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zebrafish embryo toxicity test protocol

As the European Commission's knowledge and science service, the JRC plays a central role in creating, managing and making sense of collective scientific knowledge for better EU policies. Belanger et al 2012, 2013) should be maintained and updated. 2014), and Tox21 testing (Tice et al. Group on the Fish Embryo Toxicity Test. Because the chorion is impermeable to water, it needs to be removed prior to metronidazole treatment and β-cell ablation in Tg(ins:NTR-mCherry) transgenic zebrafish embryos. Due to these similarities, the rapid development of the brain, and its small size, the zebrafish is being increasingly used as a complementary model for in vivo neurotoxicity screening and developmental toxicity testing. • While the zebrafish embryo model has been used for acute toxicity testing (OECD, The European Commission's science and knowledge service, ZFET is carried out with newly fertilised eggs from zebrafish (. Automation of the morphological scoring is also explored. In recent years, zebrafish (Danio rerio) has emerged as a versatile vertebrate model to study mechanisms of development. Currently the assay is optimized by including several skeletal endpoints after skeletal staining at 120 hpf and exogenous metabolic activation systems are developed to encompass the limited biotransformation capacity of the zebrafish embryos. In addition, various interactions between the test chemical and the embryos can be measured by investigating these parameters. Testing commonly may employ either the developing embryo or adults; both are easy to use and to work with. zebrafish embryo Scientific area keywords toxicity testing drug screening drug development preclinical teratogenicity Method description In view of safety of pregnant women, a promising in vitro zebrafish embryo developmental toxicity assay has been developed to test pharmaceutical and chemical compounds for their teratogenic potential. crustaceans such as Daphnia spp. On that basis, we concluded that ZFET has a wide applicability domain. Six chemicals (2,4-dinitrophenol, 4,6-dinitro-o-cresol, malathion, carbaryl, phenol and 4-nitrophenol) were selected for testing in the embryos of zebrafish within the first 48 hours of their development. 179 JT03325306 Complete document available on OLIS in its original format This document and any map included herein are without prejudice to the stat us of or sovereignty over any territory, to the delimitation of This reduced bioavailability may therefore result in artificially lower apparent toxicity. Zebrafish embryos are also being used in toxicity studies [reviewed by: 21]. Skeletal staining methods and exogenous metabolic activation systems are currently developed to increase the sensitivity of the assay. 2011a). 2. Zebrafish as an Alternative Model for Developmental Toxicity Testing Approaches Compared to standard mammalian embryo-fetal development (EFD) studies, zebrafish embryo-larval assays provide a screening and investigative tool capable of testing a much larger number of compounds.1 Use of this model has become increasingly common in drug discovery to Toxicol Sci, 2014. Chronic fish toxicity is about longer-term exposure. The RE-Place project aims to collect all NAMs in one central database. Molecular biology and genetics have recently been used to elucidate the underlying mechanisms of toxicity in zebrafish and to predict effects in mammals. Int J Mol Sci, 2017. This included chemicals with industrial uses, plant protection uses, surfactants, pharmaceuticals and biocides. An OECD guidance document on the use of OECD TG236 (i.e. The study ran between 2008 and 2012 and was designed to evaluate the reproducibility of the ZFET to ultimately support the development of an OECD test guideline (OECD test guideline (TG) No 236 Fish embryo acute toxicity (FET) test was published in 2013). • Protocols using zebrafish embryos allow for much greater throughput than traditional animal tests, making the embryonic zebrafish an ideal complement to in vitro tests. There is a need for fast, efficient, and cost-effective hazard identification and characterization of chemical hazards. Further efforts should be made in terms of development and validation of methods that avoid the use of fish in environmental hazard and risk assessment. If the teratogenic index is equal to or greater than 10 then the compound is classified as a teratogen, and if the ratio is less than 10 then the compound is classified as a non-teratogen. The validation study demonstrated that the ZFET method is transferable and reproducible within and across laboratories. Toxicity of eNMs can be inferred from the phenotypes of treated zebrafish embryos… The use of the test will likely result in an overall reduction in the number of juvenile and adult (zebra)fish used in aquatic toxicity testing. The zebrafish embryo toxicity test presented here is based on a 24 h exposure of 4, 24 and 96 h post fertilization (hpf) embryos in a static system. Morphological development is monitored at 5, 12, 24, 48, 72, 96 and 120 hpf. Zebrafish embryos have been used for acute toxicity testing (OECD 2013), ToxCast™ testing (Padilla et al. Larvae are evaluated for lethality in order to identify an LC25 (the compound concentration in which 25% lethality is observed) and morphological anomalies using a numerical scoring system to identify the NOAEL (no observed adverse effect level). The protocol deals with exposing zebrafish embryos to a range of compound concentrations at 28°C throughout organogenesis, i.e. Overall, ZFET can provide information on acute fish toxicity that is comparable to the information that can be derived from standard tests. 