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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.

Acute Dermal Systemic Toxicity
Table listing alternative test methods that have been accepted by regulatory authorities in the U.S. and Europe; headers spanning all columns divide testmethod areas
Method ICCVAM or ICCVAM Agency Contributions U.S. Regulatory Acceptance/ Endorsement and Applicable Regulations OECD Adoption EU Regulatory Acceptance/ Endorsement
In vitro dermal absorption methods (replacement of animal use) ICCVAM working group contributed to U.S. OECD test guideline review, expert consultation meetings Accepted by U.S. via OECD TG 428 OECD TG 428 (2004) Via OECD
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 Accepted by U.S. via OECD TG 402 OECD TG 402 (1987, revised 2017) Via OECD
Acute Oral Systemic Toxicity
Table listing alternative test methodsfor Acute Oral Systemic Toxicity that have been accepted by regulatory authorities in the U.S. and Europe
Method ICCVAM or ICCVAM Agency Contributions U.S. Regulatory Acceptance/ Endorsement and Applicable Regulations OECD Adoption EU Regulatory Acceptance/ Endorsement
Up-and-down procedure for acute oral toxicity (reduction and refinement of animal use) ICCVAM peer review and report; recommended in 2001 Accepted by U.S. in 2003; EPA OPPTS 870.1100 (2002) OECD TG 425 (2001) Via OECD
Fixed dose procedure for acute oral toxicity (reduction and refinement of animal use) ICCVAM working group contributed to test guideline development Accepted by U.S. via OECD TG 420 OECD TG 420 (2001) Via OECD
Acute toxic class method for acute oral toxicity (reduction and refinement of animal use) ICCVAM working group contributed to test guideline development Accepted by U.S. via OECD TG 423 OECD TG 423 (2001) Via OECD
Avian acute oral toxicity test (reduction of animal use) ICCVAM working group contributed to U.S. OECD test guideline review Accepted by U.S. via OECD TG 223 OECD TG 223 (2010) Via OECD
Acute Inhalation Toxicity
Table listing alternative test methodsthat have been accepted by regulatory authorities in the U.S. and Europe; headers spanning all columns divide test method areas
Method ICCVAM or ICCVAM Agency Contributions U.S. Regulatory Acceptance/ Endorsement and Applicable Regulations OECD Adoption EU Regulatory Acceptance/ Endorsement
Inhalation toxicity - acute toxic class method (reduction and refinement of animal use) ICCVAM working group contributed to U.S. OECD test guideline review Accepted by U.S. via OECD TG 436 OECD TG 436 (2009) Via OECD
Inhalation toxicity testing - fixed concentration procedure (reduction and refinement of animal use) ICCVAM contributed to U.S. OECD test guideline review Accepted by U.S. via OECD TG 433 OECD TG 433 (2017) Via OECD
Biologics Testing
Table listing alternative test methodsfor Biologics Testing that have been accepted by regulatory authorities in the U.S. and Europe
Method ICCVAM or ICCVAM Agency Contributions U.S. Regulatory Acceptance/ Endorsement and Applicable Regulations OECD Adoption EU Regulatory Acceptance/ Endorsement
ELISA test for batch potency testing of human tetanus vaccines (refinement: antibody quantification) FDA activity 21 CFR 610.10; use reviewed on a case-by-case basis NA Published in European Pharmacopeia (2003)
ToBI test for batch potency testing of human tetanus vaccines (refinement: antibody quantification) FDA activity Per 21 CFR 610.10; use reviewed on a case-by-case basis NA Published in European Pharmacopeia (2003)
Use of humane endpoints in animal testing of biological products (refinement of animal use) USDA activity Addressed in 9 CFR 117.4e, Center for Veterinary Biologics (CVB) Notice No. 04-09 (2004) NA  
Rabies vaccine, humane endpoints (refinement of animal use) USDA activity Addressed in 9 CFR 117.4e, CVB Notice No. 04-09 (2004) NA  
