Alternative Methods Accepted by US Agencies

The table below includes:

Methods for chemical safety testing that are accepted by U.S. and international regulatory authorities as replacement, reduction, or refinement alternatives to required animal tests.
Guidances to support replacement, reduction, or refinement alternatives to animal use for required testing: these documents are recommendations that do not necessarily establish legally enforceable responsibilities.

An overview of non-animal methods that have been proposed for regulatory safety or efficacy testing of chemicals or biological agents can be found in the Tracking System for Alternative Methods (TSAR) resource, provided by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM). TSAR tracks progress of an alternative method from submission for validation through to its final adoption by inclusion into the regulatory framework.

NICEATM and ICCVAM interact with EURL ECVAM through the International Cooperation on Alternative Test Methods.

Toxicity Area	Method	ICCVAM or ICCVAM Agency Contributions	Regulatory Acceptance/ Endorsement and Applicable Regulations
Ocular Corrosivity and Irritation	Framework to assess eye irritation or corrosion in new chemicals	EPA activity	U.S.: EPA guidance document (2024)
Immunotoxicity	In vitro immunotoxicity: IL-2 Luc assay (reduction and replacement of animal use)	ICCVAM contributed to U.S. OECD test guideline review	U.S.: Accepted via OECD Test Guideline 444A (2023) EU: Accepted via OECD Test Guideline 444A (2023)
Skin Sensitization	Nonclinical Evaluation of the Immunotoxic Potential of Pharmaceuticals (reduction and replacement of animal use)	FDA activity	U.S.: FDA guidance document (2023)
Multiple Toxicities	Guidance for Industry and Test Method Developers: CPSC Staff Evaluation of Alternative Test Methods and Integrated Testing Approaches and Data Generated from Such Methods to Support FHSA Labeling Requirements	CPSC activity	U.S.: CPSC guidance document (2022)
Ocular Corrosivity and Irritation	Defined approaches for serious eye damage and eye irritation (replacement of animal use)	ICCVAM contributed to U.S. OECD test guideline review	U.S.: Accepted via OECD Test Guideline 467 (2022) EU: Accepted via OECD Test Guideline 467 (2022)
Endocrine Disruptors	Rapid androgen disruption activity reporter assay (replacement and reduction of animal use)	-	U.S.: Accepted via OECD Test Guideline 251 (2022) EU: Accepted via OECD Test Guideline 251 (2022)
Skin Sensitization	Guinea pig tests for skin sensitization (update refines animal use and recommends first use of non-animal methods)	-	U.S.: Accepted via OECD Test Guideline 406 (last updated 2022) EU: Accepted via OECD Test Guideline 406 (last updated 2022)
Dermal Phototoxicity	In vitro reconstructed human epidermis method for phototoxicity	ICCVAM members contributed to a retrospective review of available information to support the guideline	U.S.: Accepted via OECD Test Guideline 498 (2021, last updated 2023) EU: Accepted via OECD Test Guideline 498 (2021, last updated 2023)
Skin Sensitization	Defined approaches on skin sensitization (replaces animal use)	NICEATM, ICCVAM, and international collaborators developed and sponsored guideline	U.S.: Accepted via OECD Guideline 497 (2021, last updated 2023) EU: Accepted via OECD Guideline 497 (2021, last updated 2023)
Endocrine Disruptors	EASZY assay - detection of endocrine active substances using zebrafish embryos (reduces/replaces animal use)	-	U.S.: Accepted via OECD Test Guideline 250 (2021) EU: Accepted via OECD Test Guideline 250 (2021)
Ecotoxicity	Fish cell line acute toxicity - RTgill-W1 cell line assay (reduces/replaces animal use)	-	U.S.: Accepted via OECD Test Guideline 249 (2021) EU: Accepted via OECD Test Guideline 249 (2021)
Biologics Testing	Cryopreservation protocol for leptospiral strains for vaccine testing (reduces animal use)	USDA activity	U.S.: USDA Center for Veterinary Biologics Protocol BBAPP0011.01 (2018)
Developmental and Reproductive Toxicity	Guidance on reproductive and developmental toxicity studies for human pharmaceuticals (includes provisions for reducing animal use)	-	U.S.: Accepted via ICH Guidance Document S5(R3) in 2021 EU: Accepted via ICH Guidance Document S5(R3) in 2021
Multiple Toxicities	Guidance on nonclinical studies for pharmaceuticals for hematologic disorders (includes provisions for reducing animal use)	FDA activity	U.S.: FDA guidance document (2019)
Developmental and Reproductive Toxicity	Guidance on reproductive toxicity testing for oncology radiopharmaceuticals (includes provisions for reducing animal use)	FDA activity	U.S.: FDA guidance document (2019)
