Alternative methods endorsed by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM), the U.S. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and/or the Organisation for Economic Co-operation and Development (OECD) are listed below.
Please see here to download a printable version of the table and all factsheets below.
A printable version of the table below can be downloaded here (A4 or 8.5×11).
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See also individual factsheets on:
EYE IRRITATION (here)
SKIN IRRITATION (here)
SKIN SENSITISATION (here)
PYROGENICITY (here)
QSARS, GROUPING & READ-ACROSS (here)
EPA OPP EYE HAZARD (here)
MEDICAL DEVICES (here)
3D TISSUE MODELS (here)
ORGANS-ON-CHIPS (here)
| Toxicity endpoint | Replacement, reduction, or refinement method | Recommendations and standard methods | |
| ICCVAM / ECVAM / Other | OECD | ||
| Skin corrosion | Integrated approach on testing and assessment (IATA) | Via OECD | OECD guidance document (GD) 203 published in 2014 |
| In vitro membrane barrier test, Corrositex | ICCVAM report in 1999, recommended to U.S. agencies in 1999; ECVAM ESAC statement in 2000 | OECD test guideline (TG) 435 revised in 2015 | |
| Reconstructed human epidermis (RHE) test, EpiSkin™ (L’Oréal, France) | ICCVAM report in 2002; ECVAM ESAC statement in 1998 | OECD TG 431 revised in 2016 | |
| RHE test, EpiDerm™ (MatTek, US) | ICCVAM report in 2002; ECVAM ESAC statement in 2000 | OECD TG 431 revised in 2016 | |
| RHE test, SkinEthic™ (L’Oréal, France) | ECVAM ESAC statement in 2006 | OECD TG 431 revised in 2016 | |
| RHE test, epiCS® (Cell Systems, Germany) | ECVAM ESAC statement in 2009 | OECD TG 431 revised in 2016 | |
| Rat skin transcutaneous electrical resistance (TER) test | ICCVAM report in 2002; ECVAM ESAC statement in 1998 | OECD TG 430 revised in 2015 | |
| Skin irritation | Integrated approach on testing and assessment (IATA) | Via OECD | OECD GD 203 published in 2014 |
| RHE test, EpiSkin™ (L’Oréal, France) | ECVAM ESAC statement in 2007 | OECD TG 439 revised in 2015 | |
| RHE test, EpiDerm™ (MatTek, US) | ECVAM ESAC statements in 2007 and in 2008 | OECD TG 439 revised in 2015 | |
| RHE test, SkinEthic™ (L’Oréal, France) | ECVAM ESAC statement in 2008 | OECD TG 439 revised in 2015 | |
| RHE test, LabCyte EPI-Model (J-TEC, Japan) | Via OECD | OECD TG 439 revised in 2015 | |
| Skin absorption / penetration | In vitro diffusion method | Via OECD | OECD TG 428 published in 2004 |
| Skin sensitization | Adverse Outcome pathway (AOP) for skin sensitisation | Via OECD | OECD Series on Testing and Assessment 168 published in 2012 |
| 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 1, Annex 2), published in 2016 | |
| ARE-Nrf2 luciferase test method (e.g., KeratinoSens™ assay) | EURL ECVAM recommendation in 2014 | OECD TG 442D published in 2015 | |
| Direct peptide reactivity assay (DPRA) | EURL ECVAM recommendation in 2013 | OECD TG 442C published in 2015 | |
| Human Cell Line Activation Test (h-CLAT) | EURL ECVAM recommendation in 2015 | OECD TG 442E published in 2016 | |
| Murine Local Lymph Node Assay (LLNA)* | ICCVAM report in 1999, recommended to U.S. agencies in 1999 | OECD TG 429 revised in 2010 | |
| Reduced LLNA (rLLNA) | ICCVAM report in 2008, recommended to U.S. agencies in 2010; ECVAM ESAC statement in 2007 | OECD TG 429 revised in 2010 | |
| LLNA: BrdU–ELISA, non-radioactive | Recommended to U.S. agencies in 2010 | OECD TG 442B published in 2010 | |
| LLNA: DA, non-radioactive | Recommended to U.S. agencies in 2010 | OECD TG 442A published in 2010 | |
| IL-8 Luc assay | — | OECD draft TG published in 2016 | |
| U937 Skin Sensitization Test (U-SENS™) | — | OECD draft TG published in 2016 | |