Relevance of the target animal safety test for batch safety testing of vaccines for veterinary use (refinement of animal use) USDA activity 9 CFR 113.4 provides for authorizing exemptions from standard requirements NA Published in European Pharmacopeia (2004)
ELISA test for batch potency testing of Leptospira interrogans serovar Pomona (replacement: antigen quantification) USDA activity USDA Supplemental Assay Method (SAM) 624 (2008, updated 2017) NA  
ELISA test for batch potency testing of Leptospira interrogans serovar Canicola (replacement: antigen quantification) USDA activity USDA SAM 625 (2008, updated 2017) NA  
ELISA test for batch potency testing of Leptospira interrogans serovar Icterohaemorrhagiae (replacement: antigen quantification) USDA activity USDA SAM 627 (2008, updated 2017) NA  
ELISA test for batch potency testing of erysipelas vaccines
(replacement: antigen quantification)
USDA activity USDA SAM 613 (2008) NA Published in European Pharmacopeia
ELISA test for batch potency testing of Leptospira kirschneri serovar Grippotyphosa (replacement: antigen quantification) USDA activity USDA SAM 626 (2009, updated 2017) NA  
Cell-based potency assay for stability and potency of botulinum neurotoxin type A products (replacement of animal use) FDA activity Allergan, Inc., method accepted by FDA in 2011 NA  
USDA guidelines on master reference qualification and requalification for vaccine potency assays (reduction of animal use) USDA activity Addressed in 9 CFR 113.8(d)(2), Veterinary Services Memorandum 800.211 (2011) NA  
USDA guidelines for validation of in vitro potency assays (replacement of animal use) USDA activity Veterinary Services Memorandum 800.112 (updated 2015): clarifies information found in 9 CFR 102.3, 9 CFR 113.8, and VS Memo 800.50. NA  
USDA guidelines on use of humane endpoints and methods in animal testing of biological products (refinement of animal use) USDA activity Addressed in 9 CFR 117.4(e), CVB Notice No. 12-12 (2012) NA  
Serum neutralization test for potency testing of inactivated veterinary rabies vaccines (reduction and refinement of animal use) ICCVAM workshop in 2011 NA NA Published in European Pharmacopoeia Monograph 0451 (2012)
Alternative test procedure for tuberculin, PPD Bovis, intradermic (reduces animal use by 65%) USDA activity Applies to 9 CFR 113.409(c), described in Veterinary Services Memorandum 800.114 (2012) NA  
USDA elimination of the upper limit LD50 for rabies vaccine potency testing (reduction of animal use) USDA activity Modification to SAM 308, CVB Notice No. 13-10 (2013) NA  
USDA provision for exemption to requirement for testing Leptospira bacterins for potency in hamsters (reduction of animal use) USDA activity Applies to 9 CFR 113.101-104, Veterinary Services Memorandum 800.102 (2013) NA  
USDA provision for option to remove back-titration hamsters for potency assays for Leptospira serogroups Canicola and Icterohaemorrhagiae (reduction of animal use) USDA activity Applies to 9 CFR 113.102 and 113.103; Center for Veterinary Biologics Notices Nos. 15-13 (2015) and 17-06 (2017) NA  
Dermal Corrosivity and Irritation
Table listing alternative test methodsfor Dermal Corrosivity that have been accepted by regulatory authorities in the U.S. and Europe
Method ICCVAM or ICCVAM Agency Contributions U.S. Regulatory Acceptance/ Endorsement and Applicable Regulations OECD Adoption EU Regulatory Acceptance/ Endorsement
Corrositex® in vitro membrane barrier skin corrosivity test (replacement of animal use) ICCVAM peer review and report; recommended in 1999 Accepted by U.S. agencies in 1999; 49 CFR 173.137 (2011) OECD TG 435 (2006, updated 2015) Via OECD
EpiSkin™ in vitro human skin model skin corrosivity test (replacement of animal use) ICCVAM review and report; recommended in 2002 Accepted by U.S. via OECD TG 431; 49 CFR 173.137 (2011) OECD TG 431 (2004, updated 2013, 2014, 2015, 2016) Via OECD
