Table of Validated & Accepted Alternative Methods

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Table of Validated & Accepted Alternative Methods

Last updated: September 30, 2014
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Validation & Regulatory Acceptance Status of Alternative Test Methods & Testing Strategies

EndpointMethod NameTest Type1Endorsement of Scientific Validity (Links to ESAC Statements; ECVAM; ICCVAM; JaCVAM)Regulatory Acceptance
Lead AuthoritySubsequent Endorsement(s)International (Links to OECD: OECD TGs; Draft TGs; GDs; Draft GDs)

National/ Regional2

Acute mammalian toxicity (oral)Acute toxic class methodIn vivoESAC (2007)OECD TG 423 (2001)
Fixed dose procedureIn vivoESAC (2007)OECD TG 420 (2001)
Up-and-down procedureIn vivoICCVAM (2001)ESAC (2007)OECD TG 425 (2006)EPA OPPTS 870.1100 (2002)
Normal human keratinocyte neutral red uptake (NHK NRU) assayIn vitro3ICCVAM (2008)JaCVAM (2011)OECD GD 129 (2010)
Balb/c 3T3 neutral red uptake assayIn vitro3ICCVAM (2008)

ECVAM (2011)

JaCVAM (2011)

OECD GD 129 (2010)
Acute mammalian toxicity (hematotoxicity)Colony Forming Unit-Granulocyte/Macrophage Assay for acute neutropenia in humansIn vitroESAC (2006)
Acute mammalian toxicity (inhalation)Acute toxic class methodIn vivo

OECD TG 436 (2009)

OECD GD No. 153 (2011)

Acute toxicity testing of pesticidesGuidance for waiving or bridging of mammalian acute toxicity tests for pesticides (acute oral, dermal, inhalation; primary eye and dermal; dermal sensitization)In vivoEPA OPP (2012)
Biologics & VaccinesELISA for swine erysipelas vaccines batch potency testingIn vitroESAC (2002)ICCVAM agency, USDA (2009)

European Pharmacopeia, Monograph 064 (2004); USDA Supplemental Assay Method 613

ELISA for human tetanus vaccines batch potency testingIn vitroESAC (2000)European Pharmacopeia (2003); FDA, 21 CFR 610.10 (accepted on case-by-case basis)
Toxin binding inhibition test for human tetanus vaccines batch potency testingIn vitroESAC (2000)European Pharmacopeia; FDA, 21 CFR 610.10 (accepted on case-by-case basis)
Batch potency testing of erythropoietin concentrated solutionIn vivoESAC (2002)European Pharmacopeia, Monograph 0064
Deletion of target-animal safety test for batch safety testing of veterinary vaccines after consistency in 10 consecutive batchesNAESAC (2002)EU: General monograph 0062
ELISA for in vitro batch potency testing of Leptospira veterinary vaccinesIn vitroICCVAM agency, USDA (2008)USDA Supplemental Assay Methods 624, 625, 626, and 627
Use of humane endpoints in animal testing of veterinary biologics, including rabies vaccinesIn vivoICCVAM agency, USDA (2004; 2012)OECD GD No. 19USDA: 9 CFR Part 113, Notice No. 04-09 (2004); 9 CFR 113, CVB Notice No. 12-12
Veterinary vaccine potency assays: exemptions from Standard Requirements tests; master reference qualification and requalificationNAICCVAM agency, USDAUSDA: 9 CFR 113.409(c), Memorandum 800.114 (2012)
Cell-based assay for stability and potency of botulinum neurotoxin type A products15In vitroICCVAM agency, FDA; ICCVAM Workshop (2006)US FDA: Allergan, Inc., method accepted (2011)
Alternative test procedure for tuberculin, PPD Bovis, intradermicIn vivoICCVAM agency, USDAUSDA: 9 CFR 113.409(c), Memorandum 800.114 (2012)
CarcinogenicitySyrian Hamster Embryo Cell Transformation Assay (SHE CTA): SHE CTA performed at pH 6.7, SHE CTA performed at pH 7.04In vitroEURL ECVAM Recommendation (2012)

Draft OECD TG (2013)

OECD Detailed Review Paper No. 31

Bhas 42 Cell Transformation Assay (Bhas 42 CTA)4In vitroEURL ECVAM Recommendation (2012)Draft OECD TG (2013)
Chronic toxicityEnding 1-year dog studies of pesticidesIn vivoESAC (2006)Revised US EPA Pesticide Data Requirements
Dermal absorption/penetrationIn vitro skin absorption methodsIn vitroOECD Expert Group (2002)

