Reports

The objective of these chronic toxicity studies is to characterize the profile of a substance in a mammalian species (primarily rodents) following prolonged and repeated exposure.The Test Guideline focuses on rodents and oral administration. Both sexes should be used. For rodents, at least 20 animals per sex per group should normally be used at each dose level, while for non-rodents a minimum of 4 per sex per group is recommended. At least three dose levels should be used in addition to the concurrent control group. Frequency of exposure normally is daily, but may vary according to the route chosen (oral, dermal or inhalation) and should be adjusted according to the toxicokinetic profile of the test substance. The duration of the exposure period should be 12 months. The study report should include: measurements (weighing) and regular detailed observations (haematological examination, urinalysis, clinical chemistry), as well as necropsy procedures and histopathology.

The Test Guideline (TG) describes the use of liver S9 sub-cellular fraction (RT-S9) of rainbow trout (Oncorhynchus mykiss) as a metabolising system to determine the clearance (CL, IN VITRO, INT ) of a test chemical using a substrate depletion approach. Introduction of the test chemical to the RT-S9 incubation medium initiates the reaction. In order to collect samples at various time points, the reaction is terminated by transferring an aliquot of the medium to a stopping solution. The decrease of the test chemical concentration from the incubation vial is measured with a validated analytical method and used to determine the CL, IN VITRO, INT.  The value obtained can then be used to improve in silico predictions of the test chemical bioaccumulation in fish.

This Test Guideline is designed to provide an evaluation of reproductive and developmental effects that may occur as a result of pre- and postnatal chemical exposure as well as an evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring. In the assay, sexually-mature males and females rodents (parental (P) generation) are exposed to graduated doses of the test substance starting 2 weeks before mating and continuously through mating, gestation and weaning of their pups (F1 generation). At weaning, pups are selected and assigned to cohorts of animals for reproductive/developmental toxicity testing (cohort 1), developmental neurotoxicity testing (cohort 2) and developmental immunotoxicity testing (cohort 3). The F1 offspring receive further treatment with the test substance from weaning to adulthood. Clinical observations and pathology examinations are performed on all animals for signs of toxicity, with special emphasis on the integrity and performance of the male and female reproductive systems and the health, growth, development and function of the offspring. Part of cohort 1 (cohort 1B) may be extended to include an F2 generation; in this case, procedures for F1 animals will be similar to those for the P animals.

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).

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.

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.

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.

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 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.