EpiDerm™ in vitro human skin model skin corrosivity test (replacement of animal use) ICCVAM review and report; recommended in 2002 Accepted by U.S. via OECD TG 431; 49 CFR 173.137 (2011) OECD TG 431 (2004, updated 2013, 2014, 2015, 2016) Via OECD
SkinEthic™ in vitro human skin model skin corrosivity test (replacement of animal use) ICCVAM contributed to U.S. OECD test guideline review Accepted by U.S. via OECD TG 431 (meets performance standards 2006) OECD TG 431 (2004, updated 2013, 2014, 2015, 2016) Via OECD
Rat TER in vitro skin corrosivity test (replacement of animal use) ICCVAM review and report; recommended in 2002 Accepted by U.S. via OECD TG 430 OECD TG 430 (2004, updated 2013, 2015) Via OECD
Reconstructed human epidermis in vitro test method for skin corrosivity testing (replacement of animal use) ICCVAM contributed to U.S. OECD test guideline review Accepted by U.S. via OECD TG 431 (meets performance standards 2009) OECD TG 431 (2004, updated 2013, 2014, 2015, 2016) Via OECD
EpiSkin™ in vitro human skin model skin irritation test (replacement of animal use) ICCVAM working group contributed to U.S. OECD test guideline review Accepted by U.S. via OECD TG 439 OECD TG 439 (2010, updated 2013, 2015) Via OECD
EpiDerm™ in vitro human skin model skin irritation test (replacement of animal use) ICCVAM working group contributed to U.S. OECD test guideline review Accepted by U.S. via OECD TG 439 OECD TG 439 (2010, updated 2013, 2015) Via OECD
SkinEthic™ in vitro human skin model skin irritation test (replacement of animal use) ICCVAM working group contributed to U.S. OECD test guideline review Accepted by U.S. via OECD TG 439 OECD TG 439 (2010, updated 2013, 2015) Via OECD
Dermal Phototoxicity
Table listing alternative test methodsfor dermal phototoxicity that have been accepted by regulatory authorities in the U.S. and Europe
Method ICCVAM or ICCVAM Agency Contributions U.S. Regulatory Acceptance/ Endorsement and Applicable Regulations OECD Adoption EU Regulatory Acceptance/ Endorsement
3T3 NRU phototoxicity test for skin photo-irritation (replacement of animal use) ICCVAM contributed to U.S. OECD test guideline review Accepted by U.S. via OECD TG 432 OECD TG 432 (2004) Via OECD
3T3 NRU phototoxicity test: application to UV filter chemicals (replacement of animal use) ICCVAM contributed to U.S. OECD test guideline review Accepted by U.S. via OECD TG 432 OECD TG 432 (2004) Via OECD
Ecotoxicity
Table listing alternative test methodsfor Biologics Testing that have been accepted by regulatory authorities in the U.S. and Europe
Method ICCVAM or ICCVAM Agency Contributions U.S. Regulatory Acceptance/ Endorsement and Applicable Regulations OECD Adoption EU Regulatory Acceptance/ Endorsement
Revised protocol for evaluation of nontoxic replacements for lead shot (reduction of animal use) Dept. of the Interior activity 50 CFR 20.134 NA  
Endocrine Disruptors
Table listing pyrogen test methodsthat have been accepted by regulatory authorities in the U.S. and Europe
Method ICCVAM or ICCVAM Agency Contributions U.S. Regulatory Acceptance/ Endorsement and Applicable Regulations OECD Adoption EU Regulatory Acceptance/ Endorsement
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 Accepted by U.S. via OECD TG 455; EPA OPPTS 890.1300 (2009) OECD TG 455 (2009, updated 2012, 2015, 2016) Via OECD
In vitro H295R steroidogenesis assay (replacement of animal use) ICCVAM working group contributed to U.S. OECD test guideline review Accepted by U.S. agencies via OECD TG 456 OECD TG 456 (2011) Via OECD
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 Accepted by U.S. agencies in 2012 OECD TG 457 (2011) Via OECD
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 Accepted by U.S. agencies in 2012 OECD TG 457 (2011) Via OECD
Performance-based test guideline for human recombinant estrogen receptor binding assays ICCVAM agency scientists contributed to contributed to OECD test guideline review Accepted by U.S. via OECD TG 493 OECD TG 493 (2015) Via OECD
