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This Test Guideline describes an in vitro procedure that may be used for the hazard identification of irritant chemicals (substances and mixtures) in accordance with the UN Globally Harmonized System of Classification and Labelling (GHS) Category 2.  It is based on reconstructed human epidermis (RhE), which in its overall design closely mimics the biochemical and physiological properties of the upper parts of the human skin. Cell viability is measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that is quantitatively measured after extraction from tissues. Irritant test substances are identified by their ability to decrease cell viability below defined threshold levels (below or equal to 50% for UN GHS Category 2). This Test Guideline also includes a set of Performance Standards for the assessment of similar and modified RhE-based test methods. There are three validated test methods that adhere to this Test Guideline. Depending on the regulatory framework and the classification system in use, this procedure may be used to determine the skin irritancy of test substances as a stand-alone replacement test for in vivo skin irritation testing, or as a partial replacement test, within a tiered testing strategy.

This Test Guideline describes procedures designed to estimate the acute oral toxicity of substances to birds, and it provides three testing options: (1) limit dose test, (2) LD50-slope test, and (3) LD50-only test. The LD50-slope and LD50-only options are sequential testing procedures. The test method selected will depend on whether or not a definitive median dose (LD50) and slope of the dose-response curve are both needed. The limit dose test is the preferred test when toxicity is expected to be low and lethality is unlikely at the limit dose. The limit dose should be adequate for assessment purposes, and it is usually 2000 mg/kg-bwt. Five or ten birds are tested at the limit dose in addition to a control group. The LD50-slope test is the preferred test when regulatory or other requirements determine that the slope of the dose-response curve and/or the confidence interval is required in addition to an estimate of the LD50. This is a 3- or 4-stage test with 24 or 34 birds in addition to a control group. The LD50-only test is the preferred test when regulatory or other requirements determine that only the median lethal dose is required but neither the slope of the dose response curve or the confidence interval for the LD50 is required. This may be the appropriate test to estimate a percentile of a species sensitivity distribution of LD50s and to provide information for product labelling purposes. This test has two stages, with 14 birds in addition to a control group. Software to be used with TG 223. Click here. Software not part of the Mutual Acceptance of Data.

This Guideline is designed to assess the effects of prolonged exposure of chemicals to the life-cycle of the sediment-dwelling freshwater dipteran Chironomus sp. First instar chironomid larvae are exposed to five concentrations of the test chemical in sediment-water systems. The test substance is spiked into the water or alternatively the sediment, and first instar larvae are subsequently introduced into test beakers in which the sediment and water concentrations have been stabilised. Chironomid emergence, time to emergence, and sex ratio of the fully emerged and alive midges are assessed. Emerged adults are transferred to breeding cages, to facilitate swarming, mating and oviposition. The number of egg ropes produced and their fertility are assessed. From these egg ropes, first instar larvae of the 2nd generation are obtained. These larvae are placed into freshly prepared test beakers (spiking procedure as for the 1st generation) to determine the viability of the 2nd generation through an assessment of their emergence, time to emergence and the sex ratio of the fully emerged and alive midges. All data are analysed either by a regression model to estimate the concentration that would cause X% reduction in the relevant endpoint, or by using hypothesis testing to determine a No Observed Effect Concentration (NOEC).

The Local Lymph Node Assay: DA (LLNA: DA) 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 (CBA/J strain), is based on measurement of the adenosine triphosphate (ATP) content by bioluminescence 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, if appropriate, a positive control group. The experimental schedule is during 8 days. The time from animal sacrifice to measurement of ATP should not exceed 30 min. The procedure from excision of lymph nodes to ATP measurement should be kept uniform for each animal and completed within 20 minutes. The luciferin/luciferase method is applied to measure the bioluminescence in Relative Luminescence Units (RLU). This study includes: measurements (weighing, RLU), and clinical daily observations. The results are expressed as the Stimulation Index (SI) obtained by calculation. The SI should be ¡Ý1.8 before further evaluation of the test material as a potential skin sensitizer is warranted.

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.

This Test Guideline describes a method to assess the effects of chemical substances in soil on the reproductive output of the soil mite species Hypoaspis (Geolaelaps) aculeifer Canestrini (Acari: Laelapidae). It can be used for water soluble or insoluble substances, but not with volatile substances. Adult females of similar age are exposed to a range of concentrations of the test substance mixed into 20 g dry mass of artificial soil 28-35 days after the start of the egg laying period. Depending on the endpoint (ECx, NOEC or both), five to twelve concentrations should be tested. At least two to four replicates for each test concentrations and six to eight control replicates, of 10 animals each, are recommended. At 20¡ãC, the test lasts 14 days after introducing the females, which usually allows the control offspring to reach the deutonymph stage. The number of surviving females (mortality ¡Ü 20% for a valid test) and the number of juveniles per test vessel (at least 50 for a valid test) are determined. The fecundity of the mites exposed to the test substance is compared to that of controls in order to determine the ECx (e.g. EC10, EC50) or the No Observed Effect Concentration (NOEC). Any observed differences between the behaviour and the morphology of the mites in the control and the treated vessels should be recorded.

This Test Guideline describes a method to estimate the developmental toxicity of a test chemical to the dung dwelling life stages of dung-dependent dipteran species. Two test species can be used. The test chemical is mixed with bovine faeces, to which either 10 eggs of Scathophaga stercoraria or 10 larvae of Musca autumnalis are added. The test will be terminated 5 days after emergence of the last adult in the control (> 18 days for S. stercoraria, >13 days for M. autumnalis). Then the possible impacts of the test chemical on the following measurement endpoints are assessed under controlled conditions: sex and total number of emerged adult flies, retardation of emergence indicated by the developmental rate and morphological change. Depending on the experimental design, the No Observed Effect Concentration (NOEC) or the Effect concentration for x percent effect (ECx) can be determined. This Guideline can be used for water soluble or insoluble substances, but is not applicable to volatile substances. If the toxicity of the chemical is unknown, five nominal test concentrations should be conducted. A positive control should be tested periodically. The test is considered valid if in the controls hatching of larvae is superior or equal to 70% of the number of introduced eggs, emergence of adults is superior or equal to 70% and superior or equal to 50% of the respectively hatched and introduced larvae and if the emergence of adult flies starts after 18 +- 2 days (S. stercoraria) or after 13 +- 2 days(M. autumnalis).