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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  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 of by Flow Cytometry (FCM). 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 ≥1.6 for the ELISA method or ≥2.7 for the FCM method for identifying the test material as a potential skin sensitizer.  

This revised Test Guideline 412 (TG 412) has been designed to fully characterize test article toxicity by the inhalation route following repeated exposure for a limited period of time (28 days), and to provide data for quantitative inhalation risk assessments.  It was updated in 2017 to enable the testing and characterisation of effects of nanomaterials tested. Groups of at least 5 male and 5 female rodents are exposed 6 hours per day for 28 days to a) the test chemical at three or more concentration levels, b) filtered air (negative control), and/or c) the vehicle (vehicle control). Animals are generally exposed 5 days per week but exposure for 7 days per week is also allowed. Males and females are always tested, but they may be exposed at different concentration levels if it is known that one sex is more susceptible to a given test article. This guideline allows the study director the flexibility to include satellite (reversibility) groups, bronchoalveolar lavage (BAL), lung burden (LB) for particles, neurologic tests, and additional clinical pathology and histopathological evaluations in order to better characterize the toxicity of a test chemical.

This method provides information on health hazard likely to arise from short-term exposure to a test chemical by inhalation. It is a principle of the method that only moderately toxic concentrations are used so that ‘evident toxicity’, rather than death/moribundity is used as an endpoint, and concentrations that are expected to be lethal are avoided. Groups of animals of a single sex are exposed for a short period of time to the test chemical in a stepwise procedure using the appropriate fixed concentrations for vapours, dusts/mists (aerosols) or gases.  Further groups of animals may be tested at higher concentrations in the absence of signs of evident toxicity or mortality at lower concentrations. This procedure continues until the concentration causing evident toxicity or no more than one death/ moribund animal is identified, or when no effects are seen at the highest concentration or when deaths/ moribundity occur at the lowest concentration.  A total of five animals of one sex will normally be used for each concentration level investigated. The results of this study include: measurements (weighing at least weekly) and daily detailed observations, as well as gross necropsy. The method provides information on the hazardous properties and allows the substance to be classified for acute toxicity according to the Globally Harmonised System of classification and labelling of chemicals.  

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 revised Test Guideline 413 (TG 413) has been designed to fully characterize test article toxicity by the inhalation route following repeated exposure for a period of 90 days, and to provide data for quantitative inhalation risk assessments.  It was updated in 2017 to enable the testing and characterisation of effects of nanomaterials tested. Groups of at least 10 male and 10 female rodents are exposed 6 hours per day for 90 days to a) the test chemical at three or more concentration levels, b) filtered air (negative control), and/or c) the vehicle (vehicle control). Animals are generally exposed 5 days per week but exposure for 7 days per week is also allowed. Males and females are always tested, but they may be exposed at different concentration levels if it is known that one sex is more susceptible to a given test chemical. The results of the study include measurement and daily and detailed observations (haematology and clinical chemistry), as well as ophthalmology, gross pathology, organ weights, and histopathology. This Test Guideline allows the flexibility to include satellite (reversibility) groups, interim sacrifices, bronchoalveolar lavage (BAL), lung burden (LB) for particles, neurologic tests, and additional clinical pathology and histopathological evaluations in order to better characterize the toxicity of a test chemical.

This test guideline describes a test procedure to gain information on dispersion stability of manufactured nanomaterials in simulated environmental media. The main purpose of this guideline is to assess the ability of a nanomaterial to attain a colloidal dispersion and to conserve this dispersion under environmentally relevant conditions. The test procedure involves a dispersion of the nanomaterial with the aid of a calibrated sonication procedure and the determination of the mass concentration of the nanomaterial in a set of test vials while the particles undergo homoagglomeration and settling in environments of different hydrochemistry

This Test Guideline describes a chronic oral toxicity test on adult worker honey bees under laboratory conditions over an exposure period of 10 days. Young bees (max. 2 days old) are exposed to 50 % (w/v) aqueous sucrose solution containing the test chemical by continuous and ad libitum feeding over a period of 10 days. Mortality and behavioural abnormalities are observed and recorded daily during the 10 day test period. The chronic effects of the test chemical are evaluated by comparing the results of the test chemical treated group to those of the respective control group. The test is designed for the determination of the following endpoints  LC50 (median Lethal Concentration) and the LDD50 (median Lethal Dietary Dose) values after 10 days of exposure, and NOEC (No Observed Effect Concentration) and NOEDD (No Observed Effect Dietary Dose).  

This test guideline is a laboratory test method, designed to assess the acute oral toxicity of pesticides and other chemicals to adult worker bumblebees. Adult worker bumblebees are exposed to 50 % (w/v) aqueous sucrose solution containing the test chemical. The test duration is at least 48 h. Mortality is recorded daily and compared with control values. Results are analysed in order to calculate the LD50 and NOED, if possible, at 24 h & 48 h and furthermore at 72 h & 96 h in case the study is prolonged.

This test guideline is a laboratory test method, designed to assess the acute contact toxicity of pesticides and other chemicals to adult worker bumblebees. Adult worker bumblebees are exposed to the test chemical dissolved in an appropriate carrier, by direct application to the dorsal thorax (droplet). The test duration is at least 48 h. Mortality is recorded daily and compared with control values. Results are analysed in order to calculate the LD50 and NOED, if possible, at 24 h & 48 h and furthermore at 72 h & 96 h in case the study is prolonged.