Integrated testing strategy to identify chemicals with the potential to interact with the estrogen receptor Developed and validated by EPA and NICEATM scientists Accepted by EPA in 2015 as an alternative to three Tier 1 tests used in the Endocrine Disruptor Screening Program NA  
Stably transfected human androgen receptor transactivation assay for detection of androgenic agonist and antagonist activity of chemicals ICCVAM agency scientists contributed to contributed to OECD test guideline review Accepted by U.S. via OECD TG 458 OECD TG 458 (2016) Via OECD
Genetic Toxicity
Table listing alternative test methodsfor Biologics Testing that have been accepted by regulatory authorities in the U.S. and Europe
Method ICCVAM or ICCVAM Agency Contributions U.S. Regulatory Acceptance/ Endorsement and Applicable Regulations OECD Adoption EU Regulatory Acceptance/ Endorsement
In vitro mammalian chromosomal aberration test   Accepted by U.S. via OECD TG 473 OECD TG 473 (1983, updated 1997, 2014, 2016) Via OECD
In vitro mammalian cell micronucleus test (replacement of animal use) ICCVAM working group contributed to U.S. OECD test guideline review Accepted by U.S. via OECD TG 487 OECD TG 487 (2010, updated 2014, 2016) Via OECD
In vivo mammalian alkaline comet assay (reduction of animal use) NICEATM and ICCVAM scientists served on validation management team Accepted by U.S. via OECD TG 489 OECD TG 489 (2014, updated 2016) Via OECD
In vitro mammalian cell gene mutation tests using the thymidine kinase assay ICCVAM agency scientists contributed to OECD test guideline review Accepted by U.S. via OECD TG 490 OECD TG 490 (2015, updated 2016) Via OECD
In vitro mammalian cell gene mutation tests using the HPRT and XPRT genes   Accepted by U.S. via OECD TG 476 OECD TG 476 (2016) Via OECD
Immunotoxicity: Allergic Contact Dermatitis
Table listing alternative test methodsfor allergic contact dermatitis that have been accepted by regulatory authorities in the U.S. and Europe
Method ICCVAM or ICCVAM Agency Contributions U.S. Regulatory Acceptance/ Endorsement and Applicable Regulations OECD Adoption EU Regulatory Acceptance/ Endorsement
Murine local lymph node assay (LLNA) for skin sensitization (reduction and refinement of animal use) ICCVAM peer review and report; recommended in 1999 Accepted by U.S. agencies in 1999; EPA OPPTS 870.2600 (2003), FDA Guidance for Industry: Immunotoxicology Evaluation of Investigational New Drugs (2002), ISO (2002) OECD TG 429 (2002, updated 2010) Via OECD
Updated LLNA protocol (requires 20% fewer animals) ICCVAM peer review and report; recommended in 2009 Accepted by U.S. agencies in 2010; EPA updated policy on the use of the LLNA for end-use pesticide products in 2011 OECD TG 429 (2010) Via OECD
Reduced LLNA protocol (requires 40% fewer animals by using only the high dose group) ICCVAM peer review and report; recommended in 2009 Accepted by U.S. agencies in 2010 OECD TG 429 (2010) Via OECD
LLNA: DA for skin sensitization testing (a nonradioisotopic LLNA test method) ICCVAM peer review and report; recommended in 2010 Accepted by U.S. agencies in 2010 OECD TG 442A (2010) Via OECD
LLNA: BrdU-ELISA for skin sensitization testing (a nonradioisotopic LLNA test method) ICCVAM peer review and report; recommended in 2010 Accepted by U.S. agencies in 2010 OECD TG 442B (2010) Via OECD
LLNA for potency categorization of skin sensitizers (refinement and reduction of animal use) ICCVAM peer review and report; recommendations in 2011 Accepted by U.S. agencies in 2012 NA NA
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 Accepted by U.S. via OECD TG 442C OECD TG 442C (2015) Via OECD
In vitro skin sensitization test (ARE-Nrf2 luciferase test: reduction or replacement of animal use) NICEATM and ICCVAM scientists commented on draft TG Accepted by U.S. via OECD TG 442D OECD TG 442D (2015) Via OECD
