Latest Documents


  • 8-September-2009

    English

    Test No. 453: Combined Chronic Toxicity/Carcinogenicity Studies

    The objective of a combined chronic toxicity/carcinogenicity study is to identify carcinogenic and the majority of chronic effects, and to determine dose-response relationships following prolonged and repeated exposure.

    The rat is typically used for this study. For rodents, each dose group and concurrent control group intended for the carcinogenicity phase of the study should contain at least 50 animals of each sex, while for the chronic toxicity phase of the study should contain at least 10 animals of each sex.  At least three dose levels should be used, in addition to the concurrent control group for both the chronic toxicity phase and the carcinogenicity phase of the study. The three main routes of administration are oral, dermal, and inhalation. The Test Guideline focuses on the oral route of administration.

    The period of dosing and duration of the study is normally 12 months for the chronic phase, and 24 months for the carcinogenicity phase. The study report should include:  measurements (weighing) and regular detailed observations (haematological examination, urinalysis, clinical chemistry), as well as necropsy procedures and histopathology. All these observations permit the detection of neoplastic effects and a determination of carcinogenic potential as well as the general toxicity.

  • 8-September-2009

    English

    Test No. 231: Amphibian Metamorphosis Assay

    This Test Guideline describes an amphibian metamorphosis assay intended to screen substances which may interfere with the normal functioning of the hypothalamo-pituitary-thyroid axis. The assay was validated with the species Xenopus laevis, which is recommended for use in the Guideline. The assay uses three test chemical concentrations and the necessary controls, including a carrier control if necessary. The assay starts with tadpoles at the development stage 51 on the Nieuwkoop and Faber scale and is extended for a duration of 21 days. Four replicate test vessels are used for each treatment level and control(s). After 7 days of exposure, a sub-set of tadpoles from each treatment level is sampled for the measurement of the length of the hind-limb. At termination of 21-day exposure period, developmental stage, snout-to-vent length and hind limb length are measured on all remaining tadpoles. A sub-set of tadpoles from each treatment level is fixed (whole-body or dissected) for histopathology of the thyroid gland.

  • 8-September-2009

    English

    Test No. 232: Collembolan Reproduction Test in Soil

    This Test Guideline is designed for assessing the effects of chemicals on the reproduction of collembolans in soil. The parthenogenetic Folsomia candida is the recommended species for use, but an alternative species such as sexually reproducing Folsomia fimetaria could also be used if they meet the validity criteria. This Guideline can be used for testing both water soluble and insoluble substances but it is not applicable to volatile ones. The Guideline aims to determine toxic effects of the test substance on adult mortality and reproductive output expressed as LCx and ECx respectively, or NOEC/LOEC value. The number of treatment concentrations varies depending on endpoints to be determined. For a combined approach to examine both the NOEC/LOEC and ECx, eight concentrations in a geometric series with four replicates for each concentration as well as eight control replicates should be used. In each test vessel, 10 juveniles F. candida (or 10 males and 10 females adults F. fimetaria) should be placed on 30 g of modified OECD artificial soil using a 5 % organic matter content. The duration of a definitive reproduction test is 4 weeks for F. candida or 3 weeks for F. fimetaria.

  • 8-September-2009

    English

    Test No. 230: 21-day Fish Assay - A Short-Term Screening for Oestrogenic and Androgenic Activity, and Aromatase Inhibition

    This Test Guideline describes an in vivo screening assay for certain endocrine active substances where sexually mature male and spawning female fish are held together and exposed to a chemical during a limited part of their life-cycle (21 days). This assay covers the screening of oestrogenic and androgenic activity, and aromatase inhibition. The assay was validated on the fathead minnow (Pimephales promelas), the Japanese medaka (Oryzias latipes) and the zebrafish (Danio rerio); however zebrafish does not allow the detection of androgenic activity. At termination of the 21-day exposure period, depending on the species used, one or two biomarker endpoint(s) are measured in males and females as indicators of oestrogenic, aromatase inhibition or androgenic activity of the test chemical; these endpoints are vitellogenin and secondary sexual characteristics. Vitellogenin is measured in fathead minnow, Japanese medaka and zebrafish, whereas secondary sex characteristics are measured in fathead minnow and Japanese medaka only.

  • 8-September-2009

    English

    Test No. 455: The Stably Transfected Human Estrogen Receptor-alpha Transcriptional Activation Assay for Detection of Estrogenic Agonist-Activity of Chemicals

    This Test Guideline describes an in vitro assay, which  provides mechanistical information, and can be used for screening and prioritization purposes. The test system utilises the hERalpha-HeLa-9903 cell line derived from a human cervical tumor and stably transfected. This cell line can measure the ability of a test chemical to induce hERalpha-mediated transactivation of luciferase gene expression. The cells are exposed to 7 non-cytotoxic concentrations of the test chemical for 20-24 hours to induce the reporter gene products. Four reference chemicals should be included in each experiment: a strong estrogen (17beta-estradiol), a weak estrogen (17alpha-estradiol), a very weak estrogen (17alpha-methyltestosterone) and a negative control (corticosterone). The activity of the luciferase enzyme is measured in a luminometer. A test chemical is considered to be positive if the maximum response induced is equal to or exceeds 10% of the response of the positive control (1 nM 17alpha-estradiol) in at least two of two or two of three runs.

