this study. This Test Guideline is designed for use with the rat. It is recommended that each group be started with at least 10 animals of each sex. Generally, at least three test groups and a control group should be used. Dose levels may be based on information from acute toxicity tests or on results from repeated dose studies. The test substance is administered orally and daily. The results of this study include clinical observations, body weight and food/water consumption, oestrous cycle monitoring, offspring parameters observation/measurement, thyroid hormone measurement, as well as gross necropsy and histopathology. The findings of this toxicity study should be evaluated in terms of the observed effects, necropsy and microscopic findings. Because of the short period of treatment of the male, the histopathology of the testis and epididymus should be considered along with the fertility data, when assessing male reproductive effects.

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