Chronic Toxicity	Guidance on nonclinical studies for oncology therapeutic radiopharmaceuticals (includes provisions for reducing animal use)	FDA activity	U.S.: FDA guidance document (2019)
Acute Oral Systemic Toxicity	Guidance on nonclinical studies of microdose radiopharmaceutical diagnostic drugs (includes provisions for reducing animal use)	FDA activity	U.S.: FDA guidance document (2018)
Chronic Toxicity	Guidance on nonclinical evaluation of cancer drugs (includes provisions for reducing animal use)	FDA activity	U.S.: FDA Q & A guidance document (2018)
Multiple Toxicities	Guidance on use of microsampling techniques in toxicokinetics studies (includes provisions for reducing and refining animal use)	FDA activity	U.S.: FDA Q & A guidance document (2018)
Skin Sensitization	Guidance on safety evaluation of immunotoxic potential of drugs and biologics (includes provisions for reducing or replacing animal use)	FDA activity	U.S.: FDA guidance (2020) provides for use of in silico, in chemico, and in vitro studies to predict human skin sensitization
Acute Dermal Systemic Toxicity	In vitro dermal absorption methods (replacement of animal use)	ICCVAM working group contributed to U.S. OECD test guideline review, expert consultation meetings	U.S.: Accepted via OECD Test Guideline 428 (2004) EU: Accepted via OECD Test Guideline 428 (2004)
Acute Dermal Systemic Toxicity	Acute dermal toxicity (includes provisions for waiving test and reducing or refining animal use)	NICEATM and ICCVAM scientists participated in drafting and editing the revised test guideline	U.S.: Accepted via OECD Test Guideline 402 (1987, revised 2017) EU: Accepted via OECD Test Guideline 402 (1987, revised 2017)
Acute Dermal Systemic Toxicity	Guidance for waiving acute dermal toxicity tests for pesticide formulations and supporting retrospective analysis (reduction of animal use)	EPA activity; NICEATM contributed to data analysis	U.S.: EPA guidance (2016, finalized 2020) provides for authorizing waivers of required dermal toxicity testing for formulated pesticide products from standard requirements under 40 CFR 158
Chronic Toxicity	Subacute inhalation toxicity: 28-day study (reduction of animal use)	-	U.S.: Accepted via OECD Test Guideline 412 (1981, revised 2009, 2018) EU: Accepted via OECD Test Guideline 412 (1981, revised 2009, 2018)
Chronic Toxicity	Subchronic inhalation toxicity: 90-day study (reduction of animal use)	-	U.S.: Accepted via OECD Test Guideline 412 (1981, revised 2009, 2018) EU: Accepted via OECD Test Guideline 412 (1981, revised 2009, 2018)
Acute Inhalation Toxicity	Inhalation toxicity - acute toxic class method (reduction and refinement of animal use)	ICCVAM working group contributed to U.S. OECD test guideline review	U.S.: Accepted via OECD Test Guideline 436 (2009) EU: Accepted via OECD Test Guideline 436 (2009)
Acute Inhalation Toxicity	Inhalation toxicity testing - fixed concentration procedure (reduction and refinement of animal use)	ICCVAM contributed to U.S. OECD test guideline review	U.S.: Accepted via OECD Test Guideline 433 (2017, revised 2018) EU: Accepted via OECD Test Guideline 433 (2017, revised 2018)
Chronic Toxicity	Repeated dose 90-day oral toxicity study in rodents (reduction of animal use)	-	U.S.: Accepted via OECD Test Guideline 408 (revised 2018) EU: Accepted via OECD Test Guideline 408 (revised 2018)
Chronic Toxicity	Chronic toxicity studies (reduction of animal use)	-	U.S.: Accepted via OECD Test Guideline 452 (1981, revised 2018) EU: Accepted via OECD Test Guideline 452 (1981, revised 2018)
Chronic Toxicity	Combined chronic toxicity/carcinogenicity studies (reduction of animal use)	-	U.S.: Accepted via OECD Test Guideline 453 (1981, revised 2018) EU: Accepted via OECD Test Guideline 453 (1981, revised 2018)
Chronic Toxicity	Guidance for waiving subacute avian dietary tests for pesticide registration and supporting retrospective analysis	EPA activity	U.S.: EPA guidance (2019, finalized 2020) provides for authorizing waivers of required subacute avian dietary toxicity testing for formulated pesticide products from standard requirements under 40 CFR 158.630
Acute Oral Systemic Toxicity	Up-and-down procedure for acute oral toxicity (reduction and refinement of animal use)	ICCVAM peer review and report; recommended in 2001	U.S.: Accepted via OECD Test Guideline 425 (2001, last revised 2021); EPA OPPTS 870.1100 (2002) EU: Accepted via OECD Test Guideline 425 (2001, last revised 2021)
