Table of Validated and Accepted Alternative Methods

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Table of Validated and Accepted Alternative Methods

Last updated: December 21, 2013

Validation & Regulatory Acceptance Status of Alternative Test Methods & Testing Strategies
Endpoint Method Name Test Type1 Endorsement of Scientific Validity(Links to ESAC Statements; ECVAM; ICCVAM; JaCVAM) Regulatory Acceptance
Lead Authority Subsequent Endorsement(s) International (Links to OECD: approved OECD TGs; Draft TGs; GDs; Draft GDs) National/Regional2
Acute mammalian toxicity (oral) Acute toxic class method

In vivo

ESAC (2007)

OECD TG 423 (2001)

Fixed dose procedure

In vivo

ESAC (2007)

OECD TG 420 (2001)

Up-and-down procedure

In vivo

ICCVAM (2001)

ESAC (2007)

OECD TG 425 (2006)

EPA OPPTS 870.1100 (2002)
Normal human keratinocyte neutral red uptake (NHK NRU) assay

In vitro3

ICCVAM (2008)

JaCVAM (2011)

OECD GD 129 (2010)

Balb/c 3T3 neutral red uptake assay

In vitro3

ICCVAM (2008)

ECVAM (2011)JaCVAM (2011)

OECD GD 129 (2010)

Acute mammalian toxicity (hematotoxicity) Colony Forming Unit-Granulocyte/Macrophage Assay for acute neutropenia in humans

In vitro

ESAC (2006)
Acute mammalian toxicity (inhalation) Acute toxic class method

In vivo

OECD TG 436 (2009)

OECD GD No. 153 (2011)

Acute toxicity testing of pesticides Guidance for waiving or bridging of mammalian acute toxicity tests for pesticides (acute oral, dermal, inhalation; primary eye and dermal; dermal sensitization)

In vivo

EPA OPP (2012)
Biologics & Vaccines ELISA for swine erysipelas vaccines batch potency testing

In vitro

ESAC (2002)

ICCVAM agency, USDA (2009)

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

ELISA for human tetanus vaccines batch potency testing

In vitro

ESAC (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 testing

In vitro

ESAC (2000)

European Pharmacopeia; FDA, 21 CFR 610.10 (accepted on case-by-case basis)

Batch potency testing of erythropoietin concentrated solution

In vivo

ESAC (2002)

European Pharmacopeia, Monograph 0064

Deletion of target-animal safety test for batch safety testing of veterinary vaccines after consistency in 10 consecutive batches

NA

ESAC (2002)

EU: General monograph 0062

ELISA for in vitro batch potency testing of Leptospira veterinary vaccines

In vitro

ICCVAM agency, USDA (2008)

USDA Supplemental Assay Methods 624, 625, 626, and 627

Use of humane endpoints in animal testing of veterinary biologics, including rabies vaccines

In vivo

ICCVAM agency, USDA (2004; 2012)

OECD GD No. 19

USDA: 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 requalification

NA

ICCVAM agency, USDA

USDA: 9 CFR 113.409(c), Memorandum 800.114 (2012)

Cell-based assay for stability and potency of botulinum neurotoxin type A products15

In vitro

ICCVAM agency, FDA; ICCVAM Workshop (2006)

US FDA: Allergan, Inc., method accepted (2011)

Alternative test procedure for tuberculin, PPD Bovis, intradermic

In vivo

ICCVAM agency, USDA

USDA: 9 CFR 113.409(c), Memorandum 800.114 (2012)

Carcinogenicity Three cell transformation assays (CTA): (Syrian Hamster Embryo (SHE) CTA performed at pH 6.7, SHE CTA performed at pH 7.0, and BALB/c 3T3 CTA)4

In vitro

EURL ECVAM Recommendation (2012)

Draft OECD TG (2012) (for SHE CTA)OECD Detailed Review Paper No. 31
Chronic toxicity Ending 1-year dog studies of pesticides

In vivo

ESAC (2006)

Revised US EPA Pesticide Data Requirements

Dermal absorption/penetration In vitro skin absorption methods

In vitro

OECD Expert Group (2002)

OECD TG 428 (2004)

OECD GD 28 (2004)

OECD Guidance Notes No. 156

Ecotoxicity Acute aquatic toxicity: Upper threshold concentration step-down approach

In vivo

ESAC (2006)

OECD GD 126 (2010)

Acute avian toxicity (oral): Sequential testing procedure to minimizenumbers of birds used

In vivo

OECD TG 223 (2010)

