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ShareBelow is a non-exhaustive list of in silico, in chemico, in vitro, and ex vivo test methods that could be used to meet regulatory testing requirements. To download a pdf version, click on the format option here: A4 or 8.5×11.
See here for an 11×17 printable poster describing non-animal test strategies for eye irritation, skin irritation, and skin sensitisation.
See also individual factsheets on:
3D TISSUE MODELS (here)
EPA OPP EYE HAZARD (here)
EYE IRRITATION (here)
GENOTOXICITY (here)
MEDICAL DEVICES (here)
ORGANS-ON-CHIPS (here)
PHOTOTOXICITY (here)
PYROGENICITY (here)
QSARS, GROUPING & READ-ACROSS (here)
SKIN IRRITATION (here, training tool also available here)
SKIN SENSITISATION (here)
The following factsheets are available in French:
Les fiches de renseignements suivantes sont disponibles en français:
IRRITATION OCULAIRE (ici)
IRRITATION DE LA PEAU (ici)
SENSIBILISATION DE LA PEAU (ici)
PYROGÉNICITÉ (ici)
| TOXICITY ENDPOINT | TEST METHODS AND APPROACHES | RECOMMENDATIONS AND STANDARD METHODS | ||
|---|---|---|---|---|
| OECD | OTHER AUTHORITY | |||
| SERIOUS EYE DAMAGE AND IRRITATION | Integrated approach on testing and assessment (IATA) for serious eye damage and irritation | OECD guidance document (GD) 263, published in 2023 | _ | |
| Guideline on defined approaches on eye irritation | OECD test guideline (TG) 467, published in 2022 | _ | ||
| Chemical toxicity assessment strategy | _ | European Chemicals Agency guidance Chapter R.7a., R.7.2.8–R.7.2.11 (2017) | ||
| Use of a testing framework employing cytosensor microphysiometer (CM), BCOP, and the EpiOcular™ model for classification of pesticide products | _ | US Environmental Protection Agency policy (2015) | ||
| Reconstructed human cornea-like epithelium (RhCE) test method | EpiOcular™ (MatTek, US) | OECD TG 492, revised in 2019 | ESAC statement (2014); JaCVAM statements (2017 and 2018); KoCVAM guideline (2016) | |
| SkinEthic™ (L’Oréal, France) | ||||
| LabCyte (J-TEC, Japan) | ||||
| MCTT HCE™ (Biosolution, South Korea) | ||||
| Reconstructed human cornea-like epithelium (RhCE) test method (SkinEthic™) | OECD TG 492B, published in 2022 | _ | ||
| Fluorescein leakage (FL) test method | OECD TG 460, revised in 2017 | ESAC statement (2009); JaCVAM statement (2013) | ||
| Short time exposure (STE) in vitro test method | OECD TG 491, revised in 2020 | ICCVAM report (2013); JaCVAM statement (2016); KoCVAM guideline (2017) | ||
| Vitrigel-eye irritancy test (EIT) method | OECD TG 494, revised in 2021 | - | ||
| In vitro macromolecular test method | OECD TG 496, published in 2019 | - | ||
| Bovine corneal opacity and permeability (BCOP) test method | OECD TG 437, revised in 2020 | ICCVAM report (2006); ESAC statement (2007); JaCVAM statements (2009 and 2014); KoCVAM guideline (2011) | ||
| Isolated chicken eye (ICE) test method | OECD TG 438, revised in 2018 | ICCVAM report (2006); ESAC statement (2007); JaCVAM statement (2009) | ||
| Cytosensor microphysiometer (CM) assay | - | ESAC statement (2009); ICCVAM report (2010) | ||
| SKIN CORROSION AND IRRITATION | Integrated approach on testing and assessment (IATA) for skin corrosion and irritation | OECD GD 203, published in 2014 | - | |
| Chemical toxicity assessment strategy for skin corrosion and irritation | - | European Chemicals Agency guidance Chapter R.7a., R.7.2 (2017) | ||
| In vitro membrane barrier test Corrositex for skin corrosion | OECD TG 435, revised in 2015 | ICCVAM report (1999); ESAC statement (2000); JaCVAM statement (2017) | ||
| In vitro skin corrosion: Reconstructed human epidermis (RhE) test | EpiSkin™ (L’Oréal, France) | OECD TG 431, revised in 2019 | ICCVAM report (2002); ESAC statement (1998); JaCVAM statement (2017) | |