Acute Oral Systemic Toxicity	Fixed dose procedure for acute oral toxicity (reduction and refinement of animal use)	ICCVAM working group contributed to test guideline development	U.S.: Accepted via OECD Test Guideline 420 (2001) EU: Accepted via OECD Test Guideline 420 (2001)
Acute Oral Systemic Toxicity	Acute toxic class method for acute oral toxicity (reduction and refinement of animal use)	ICCVAM working group contributed to test guideline development	U.S.: Accepted via OECD Test Guideline 423 (2001) EU: Accepted via OECD Test Guideline 423 (2001)
Acute Oral Systemic Toxicity	Avian acute oral toxicity test (reduction of animal use)	ICCVAM working group contributed to U.S. OECD test guideline review	U.S.: Accepted via OECD Test Guideline 223 (2010) EU: Accepted via OECD Test Guideline 223 (2010)
Acute Oral Systemic Toxicity	Acute toxicity in vitro starting dose procedure, 3T3 cells (reduction and refinement of animal use)	ICCVAM 2001 workshop report; ICCVAM 2006 peer review and report; recommended in 2008	U.S.: Accepted by agencies in 2008 EU: Accepted via OECD Guidance Document 129 (2010)
Acute Oral Systemic Toxicity	Acute toxicity in vitro starting dose procedure, NHK cells (reduction and refinement of animal use)	ICCVAM 2001 workshop report; ICCVAM 2006 peer review and report; recommended in 2008	U.S.: Accepted by agencies in 2008 EU: Accepted via OECD Guidance Document 129 (2010)
Multiple Toxicities	Harmonized guidance for nonclinical safety studies for pharmaceuticals (reduction of animal use)	FDA activity	U.S.: Accepted via ICH Guidance Document M3(R2) in 2010 EU: Accepted via ICH Guidance Document M3(R2) in 2010
Multiple Toxicities	Harmonized guidance for preclinical safety evaluation of biotechnology-derived pharmaceuticals (reduction of animal use)	FDA activity	U.S.: Accepted via ICH Guidance Document S6(R1) in 2012 EU: Accepted via ICH Guidance Document S6(R1) in 2012
Acute Oral Systemic Toxicity	In vitro assays for paralytic shellfish toxin detection (replacement of animal use)	FDA activity	U.S.: Receptor binding assay listed as approved method in National Shellfish Sanitation Program Guide for Control of Molluscan Shellfish (2013)
Biologics Testing	ELISA test for batch potency testing of human tetanus vaccines (refinement: antibody quantification)	FDA activity	U.S.: 21 CFR 610.10; use reviewed on a case-by-case basis EU: Published in European Pharmacopeia (2003)
Biologics Testing	ToBI test for batch potency testing of human tetanus vaccines (refinement: antibody quantification)	FDA activity	U.S.: 21 CFR 610.10; use reviewed on a case-by-case basis EU: Published in European Pharmacopeia (2003)
Biologics Testing	Use of humane endpoints in animal testing of biological products (refinement of animal use)	USDA activity	U.S.: Addressed in 9 CFR 117.4e; CVB Notice No. 04-09 (2004)
Biologics Testing	Relevance of the target animal safety test for batch safety testing of vaccines for veterinary use (refinement of animal use)	USDA activity	U.S.: 9 CFR 113.4 provides for authorizing exemptions from standard requirements EU: Published in European Pharmacopeia (2004)
Biologics Testing	ELISA test for batch potency testing of Leptospira interrogans serovar Pomona (replacement: antigen quantification)	USDA activity	U.S.: USDA SAM 624 (2008, updated 2017)
Biologics Testing	ELISA test for batch potency testing of Leptospira interrogans serovar Canicola (replacement: antigen quantification)	USDA activity	U.S.: USDA SAM 625 (2008, updated 2017)
Biologics Testing	ELISA test for batch potency testing of Leptospira interrogans serovar Icterohaemorrhagiae (replacement: antigen quantification)	USDA activity	U.S.: USDA SAM 627 (2008, updated 2017)
Biologics Testing	ELISA test for batch potency testing of erysipelas vaccines (replacement: antigen quantification)	USDA activity	U.S.: USDA SAM 613 (2008) EU: Published in European Pharmacopeia
Biologics Testing	ELISA test for batch potency testing of Leptospira kirschneri serovar Grippotyphosa (replacement: antigen quantification)	USDA activity	U.S.: USDA SAM 626 (2009, updated 2017)
Biologics Testing	Cell-based potency assay for stability and potency of botulinum neurotoxin type A products (replacement of animal use)	FDA activity	U.S.: Allergan, Inc., method accepted by FDA in 2011
Biologics Testing	USDA guidelines on master reference qualification and requalification for vaccine potency assays (reduction of animal use)	USDA activity	U.S.: Addressed in 9 CFR 113.8(d)(2); VS Memorandum 800.211 (2011, updated 2023)
Biologics Testing	USDA guidelines for validation of in vitro potency assays (replacement of animal use)	USDA activity	U.S.: VS Memorandum 800.112 (updated 2015): clarifies information found in 9 CFR 102.3, 9 CFR 113.8, and VS Memo 800.50.