OECD TG 428 (2004)

OECD GD 28 (2004)

OECD Guidance Notes No. 156

EcotoxicityAcute aquatic toxicity: Upper threshold concentration step-down approachIn vivoESAC (2006)OECD GD 126 (2010)
Acute avian toxicity (oral): Sequential testing procedure to minimizenumbers of birds usedIn vivoOECD TG 223 (2010)
Fish embryo (Zebrafish) toxicity18In vivoESAC (2013)OECD TG 236 (2013)
Endocrine active substancesAndrogen receptor binding assay (rat prostate cytosol)5Ex vivoOPPTS TG 890.1150 (EPA, 2009)
Aromatase inhibition assay (human recombinant)5In vitroOPPTS TG 890.1200 (EPA, 2009)
Stably Transfected Transactivation In Vitro Assays to detect Estrogen Receptor Agonists (ER TA assays)5,6

Validated reference test methods are:

  • Stably Transfected TA (STTA) assay (2) using the (h) ERα-HeLa-9903 cell line
  • BG1Luc ER TA assay using the BG1Luc-4E2 cell line
In vitroOECD/ US EPAUpdated OECD TG 455 (2012)OPPTS TG 890.1300
Estrogen receptor binding assay rat uterine cytosol (ER-RUC)5Ex vivoOPPTS TG 890.1250 (EPA, 2009)
H295R steroidogenesis assay5In vitroOECD/EPAOECD TG 456 (2011)OPPTS TG 890.1550 (EPA, 2009)
US EPA Tier 1 Screening Battery5In vitro/In vivoUS EPA (2009)
BG1Luc ER TA test method for estrogen agonists and antagonists5, 6In vitroICCVAM (2012)

OECD TG 457 (2012)

Draft Performance Standards for TG 457 (2012)

Eye corrosionBovine corneal opacity permeability (BCOP) testEx vivoICCVAM (2007)

ESAC (2007)

JaCVAM (2009)

OECD TG 437 (2009; revised 2013)10
Cytosensor Microphysiometer modifiedIn vitroESAC 40 (2009)7ICCVAM (2010)8Draft OECD TG (2012)
Fluorescein LeakageIn vitroESAC (2009)9JaCVAM (2012)

OECD TG 460 (2012)

OECD Summary Document No. 180

Hen’s egg test-chorioallantoic membrane (HET-CAM)In vitroEU Competent Authorities for Dangerous Substances Directive
Isolated chicken eye (ICE) testEx vivoICCVAM (2007)

ESAC (2007)

JaCVAM (2009)

OECD TG 438 (2009; revised 2013)10
Isolated rabbit eye test

Ex vivo

EU Competent Authorities for Dangerous Substances Directive

Short Time Exposure (STE) test method21In vitroDraft OECD TG (2014)
Routine use of topical anesthetics, systemic analgesics, and humane endpointsIn vivoICCVAM (2010)

Updated OECD TG 405 (2012)

OECD GD No. 19

Sequential testing strategy for eye irritation and corrosionIn vitro/Ex vivo/In vivoICCVAM16Updated OECD TG 405 (2012)
Eye irritationCytosensor Microphysiometer modifiedIn vitroESAC (2009)7ICCVAM (2010)8Draft OECD TG (2012)
Short Time Exposure (STE) test method21In vitroICCVAM (2013)Draft OECD TG (2014)
Reconstructed Human Cornea-like Epithelium (RhCE) Test Method22In vitroEURL ECVAMDraft OECD TG (2014)
Bovine Corneal Opacity Permeability (BCOP) TestEx vivoICCVAM (2007)

ESAC (2007)

JaCVAM (2009)

OECD TG 437 (2009; revised 2013)10
Isolated chicken eye (ICE) testEx vivoICCVAM (2007)

ESAC (2007)

JaCVAM (2009)

OECD TG 438 (2009; revised 2013)10
Routine use of topical anesthetics, systemic analgesics, and humane endpointsIn vivoICCVAM (2010)

Updated OECD TG 405 (2012)

OECD GD No. 19

Sequential testing strategy for eye irritation and corrosionIn vitro/Ex vivo/In vivoICCVAM16Updated OECD TG 405 (2012)
GenotoxicityBacterial reverse mutation (Ames) testIn vitroOECD TG 471 (1997)
In vitro cell gene mutation testIn vitro

OECD TG 476 (1997)

Draft Updated TG 476 (2014)