    Software to be used with TG 425, 432, 455. Click here. Software not part of the Mutual Acceptance of Data.

  • 8-September-2009

    English

    Test No. 451: Carcinogenicity Studies

    The objective of a long-term carcinogenicity study is to observe test animals for a major portion of their life span for the development of neoplastic lesions during or after exposure to various doses of a test substance by an appropriate route of administration.

    This Test Guideline is intended primarily for use with rats and mice, and for oral administration. Both sexes should be used. Each dose group and concurrent control group should contain at least 50 animals of each sex. At least three dose levels and a concurrent control should be used. Animals are dosed with the test substance daily (oral, dermal or inhalation administration) and the mode of exposure should be adjusted according to the toxicokinetic profile of the test substance. The duration of the study will normally be 24 months for rodents. For specific strains of mice, duration of 18 months may be more appropriate. Termination of the study should be considered when the number of survivors in the lower dose groups or the control group falls below 25 per cent. The results of these studies include: measurements (weighing, food consumption), and, at least, daily and detailed observations, as well as gross necropsy and histopathology.

  • 8-September-2009

    English

    Test No. 412: Subacute Inhalation Toxicity: 28-Day Study

    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.

    Groups of at least 5 male and 5 female rodents are exposed 6 hours per day for 28 days to a) the test article 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), neurologic tests, and additional
    clinical pathology and histopathological evaluations in order to better characterize the toxicity of a test
    article.

  • 8-September-2009

    English

    Test No. 441: Hershberger Bioassay in Rats - A Short-term Screening Assay for (Anti)Androgenic Properties

    The Hershberger Bioassay is an in vivo short–term screening test. It evaluates the ability of a chemical to elicit biological activities consistent with androgen agonists, antagonists or 5 á-reductase inhibitors. The current bioassay is based on the changes in weight of five androgen-dependent tissues in the castrate-peripubertal male rat: the ventral prostate, seminal vesicle (plus fluids and coagulating glands), levator ani-bulbocavernosus muscle, paired Cowper’s glands and the glans penis. In order to establish whether a test substance can have androgenic or antiandrogenic action, two – respectively three - dose groups of the test substance, plus positive and vehicle (negative) controls are normally sufficient. The test substance is administered by gavage or subcutaneous injection daily for 10 consecutive days. To test for antiandrogens, the test substance is administered together with a reference androgen agonist. Each treated and control group should include a minimum of 6 animals.  The animals are necropsied approximately 24 hours after the last administration of the test substance. The tissues are excised and their fresh weights determined.  A statistically significant increase (androgenic) or decrease (antiandrogenic) in the weights of two of the five tissues indicates a positive response in this assay.

  • 8-September-2009

    English

    Test No. 403: Acute Inhalation Toxicity

    This method provides information on health hazard likely to arise from short-term exposure to a test article (gas, vapour or aerosol/particulate test article) by inhalation.

    The revised Test Guideline describes two studies: a traditional LC50 protocol and a Concentration x Time (C x t) protocol. It can be used to estimate a median lethal concentration (LC50), non-lethal threshold concentration (LC01) and slope, and to identify possible sex susceptibility. This Test Guideline enables a test article quantitative risk assessment and classification according to the Globally Harmonized System for the Classification and Labelling of Chemicals. In the traditional LC50 protocol, animals are exposed to one limit concentration or to three concentrations, at least, for a predetermined duration, generally of 4 hours. Usually 10 animals should be used for each concentration. In the C x T protocol, animals are exposed to one limit concentration or a series of concentrations over multiple time durations. Usually 2 animals per C x t interval are used. Animals (the preferred species is the rat) should be observed for at least 14 days. The study includes measurements (including weighing), daily and detailed observations, as well as gross necropsy.

  • 8-September-2009

    English

    Test No. 438: Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants

    The Isolated Chicken Eye  (ICE) test method is an in vitro test method that can be used to classify substances as “ocular corrosives and severe irritants”. The ICE method uses eyes collected from chickens obtained from slaughterhouses where they are killed for human consumption, thus eliminating the need for laboratory animals. The eye is enucleated and mounted in an eye holder with the cornea positioned horizontally. The test substance and negative/positive controls are applied to the cornea. Toxic effects to the cornea are measured by a qualitative assessment of opacity, a qualitative assessment of damage to epithelium based on fluorescein retention, a quantitative measurement of increased thickness (swelling), and a qualitative evaluation of macroscopic morphological damage to the surface. The endpoints are evaluated separately to generate an ICE class for each endpoint, which are then combined to generate an Irritancy Classification for each test substance.

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