Fish embryo toxicity18

In vivo

OECD TG 236 (2013)

Endocrine active substances Androgen receptor binding assay (rat prostate cytosol)5

Ex vivo

OPPTS TG 890.1150 (EPA, 2009)

Aromatase inhibition assay (human recombinant)5

In vitro

OPPTS TG 890.1200 (EPA, 2009)

Stably transfected transactivation in vitro assays to detect estrogen receptor agonists5

In vitro

OECD/EPA

Updated OECD TG 455 (2012)Draft Performance Standards for TG 455 (2011)

OPPTS TG 890.1300

Estrogen receptor binding assay rat uterine cytosol (ER-RUC)5

Ex vivo

OPPTS TG 890.1250 (EPA, 2009)

H295R steroidogenesis assay5

In vitro

OECD/EPA

OECD TG 456 (2011)

OPPTS TG 890.1550 (EPA, 2009)

US EPA Tier 1 Screening Battery5

In vitro/In vivo

US EPA (2009)

BG1Luc ER TA test method for estrogen agonists and antagonists5, 6

In vitro

ICCVAM (2012)

OECD TG 457 (2012)Draft Performance Standards for TG 457 (2012)
Eye corrosion Bovine corneal opacity permeability (BCOP) test

Ex vivo

ICCVAM (2007)

ESAC (2007)

JaCVAM (2009)

OECD TG 437 (2009)

Draft Revised TG 437 (2012)

OECD Proficiency Standards

OECD GD No. 160

Cytosensor Microphysiometer modified

In vitro

ESAC 40 (2009)7

ICCVAM (2010)8

Draft OECD TG (2012)

Fluorescein Leakage

In vitro

ESAC (2009)9

JaCVAM (2012)

OECD TG 460 (2012)

OECD Summary Document No. 180

Hen’s egg test-chorioallantoic membrane (HET-CAM)

In vitro

EU Competent Authorities for Dangerous Substances Directive

Isolated chicken eye (ICE) test

Ex vivo

ICCVAM (2007)

ESAC (2007)

JaCVAM (2009)

OECD TG 438 (2009)

Draft Revised TG 438 (2012)

OECD Proficiency Standards

OECD GD No. 160

Isolated rabbit eye test

Ex vivo

EU Competent Authorities for Dangerous Substances Directive

Routine use of topical anesthetics, systemic analgesics, and humane endpoints

In vivo

ICCVAM (2010)

Updated OECD TG 405 (2012)

OECD GD No. 19

Sequential testing strategy for eye irritation and corrosion

In vitro/Ex vivo/In vivo

ICCVAM16

Updated OECD TG 405 (2012)

Eye irritation Cytosensor Microphysiometer modified

In vitro

ESAC (2009)7

ICCVAM (2010)8

Draft OECD TG (2012)

Rabbit low-volume eye test (LVET)

In vivo

ESAC (2009)10

ICCVAM (2009)10

Routine use of topical anesthetics, systemic analgesics, and humane endpoints

In vivo

ICCVAM (2010)

Updated OECD TG 405 (2012)

OECD GD No. 19

Sequential testing strategy for eye irritation and corrosion

In vitro/Ex vivo/In vivo

ICCVAM16

Updated OECD TG 405 (2012)

Genotoxicity Bacterial reverse mutation (Ames) test

In vitro

OECD TG 471 (1997)

In vitro cell gene mutation test

In vitro

OECD TG 476 (1997)

In vitro chromosomal aberration test

In vitro

OECD TG 473 (1997)

Draft updated TG 473 (2012)

In vitro mammalian cell micronucleus test

In vitro

ESAC (2006)

ICCVAM16

OECD TG 487 (2010)

Draft Updated TG 487 (2012)

ICH (2011)

In vitro sister chromatid exchange test

In vitro

OECD TG 479 (1986)

In vitro unscheduled DNA synthesis test

In vitro

OECD TG 482 (1986)

Saccharomyces cerevisiae gene mutation assay

In vitro

OECD TG 480 (1986)

Saacharomyces cerevisiae mitotic recombination assay

In vitro

OECD TG 481 (1986)

Immunotoxicity/Skin Sensitization Local lymph node assay (LLNA)

In vivo

ICCVAM (1999)

ICCVAM Protocol and Performance Standards (2009)

ESAC (2000)

Updated OECD TG 429 (2010)

Reduced LLNA: rLLNA11

In vivo

ESAC (2007)

Performance Standards: ESAC (2008)

ICCVAM (2009)

Performance Standards (2009)

JaCVAM (2012)