In vitro skin sensitization test (human cell line activation test: reduction or replacement of animal use) NICEATM and ICCVAM scientists commented on draft TG Accepted by U.S. via OECD TG 442E OECD TG 442E (2016, updated 2017) Via OECD
Ocular Corrosivity and Irritation
Table listing ocular corrosivity and irritation test methodsthat have been accepted by regulatory authorities in the U.S. and Europe
Method ICCVAM or ICCVAM Agency Contributions U.S. Regulatory Acceptance/ Endorsement and Applicable Regulations OECD Adoption EU Regulatory Acceptance/ Endorsement
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 Accepted by U.S. agencies in 2008; 2013 update accepted via OECD TG 437 OECD TG 437 (2009, updated 2013, 2017) Via OECD
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 Accepted by U.S. agencies in 2008; 2013 update accepted via OECD TG 438 OECD TG 438 (2009, updated 2013, 2017) Via OECD
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 New OECD test guideline under consideration  
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 Accepted by U.S. agencies in 2011 Updated OECD TG 405 (2012, further revised 2017) Via OECD
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 Accepted by U.S. agencies via updated OECD TG 405 Updated OECD TG 405 (2017) Via OECD
In vitro fluorescein leakage test method for identifying ocular corrosives and severe irritants (replacement of animal use) ICCVAM contributed to OECD test guideline review Accepted by U.S. agencies via OECD TG 460 OECD TG 460 (2012, updated 2017) Via OECD
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 Accepted by U.S. agencies via OECD TG 491 OECD TG 491 (2015, updated 2017) Via OECD
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 Accepted by U.S. agencies via OECD TG 492 OECD TG 492 (2015, updated 2017) Via OECD
Pyrogen Testing
Table listing pyrogen test methodsthat have been accepted by regulatory authorities in the U.S. and Europe
Method ICCVAM or ICCVAM Agency Contributions U.S. Regulatory Acceptance/ Endorsement and Applicable Regulations OECD Adoption EU Regulatory Acceptance/ Endorsement
Human whole blood/interleukin-1β in vitro pyrogen test (replacement of animal use) ICCVAM peer review and report; recommended in 2008 Accepted by FDA in 2009; use addressed in June 2012 FDA guidance on pyrogen testing NA Published in European Pharmacopeia
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 Accepted by FDA in 2009; use addressed in June 2012 FDA guidance on pyrogen testing NA Published in European Pharmacopeia
Human whole blood/interleukin-6 in vitro pyrogen test (replacement of animal use) ICCVAM peer review and report; recommended in 2008 Accepted by FDA in 2009; use addressed in June 2012 FDA guidance on pyrogen testing NA Published in European Pharmacopeia
Human peripheral blood mononuclear cell/interleukin-6 in vitro pyrogen test (replacement of animal use) ICCVAM peer review and report; recommended in 2008 Accepted by FDA in 2009; use addressed in June 2012 FDA guidance on pyrogen testing NA Published in European Pharmacopeia
Monocytoid cell line Mono Mac 6/interleukin-6 in vitro pyrogen test (replacement of animal use) ICCVAM peer review and report; recommended in 2008 Accepted by FDA in 2009; use addressed in June 2012 FDA guidance on pyrogen testing NA Published in European Pharmacopeia
Reproductive and Developmental Toxicity
Table listing alternative test methodsthat have been accepted by regulatory authorities in the U.S. and Europe; headers spanning all columns divide testmethod areas
Method ICCVAM or ICCVAM Agency Contributions U.S. Regulatory Acceptance/ Endorsement and Applicable Regulations OECD Adoption EU Regulatory Acceptance/ Endorsement
Extended one-generation reproductive toxicity study (reduction of animal use) ICCVAM agencies contributed to U.S. OECD test guideline review Accepted by U.S. via OECD TG 443 OECD TG 443 (2012) Via OECD