Latest Documents


  • 23-July-2010

    English

    Test No. 442B: Skin Sensitization - Local Lymph Node Assay: BrdU-ELISA

    The Local Lymph Node Assay: BrdU-ELISA (LLNA:BrdU-ELISA) is a non-radioactive modification to the LLNA method for identifying potential skin sensitizing test substances and measuring the proliferation of lymphocytes they induce in the auricular lymph nodes. The method described in mouse (CBA/JN strain) is based on the use of measuring 5-bromo-2-deoxyuridine (BrdU) content, an analogue of thymidine, as an indicator of this proliferation. A minimum of four animals is used per dose group, with a minimum of three concentrations of the test substance, plus a concurrent negative control group and a positive control group. The experimental schedule is during 6 days. Thereafter, the animals are killed and a single cell suspension of lymph node cells (LNC) is prepared. The procedure for preparing the LNC is crucial, in particular for the small lymph nodes in NC animals. Then the BrdU content in DNA of lymphocytes is measured by ELISA using a commercial kit. This study includes: measurements (weighing, BrdU) and clinical daily observations. The results are expressed as the Stimulation Index (SI) obtained by calculation from the mean BrdU labelling index. The SI should be ¡Ý1.6 before further evaluation of the test material as a potential skin sensitizer is warranted.

  • 23-July-2010

    English

    Test No. 233: Sediment-Water Chironomid Life-Cycle Toxicity Test Using Spiked Water or Spiked Sediment

    This Guideline is designed to assess the effects of prolonged exposure of chemicals to the life-cycle of the sediment-dwelling freshwater dipteran Chironomus sp. First instar chironomid larvae are exposed to five concentrations of the test chemical in sediment-water systems. The test substance is spiked into the water or alternatively the sediment, and first instar larvae are subsequently introduced into test beakers in which the sediment and water concentrations have been stabilised. Chironomid emergence, time to emergence, and sex ratio of the fully emerged and alive midges are assessed. Emerged adults are transferred to breeding cages, to facilitate swarming, mating and oviposition. The number of egg ropes produced and their fertility are assessed. From these egg ropes, first instar larvae of the 2nd generation are obtained. These larvae are placed into freshly prepared test beakers (spiking procedure as for the 1st generation) to determine the viability of the 2nd generation through an assessment of their emergence, time to emergence and the sex ratio of the fully emerged and alive midges. All data are analysed either by a regression model to estimate the concentration that would cause X% reduction in the relevant endpoint, or by using hypothesis testing to determine a No Observed Effect Concentration (NOEC).

  • 23-July-2010

    English

    Test No. 487: In Vitro Mammalian Cell Micronucleus Test

    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.

  • 23-July-2010

    English

    Test No. 209: Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)

    This Test Guideline is designed to assess the effects of a substance on micro-organisms from activated sludge of waste-water treatment plants by measuring their respiration rate (carbon and/or ammonium oxidation) as oxygen consumption. The test results may also serve as an indicator of suitable non-inhibitory concentrations of test substances to be used in biodegradability tests. The test allows the determination of ECx and/or NOEC values of the test substance. The inhibition of three different oxygen uptakes may be determined, i.e. total, heterotrophic only, and that due to nitrification in the absence and presence of N-allylthiourea, a specific nitrification inhibitor. For obtaining both NOEC and ECx, six controls and five treatment concentrations in a geometric series with five replicates are recommended. In each test vessel, test mixtures containing water, synthetic sewage feed and the test substance are incubated at the pH of 7.5 ±0.5 and the temperature within 20±2°C under forced aeration to keep the dissolved oxygen concentration above 60-70% saturation. The oxygen consumption is measured after 3 hours of exposure and additional measurements at 30 minutes of exposure can be performed in the case that the test substance is rapidly degraded.

  • 23-July-2010

    English

    Test No. 439: In Vitro Skin Irritation - Reconstructed Human Epidermis Test Method

    This Test Guideline describes an in vitro procedure that may be used for the hazard identification of irritant chemicals (substances and mixtures) in accordance with the UN Globally Harmonized System of Classification and Labelling (GHS) Category 2.  It is based on reconstructed human epidermis (RhE), which in its overall design closely mimics the biochemical and physiological properties of the upper parts of the human skin. Cell viability is measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that is quantitatively measured after extraction from tissues. Irritant test substances are identified by their ability to decrease cell viability below defined threshold levels (below or equal to 50% for UN GHS Category 2). This Test Guideline also includes a set of Performance Standards for the assessment of similar and modified RhE-based test methods. There are three validated test methods that adhere to this Test Guideline. Depending on the regulatory framework and the classification system in use, this procedure may be used to determine the skin irritancy of test substances as a stand-alone replacement test for in vivo skin irritation testing, or as a partial replacement test, within a tiered testing strategy.