| EpiDerm™ (MatTek, US) | ICCVAM report (2002); ESAC statement (2000); JaCVAM statement (2017) | |||
| SkinEthic™ (L’Oréal, France) | ESAC statement (2006); JaCVAM statement (2017) | |||
| epiCS® (Phenion, Germany) | ESAC statement (2009); JaCVAM statement (2017) | |||
| LabCyte EPI-MODEL24 SCT (J-TEC, Japan) | - | |||
| Vitrolife-Skin™ | - | JaCVAM statement (2008) | ||
| In vitro skin irritation: Reconstructed human epidermis (RhE) test | EpiSkin™ (L’Oréal, France) | OECD TG 439, revised in 2021 | ESAC statement (2007); JaCVAM statement (2010); KoCVAM guideline (2014) | |
| EpiDerm™ (MatTek, US) | ESAC statement (2008); JaCVAM statement (2013); KoCVAM guideline (2017) | |||
| SkinEthic™ (L’Oréal, France) | ESAC statement (2008); JaCVAM statement (2013); KoCVAM guideline (2017) | |||
| LabCyte EPI-MODEL24 SIT (J-TEC, Japan) | JaCVAM statement (2013); KoCVAM guideline (2017) | |||
| Skin+® (Sterlab, France) | - | |||
| epiCS® (Phenion, Germany) | ||||
| KeraSkin™ (Biosolution, South Korea) | ||||
| SKIN SENSITISATION | Adverse outcome pathway (AOP) for skin sensitisation | OECD GD 168 (Part 1, Part 2), published in 2012 | - | |
| Guideline on defined approaches on skin sensitisation | OECD Guideline 497, published in 2021 | - | ||
| Guidance on reporting of defined approaches and individual information sources to be used within integrated approaches to testing and assessment (IATA) | OECD GD 256 (Annex I, Annex II), published in 2016 | - | ||
| Use of alternative approaches for skin sensitisation as a replacement for animal testing | - | US Environmental Protection Agency policy (2018) | ||
| Chemical toxicity assessment strategy | - | European Chemicals Agency guidance Chapter R.7a., R.7.3.4–R.7.3.7 (2017) | ||
| OECD QSAR Toolbox | Implementing AOP workflow for skin sensitisation | OECD training manual, released in 2017 | - | |
| Example for predicting skin sensitisation of a mixture | ||||
| Example of how to predict the skin sensitisation potential of a chemical by read-across based on an analogue approach |
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| In chemico assays addressing the adverse outcome pathway (AOP) key event on covalent binding to proteins | Direct peptide reactivity assay (DPRA) | OECD TG 442C, revised in 2022 | EURL ECVAM recommendation (2013); JaCVAM statement (2015); KoCVAM guideline (2016) | |
| Kinetic DPRA | ||||
| Amino acid derivative reactivity assay (ADRA) | ||||
| ARE-Nrf2 luciferase test method | KeratinoSens™ | OECD TG 442D, revised in 2018 | EURL ECVAM recommendation (2014); JaCVAM statement (2015); KoCVAM guideline (2017) | |
| LuSens | ||||
| In vitro assays addressing the adverse outcome pathway (AOP) key event on activation of dendritic cells | Human cell line activation test (h-CLAT) | OECD TG 442E, revised in 2022 | EURL ECVAM recommendation (2015); JaCVAM statement (2017); KoCVAM guideline (2017) | |
| IL-8 Luc assay | - | |||
| U937 skin sensitization test (U-SENS™) | ||||
| GARD™ skin assay | ||||
| PHOTOTOXICITY | 3T3 neutral red uptake (NRU) phototoxicity test | OECD TG 432, revised in 2019 | ESAC statement (1997); ICH S10; KoCVAM guideline (2007) | |
| Reactive oxygen species (ROS) assay | OECD TG 495, published in 2019 | JaCVAM statement (2016); ICH S10 | ||
| Reconstructed human epidermis phototoxicity test method | OECD TG 498, published in 2021 | ICH S10 | ||
| SKIN ABSORPTION/PENETRATION | In vitro diffusion method | OECD TG 428, published in 2004 | JaCVAM statement (2014); KoCVAM guideline (2009) | |
| ACUTE SYSTEMIC TOXICITY | Guidance for waiving tests for pesticide formulations | - | Canada Pest Management Regulatory Agency guidance (2013); US Environmental Protection Agency guidance for acute dermal toxicity tests (2016) | |