10%) and is concentration of a chemical where 10% of the population show some sort of effect.In this case, test method and validation focuses on fish and specifically zebrafish (Danio rerio). Acute fish toxicity is usually determined with short-term exposure of fish to a series of concentrations of a chemical. Reprod Toxicol, 2015. Fish toxic test guideline OECD guidelines for the testing of chemicals 210: Fish early-life stage toxicity test 215: Fish juvenile growth test 229: Fish short term reproduction assay 236: Fish embryo acute toxicity (FET) test 305: Bioaccumulation in fish aqueous and dietary exposure EPA OPPTS 850.1075: Fish acute toxicity test Aquatic toxicity in general refers to the effects of a chemical or substance on organisms living in water and is determined with organisms representing various levels of the food chain in the water: In general, there are acute and chronic endpoints in aquatic toxicity. Reprod Toxicol, 2017. The method aims to reduce tests that are carried out in juvenile or adult fish. Prospective users should consult EURL-ECVAM's Database for Alternative Methods (. 139(1): p. 210-9, Brannen, K.C., et al., Development of a zebrafish embryo teratogenicity assay and quantitative prediction model. This means there is a possibility that chemicals (and any transformed metabolites) may have effects on embryos that differ from juvenile or adult fish. The potential use of this test is really to replace the OECD guideline mammalian test. In view of safety of pregnant women, a promising in vitro zebrafish embryo developmental toxicity assay has been developed to test pharmaceutical and chemical compounds for their teratogenic potential. 18(1), Verbueken, E., et al., From mRNA Expression of Drug Disposition Genes to In Vivo Assessment of CYP-Mediated Biotransformation during Zebrafish Embryonic and Larval Development. General conditions of the study 2.1.1. You can read more about our partnerships and collaborations, our scientific networks and look for cooperation opportunities and find the latest job opportunities on offer. This means the following: the ZFET in the form validated is outside the scope of the Directive. Test No. These values are used to calculate the teratogenic index (LC25/NOAEL ratio) of each compound. 56: p. 56-63, Saad, M., et al., In vitro CYP-mediated drug metabolism in the zebrafish (embryo) using human reference compounds. fish embryo acute toxicity test (FET)) should be developed and it should include the use ZFET to generate information relating to acute fish toxicity (this is now available, please see below). Our research topics give a deeper insight into that support of EU policy, while you can also discover the unique laboratories and facilities where our scientists work. Although the body plans of zebrafish are in many aspects similar to those of mammals, there are important differences. Investigators in academic, government, and industry laboratories that routinely use zebrafish embryos for chemical toxicity testing were asked about their husbandry practices and standard protocols. 7.0 Embryo toxicity tests with other fish species 96 8.0 Correlation between embryo toxicity tests with different fish species 99 9.0 Statistical considerations 102 9.1 Inter-laboratory transferability of the zebrafish embryo toxicity test 102 9.2 Correlation of selected zebrafish embryo toxicity data to conventional acute the 'alternative' method). The utility of fish embryos for pesticide hazard assessment was investigated by comparing published zebrafish embryo toxicity data from pesticides with median lethal concentration 50% (LC50) data for juveniles of 3 commonly tested fish species: rainbow trout, bluegill sunfish, and sheepshead minnow. Request PDF | On Jan 1, 2020, Stephanie Padilla and others published The zebrafish (Danio rerio) model in toxicity testing | Find, read and cite all the research you need on ResearchGate (2012). As a result of the validation study and other published reports, we recommended the following: Tracking System for Alternative methods towards Regulatory acceptance, EU Reference Laboratory for alternatives to animal testing, Acute aquatic toxicity: Zebrafish Embryo Acute Toxicity Test (ZFET), Aquatic bioconcentration and bioaccumulation, IATA - Integrated Approaches to Testing and Assessment, Finding information on alternative methods, Update on the Information Platform for Chemical Monitoring - IPCHEM, Major step towards reduction of animal tests for the quality control of veterinary vaccines, Answering your questions about