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Alternative Methods Accepted by US Agencies

View a list of guidance documents on a separate webpage

This page lists alternative safety testing methods that are accepted by U.S. and international regulatory authorities, organized by testing category. Appropriate use of these methods and approaches can reduce animal use and improve animal welfare.

Toxicity Area Method ICCVAM or ICCVAM Agency Contributions Regulatory Acceptance/ Endorsement and Applicable Regulations
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 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, revised 2008); EPA OPPTS 870.1100 (2002)
  • EU: Accepted via OECD Test Guideline 425 (2001, revised 2008)
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 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)
  • EU: Accepted via OECD Test Guideline 433 (2017)
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; Center for Veterinary Biologics (CVB) Notice No. 04-09 (2004)
Biologics Testing Rabies vaccine, humane endpoints (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 Supplemental Assay Method (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); Veterinary Services Memorandum 800.211 (2011)
Biologics Testing USDA guidelines for validation of in vitro potency assays (replacement of animal use) USDA activity
  • U.S.: Veterinary Services 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 Veterinary Services Memorandum 800.114 (2012)
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 testing Leptospira bacterins for potency in hamsters (reduction of animal use) USDA activity
  • U.S.: Applies to 9 CFR 113.101-104; Veterinary Services Memorandum 800.102 (2013)
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; Center for Veterinary Biologics Notices Nos. 15-13 (2015) and 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 436 (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); 49 CFR 173.137 (2011)
  • EU: Accepted via OECD Test Guideline 431 (2004, updated 2013, 2014, 2015, 2016)
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); 49 CFR 173.137 (2011)
  • EU: Accepted via OECD Test Guideline 431 (2004, updated 2013, 2014, 2015, 2016)
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)
  • EU: Accepted via OECD Test Guideline 431 (2004, updated 2013, 2014, 2015, 2016)
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, updated 2013, 2014, 2015, 2016)
  • EU: Accepted via OECD Test Guideline 431 (2004, updated 2013, 2014, 2015, 2016)
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, updated 2013, 2015)
  • EU: Accepted via OECD Test Guideline 439 (2010, updated 2013, 2015)
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, updated 2013, 2015)
  • EU: Accepted via OECD Test Guideline 439 (2010, updated 2013, 2015)
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, updated 2013, 2015)
  • EU: Accepted via OECD Test Guideline 439 (2010, updated 2013, 2015)
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)
  • EU: Accepted via OECD Test Guideline 432 (2004)
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)
  • EU: Accepted via OECD Test Guideline 432 (2004)
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
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, updated 2012, 2015, 2016); EPA OPPTS 890.1300 (2009)
  • EU: Accepted via OECD Test Guideline 455 (2009, updated 2012, 2015, 2016)
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)
  • EU: Accepted via OECD Test Guideline 456 (2011)
Endocrine Disruptors In vitro BG1Luc ER TA agonist assay to identify substances that induce human ER activity (replacement of animal use) ICCVAM peer review and report; recommendations in 2012
  • U.S.: Accepted by agencies in 2012
  • EU: Accepted via OECD Test Guideline 457 (2011)
Endocrine Disruptors In vitro BG1Luc ER TA antagonist assay to identify substances that inhibit human ER activity (replacement of animal use) ICCVAM peer review and report; recommendations in 2012
  • U.S.: Accepted by agencies in 2012
  • EU: Accepted via OECD Test Guideline 457 (2011)
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 androgen receptor transactivation 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)
  • EU: Accepted via OECD Test Guideline 458 (2016)
Genetic Toxicity In vitro mammalian chromosomal aberration test  
  • U.S.: Accepted via OECD Test Guideline 473 (1983, updated 1997, 2014, 2016)
  • EU: Accepted via OECD Test Guideline 473 (1983, updated 1997, 2014, 2016)
Genetic Toxicity 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, updated 2014, 2016)
  • EU: Accepted via OECD Test Guideline 487 (2010, updated 2014, 2016)
Genetic Toxicity 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 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 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)
Immunotoxicity: Allergic Contact Dermatitis Murine local lymph node assay (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)
Immunotoxicity: Allergic Contact Dermatitis 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)
Immunotoxicity: Allergic Contact Dermatitis 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)
Immunotoxicity: Allergic Contact Dermatitis 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)
Immunotoxicity: Allergic Contact Dermatitis 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
  • EU: Accepted via updated OECD Test Guideline 442B (2010)
Immunotoxicity: Allergic Contact Dermatitis 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
Immunotoxicity: Allergic Contact Dermatitis In chemico skin sensitization test (direct peptide reactivity assay: reduction or replacement of animal use) NICEATM and ICCVAM scientists served on validation management team and commented on draft TG
  • U.S.: Accepted via OECD Test Guideline 442C (2015)
  • EU: Accepted via OECD Test Guideline 442C (2015)
Immunotoxicity: Allergic Contact Dermatitis In vitro skin sensitization test (ARE-Nrf2 luciferase test: reduction or replacement of animal use) NICEATM and ICCVAM scientists commented on draft TG
  • U.S.: Accepted via OECD Test Guideline 442D (2015)
  • EU: Accepted via OECD Test Guideline 442D (2015)
Immunotoxicity: Allergic Contact Dermatitis In vitro skin sensitization test (human cell line activation test: reduction or replacement of animal use) NICEATM and ICCVAM scientists commented on draft TG
  • U.S.: Accepted via OECD Test Guideline 442E (2016, updated 2017)
  • EU: Accepted via OECD Test Guideline 442E (2016, updated 2017)
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, updated 2013, 2017)
  • EU: Accepted via OECD Test Guideline 437 (2009, updated 2013, 2017)
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, updated 2013, 2017)
  • EU: Accepted via OECD Test Guideline 438 (2009, updated 2013, 2017)
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, further revised 2017)
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)
  • EU: Accepted via updated OECD Test Guideline 405 (2017)
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, updated 2017)
  • EU: Accepted via OECD Test Guideline 460 (2012, updated 2017)
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 TG
  • U.S.: Accepted via OECD Test Guideline 491 (2015, updated 2017)
  • EU: Accepted via OECD Test Guideline 491 (2015, updated 2017)
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 TG
  • U.S.: Accepted via OECD Test Guideline 492 (2015, updated 2017)
  • EU: Accepted via OECD Test Guideline 492 (2015, updated 2017)
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
Reproductive and Developmental Toxicity Testing 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)
  • EU: Accepted via OECD Test Guideline 443 (2012)