In vitro mammalian cell gene mutation assays using the thymidine kinase geneIn vitroDraft OECD TG (2014)19
In vitro chromosomal aberration testIn vitroUpdated OECD TG 473 (2014)
In vitro mammalian cell micronucleus testIn vitroESAC (2006)ICCVAM16

Updated OECD TG 487 (2014)

ICH (2011)

In vitro sister chromatid exchange testIn vitroOECD TG 479 (1986)
In vitro unscheduled DNA synthesis testIn vitroOECD TG 482 (1986)
Saccharomyces cerevisiae gene mutation assayIn vitroOECD TG 480 (1986)
Saacharomyces cerevisiae mitotic recombination assayIn vitroOECD TG 481 (1986)
Phototoxicity3T3 Neutral Red Uptake Phototoxicity TestIn vitroESAC (1997)OECD TG 432 (2004)
3T3 NRU Phototoxicity Test: Application to UV filter chemicalsIn vitroESAC (1998)OECD TG 432 (2004)
Reactive Oxygen Species (ROS) Photosafety Assay (non-biological method)23In vitroJaCVAM (2013)ICH
Preclinical and nonclinical safety studies for drug development (harmonized guidance can reduce use of animals)Guidance on nonclinical safety studies for the conduct of human clinical trials and marketing authorization for pharmaceuticals M3(R2)In vivoICH (2010)FDA; EMA; PMDA
Preclinical safety evaluation of biotechnology-derived pharmaceuticals, ICH S6(R1)In vivoICH (1997; updated 2011)FDA; EMA; PMDA
PyrogenicityHuman whole blood IL-1In vitroESAC (2006)ICCVAM (2008)12European Pharmacopeia; FDA
Human whole blood IL-6In vitroESAC (2006)ICCVAM (2008)12European Pharmacopeia; FDA
Human cryopreserved whole blood IL-1In vitroESAC (2006)ICCVAM (2008)12European Pharmacopeia; FDA
PBMC IL-6In vitroESAC (2006)ICCVAM (2008)12European Pharmacopeia; FDA
MM6 IL-6In vitroESAC (2006)ICCVAM (2008)12European Pharmacopeia; FDA
Limulus amebocyte lysate (LAL) testIn vivo/In vitroEU, US, and Japanese Pharmacopeia (2005); ICH harmonized text (2010)
Reproductive & developmental toxicityEmbryonic stem cell test for embryotoxicity17In vitroESAC (2002)
Micromass embryotoxicity assayEx vivoESAC (2002)
Whole rat embryotoxicity assayEx vivoESAC (2002)
Extended one-generation reproductive toxicity studyIn vivoICCVAM16Updated OECD TG 443 (2012)
Skin corrosionEST-1000 human reconstructed epidermisIn vitroESAC (2009)13Updated OECD TG 431 (2014)
Membrane Barrier Corrosivity Test Method (Corrositex®)In vitroICCVAM (1999)ESAC (2000)

OECD TG 435 (2006)

Draft Revised TG 435 (2014)

EpiSkin® human skin modelIn vitro

ESAC (1998)

ESAC (2009)13

ICCVAM (2002)

Performance Standards (2004)

OECD TG 431 (2004; revised 2013)

Draft Revised TG 431 (2014)

EpiDermTM human skin modelIn vitro

ESAC (2000)

ESAC (2009)13

ICCVAM (2002)

Performance Standards (2004)

OECD TG 431 (2004; revised 2013)

Draft Revised TG 431 (2014)

Rat skin transcutaneous electrical resistance (TER)assayEx vivoESAC (1998)

ICCVAM (2002)

Performance Standards (2004)

OECD TG 430 (2004; revised 2013)

Draft Revised TG 430 (2014)

SkinEthicTM human skin modelIn vitro

ESAC (2006)

ESAC (2009)13

ICCVAM (meets performance standards)

OECD TG 431 (2004; revised 2013)

Draft Revised TG 431 (2014)

Vitrolife-SkinTM human reconstructed epidermisIn vitroJaCVAM (2008)

OECD TG 431 (2004; revised 2013)

Draft Revised TG 431 (2014)

Skin irritationEpiSkin® skin irritation test (with MTT reduction)In vitro

ESAC (2007)

ESAC (2009) (Performance under UN GHS; Reference Chemicals; Performance Standards)

ESAC (2009) (Updated Performance Standards)

JaCVAM (2010)

Updated TG 439 (2013)

Draft Revised TG 439 (2014)