| *The murine local lymph node assay (LLNA) should only be used when required by regulatory agencies. Researchers should make every effort to use available non-animal methods. | |||
| Eye corrosion | Fluorescein leakage (FL) test method | Via OECD | OECD TG 460 published in 2012 |
| Cytosensor microphysiometer (CM) assay | ESAC statement in 2009; ICCVAM report in 2010, recommended to U.S. agencies as a screening test in 2010 | — | |
| Reconstituted human cornea-like epithelium (RhCE) test method | Via OECD | OECD TG 492 published in 2015 | |
| Short Time Exposure (STE) in vitro test method | Via OECD | OECD TG 491 published in 2015 | |
| Bovine Corneal Opacity and Permeability (BCOP) test method | ICCVAM report in 2006; recommended to U.S. agencies in 2007 | OECD TG 437 revised in 2013 | |
| Isolated Chicken Eye (ICE) test method | ICCVAM report in 2006; recommended to U.S. agencies in 2007 | OECD TG 438 revised in 2013 | |
| Eye irritation | Fluorescein leakage (FL) test method | Via OECD | OECD TG 460 published in 2012 |
| Cytosensor microphysiometer (CM) assay* | ESAC statement in 2009; ICCVAM report in 2010, recommended to U.S. agencies as a screening test in 2010 | — | |
| Reconstructed human cornea-like epithelium (RhCE) test method (e.g., EpiOcular)* | Via OECD | OECD TG 492 published in 2015 | |
| Short time exposure (STE) in vitro method | Via OECD | OECD TG 491 published in 2015 | |
| Bovine Corneal Opacity and Permeability (BCOP) test method* | ECVAM ESAC statement in 2007 | OECD TG 437 revised in 2013 | |
| Isolated Chicken Eye (ICE) test method | ECVAM ESAC statement in 2007 | OECD TG 438 revised in 2013 | |
| Always use anesthetics, analgesics and humane endpoints in the Draize eye test | Recommended to U.S. agencies in 2010 | — | |
| *EPA has established an in vitro irritation testing approach for antimicrobial cleaning products employing CM, BCOP and the EpiOcular model. More information is available at the link at the end of this table. | |||
| Miscellaneous ocular toxicity | Always use anesthetics, analgesics and humane endpoints in the Draize eye test | Recommended to U.S. agencies in 2010 | — |
| Acute systemic toxicity | 3T3 neutral red uptake (NRU) cytotoxicity test to identify substances not requiring classification | EURL ECVAM recommendation in 2013 | — |
| 3T3 NRU cytotoxicity test to estimate starting doses for oral acute systemic toxicity | ICCVAM report in 2006; recommended to US agencies in 2008 | OECD GD 129 published in 2010 | |
| NHK NRU cytotoxicity test to estimate starting doses for oral acute systemic toxicity | ICCVAM report in 2006; recommended to U.S. agencies in 2008 | OECD GD 129 published in 2010 | |
| Up and down procedure (UDP) | ICCVAM report in 2001, recommended to U.S. agencies in 2003; ECVAM ESAC statement in 2007 | OECD TG 425 revised in 2008. OECD TG 401 has been deleted. | |
| Fixed dose procedure (FDP) | Via OECD; ECVAM ESAC statement in 2007 | OECD TG 420 revised in 2002 | |
| Acute Toxic Class (ATC) method | Via OECD; ECVAM ESAC statement in 2007 | OECD TG 423 revised in 2002 | |
| Inhalation toxicity acute toxic class (ATC) method | Via OECD | OECD TG 436 published in 2009 | |
| Genotoxicity / Mutagenicity | In vitro mammalian cell gene mutation test using thymidine kinase gene | Via OECD | OECD TG 490 published in 2016 |
| In vitro micronucleus test | ECVAM ESAC statement in 2006 | OECD TG 487 revised in 2016 | |
| Bacterial reverse mutation test | Via OECD | OECD TG 471 revised in 1997 | |
| In vitro mammalian chromosome aberration test | Via OECD | OECD TG 473 revised in 2016 | |
| In vitro mammalian cell gene mutation test | Via OECD | OECD TG 476 revised in 2016 | |