Biologics Testing	USDA guidelines on use of humane endpoints and methods in animal testing of biological products (refinement of animal use)	USDA activity	U.S.: Addressed in 9 CFR 117.4(e); CVB Notice No. 12-12 (2012)
Biologics Testing	Serum neutralization test for potency testing of inactivated veterinary rabies vaccines (reduction and refinement of animal use)	ICCVAM workshop in 2011	EU: Published in European Pharmacopoeia Monograph 0451 (2012)
Biologics Testing	Alternative test procedure for tuberculin, PPD Bovis, intradermic (reduces animal use by 65%)	USDA activity	U.S.: Applies to 9 CFR 113.409(c); described in VS Memorandum 800.114 (2012)
Biologics Testing	FDA guidance on preclinical assessment of investigational cellular and gene therapy products (minimization of animal use)	FDA activity	U.S.: Guidance (2013) applies to products regulated under 42 U.S.C. 262(i)
Biologics Testing	USDA guidance for obtaining an exemption from in vivo testing of Leptospira bacterins	USDA activity	U.S.: Applies to 9 CFR 113.101-104; VS Memorandum 800.102 (2013)
Biologics Testing	USDA elimination of the upper limit LD50 for rabies vaccine potency testing (reduction of animal use)	USDA activity	U.S.: Modification to SAM 308; CVB Notice No. 13-10 (2013)
Biologics Testing	USDA provision for exemption to requirement for animal safety testing of veterinary biologics (reduction of animal use)	USDA activity	U.S.: Applies to 9 CFR 113.4; VS Memorandum 800.116 (2013, updated 2017) EU: Accepted via VICH Guideline 50 (2014)
Biologics Testing	USDA provision for option to remove back-titration hamsters for potency assays for Leptospira serogroups Canicola and Icterohaemorrhagiae (reduction of animal use)	USDA activity	U.S.: Applies to 9 CFR 113.102 and 113.103; CVB Notice No. 15-13 (2015)
Biologics Testing	USDA provision for option to remove back-titration hamsters for potency assays for Leptospira serogroups Pomona and Grippotyphosa (reduction of animal use)	USDA activity	U.S.: Applies to 9 CFR 113.102 and 113.103; CVB Notice No. 17-06 (2017)
Dermal Corrosivity and Irritation	Corrositex® in vitro membrane barrier skin corrosivity test (replacement of animal use)	ICCVAM peer review and report; recommended in 1999	U.S.: Accepted by U.S. agencies in 1999; 49 CFR 173.137 (2011) EU: Accepted via OECD Test Guideline 435 (2006, updated 2015)
Dermal Corrosivity and Irritation	EpiSkin™ in vitro human skin model skin corrosivity test (replacement of animal use)	ICCVAM review and report; recommended in 2002	U.S.: Accepted via OECD Test Guideline 431 (2004, updated 2013, 2014, 2015, 2016, 2019); 49 CFR 173.137 (2011) EU: Accepted via OECD Test Guideline 431 (2004, updated 2013, 2014, 2015, 2016, 2019)
Dermal Corrosivity and Irritation	EpiDerm™ in vitro human skin model skin corrosivity test (replacement of animal use)	ICCVAM review and report; recommended in 2002	U.S.: Accepted via OECD Test Guideline 431 (2004, updated 2013, 2014, 2015, 2016, 2019); 49 CFR 173.137 (2011) EU: Accepted via OECD Test Guideline 431 (2004, updated 2013, 2014, 2015, 2016, 2019)
Dermal Corrosivity and Irritation	SkinEthic™ in vitro human skin model skin corrosivity test (replacement of animal use)	ICCVAM contributed to U.S. OECD test guideline review	U.S.: Accepted via OECD Test Guideline 431 (2004, updated 2013, 2014, 2015, 2016, 2019) EU: Accepted via OECD Test Guideline 431 (2004, updated 2013, 2014, 2015, 2016, 2019)
Dermal Corrosivity and Irritation	Rat TER in vitro skin corrosivity test (replacement of animal use)	ICCVAM review and report; recommended in 2002	U.S.: Accepted via OECD Test Guideline 430 (2004, updated 2013, 2015) EU: Accepted via OECD Test Guideline 430 (2004, updated 2013, 2015)
Dermal Corrosivity and Irritation	Reconstructed human epidermis in vitro test method for skin corrosivity testing (replacement of animal use)	ICCVAM contributed to U.S. OECD test guideline review	U.S.: Accepted via OECD Test Guideline 431 (2004, last updated 2019) EU: Accepted via OECD Test Guideline 431 (2004, last updated 2019)
Dermal Corrosivity and Irritation	EpiSkin™ in vitro human skin model skin irritation test (replacement of animal use)	ICCVAM working group contributed to U.S. OECD test guideline review	U.S.: Accepted via OECD Test Guideline 439 (2010, last updated 2021) EU: Accepted via OECD Test Guideline 439 (2010, last updated 2021)
Dermal Corrosivity and Irritation	EpiDerm™ in vitro human skin model skin irritation test (replacement of animal use)	ICCVAM working group contributed to U.S. OECD test guideline review	U.S.: Accepted via OECD Test Guideline 439 (2010, last updated 2021) EU: Accepted via OECD Test Guideline 439 (2010, last updated 2021)