| Strategy to replace, reduce, and refine the use of animals in the assessment of acute mammalian systemic toxicity | - | EURL ECVAM guidance (2014) | ||
| Collaborative Acute Toxicity Modeling Suite (CATMoS) | - | NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) (2019) | ||
| 3T3 neutral red uptake (NRU) cytotoxicity test to identify substances not requiring classification | - | EURL ECVAM recommendation (2013) | ||
| GENOTOXICITY/MUTAGENICITY | OECD QSAR Toolbox: Example for predicting Ames mutagenicity using read-across | OECD training manual, released in 2017 | - | |
| In vitro micronucleus test | OECD TG 487, revised in 2016 | ESAC statement (2006); ICH S2(R1) | ||
| Bacterial reverse mutation test | OECD TG 471, revised in 2020 | ICH S2(R1) | ||
| In vitro mammalian chromosome aberration test | OECD TG 473, revised in 2016 | ICH S2(R1) | ||
| In vitro mammalian cell gene mutation test | OECD TG 476, revised in 2016 | - | ||
| In vitro mammalian cell gene mutation tests using the thymidine kinase gene | OECD TG 490, revised in 2016 | ICH S2(R1) | ||
| CARCINOGENICITY | In vitro cell transformation assays (CTA) | OECD GD 214, published in 2015; OECD GD 231, published in 2016 | EURL ECVAM recommendations (2012 and 2013) | |
| DEVELOPMENTAL NEUROTOXICITY | Initial recommendations on evaluation of data from the developmental neurotoxicity (DNT) in vitro testing battery | OECD GD 377, published in 2023 | - | |
| IMMUNOTOXICITY | In Vitro Immunotoxicity | IL-2 Luc Assay | OECD TG 444A, published in 2023 | - |
| PYROGENICITY | In vitro monocyte activation tests (MAT) | - | ICCVAM report (2008); ESAC statement (2006); European Pharmacopoeia general chapter 2.6.30; US Food and Drug Administration guidance (2012) | |
| HAEMATOTOXICITY | CFU-GM assay | - | ESAC statement (2006) | |
| REPRODUCTIVE TOXICITY | Embryonic stem cell test (EST) | - | ESAC statement (2001) | |
| Micromass embryotoxicity assay (Note: Animal embryos are used, therefore this test should be used only if replacing a regulatory requirement for a live-animal test using later life stages.) | ||||
| Whole rat embryotoxicity assay (Note: Animal embryos are used, therefore this test should be used only if replacing a regulatory requirement for a live-animal test using later life stages.) | ||||
| ENDOCRINE DISRUPTOR SCREENING | Stably transfected transactivation in vitro assays to detect oestrogen receptor agonists and antagonists | OECD TG 455, revised in 2021 | JaCVAM statement (2016) | |
| H295R steroidogenesis assay | OECD TG 456, published in 2011 | - | ||
| Stably transfected human androgen receptor transcriptional activation assay | OECD TG 458, revised in 2020 | - | ||
| Human recombinant oestrogen receptor (hrER) in vitro assays to detect chemicals with ER binding affinity | OECD TG 493, published in 2015 | - | ||
| Xenopus eleutheroembryonic thyroid assay (XETA) (Note: Animal embryos are used, therefore this test should be used only if replacing a regulatory requirement for a live-animal test using later life stages.) | OECD TG 248, published in 2019 | - | ||
| AQUATIC TOXICITY | OECD QSAR Toolbox: Example for predicting acute aquatic toxicity to fish of mixture with known components | OECD training manual, released in 2017 | - | |
| EnviroTox database to calculate threshold values | - | Health and Environmental Sciences Institute (2018) | ||
| Freshwater alga and cyanobacteria growth inhibition test | OECD TG 201, published in 2011 | - | ||
| Daphnia sp acute immobilisation test | OECD TG 202, published in 2004 | - | ||
| Fish cell line acute toxicity - the RTgill-W1 cell line assay | OECD TG 249, published in 2021 | ISO 21115 standard (2019) | ||