Updated OECD TG 429 (2010)

Nonradiolabelled LLNA: DA

In vivo

ICCVAM (2010)

JaCVAM (2012)

OECD TG 442A (2010)

Nonradiolabelled LLNA: BrdU-ELISA

In vivo

ICCVAM (2010)

JaCVAM (2012)

OECD TG 442B (2010)

LLNA for Potency Categorization of Skin Sensitizers

In vivo

ICCVAM (2011)

UN GHS (2009)

Some US agencies (2012)

Phototoxicity 3T3 Neutral Red Uptake Phototoxicity Test

In vitro

ESAC (1997)

OECD TG 432 (2004)

3T3 NRU Phototoxicity Test: Application to UV filter chemicals

In vitro

ESAC (1998)

OECD TG 432 (2004)

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 vivo

ICH (2010)

FDA; EMA; PMDA

Preclinical safety evaluation of biotechnology-derived pharmaceuticals, ICH S6(R1)

In vivo

ICH (1997; updated 2011)

FDA; EMA; PMDA

Pyrogenicity Human whole blood IL-1

In vitro

ESAC (2006)

ICCVAM (2008)12

European Pharmacopeia; FDA

Human whole blood IL-6

In vitro

ESAC (2006)

ICCVAM (2008)12

European Pharmacopeia; FDA

Human cryopreserved whole blood IL-1

In vitro

ESAC (2006)

ICCVAM (2008)12

European Pharmacopeia; FDA

PBMC IL-6

In vitro

ESAC (2006)

ICCVAM (2008)12

European Pharmacopeia; FDA

MM6 IL-6

In vitro

ESAC (2006)

ICCVAM (2008)12

European Pharmacopeia; FDA

Limulus amebocyte lysate (LAL) test

In vivo/In vitro

EU, US, and Japanese Pharmacopeia (2005); ICH harmonized text (2010)

Reproductive & developmental toxicity Embryonic stem cell test for embryotoxicity17

In vitro

ESAC (2002)

Micromass embryotoxicity assay

Ex vivo

ESAC (2002)

Whole rat embryotoxicity assay

Ex vivo

ESAC (2002)

Extended one-generation reproductive toxicity study

In vivo

ICCVAM16

Updated OECD TG 443 (2012)

Skin corrosion EST-1000 human reconstructed epidermis

In vitro

ESAC (2009)

ESAC (2009)13

OECD TG 431 (2004)

Draft Updated TG 431 (2012)

Membrane Barrier Corrosivity Test Method (Corrositex®)

In vitro

ICCVAM (1999)

ESAC (2000)

OECD TG 435 (2006)

EpiSkin® human skin model

In vitro

ESAC (1998)

ESAC (2009)13

ICCVAM (2002)

Performance Standards (2004)

OECD TG 431 (2004)

Draft Updated TG 431 (2012)

EpiDermTM human skin model

In vitro

ESAC (2000)

ESAC (2009)13

ICCVAM (2002)

Performance Standards (2004)

OECD TG 431 (2004)

Draft Updated TG 431 (2012)

Rat skin transcutaneous electrical resistance (TER)assay

Ex vivo

ESAC (1998)

ICCVAM (2002)

Performance Standards (2004)

OECD TG 430 (2004)

Draft Updated TG 430 (2012)

SkinEthicTM human skin model

In vitro

ESAC (2006)

ESAC (2009)13

ICCVAM (meets performance standards)

OECD TG 431 (2004)

Draft Updated TG 431 (2012)

Vitrolife-SkinTM human reconstructed epidermis

In vitro

JaCVAM (2008)

OECD TG 431 (2004)

Draft Updated TG 431 (2012)

Skin irritation EpiSkin® 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 (2012)

OECD Background Document No. 137 (2010)

EpiDermTM skin irritation test (with MTT reduction)

In vitro

ESAC (2007)14

EU 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 (2012)

OECD Background Document No. 137 (2010)

SkinEthic RHE model

In vitro

ESAC (2008)

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

ESAC (2009) (Updated Performance Standards)

JaCVAM (2012)

Updated TG 439 (2012)

OECD Background Document No. 137 (2010)

 

 1 In vitro and ex vivo methods, and in vivo methods proposed to reduce or refine animal use, are listed
 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 Three cell-based CTA assays can be used to partially replace or reduce animal use
 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 Restricted to use of existing data only; retrospective LVET data can be used in weight-of-evidence approach to identify potential ocular irritants
 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).”

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 and accepted by US regulatory authorities; 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