Dermal Corrosivity and Irritation	SkinEthic™ in vitro human skin model skin irritation test (replacement of animal use)	ICCVAM working group contributed to U.S. OECD test guideline review	U.S.: Accepted via OECD Test Guideline 439 (2010, last updated 2021) EU: Accepted via OECD Test Guideline 439 (2010, last updated 2021)
Dermal Corrosivity and Irritation	Guidance document on an integrated approach for testing and assessment for skin corrosion and irritation (replacement of animal use)	-	U.S.: Accepted via OECD Guidance Document 203 (2014) EU: Accepted via OECD Guidance Document 203 (2014)
Dermal Phototoxicity	3T3 NRU phototoxicity test for skin photo-irritation (replacement of animal use)	ICCVAM contributed to U.S. OECD test guideline review	U.S.: Accepted via OECD Test Guideline 432 (2004, updated 2019) EU: Accepted via OECD Test Guideline 432 (2004, updated 2019)
Dermal Phototoxicity	3T3 NRU phototoxicity test: application to UV filter chemicals (replacement of animal use)	ICCVAM contributed to U.S. OECD test guideline review	U.S.: Accepted via OECD Test Guideline 432 (2004, updated 2019) EU: Accepted via OECD Test Guideline 432 (2004, updated 2019)
Dermal Phototoxicity	FDA guidance on photosafety evaluation of pharmaceuticals (reduction or replacement of animal use)	FDA activity	U.S.: Accepted via ICH Guideline S10 (2015) EU: Accepted via ICH Guideline S10 (2015)
Dermal Phototoxicity	Reactive oxygen species assay for photoreactivity (replacement of animal use)	-	U.S.: Accepted via OECD Test Guideline 495 (2019) EU: Accepted via OECD Test Guideline 495 (2019)
Developmental and Reproductive Toxicity	Extended one-generation reproductive toxicity study (reduction of animal use)	ICCVAM agencies contributed to U.S. OECD test guideline review	U.S.: Accepted via OECD Test Guideline 443 (2012, updated 2018) EU: Accepted via OECD Test Guideline 443 (2012, updated 2018)
Ecotoxicity	Revised protocol for evaluation of nontoxic replacements for lead shot (reduction of animal use)	Dept. of the Interior activity	U.S.: 50 CFR 20.134
Ecotoxicity	Determination of in vitro intrinsic clearance using cryopreserved rainbow trout hepatocytes	-	U.S.: Accepted via OECD Test Guideline 319A (2018) EU: Accepted via OECD Test Guideline 319A (2018)
Ecotoxicity	Determination of in vitro intrinsic clearance using rainbow trout liver S9 sub-cellular fraction	-	U.S.: Accepted via OECD Test Guideline 319B (2018) EU: Accepted via OECD Test Guideline 319B (2018)
Endocrine Disruptors	Stably transfected transactivation in vitro assay to detect estrogen receptor agonists and antagonists (replacement of animal use)	ICCVAM working group contributed to U.S. OECD test guideline review, expert consultation meetings	U.S.: Accepted via OECD Test Guideline 455 (2009, last updated 2021); EPA OPPTS 890.1300 (2009) EU: Accepted via OECD Test Guideline 455 (2009, last updated 2021)
Endocrine Disruptors	In vitro H295R steroidogenesis assay (replacement of animal use)	ICCVAM working group contributed to U.S. OECD test guideline review	U.S.: Accepted via OECD Test Guideline 456 (2011, last updated 2023) EU: Accepted via OECD Test Guideline 456 (2011, last updated 2023)
Endocrine Disruptors	In vitro VM7Luc ER TA agonist assay to identify substances that induce human ER activity (replacement of animal use) Link to TSAR Test Method TM2016-09	ICCVAM peer review and report; recommendations in 2012	U.S.: Accepted by agencies in 2012 EU: Accepted via OECD Test Guideline 457 (2011) OECD Test Guideline 457 was deleted in 2018 as the method now falls within Test Guideline 455
Endocrine Disruptors	In vitro VM7Luc ER TA antagonist assay to identify substances that inhibit human ER activity (replacement of animal use) Link to TSAR Test Method TM2016-09	ICCVAM peer review and report; recommendations in 2012	U.S.: Accepted by agencies in 2012 EU: Accepted via OECD Test Guideline 457 (2011) OECD Test Guideline 457 was deleted in 2018 as the method now falls within Test Guideline 455
Endocrine Disruptors	Performance-based test guideline for human recombinant estrogen receptor binding assays	ICCVAM agency scientists contributed to OECD test guideline review	U.S.: Accepted via OECD Test Guideline 493 (2015) EU: Accepted via OECD Test Guideline 493 (2015)
Endocrine Disruptors	Integrated testing strategy to identify chemicals with the potential to interact with the estrogen receptor	Developed and validated by EPA and NICEATM scientists	U.S.: Accepted by EPA in 2015 as an alternative to three Tier 1 tests used in the Endocrine Disruptor Screening Program