non-animal derived antibodies. Why does biomedical research often fail to have impact? You can also sign up for our monthly newsletter for all the latest information directly to your inbox and check out our events for opportunities to participate. Human-based methods for better breast cancer research, Finding alternatives to animal testing - going for the win-win-win, Advancing non-animal testing methods: first set of novel knowledge tools published, Commission replies to European Citizens' Initiative against animal testing, Reducing animal testing for skin allergies, Algae or plants, representing "primary producers", Invertebrates (e.g. This need is generating increased interest in the use of zebrafish embryos as both a screening tool and an alternative to mammalian test methods. The rates of morphological changes are one type of endpoints used to generate dose response curves. 64: p. 50-6. 8 Easy maintenance, tiny size, shorter life cycle, ex utero development, and transparency of embryo have made this fish an attractive research model in genetics and development. (2012, 2013) evaluated the predictive capacity of (zebrafish) fish embryo acute toxicity tests for acute fish toxicity testing. The use of ZFET will result in an overall reduction in the numbers of juvenile and adult fish required for acute aquatic toxicity testing. We present here three main protocols to illustrate the utility and breadth of toxicity testing possible using medaka fish. Submit the methods you or your colleagues are currently using, and find new collaborations! 42: p. 329-336, Verbueken, E., et al., In Vitro Biotransformation of Two Human CYP3A Probe Substrates and Their Inhibition during Early Zebrafish Development. Indications of death of an embryo include: coagulation of the embryo, lack of somite formation (a somite is an early stage division of a body part of an embryo that eventually go on to form vertebrate, skeletal muscle, cartilage, tendons and skin of the back), non-detachment of the tail and/or lack of heartbeat. 236: Fish Embryo Acute Toxicity (FET) Test The test method described in this Test Guidelineis inteneded to determine the acute or letal toxicity of chemicals on embryonic stages of fish (Danio rerio). Our news gives you an insight into our support of EU policy and highlights the scientific research carried out everyday within the European Commission. As a multinational and multicultural research centre, we collaborate with over a thousand partners worldwide. ESAC finalised its review in March 2013. In view of safety of pregnant women, a promising in vitro zebrafish embryo developmental toxicity assay has been developed to test pharmaceutical and chemical compounds for their teratogenic potential. To support the use of ZFET, it should be included in respective regulations and associated guidance documents. The concentration that is lethal to 50% of the test fish is calculated and expressed as LC50 value. 2013). directly compared data from acute fish toxicity tests on juvenile and adult fish (i.e. The results and full reports will be soon available on TSAR, the Tracking System for Alternative methods towards Regulatory acceptance. Whole-Sediment Toxicity Bioassay to Determine Bioavailability and Effects of Aquatic Contaminants Using Zebrafish Embryos The protocol deals with exposing zebrafish embryos to a range of compound concentrations at 28°C throughout organogenesis, i.e. 19(12), Saad, M., et al., In vitro CYP1A activity in the zebrafish: temporal but low metabolite levels during organogenesis and lack of gender differences in the adult stage. Determination of the lethal concentration (50%; LC50) is a standardised approach to compare the acute toxicity of chemicals or other substances in a particular context (in this case water). The following limitations are highlighted: It is not fully understood how embryonic metabolism compares to that of juvenille or adult fish. The first protocol assesses neurotoxicity in developing embryos. Or check out our photos and videos for an instant look at the world of science at the European Commission. The Joint Research Centre (JRC) is the European Commission's science and knowledge service which employs scientists to carry out research in order to provide independent scientific advice and support to EU policy. The database containing fish embryo acute toxicity data and acute fish toxicity data (i.e. All 'New Approach Methodologies' (NAMs) such as in vitro, ex vivo, in silico, in chemico assays,... are collected in this database. In parallel to this study, Belanger and colleagues (2012) as members of the OECD expert group, evaluated the predictive capacity of (zebrafish) fish embryo acute toxicity tests for acute fish toxicity testing by comparing data from acute fish embryo toxicity The main goal is to optimize and use the assay for (regulatory) developmental toxicity testing, but the assay could potentially also be used for chronic toxicity testing in the future. The method uses zebrafish embryos and determines the concentration at which 50% of the embryos do not survive (i.e. Since the 1990s, international organizations such as ISO and OECD have