OECD Background Document No. 137 (2010)

EpiDermTM skin irritation test (with MTT reduction)In vitroESAC (2007)14EU test method B.46 in COM regulation 440/2008/EC
EpiDermTM SIT model (EPI-200)In vitro

ESAC (2008)

ESAC (2009) (Performance under UN GHS; Reference Chemicals; Performance Standards)

ESAC (2009) (Updated Performance Standards)

JaCVAM (2012)

Updated TG 439 (2013)

Draft Revised TG 439 (2014)

OECD Background Document No. 137 (2010)

SkinEthic RHE modelIn vitro

ESAC (2008)

ESAC (2009) (Performance under UN GHS; Reference Chemicals; Performance Standards)

ESAC (2009) (Updated Performance Standards)

JaCVAM (2012)

Updated TG 439 (2013)

Draft Revised TG 439 (2014)

OECD Background Document No. 137 (2010)

LabCyte EPI-MODEL24 SITIn vitroJaCVAM (2013)

Updated TG 439 (2013)

Draft Revised TG 439 (2014)

OECD Background Document No. 137 (2010)

Skin SensitizationLocal lymph node assay (LLNA)In vivo

ICCVAM (1999)

ICCVAM Protocol and Performance Standards (2009)

ESAC (2000)Updated OECD TG 429 (2010)
Reduced LLNA: rLLNA11In vivo

ESAC (2007)

Performance Standards: ESAC (2008)

ICCVAM (2009)

Performance Standards (2009)

JaCVAM (2012)

Updated OECD TG 429 (2010)
Nonradiolabelled LLNA: DAIn vivoICCVAM (2010)JaCVAM (2012)OECD TG 442A (2010)
Nonradiolabelled LLNA: BrdU-ELISAIn vivoICCVAM (2010)JaCVAM (2012)OECD TG 442B (2010)
LLNA for Potency Categorization of Skin SensitizersIn vivoICCVAM (2011)UN GHS (2009)Some US agencies (2012)
ARE-Nrf2 Luciferase Test Method20

In vitro

Revised Draft OECD TG (2014)
Direct Peptide Reactivity Assay (DPRA)20In chemicoDraft OECD TG (2014)
Human Cell Line Activation Test (h-CLAT)20In vitroEURL ECVAM and JaCVAMDraft OECD TG (2014)
KeratinoSens™ in vitro test method for skin sensitisationIn vitroEURL ECVAM (2014)