Endocrine Disruptors	Stably transfected human AR TA assay for detection of androgenic agonist and antagonist activity of chemicals	ICCVAM agency scientists contributed to OECD test guideline review	U.S.: Accepted via OECD Test Guideline 458 (2016, last updated 2023) EU: Accepted via OECD Test Guideline 458 (2016, last updated 2023)
Endocrine Disruptors	Aromatase (human recombinant)	EPA activity	U.S.: EPA EDSP Test Guideline OPPTS 890.1200
Genetic Toxicity/ Carcinogenicity	Guidance on testing for carcinogenicity of pharmaceuticals (reduction of animal use)	FDA activity	U.S.: Accepted via ICH Guidance Document S1B(R1) in 2022 EU: Accepted via ICH Guidance Document S1B(R1) in 2022
Genetic Toxicity/ Carcinogenicity	Carcinogenicity studies (reduction of animal use)	-	U.S.: Accepted via OECD Test Guideline 451 (1981, updated 2018) EU: Accepted via OECD Test Guideline 451 (1981, updated 2018)
Genetic Toxicity/ Carcinogenicity	In vitro mammalian chromosomal aberration test	-	U.S.: Accepted via OECD Test Guideline 473 (1983, last updated 2016) EU: Accepted via OECD Test Guideline 473 (1983, last updated 2016)
Genetic Toxicity/ Carcinogenicity	In vitro mammalian cell micronucleus test (replacement of animal use)	ICCVAM working group contributed to U.S. OECD test guideline review	U.S.: Accepted via OECD Test Guideline 487 (2010, last updated 2023) EU: Accepted via OECD Test Guideline 487 (2010, last updated 2023)
Genetic Toxicity/ Carcinogenicity	In vivo mammalian alkaline comet assay (reduction of animal use)	NICEATM and ICCVAM scientists served on validation management team	U.S.: Accepted via OECD Test Guideline 489 (2014, updated 2016) EU: Accepted via OECD Test Guideline 489 (2014, updated 2016)
Genetic Toxicity/ Carcinogenicity	In vitro mammalian cell gene mutation tests using the thymidine kinase assay	ICCVAM agency scientists contributed to OECD test guideline review	U.S.: Accepted via OECD Test Guideline 490 (2015, updated 2016) EU: Accepted via OECD Test Guideline 490 (2015, updated 2016)
Genetic Toxicity/ Carcinogenicity	In vitro mammalian cell gene mutation tests using the HPRT and XPRT genes	-	U.S.: Accepted via OECD Test Guideline 476 (2016) EU: Accepted via OECD Test Guideline 476 (2016)
Genetic Toxicity/ Carcinogenicity	Bacterial reverse mutation test	-	U.S.: Accepted via OECD Test Guideline 471 (2020) EU: Accepted via OECD Test Guideline 471 (2020)
Genetic Toxicity/ Carcinogenicity	In vitro BALB/c 3T3 cell transformation assay	-	U.S.: Included on EPA list of alternative methods and strategies considered for use under TSCA (2019) EU: Evaluated by EURL ECVAM (2012)
Multiple Toxicities	Guidance document on developing and assessing adverse outcome pathways	-	U.S.: Accepted via OECD Guidance Document 184 (2013) EU: Accepted via OECD Guidance Document 184
Multiple Toxicities	Guidance document on the reporting of defined approaches to be used within integrated approaches to testing and assessment	NICEATM and ICCVAM contributed to development of the guidance document	U.S.: Accepted via OECD Guidance Document 255 (2016) EU: Accepted via OECD Guidance Document 255
Multiple Toxicities	Guidance for waiving or bridging of mammalian acute toxicity tests for pesticides and pesticide products (reduction of animal use)	EPA activity	U.S.: EPA guidance (2012) provides for authorizing exemptions from standard requirements under 40 CFR 158.500, 40 CFR 161.340, other regulations and notices
Multiple Toxicities	Guidance regarding replacement, reduction, and refinement of animal use for testing done under the Federal Hazardous Substances Act	CPSC activity	U.S.: 15 U.S.C. 1261–1278
Multiple Toxicities	Process for evaluating and implementing alternative approaches to traditional in vivo acute toxicity studies for FIFRA regulatory use (replacement and reduction of animal use)	EPA activity	U.S.: 40 CFR 158
Ocular Corrosivity and Irritation	Bovine corneal opacity and permeability in vitro test method to identify severe eye irritants/corrosives or chemicals not requiring eye hazard classification (replacement of animal use)	ICCVAM review and report; recommended in 2007	U.S.: Accepted by agencies in 2008; 2013 update accepted via OECD Test Guideline 437 (2009, last updated 2023) EU: Accepted via OECD Test Guideline 437 (2009, last updated 2023)
