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This Test Guideline describes a method to assess effects of a test chemical on the phagocytotic activity of activated sludge containing protozoan organisms under defined conditions in the presence of different concentrations of the test chemical. The principle of biological sewage-treatment plants (STP) is to transform the organic matter of incoming waste-water in microbial biomass, which in turn is separated from the liquid yielding a purified effluent. The purpose of the test is to provide a means to record effects of test chemicals on ciliated protozoa in sewage treatment plants, which due to their grazing on bacteria considerably contribute to the functioning of STPs.

The purpose of the in vitro chromosome aberration test is to identify agents that cause structural chromosome aberrations in cultured mammalian somatic cells. Structural aberrations may be of two types: chromosome or chromatid. The in vitro chromosome aberration test may employ cultures of established cell lines, cell strains or primary cell cultures. Cell cultures are exposed to the test substance (liquid or solid) both with and without metabolic activation during about 1.5 normal cell cycle lengths. At least three analysable concentrations of the test substance should be used. At each concentration duplicate cultures should normally be used. At predetermined intervals after exposure of cell cultures to the test substance, the cells are treated with a metaphase-arresting substance, harvested, stained. Metaphase cells are analysed microscopically for the presence of chromosome aberrations.

The in vitro mammalian cell gene mutation test can be used to detect gene mutations induced by chemical substances. In this test, the used genetic endpoints measure mutation at hypoxanthine-guanine phosphoribosyl transferase (HPRT), and at a transgene of xanthineguanine phosphoribosyl transferase (XPRT). The HPRT and XPRT mutation tests detect different spectra of genetic 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 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 in vivo alkaline single cell gel electrophoresis assay, also called alkaline Comet Assay is a method measuring DNA strand breaks in eukaryotic cells. Each treated group is composed of a minimum of 5 animals of one sex (or of each sex as appropriate). A positive and a vehicle control group are also used. Administration of the treatment consists of daily doses over duration of 2 days or more, ensuring the test chemical reaches the target tissue which can be the liver, the kidney or other tissues if justified. Tissues of interest are dissected and single cells/nuclei suspensions are prepared and embedded in agarose on slides. Cells/nuclei are treated with lysis buffer to remove cellular and/or nuclear membranes. The nuclear DNA in the agar is then subjected to electrophoresis at high pH. This results in structures resembling comets which by using suitable fluorescent stain, can be observed by fluorescent microscopy. Based on their size DNA fragments migrate away from the head to the tail, and the intensity of the comet tail relative to the total intensity (head plus tail) reflects the amount of DNA breakage.

The Potamopyrgus antopodarumon reproduction test is designed to assess potential effects of prolonged exposure to chemicals on reproduction and survival of parthenogenetic lineages of the freshwater mudsnail Potamopyrgus antipodarum. Adult female P. antipodarum are exposed to a concentration range of the test chemical. The test chemical is dispersed into the reconstituted dilution water, added to test beakers, and adult snails are subsequently introduced into the test beakers. When testing 'difficult chemicals' (i.e. volatile, unstable, readily biodegradable and adsorbing chemicals) the test can be conducted under flow-through conditions as an alternative to the semi-static design with fixed renewal periods of the medium (see paragraph 29). P. antipodarum survival over the 28 days exposure period and reproduction at the end of the test after 28 days are examined. Reproduction is evaluated by counting the number of embryo in the brood pouch (without distinction of developmental stages) at the end of 28 days exposure. The toxic effect of the test chemical on embryo numbers is expressed as ECX by fitting an appropriate regression model in order to estimate the concentration that would cause x % reduction in embryo numbers or alternatively as the No Observed Effect Concentration and Lowest Observed Effect Concentration (NOEC/LOEC) value (2).

This screening Test Guideline describes the effects of a test chemical on male and female reproductive performance. It has been updated with endocrine disruptor endpoints, in particular measure of anogenital distance and male nipple retention in pups and thyroid examination. The test substance is administered in graduated doses to several groups of males and females. Males should be dosed for a minimum of four weeks. Females should be dosed throughout the study, so approximately 63 days. Matings 'one male to one female' should normally be used in this study. This Test Guideline is designed for use with the rat. It is recommended that each group be started with at least 10 animals of each sex. Generally, at least three test groups and a control group should be used. Dose levels may be based on information from acute toxicity tests or on results from repeated dose studies. The test substance is administered orally and daily. The results of this study include clinical observations, body weight and food/water consumption, oestrous cycle monitoring, offspring parameters observation/measurement, thyroid hormone measurement, as well as gross necropsy and histopathology. The findings of this toxicity study should be evaluated in terms of the observed effects, necropsy and microscopic findings. Because of the short period of treatment of the male, the histopathology of the testis and epididymus should be considered along with the fertility data, when assessing male reproductive effects.