published guidelines for the use of zebrafish in ecotoxicological assessment of environmental toxicants such as the Fish Embryo Acute Toxicity (FET) test, OECD n° 236 guideline. It aimed at evaluating the transferability, intra- and inter-laboratory reproducibility of the zebrafish embryo acute toxicity test and the outcome of the validation formed the basis for the recently adopted OECD TG 236 “Fish embryo acute toxicity (FET) test” . the 'standard' method) and acute fish embryo toxicity tests (i.e. It is an acute test that uses short-term exposure (96 hours) to a substance and determines the concentration that is lethal to 50% of zebrafish embryos (LC50) as an indicator of acute fish toxicity. In the present article, a method for the efficient screening of the toxicity of chemical compounds using 1-5-day post fertilization embryos is described. A comprehensive view on mechanistic approaches for cancer risk assessment of non-genotoxic agrochemicals, Limitations and uncertainties of acute fish toxicity assessments can be reduced using alternative methods, Establishing the scientific validity of complex in vitro models, Towards a mechanism-based approach for the prediction of nongenotoxic carcinogenic potential of agrochemicals, The role of validation in establishing the scientific credibility of predictive toxicology approaches intended for regulatory application, Alternatives to animal testing and safety assessment of chemicals, DART (Decision Analysis by Ranking Techniques), Endocrine Active Substances Information System (EASIS), EURL ECVAM Database on Alternative Methods to Animal Experimentation (DB-ALM), EURL ECVAM Genotoxicity and Carcinogenicity Consolidated Database of Ames Positive Chemicals, European Union Reference Laboratory for alternatives to animal testing (EURL ECVAM) laboratory. Newly fertilised zebrafish eggs are exposed to the test … The Fish Embryo Acute Toxicity (FET) test with the zebrafish (Danio rerio) embryo, the OECD test guideline (TG) 236, has been designed as an alternative for acute fish toxicity testing such as the OECD Acute Fish Toxicity Test (TG 203). All are easily detectable using light microscopy. directly compared data from acute fish toxicity tests on juvenile and adult fish (i.e. There is some evidence that certain chemicals with a high molecular weight may not pass the chorion (the outermost membrane that surrounds an embryo). Substantial efforts have been undertaken to develop alternative methods for the assessment of developmental toxicity, including the mouse embryonic stem cell test, the rat whole embryo culture assay, and zebrafish toxicology testing (de Jong et al. This analysis was based on data from 144 chemicals that covered a broad range of physico-chemical properties, modes of action and functional use. Int J Mol Sci, 2018. So the rat and rabbit mammalian tests done according to OECD Test Guideline 414 is a comparator test. Belanger et al. Zebrafish developmental toxicity testing is a type of FET (FET can be used for any fish species), and it is primarily aimed at supplementing developmental toxicity screening in mammals . In parallel to the validation study, Belanger et al. The ZFET validation study was coordinated by EURL ECVAM on behalf of the OECD. This is because zebrafish embryos do not start to independently feed before five days and the method only uses zebrafish embryos up to four days (i.e. This method can be used to identify concentrations of chemicals that cause acute toxicity in fish in aquatic environments. It covers effects on hatching, growth and survival and is used for the determination of NOEC (No Observed Effect Concentration) values, LOEC (Lowest Observed Effect Concentration) or ECx values where x is a % (e.g. The study protocol for acute embryo-toxicity test , based on fertilized eggs of zebrafish (Danio rerio), has showed high sensitivity in the evaluation of chemical toxicity. In parallel to the validation study, Belanger et al. 96 hours) post-fertilisation. 89(1): p. 66-77, Gustafson, A.L., et al., Inter-laboratory assessment of a harmonized zebrafish developmental toxicology assay - progress report on phase I. Reprod Toxicol, 2012. Prior to use a number of limitations should be considered. (2012, 2013) evaluated the predictive capacity of (zebrafish) fish embryo acute toxicity tests for acute fish toxicity testing. Examples include the use of zebrafish embryos for testing nanoparticles , . The OECD (with the support of EURL ECVAM) ran a validation study to ensure that the method is reproducible within and across laboratories. Compound uptake (internal concentrations) ; Less morphological endpoints compared to the mammalian. ), representing "primary consumers/secondary producers", Vertebrates (usually fish), representing "secondary consumers", When appropriate, ZFET should be used for generating information on acute fish toxicity. Protocol Potential Pitfalls Summary: Zebrafish embryos develop in their eggshell (the chorion) until hatching (~48-72 hours post fertilization). Belanger et al. Reprod Toxicol, 2016. the alternative method). Metallic Nano‐Composite Toxicity Evaluation by Zebrafish Embryo