| Carcinogenicity | In vitro cell transformation assays (CTA) | EURL ECVAM recommendation in 2012 and 2013 | OECD GD 214, published in 2015; OECD GD 231, published in 2016 |
| Combined chronic toxicity and carcinogenicity studies | Via OECD | OECD TG 453 revised in 2009 | |
| Pyrogenicity | In vitro monocyte activation tests (MAT) | ICCVAM report in 2008; ECVAM ESAC statement in 2006, adopted into European Pharmacopoeia in 2009 | — |
| Limulus amoebocyte lysate (LAL) test | US Pharmacopeia general chapter 85; European Pharmacopoeia general chapter 2.6.14; ICH Annex 14 | — | |
| Hematotoxicity | CFU-GM assay | ECVAM ESAC statement in 2006 | — |
| Acute phototoxicity | 3T3 neutral red uptake (NRU) phototoxicity test | ECVAM ESAC statement in 1997 | OECD TG 432 published in 2004 |
| Reproductive toxicity
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Embryonic stem cell test | ECVAM ESAC statement in 2001 | — |
| Micromass embryotoxicity assay | ECVAM ESAC statement in 2001 | — | |
| Whole rat embryotoxicity assay | ECVAM ESAC statement in 2001 | — | |
| Extended one-generation reproductive toxicity study (EOGRTS) | Via OECD | OECD TG 443 published in 2012 | |
| Combined repeated dose toxicity study with the reproductive/developmental toxicity screening test | Via OECD | OECD TG 422 revised in 2015 | |
| Endocrine disruptor screening | Human recombinant estrogen receptor in vitro assays | Via OECD | OECD TG 493 published in 2015 |
| Estrogen receptor-alpha transcriptional activation assay | Via OECD | OECD TG 455 revised in 2016 | |
| H295R steroidogenesis assay | Via OECD | OECD TG 456 published in 2011 | |
| BG1Luc estrogen receptor method | Via OECD | OECD TG 457 published in 2012 | |
| Stably transfected human androgen receptor transcriptional activation assay | Via OECD | OECD TG 458 published in 2016 | |
| Aquatic toxicity | Fish embryo acute toxicity test (FET) | Via OECD; EURL ECVAM recommendation in 2014 | OECD TG 236 published in 2013 |
| Threshold approach, tiered testing strategy | ECVAM ESAC statement in 2006 | OECD GD 126 published in 2010 | |
| Freshwater alga and cyanobacteria growth inhibition test | Via OECD | OECD TG 201 published in 2011 | |
| Daphnia sp. acute immobilisation test | Via OECD | OECD TG 202 published in 2004 | |
| All endpoints | Guidance on the grouping of chemicals | Via OECD | OECD GD 194 published in 2014 |
| Clinical signs as humane endpoints | Various ICCVAM and ECVAM recommended methods | OECD GD 19 published in 2000 | |
| Guidance on considerations for waiving or bridging of mammalian acute toxicity tests | — | OECD GD 237, published in 2016 | |
| Guidance for describing non-guideline in vitro test methods | — | OECD GD 211, published in 2014 | |
| Guidance on the reporting of defined approaches to be used within integrated approaches to testing and assessment | — | OECD GD 255, published in 2016 | |
| Toxicity endpoint | Replacement or refinement methods | 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 and monoclonal antibody production methods | USDA, NIH and EURL ECVAM endorsement (expanded recombinant antibody information presented on the following pages) | |
| Humane endpoints for vaccine-challenge methods | USDA CVB Notice 12-12 | |
| Anesthesia for rabies vaccine challenge assay | ||
| Veterinary target animal batch safety test (TABST) | Can be waived following demonstration of compliance, USDA CVB memorandum 800.116 |
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For all endpoints, in vitro methods developed in-house should always be used.
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Detailed information on the guidance documents and test methods described in this table can be found at the following sites:
For information on alternatives to animal-derived antibodies, see here.