Ocular Corrosivity and Irritation	Isolated chicken eye in vitro test method to identify severe eye irritants/corrosives or chemicals not requiring eye hazard classification (replacement of animal use)	ICCVAM review and report; recommended in 2007	U.S.: Accepted by agencies in 2008; 2013 update accepted via OECD Test Guideline 438 (2009, last updated 2023) EU: Accepted via OECD Test Guideline 438 (2009, last updated 2023)
Ocular Corrosivity and Irritation	Cytosensor microphysiometer in vitro test method for eye safety testing (replacement of animal use)	ICCVAM peer review and report; recommended in 2010	Accepted by U.S. agencies in 2011 (OECD test guideline under consideration)
Ocular Corrosivity and Irritation	Use of anesthetics, analgesics, and humane endpoints for in vivo eye safety testing (refinement of animal use)	ICCVAM peer review and report; recommended in 2010	U.S.: Accepted by agencies in 2011 EU: Accepted via updated OECD Test Guideline 405 (2012, last revised 2023)
Ocular Corrosivity and Irritation	Histopathological evaluation and collection of data for identification of nonsevere irritants using in vitro methods	-	U.S.: Accepted via OECD Guidance Document 160 (2011) EU: Accepted via OECD Guidance Document 160 (2011)
Ocular Corrosivity and Irritation	Non-animal testing scheme for assessing eye irritation potential of antimicrobial cleaning products	EPA activity	U.S.: EPA guidance (2013, updated 2015) describes a non-animal testing scheme to satisfy the in vivo data requirement for eye irritation in 40 CFR 158 for antimicrobial cleaning products
Ocular Corrosivity and Irritation	Recommendation to perform weight-of-evidence analysis and non-animal testing strategies before in vivo testing	NICEATM and ICCVAM scientists served on expert panel and commented on draft test guideline	U.S.: Accepted via updated OECD Test Guideline 405 (2017, last updated 2023) EU: Accepted via updated OECD Test Guideline 405 (2017, last updated 2023)
Ocular Corrosivity and Irritation	In vitro fluorescein leakage test method for identifying ocular corrosives and severe irritants (replacement of animal use)	ICCVAM contributed to OECD test guideline review	U.S.: Accepted via OECD Test Guideline 460 (2012, last updated 2023) EU: Accepted via OECD Test Guideline 460 (2012, last updated 2023)
Ocular Corrosivity and Irritation	Short time exposure test for identification of ocular corrosives and substances not requiring ocular hazard labeling (replacement of animal use)	ICCVAM working group scientists served on validation management team and commented on draft test guideline	U.S.: Accepted via OECD Test Guideline 491 (2015, last updated 2023) EU: Accepted via OECD Test Guideline 491 (2015, last updated 2023)
Ocular Corrosivity and Irritation	Reconstructed human cornea-like epithelium test for identification of substances not requiring ocular hazard labeling (replacement of animal use)	ICCVAM working group scientists served on validation management team and commented on draft test guideline	U.S.: Accepted via OECD Test Guideline 492 (2015, last updated 2023) EU: Accepted via OECD Test Guideline 492 (2015, last updated 2023)
Ocular Corrosivity and Irritation	Vitrigel-Eye Irritancy test method for identifying chemicals not requiring ocular hazard labeling (replacement of animal use)	NICEATM scientists served on the management team for the validation study; ICCVAM scientist participated in the peer review	U.S.: Accepted via OECD Test Guideline 494 (2019, updated 2021) EU: Accepted via OECD Test Guideline 494 (2019, updated 2021)
Ocular Corrosivity and Irritation	In vitro macromolecular test method for identifying chemicals inducing serious eye damage and chemicals not requiring classification for eye irritation or serious eye damage	-	U.S.: Accepted via OECD Test Guideline 496 (2019, last updated 2023) EU: Accepted via OECD Test Guideline 496 (2019, last updated 2023)
Pyrogen Testing	Human whole blood/interleukin-1β in vitro pyrogen test (replacement of animal use)	ICCVAM peer review and report; recommended in 2008	U.S.: Accepted by FDA in 2009; use addressed in June 2012 FDA guidance on pyrogen testing EU: Published in European Pharmacopeia
Pyrogen Testing	Human whole blood/interleukin-1β in vitro pyrogen test: application of cryopreserved human whole blood (replacement of animal use)	ICCVAM peer review and report; recommended in 2008	U.S.: Accepted by FDA in 2009; use addressed in June 2012 FDA guidance on pyrogen testing EU: Published in European Pharmacopeia
Pyrogen Testing	Human whole blood/interleukin-6 in vitro pyrogen test (replacement of animal use)	ICCVAM peer review and report; recommended in 2008	U.S.: Accepted by FDA in 2009; use addressed in June 2012 FDA guidance on pyrogen testing EU: Published in European Pharmacopeia