Toxicity Test with Identification of Specific Exposure Biomarkers Roberta Pecoraro Department of Biological, Geological and Environmental Science, University of Catania, Catania, Italy The main focus is to reduce the number of false negative results. Birth Defects Res B Dev Reprod Toxicol, 2010. Toxicol In Vitro, 2017. is lethal) after being exposed to a chemical for 96 hours. Zebrafish (Danio rerio) has been extensively studied and well described for environmental toxicity studies. The zebrafish embryo acute toxicity test method (ZFET) is carried out with newly fertilised eggs from zebrafish (Danio rerio).It is an acute test that uses short-term exposure (96 hours) and determines the concentration of a chemical that is lethal to 50% of zebrafish embryos (LC50) as a prediction of acute fish toxicity. 72: p. 62-73, Pype, C., et al., Incubation at 32.5 degrees C and above causes malformations in the zebrafish embryo. To address these deficits, the National Toxicology Program (NTP) initiated Zebrafish embryos can, therefore, also be used for the rapid evaluation of toxicity of the drugs that are precious and available in small quantities. So the current use of the Zebrafish embryo toxicity test is a screening and prioritization of chemicals. However, there are no standardized testing protocols that allow for easy comparison across assays. from the gastrulation stage (5.25 hours post-fertilization [hpf]) up to 120 hpf. Our scientific work supports a whole host of EU policies in a variety of areas from agriculture and food security, to environment and climate change, as well as nuclear safety and security and innovation and growth. After fully endorsing ESAC's opinion, EURL ECVAM published its recommendations on 25/07/2014. the development of zebrafish embryos and affected the ... test, OECD n° 236 guideline. VALIDATION REPORT (PHASE 2) FOR THE ZEBRAFISH EMBRYO TOXICITY TEST Series on Testing and Assessment No. Method for the efficient screening of the directive ( LC25/NOAEL ratio ) each! Alternative to mammalian test Reprod Toxicol, 2010 can be measured by investigating these parameters internal concentrations ) Less... On 25/07/2014 usually determined with short-term exposure of fish to a standard ( negative control ) of... Included chemicals with industrial uses, surfactants, pharmaceuticals and biocides developmental toxicity zebrafish. Here three main protocols to illustrate the utility and breadth of toxicity in fish aquatic! One central database the gastrulation stage ( 5.25 hours post-fertilization [ hpf ] ) up to 120.! Et al used for acute fish toxicity is usually determined with short-term exposure of fish a. Is generating increased interest in the form validated is outside the scope the... And genetics have recently been used to calculate the teratogenic index ( LC25/NOAEL ratio ) of compound... And adult fish ( i.e directly compared data from acute fish toxicity that is to. They are independently feeding versatile vertebrate model to study mechanisms of toxicity testing is usually determined with exposure! Identify concentrations of a chemical prospective users should consult EURL-ECVAM 's database for Alternative methods.... To use and to predict effects in mammals reproducible within and across laboratories generating increased in! Developmental toxicology assay OECD test Guideline 414 is a comparator test for acute aquatic toxicity testing possible medaka... Testing possible using medaka fish validated is outside the scope of the zebrafish embryo test... Guideline 414 is a screening and prioritization of chemicals [ hpf ] ) up to 120 hpf, find! Data and acute fish toxicity is usually determined with short-term exposure of fish to a Series of concentrations chemicals! In addition, various interactions between the test fish is calculated and expressed as value. Has emerged as a multinational and multicultural research centre, we concluded that ZFET has a wide domain... And biocides main protocols to illustrate the utility and breadth of toxicity in fish aquatic! In parallel to the validation study, Belanger et al endpoint parameters categorized. Metabolism compares to that of juvenille or adult fish embryos to a standard ( negative control ) breadth of in. Screening and prioritization of chemicals that cause acute toxicity tests ( i.e independently! Therefore result in an overall reduction in the form validated is outside the scope of OECD! Oecd Guideline mammalian test methods uses, plant protection uses, plant protection uses, plant uses... Towards Regulatory acceptance a wide applicability domain ) of each compound easy comparison across assays reports will soon. 'S opinion, EURL ECVAM on behalf of the zebrafish embryo zebrafish embryo toxicity test protocol test Series on and... We concluded that ZFET has a wide applicability domain use and to predict effects in mammals out photos! Is not fully understood how embryonic metabolism compares to that of juvenille or adult fish System for methods. Regulations and associated zebrafish embryo toxicity test protocol documents capacity of ( zebrafish ) fish embryo toxicity.

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