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The purpose of this guidance is to harmonise the way non-guideline in vitro methods are described and thereby facilitate an assessment of the relevance of test methods for biological activities and responses of interest, and an assessment of the quality of data produced, irrespective of whether these tests are based on manual protocols or assay protocols adapted for use on automated platforms or high-throughput screening systems (HTS). This guidance outlines the elements considered relevant for providing a comprehensive description of an in vitro method to facilitate the interpretation of results and support scientifically defensible fit-for-purpose applications.    

This document presents a harmonized method for characterising, for assessment purposes, a specific subcategory of UVCBs (Substances of Unknown or Variable composition, Complex reaction products or Biological materials): oleochemical substances. Many oleochemicals are UVCBs, due to the variability in the composition of the starting materials. The method presented in this document gives guidance on how oleochemical substances can be characterised in a way that their composition is accurately and consistently reflected to ensure that substances with the same chemical composition, manufactured in different countries, can be characterised with the same description for hazard assessment purposes. A common understanding and approach to characterising UVCBs would enable regulatory authorities to increase cooperation in the field of hazard assessment and help industry deal with regulatory requirements from multiple jurisdictions.  

This guidance document is part of the OECD effort to provide guidance for assessing the hazards of chemical substances while gaining efficiencies and improving animal welfare. The approach described in this guidance document is to consider closely related chemicals as a group, or category, rather than as individual chemicals. While the first edition was published in 2007, This edition has been augmented with experience and examples encountered in the OECD Cooperative Chemicals Assessment Programme, formerly the HPV Chemicals Programme since 2007, the second edition also intends to introduce new or revised guidance on: elaborating the analogue and category approach, quantitative and qualitative read-across, justifying read-across, using bioprofiling results for grouping chemicals, and specific types of category approaches (e.g. chemicals of variable composition, and metals).

Several models, tools and methods have been published in the past 20 years to include bioavailability in risk assessment and several OECD member countries already have developed frameworks and published guidance documents for taking metal specificities into account in environmental risk assessment. The aim of the current guidance is not to replace the aforementioned frameworks or guidance documents, but rather, to provide an overarching framework on how to apply these tools depending on which data are actually available/needed to assess the bioavailability of the metal under scrutiny. Further harmonisation of these approaches and methodology, where appropriate, over the different OECD countries is recommended and could facilitate a more worldwide application and the Mutual Acceptance of Data since using common assessment approaches may help comparing and exchanging data sets, which could result in significant resource savings.  

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.

The mammalian in vivo chromosome aberration test is used for the detection of structural chromosome aberrations induced by test compounds in bone marrow cells of animals, usually rodents (rats, mice and Chinese hamsters). Structural chromosome aberrations may be of two types: chromosome or chromatid. Animals are exposed to the test substance (liquid or solid) by an appropriate route of exposure (usually by gavage using a stomach tube or a suitable intubation cannula, or by intraperitoneal injection) and are sacrificed at appropriate times after treatment. Prior to sacrifice, animals are treated with a metaphase-arresting agent. Chromosome preparations are then made from the bone marrow cells and stained, and metaphase cells are analysed for chromosome aberrations. Each treated and control group must include at least 5 analysable animals per sex. The limit dose is 2000 mg/kg/body weight/day for treatment up to 14 days, and 1000 mg/kg/body weight/day for treatment longer than 14 days.

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.

The mammalian in vivo micronucleus test is used for the detection of damage induced by the test substance to the chromosomes or the mitotic apparatus of erythroblasts, by analysis of erythrocytes as sampled in bone marrow and/or peripheral blood cells of animals, usually rodents (mice or rats). The purpose of the micronucleus test is to identify substances (liquid or solid) that cause cytogenetic damage which results in the formation of micronuclei containing lagging chromosome fragments or whole chromosomes. An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals is an indication of induced chromosome damage. Animals are exposed to the test substance by an appropriate route (usually by gavage using a stomach tube or a suitable intubation cannula, or by intraperitoneal injection). Bone marrow and/or blood cells are collected, prepared and stained. Preparations are analyzed for the presence of micronuclei. Each treated and control group must include at least 5 analysable animals per sex. Administration of the treatments consists of a single dose of test substance or two daily doses (or more). The limit dose is 2000 mg/kg/body weight/day for treatment up to 14 days, and 1000 mg/kg/body weight/day for treatment longer than 14 days.