Pyrogen Testing	Human peripheral blood mononuclear cell/interleukin-6 in vitro pyrogen test (replacement of animal use)	ICCVAM peer review and report; recommended in 2008	U.S.: Accepted by FDA in 2009; use addressed in June 2012 FDA guidance on pyrogen testing EU: Published in European Pharmacopeia
Pyrogen Testing	Monocytoid cell line Mono Mac 6/interleukin-6 in vitro pyrogen test (replacement of animal use)	ICCVAM peer review and report; recommended in 2008	U.S.: Accepted by FDA in 2009; use addressed in June 2012 FDA guidance on pyrogen testing EU: Published in European Pharmacopeia
Pyrogen Testing	In vitro monocyte activation type pyrogen test (replacement of animal use)	FDA activity	U.S.: FDA guidance (2012) provides for use in place of USP methods
Skin Sensitization	LLNA for skin sensitization (reduction and refinement of animal use)	ICCVAM peer review and report; recommended in 1999	U.S.: Accepted by agencies in 1999; EPA OPPTS 870.2600 (2003), FDA Guidance for Industry: Immunotoxicology Evaluation of Investigational New Drugs (2002), ISO (2002) EU: Accepted via OECD Test Guideline 429 (2002, updated 2010)
Skin Sensitization	Updated LLNA protocol (requires 20% fewer animals)	ICCVAM peer review and report; recommended in 2009	U.S.: Accepted by agencies in 2010; EPA updated policy on the use of the LLNA for end-use pesticide products in 2011 EU: Accepted via updated OECD Test Guideline 429 (2010)
Skin Sensitization	Reduced LLNA protocol (requires 40% fewer animals by using only the high dose group)	ICCVAM peer review and report; recommended in 2009	U.S.: Accepted by agencies in 2010 EU: Accepted via updated OECD Test Guideline 429 (2010)
Skin Sensitization	LLNA: DA for skin sensitization testing (a nonradioisotopic LLNA test method)	ICCVAM peer review and report; recommended in 2010	U.S.: Accepted by agencies in 2010 EU: Accepted via updated OECD Test Guideline 442A (2010)
Skin Sensitization	LLNA: BrdU-ELISA for skin sensitization testing (a nonradioisotopic LLNA test method)	ICCVAM peer review and report; recommended in 2010	U.S.: Accepted by agencies in 2010; updates accepted via OECD Test Guideline 442B (2018) EU: Accepted via OECD Test Guideline 442B (2010, updated 2018)
Skin Sensitization	LLNA for potency categorization of skin sensitizers (refinement and reduction of animal use)	ICCVAM peer review and report; recommendations in 2011	U.S.: Accepted by agencies in 2012
Skin Sensitization	Adverse outcome pathway for skin sensitization initiated by covalent binding to proteins (Part 1) (Part 2)	-	U.S.: Accepted via OECD Guidance Document 168 (2012) EU: Accepted via OECD Guidance Document 168 (2012)
Skin Sensitization	CPSC guidance to clarify the definition of “strong sensitizer” (reduction or replacement of animal use)	CPSC activity	U.S.: CPSC guidance (2014) clarifies acceptability of in vitro test data for testing required under 15 U.S.C. 1261–1278 (Federal Hazardous Substances Act)
Skin Sensitization	In chemico skin sensitization test (reduction or replacement of animal use)	NICEATM and ICCVAM scientists served on validation management team and commented on draft and updated test guideline	U.S.: Accepted via OECD Test Guideline 442C (2015, last updated 2023) EU: Accepted via OECD Test Guideline 442C (2015, last updated 2023)
Skin Sensitization	In vitro skin sensitization test (ARE-Nrf2 luciferase test: reduction or replacement of animal use)	NICEATM and ICCVAM scientists commented on draft test guideline	U.S.: Accepted via OECD Test Guideline 442D (2015, last updated 2022) EU: Accepted via OECD Test Guideline 442D (2015, last updated 2022)
Skin Sensitization	Guidance document on the reporting of defined approaches and individual information sources to be used within IATA for skin sensitisation	NICEATM and ICCVAM submitted a case study included in the guidance document	U.S.: Accepted via OECD Guidance Document 256 (2016) EU: Accepted by via OECD Guidance Document 256 (2016)
Skin Sensitization	In vitro skin sensitization test (human cell line activation test: reduction or replacement of animal use)	NICEATM and ICCVAM scientists commented on draft test guideline	U.S.: Accepted via OECD Test Guideline 442E (2016, last updated 2023) EU: Accepted via OECD Test Guideline 442E (2016, last updated 2023)
Skin Sensitization	Policy on use of non-animal defined approaches to identify potential skin sensitizers	EPA activity	U.S.: EPA interim policy (2018) applies to testing of single chemicals required under FIFRA and TSCA