  • 23-July-2010

    English

    Test No. 417: Toxicokinetics

    This Test Guideline describes in vivo studies that provide information on mass balance, absorption, bioavailability, tissue distribution, metabolism, excretion, and basic toxicokinetic parameters [e.g. AUC], as well as supplemental approaches that may provide useful information on toxicokinetics. Information from toxicokinetic studies helps to relate concentration or dose to the observed toxicity and to understand its mechanism of toxicity. The test substance ("unlabelled" or "radiolabelled" forms) is normally administered by an oral route, but other routes of administration may be applicable. Single dose administration of the substance (preferably a minimum of two dose levels) may be adequate, but repeated dose may be needed in some circumstances. Toxicokinetic studies should preferably be carried out in the same species as that used in other toxicological studies performed with the substance (normally the rat, a minimum of 4 animals of one sex for each dose). Initial estimation of absorption can be achieved by mass balance determination, but further investigations such as intravenous (IV) administration and biliary excretion studies might be necessary. Bioavailability can be determined from plasma/blood kinetics of oral and IV groups. The percent of the total dose in tissues should at a minimum be measured at the termination of experiment,but additional time points may also be needed. Metabolites present at 5 % or greater of the administered dose should be identified. The rate and extent of excretion of the administered dose should be determined by measuring the percent recovered dose from urine, faeces and expired air.

  • 23-July-2010

    English

    Test No. 317: Bioaccumulation in Terrestrial Oligochaetes

    This Test Guideline describes procedures designed to assess bioaccumulation of chemicals in soil oligochaetes. The parameters which characterise the bioaccumulation of a substance include the bioaccumulation factor (BAF), the uptake rate constant (ks) and the elimination rate constant (ke). The test consists of two phases: the uptake (exposure) phase and the elimination (post-exposure) phase. An elimination phase is always required unless uptake of the test substance during the exposure phase has been insignificant. The test organisms are exposed to the test substance during the uptake phase. The test substance is incorporated into the soil; it is recommended to use the artificial soil described in the OECD Test Guideline 207 (Earthworm, acute toxicity test). The uptake phase should be of 14 days (enchytraeids) or 21 days (earthworms) unless it is demonstrated that steady state has been reached. For the elimination phase, the worms are transferred to a soil free of test substance. The elimination phase is generally of 21 days.

  • 23-July-2010

    English

    Test No. 429: Skin Sensitisation - Local Lymph Node Assay

    The basic principle underlying the Local Lymph Node Assay (LLNA) in mouse is that sensitizers induce a primary proliferation of lymphocytes in the auricular lymph nodes draining the site of chemical application. This proliferation is proportional to the dose applied and provides a measurement of sensitisation. The method described is based on the use of radioactive labelling to measure cell proliferation. A minimum of four animals is used per dose group, with a minimum of three concentrations of the test substance, plus a negative control group treated with the vehicle only, and a positive control, as appropriate. The experimental schedule of the assay is during 6 days. Thereafter, the animals are killed and a cell suspension of lymph node cells is prepared. The incorporation of 3H-methyl thymidine is measured by ¦Â-scintillation counting as disintegrations per minute (DPM). The Test Guideline includes performance standards that can be used to evaluate the validation status of new and/or modified test methods that are functionally and mechanistically similar to the LLNA. A reduced LLNA approach which could use up to 40% fewer animals is also described as an option. This study includes: measurements (weighing, DPM), and clinical daily observations. Results are expressed as the Stimulation Index (SI).The SI is obtained by calculation and should be ¡Ý3 before classification of the test material as a skin sensitizer is warranted.

  • 23-July-2010

    English

    Test No. 442A: Skin Sensitization - Local Lymph Node Assay: DA

    The Local Lymph Node Assay: DA (LLNA: DA) is a non-radioactive modification to the LLNA method for identifying potential skin sensitizing test substances and measuring the proliferation of lymphocytes they induce in the auricular lymph nodes. The method, described in mouse (CBA/J strain), is based on measurement of the adenosine triphosphate (ATP) content by bioluminescence as an indicator of this proliferation. A minimum of four animals is used per dose group, with a minimum of three concentrations of the test substance, plus a concurrent negative control group and, if appropriate, a positive control group. The experimental schedule is during 8 days. The time from animal sacrifice to measurement of ATP should not exceed 30 min. The procedure from excision of lymph nodes to ATP measurement should be kept uniform for each animal and completed within 20 minutes. The luciferin/luciferase method is applied to measure the bioluminescence in Relative Luminescence Units (RLU). This study includes: measurements (weighing, RLU), and clinical daily observations. The results are expressed as the Stimulation Index (SI) obtained by calculation. The SI should be ¡Ý1.8 before further evaluation of the test material as a potential skin sensitizer is warranted.

  • 23-July-2010

    English

    Test No. 223: Avian Acute Oral Toxicity Test

    This Test Guideline describes procedures designed to estimate the acute oral toxicity of substances to birds, and it provides three testing options: (1) limit dose test, (2) LD50-slope test, and (3) LD50-only test. The LD50-slope and LD50-only options are sequential testing procedures. The test method selected will depend on whether or not a definitive median dose (LD50) and slope of the dose-response curve are both needed. The limit dose test is the preferred test when toxicity is expected to be low and lethality is unlikely at the limit dose. The limit dose should be adequate for assessment purposes, and it is usually 2000 mg/kg-bwt. Five or ten birds are tested at the limit dose in addition to a control group. The LD50-slope test is the preferred test when regulatory or other requirements determine that the slope of the dose-response curve and/or the confidence interval is required in addition to an estimate of the LD50. This is a 3- or 4-stage test with 24 or 34 birds in addition to a control group. The LD50-only test is the preferred test when regulatory or other requirements determine that only the median lethal dose is required but neither the slope of the dose response curve or the confidence interval for the LD50 is required. This may be the appropriate test to estimate a percentile of a species sensitivity distribution of LD50s and to provide information for product labelling purposes. This test has two stages, with 14 birds in addition to a control group.

    Software to be used with TG 223. Click here. Software not part of the Mutual Acceptance of Data.

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