| In vitro intrinsic clearance test using cryopreserved rainbow trout hepatocytes (Note: Animal primary cells are used, therefore this test should be used only if replacing a regulatory requirement for a live-animal test.) | OECD TG 319A, published in 2018; OECD GD 280, published in 2018 | - | ||
| In vitro intrinsic clearance test using rainbow trout liver S9 sub-cellular fraction (Note: Animal primary cells are used, therefore this test should be used only if replacing a regulatory requirement for a live-animal test.) | OECD TG 319B, published in 2018; OECD GD 280, published in 2018 | - | ||
| Fish embryo acute toxicity test (FET) (Note: Animal embryos are used, therefore this test should be used only if replacing a regulatory requirement for a live-animal test using later life stages.) | OECD TG 236, published in 2013 | EURL ECVAM recommendation (2014) | ||
| MULTIPLE ENDPOINTS | Guidance on considerations for waiving or bridging of mammalian acute toxicity tests | OECD GD 237, published in 2016 | - | |
| Guidance for waiving or bridging of acute toxicity tests for pesticides | - | Health Canada guidance (2013) | ||
| Guidance on the reporting of defined approaches to be used within integrated approaches to testing and assessment | OECD GD 255, published in 2016 | - | ||
| Guidance on the validation of (Q)SAR models | OECD GD 69, published in 2007 | - | ||
| OECD QSAR Toolbox: Guidance documents and training materials | OECD, revised in 2018 | - | ||
| QSAR Model Database | - | Maintained by the European Commission Joint Research Centre | ||
| Various modelling programs | - | For example, programs from Lhasa Limited, Instem, ScitoVation, and Simulations Plus | ||
| Guidance on the grouping of chemicals | OECD GD 194, published in 2014 | - | ||
| Read-across assessment framework | - | European Chemicals Agency guidance (2017) | ||
| Guidance on good in vitro method practices | OECD GD 286, published in 2018 | - | ||
| Guidance for describing non-guideline in vitro test methods | OECD GD 211, published in 2014 | - | ||
| Classification of mixtures based on the toxicity of ingredients | - | United Nations “Globally Harmonized System of Classification and Labelling of Chemicals” guidance (2015); US Environmental Protection Agency pilot program | ||
| ENDPOINT | REPLACEMENT METHOD OR STRATEGY | REGULATORY ACCEPTANCE |
| Biologics testing | In vitro leptospirosis vaccine potency assay | USDA Supplemental Assay Methods (SAM) 624, 625, 626 and 627 |
| In vitro erysipelas vaccine potency assay | USDA SAM 612 and 613 | |
| In vitro clostridial vaccine potency assay | USDA draft SAM 220 | |
| In vitro tetanus toxoid potency assay | USDA SAM 217 | |
| In vitro recombinant antibody production methods | www.thepsci.eu/antibodies/ | |
| Veterinary target animal batch safety test (TABST) | Can be waived following demonstration of compliance; USDA CVB memorandum 800.116 | |
| Revocation of general safety tests (GST)/abnormal toxicity tests (ATT) | FDA amended biologics regulations to revoke GST (2015); all European Pharmacopoeia monographs revised to revoke the ATT (2017) |
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For all endpoints, in vitro methods developed in-house should always be used. Researchers should make every effort to use available non-animal methods. If these methods are not accepted by regulatory agencies, information on additional replacement, reduction, and refinement methods can be found here: NICEATM Accepted Alternative Methods EURL ECVAM Tracking System for Alternative Methods Towards Regulatory Acceptance EURL ECVAM Dataset on Alternative Methods to Animal Experimentation
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Detailed information on the guidance documents and test methods described in this document can be found at the following sites: