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Non-animal Methods for Toxicity Testing

Table of Validated and Accepted Alternative Methods


Last Updated: September 27, 2011
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Validation & Regulatory Acceptance Status of Alternative Test Methods & Testing Strategies

 

Endpoint Method Name Test Type1 Endorsement of Scientific Validity Regulatory Acceptance
      Lead Authority Subsequent Endorsement(s) International (click here for approved OECD TGs, here for Draft TGs, and here for GDs) National/ Regional (for methods not yet accepted internationally)
Acute aquatic toxicity Upper threshold concentration step-down approach

In vivo

ESAC 17 (2006)

 

OECD GD 126 (2010)

 
Acute mammalian toxicity (oral) Acute toxic class method

In vivo

 

ESAC 33 (2007)

OECD TG 423 (2001)

 
  Fixed dose procedure

In vivo

 

ESAC 32 (2007)

OECD TG 420 (2001)

 
  Up-and-down procedure

In vivo

ICCVAM (2001)

ESAC 34 (2007)

OECD TG 425 (2006)

 
  Normal human keratinocyte neutral red uptake (NHK NRU) assay

In vitro2

ICCVAM (2006)

 

OECD GD 129 (2010)

US agencies (2008)

  Balb/c 3T3 neutral red uptake assay

In vitro2

ICCVAM (2006)

 

OECD GD 129 (2010)

US agencies (2008)

Acute mammalian toxicity (inhalation) Acute toxic class method

In vivo

   

OECD TG 436 (2009)

 
  Fixed concentration procedure

In vivo

   

Draft TG OECD 433

 
Biologics & Vaccines ELISA for erysipelas vaccines batch potency testing

In vitro

ESAC 14 (2002)

   

European Pharmacopeia

  ELISA for human tetanus vaccines batch potency testing

In vitro

ESAC 9 (2000)

   

European Pharmacopeia

  Toxin binding inhibition test for human tetanus vaccines batch potency testing

In vitro

ESAC 10 (2000)

   

European Pharmacopeia

Chronic toxicity Ending 1-year dog studies of pesticides

In vivo

ESAC 26 (2006)

   

Revised US EPA Pesticide Data Requirements

Dermal penetration In vitro skin absorption methods

In vitro

OECD Expert Group (2002)

 

OECD TG 428 (2004)

OECD GD 28 (2004)

Draft OECD Guidance Notes (2010)

 
Endocrine active substances3 Androgen receptor binding assay (rat prostate cytosol)

Ex vivo

     

OPPTS TG 890.1150 (EPA, 2009)

  Aromatase inhibition assay (human recombinant)

In vitro

     

OPPTS TG 890.1200 (EPA, 2009)

  Estrogen receptor (ER)-alpha transcriptional activation assay for estrogen agonists (STTA)

In vitro

OECD/EPA

 

OECD TG 455 (2009)

OPPTS TG 890.1300

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

Ex vivo

     

OPPTS TG 890.1250 (EPA, 2009)

  H295R steroidogenesis assay

In vitro

OECD/EPA

 

OECD TG 456 (2011)

OPPTS TG 890.1550 (EPA, 2009)

  US EPA Tier 1 Screening Battery

In vitro/In vivo

     

US EPA (2009)

  BG1Luc ER TA test method for estrogen agonists and antagonists

In vitro

ICCVAM (expected 2011)

     
Eye corrosion Bovine corneal opacity permeability (BCOP) test

In vitro

ICCVAM (2007)

ESAC 27 (2007)

INVITTOX Protocol 127

JaCVAM (2009)

JaCVAM Regulatory Acceptance Board (2009)

OECD TG 437 (2009)

OECD Proficiency Standards

Draft OECD GD on Supplement to TG 437 and 438 (histopathology) (2009)

 
  Cytosensor Microphysiometer modified (cytotoxicity/cell-based assay)

In vitro

ESAC 40 (2009)4

INVITTOX Protocol 102 modified

ICCVAM (2010)5

Draft OECD TG (2010)

 
  Fluorescein Leakage (cytotoxicity/cell-based assay)

In vitro

ESAC 40 (2009)6

INVITTOX Protocol 71

 

Draft OECD TG (2010)

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

In vitro

     

EU Competent Authorities for Dangerous Substances Directive

  Isolated chicken eye (ICE) test

In vitro or Ex vivo

ICCVAM (2007)

ESAC 28 (2007)

INVITTOX Protocol 80

JaCVAM (2009)

JaCVAM Regulatory Acceptance Board (2009)

OECD TG 438 (2009)

OECD Proficiency Standards

Draft OECD GD on Supplement to TG 437 and 438 (histopathology) (2009)

 
  Isolated rabbit eye test

In vitro or Ex vivo

     

EU Competent Authorities for Dangerous Substances Directive

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

In vivo

ICCVAM (2009)

 

Expected 2012

 
Eye irritation Cytosensor Microphysiometer modified (cytotoxicity/cell-function based in vitro assay)

In vitro

ESAC 40 (2009)4

INVITTOX Protocol 102 modified

ICCVAM (2010)5

Draft OECD TG (2010)

 
  Rabbit low-volume eye test (LVET)

In vivo

ESAC 41 (2009)7

ICCVAM (2009)8

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

In vivo

ICCVAM (2009)

 

Expected 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)

 
  In vitro mammalian cell micronucleus test

In vitro

ESAC 24 (2006)

ICCVAM comments to OECD

OECD TG 487 (2010)

 
  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)

 
Hematotoxicity: acute neutropenia Colony forming unit granulocyte macrophage (CFU-GM) assay

In vitro

ESAC 18 (2006)

   

Submitted to EMEA

Immunotoxicity/Skin Sensitization Local lymph node assay (LLNA)

In vivo

ICCVAM (1999)

ICCVAM Protocol and Performance Standards (2009)

ESAC 6 (2000)

OECD TG 429 (2002)

Updated OECD TG 429 (2010)

 
  Reduced LLNA: rLLNA9

In vivo

ESAC 29 (2007)

Performance Standards: ESAC 37 (2008)

ICCVAM (2009)

Performance Standards (2009)

Updated OECD TG 429 (2010)

 
  Nonradiolabelled LLNA: DA

In vivo

JaCVAM (2008)

JaCVAM Regulatory Acceptance Board (2008)

ICCVAM (2009)10

OECD TG 442A (2010)

 
  Nonradiolabelled LLNA: BrdU-ELISA

In vivo

ICCVAM (2009)10

JaCVAM (2010)

JaCVAM Regulatory Acceptance Board (2010)

OECD TG 442B (2010)

 
  LLNA for Potency Categorization

In vivo

ICCVAM (2009)

 

UN GHS (2009)

US agencies (anticipated 2011)

Phototoxicity 3T3 Neutral Red Uptake Phototoxicity Test

In vitro

ESAC 1 (1997)

 

OECD TG 432 (2004)

 
  3T3 NRU Phototoxicity Test: Application to UV filter chemicals

In vitro

ESAC 4 (1998)

 

OECD TG 432 (2004)

 
Pyrogenicity Human whole blood IL-1

In vitro

ESAC 19 (2006)

ICCVAM (2008)11

 

EMEA; European Pharmacopeia; US agencies

  Human whole blood IL-6

In vitro

ESAC 20 (2006)

ICCVAM (2008)11

 

EMEA; European Pharmacopeia; US agencies

  Human cryopreserved whole blood IL-1

In vitro

ESAC 23 (2006)

ICCVAM (2008)11

 

EMEA; European Pharmacopeia; US agencies

  PBMC IL-6

In vitro

ESAC 21 (2006)

ICCVAM (2008)11

 

EMEA; European Pharmacopeia; US agencies

  MM6 IL-6

In vitro

ESAC 22 (2006)

ICCVAM (2008)11

 

EMEA; European Pharmacopeia; US agencies

  Limulus amebocyte lysate (LAL) test

In vitro

     

European Pharmacopeia (5.0); US Pharmacopeia (85)

Reproductive & developmental toxicity Embryonic stem cell test for embryotoxicity

In vitro

ESAC 13 (2002)

 

See Draft OECD GD 43, page 18

 
  Micromass embryotoxicity assay

Ex vivo

ESAC 11 (2002)

     
  Whole rat embryotoxicity assay

Ex vivo

ESAC 12 (2002)

     
  Extended one-generation reproductive toxicity study

In vivo

   

OECD TG 443 (2011)

 
Skin corrosion EST-1000 human reconstructed epidermis

In vitro

ESAC 39 (2009)

ESAC 43 (2009)12

 

OECD TG 431 (2004)

Draft Updated TG 431 (2009)

 
  Corrositex® noncellular membrane

In vitro

ICCVAM (1999)

ESAC 8 (2000)

OECD TG 435 (2006)

 
  EpiSkin® human skin model

In vitro

ESAC 2 (1998)

ESAC 43 (2009)12

ICCVAM (2002)

Performance Standards (2004)

OECD TG 431 (2004)

Draft Updated TG 431 (2009)

 
  EpiDermTM human skin model

In vitro

ESAC 7 (2000)

ESAC 43 (2009)12

ICCVAM (2002)

Performance Standards (2004)

OECD TG 431 (2004)

Draft Updated TG 431 (2009)

 
  Rat skin transcutaneous electrical resistance (TER)assay

Ex vivo

ESAC 3 (1998)

ICCVAM (2002)

Performance Standards (2004)

OECD TG 430 (2004)

Draft Updated TG 430 (2009)

 
  SkinEthicTM human skin model

In vitro

ESAC 25 (2006)

ESAC 43 (2009)12

ICCVAM

OECD TG 431 (2004)

Draft Updated TG 431 (2009)

 
  Vitrolife-Skin human reconstructed epidermis

In vitro

JaCVAM (2008)

JaCVAM Regulatory Acceptance Board (2008)

ESAC 43 (2009)12

OECD TG 431 (2004)

Draft Updated TG 431 (2009)

 
Skin irritation EpiSkin® skin irritation test (with MTT reduction)

In vitro

ESAC 31 (2007)

ESAC 38 (2009) (Performance under UN GHS; Reference Chemicals; Performance Standards)

ESAC 42 (2009) (Updated Performance Standards)

JaCVAM (2010)

JaCVAM Regulatory Acceptance Board (2010)

OECD TG 439 (2010)

 
  EpiDermTM skin irritation test (with MTT reduction)

In vitro

ESAC 30 (2007)13

   

EU test method B.46 in COM regulation 440/2008/EC

  EpiDermTM SIT model (EPI-200)

In vitro

ESAC 36 (2008)

ESAC 38 (2009) (Performance under UN GHS; Reference Chemicals; Performance Standards)

ESAC 42 (2009) (Updated Performance Standards)

 

OECD TG 439 (2010)

 
  SkinEthic RHE model

In vitro

ESAC 36 (2008)

ESAC 38 (2009) (Performance under UN GHS; Reference Chemicals; Performance Standards)

ESAC 42 (2009) (Updated Performance Standards)

 

OECD TG 439 (2010)

 

 1 All in vitro and ex vivo methods listed; in vivo methods proposed to reduce or refine animal use also listed
 2 Replaces animal use for initial dose setting, but in vivo test required to complete assessment
 3 Screening assays to be used as part of a broader testing strategy
 4 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
 5 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
 6 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
 7 Restrictions include use of existing data only
 8 Retrospective LVET data can be used in a weight-of-evidence approach to identify potential ocular irritants
 9 rLLNA can be used for hazard classification when dose-response information is not needed
 10 ICCVAM recommendations on use of LLNA for skin sensitization potency categorization anticipated in 2011
 11 Subject to product-specific validation to demonstrate equivalence to the rabbit pyrogen test (RPT)
 12 Statement on 2 reference chemicals for in vitro skin corrosion testing
 13 Recommended as screening test or as part of sequential testing strategy; only positive test results accepted in the 2007 endorsement


Supplemental Information

A. ECVAM: Summary of test methods evaluated by ECVAM and accepted by EU regulatory authorities; ESAC statements; TSAR (Tracking System for Alternative test methods Review, Validation and Approval in the Context of EU Regulations on Chemicals)

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

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

F. VICH test guidelines


Acronyms

EMEA: European Medicines Agency
EPA: US Environmental Protection Agency
ESAC: ECVAM Scientific Advisory Committee
GD: OECD Guidance Document
GHS: Globally Harmonized System of Classification and Labelling of Chemicals
ICCVAM: US Interagency Coordinating Committee on the Validation of Alternative Methods
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
TG: OECD Test Guideline