1 Table content is limited to in vitro, ex vivo, in chemico and/or in silico test methods, and in vivo methods or test schemes proposed to reduce or refine animal use.
2 National and regional regulatory acceptance is not fully documented here
3 Replaces animal use for initial dose setting, but in vivo test required to complete assessment
4 In vitro cell transformation assays (CTAs) can detect both genotoxic and non-genotoxic carcinogens, which can be distinguished by parallel use of genotoxicity test(s). CTA results are recommended for use as part of a testing strategy and/or in a weight of evidence approach for the assessment of carcinogenic potential, and may reduce the use of in vivo testing.
5 Screening assays to be used as part of a broader testing strategy
6 BG1Luc ER TA test method has been adapted to a high throughput screening (HTS) format and it is expected to be incorporated into Tox21 screening
7 Recommended for use as initial step within a Top-Down Approach to identify ocular corrosives and severe irritants (EU R41, GHS Category 1, and EPA Category I) for water-soluble chemicals and/or as initial step within a Bottom-Up Approach to identify non-irritants (EU:NC; GHS: NC; EPA: cat IV) for water-soluble surfactants and water-soluble surfactant-containing mixtures; does NOT correctly identify moderate and mild ocular irritants (EU: R36; GHS: Cat 2A/B; EPA: Cat II/III) so can be used for only two of the three EU and GHS classification categories for ocular irritation; cannot be used for default categorization; additional limitations on equipment availability
8 Can be used as screening test to distinguish water-soluble surfactant chemicals and certain types of surfactant-containing formulations that are not labeled as irritants (i.e., EPA Category IV, EU Not Labeled, FHSA Not Labeled) from all other hazard categories (i.e., EPA Category I, II, III; EU R41, R36; FHSA Irritant) for hazard classification and labeling under EPA, EU and FHSA classification systems; high false negative rate (24%-40%) for non-surfactant substances and formulations; high false positive rate (50% to 69%) for substances not labeled as irritants. Can be used as a screening test to identify water soluble substances as ocular corrosives and severe irritants (i.e., EPA Category I, EU R41, GHS Category 1) in tiered-testing strategy as part of weight-of-evidence approach; negative results need to be tested in another test method
9 Recommended for use as initial step within a Top-Down Approach to identify ocular corrosives and severe irritants (EU R41, GSH Category 1, and EPA Category I) for water-soluble chemicals; further refinement with respect to variability and applicability domain recommended
10 BCOP and ICE methods can be used to identify chemicals as either 1) causing “serious eye damage” (category 1 of GHS, or 2) not requiring classification for eye irritation or serious eye damage according to GHS (new to the 2013 TG updates).
11 rLLNA can be used for hazard classification when dose-response information is not needed
12 Subject to product-specific validation to demonstrate equivalence to the rabbit pyrogen test (RPT)
13 Statement on 2 reference chemicals for in vitro skin corrosion testing
14 Recommended as screening test or as part of sequential testing strategy; only positive test results accepted in the 2007 endorsement
15 Cell-based assay for botulinum toxin potency testing is limited to products produced using the Allergan method
16 Contributed to OECD TG development and/or review
17 Draft OECD GD 43, see page 18
18 EURL ECVAM reports that: “TG236 does not indicate whether the fish embryo acute toxicity test can be used as an alternative to the OECD TG203; however, several recently published papers demonstrate that the LC50 values produced with the fish embryo acute toxicity test correlate well with those observed in juvenile or adult fish (Lammer et al, 2009; Knoebel et al 2012; Belanger et al (2013).”
19 The assays using the thymidine kinase (TK) locus were originally contained in TG 476.
20 In vitro and in chemico methods endorsed as valid for supporting the discrimination between skin sensitizers and non-sensitizers in accordance with the UN GHS. The guidelines explain it is likely that combinations of non-animal methods using integrated testing approaches will be needed to substitute for the animal tests.
21 STE can be used with specific limitations for hazard classification and labeling of chemicals that induce serious eye damage and those that do not cause either serious eye damage or eye irritation, as defined by the United Nations Globally Harmonized System of Classification and Labeling of Chemicals (GHS).
22 The TG describes an in vitro procedure for the identification of chemicals not requiring classification and labelling for eye irritation or serious eye damage in accordance with UN GHS. The only method currently covered by this TG is the EpiOcular™ Eye Irritation Test (EIT) developed using human skin cells.
23 ROS assay can be used in an integrated photosafety testing and decision strategy for drug research and development where negative results would not require further testing, while positive and inconclusive results would proceed to the next level of testing in an in vitro test system such as the 3T3 Phototoxicity Assay.

Supplemental Information

A. ECVAM: Summary of test methods endorsed by ECVAM and accepted by EU regulatory authorities; ESAC statements; DB-ALM (DataBase service on ALternative Methods); TSAR (Tracking System for Alternative test methods Review)

B. ICCVAM: Summary of test methods evaluated by ICCVAM; table showing validation and acceptance status of all methods reviewed; ICCVAM-recommended protocols

C. JaCVAM: Summary of test methods evaluated and accepted by Japan’s regulatory authorities

D. OECD Test Guidelines (TGs), Guidance Documents, Draft Test Guidelines

E. ICH test guidelines: technical requirements for medicinal products containing new drugs accepted by the regulatory bodies of EU, Japan, and USA

F. VICH test guidelines: EU-Japan-USA programme aimed at harmonising technical requirements for veterinary product registration

G. ISO: toxicity test guidelines for the biological evaluation of medical devices and ecotoxicity (soil quality, water quality, fire effluents, biocides used for anti-fouling systems on ships)

Acronyms:

CFR: Code of Federal Regulations

EMA: European Medicines Agency

EPA: US Environmental Protection Agency

ESAC: ECVAM Scientific Advisory Committee

EURL ECVAM: European Union Reference Laboratory for alternatives to animal testing

FDA: United States Food and Drug Administration

GD: OECD Guidance Document

GHS: Globally Harmonized System of Classification and Labeling of Chemicals

ICCVAM: US Interagency Coordinating Committee on the Validation of Alternative Methods

ICH: International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use

ISO: International Organization for Standardization

JaCVAM: Japanese Center for the Validation of Alternative Methods

OECD: Organisation for Economic Cooperation and Development

OPPTS: US EPA’s Office of Prevention, Pesticides and Toxic Substances

PMDA: Japan’s Pharmaceuticals and Medical Devices Agency

TG: OECD Test Guideline

USDA: United States Department of Agriculture

VICH: International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products