Test Methods For Regulatory Use

Toxicity Endpoint: Eye Irritation and Corrosion

Test Methods and Approaches	OECD Guidelines and Guidance	Other Authority Recommendations
Integrated approach on testing and assessment (IATA) for serious eye damage and irritation	OECD guidance document (GD) 263, published in 2017	–
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™, SkinEthic™, LabCyte, MCTT HCE™)	OECD test guideline (TG) 492, revised in 2019	ESAC statement (2014); JaCVAM statements (2017 and 2018); KoCVAM guideline (2016)
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)

Toxicity Endpoints: Skin Corrosion and Irritation

Test Methods and Approaches	OECD Guidelines and Guidance	Other Authority Recommendations
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™, EpiDerm™, SkinEthic™, epiCS®, LabCyte EPI-MODEL24 SCT)	OECD TG 431, revised in 2019	ICCVAM report (2002); ESAC statement (1998, 2000, 2006 2009); JaCVAM statement (2017)
In vitro skin corrosion: RhE test (Vitrolife-Skin™)	–	JaCVAM statement (2008)
In vitro skin irritation: Reconstructed human epidermis (RhE) test (EpiSkin™, EpiDerm™, SkinEthic™, LabCyte EPI-MODEL24 SIT, Skin+®, epiCS®, KeraSkin)	OECD TG 439, revised in 2021	ESAC statement (2007, 2008); JaCVAM statement (2010, 2013); KoCVAM guideline (2014, 2017)

Toxicity Endpoint: Skin Sensitization

Test Methods and Approaches	OECD Guidelines and Guidance	Other Authority Recommendations
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 sensitization, 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)	OECD training manual, released in 2017	–
In chemico assays addressing the adverse outcome pathway (AOP) key event on covalent binding to proteins (direct peptide reactivity assay, DPRA; kinetic DPRA; amino acid derivative reactivity assay, ADRA)	OECD TG 442C, revised in 2022	EURL ECVAM recommendation (2013); JaCVAM statement (2015); KoCVAM guideline (2016)
ARE-Nrf2 luciferase test method (KeratinoSens™, LuSens)	OECD TG 442D, revised in 2018	EURL ECVAM recommendation (2014); JaCVAM statement (2015); KoCVAM guideline (2017)
In vitro assays addressing the adverse outcome pathway (AOP) key event on activation of dendritic cells (human cell line activation test, h-CLAT; IL-8 Luc assay; U937 skin sensitization test, U-SENS™, GARD™ skin assay)	OECD TG 442E, revised in 2022	EURL ECVAM recommendation (2015); JaCVAM statement (2017); KoCVAM guideline (2017)

Toxicity Endpoint: Phototoxicity

Test Methods and Approaches	OECD Guidelines and Guidance	Other Authority Recommendations
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

Toxicity Endpoint: Skin Absorption / Penetration

Test Methods and Approaches	OECD Guidelines and Guidance	Other Authority Recommendations
In vitro diffusion method	OECD TG 428, published in 2004	JaCVAM statement (2014); KoCVAM guideline (2009)

Toxicity Endpoint: Acute systemic toxicity

Test Methods and Approaches	OECD Guidelines and Guidance	Other Authority Recommendations
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)

Toxicity Endpoint: Genotoxicity / Mutagenicity

Test Methods and Approaches	OECD Guidelines and Guidance	Other Authority Recommendations
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)

Toxicity Endpoint: Carcinogenicity

Test Methods and Approaches	OECD Guidelines and Guidance	Other Authority Recommendations
In vitro cell transformation assays (CTA)	OECD GD 214, published in 2015; OECD GD 231, published in 2016	EURL ECVAM recommendations (2012 and 2013)

Toxicity Endpoint: Pyrogenicity

Test Methods and Approaches	OECD Guidelines and Guidance	Other Authority Recommendations
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)

Toxicity Endpoint: Hematotoxicity

Test Methods and Approaches	OECD Guidelines and Guidance	Other Authority Recommendations
CFU-GM assay	–	ESAC statement (2006)

Toxicity Endpoint: Reproductive toxicity

Test Methods and Approaches	OECD Guidelines and Guidance	Other Authority Recommendations
Embryonic stem cell test	–	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.)		ESAC statement (2001)
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.)		ESAC statement (2001)

Toxicity Endpoint: Endocrine disruptor screening

Test Methods and Approaches	OECD Guidelines and Guidance	Other Authority Recommendations
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	–

Toxicity Endpoint: Aquatic toxicity

Test Methods and Approaches	OECD Guidelines and Guidance	Other Authority Recommendations
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)

Toxicity Endpoint: All endpoints

Test Methods and Approaches	OECD Guidelines and Guidance	Other Authority Recommendations
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

Toxicity Endpoint: Biologics testing

Replacement Method or Strategy	Regulatory acceptance
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	See 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)

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