Table of Validated & Accepted Alternative Methods

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Table of Validated & Accepted Alternative Methods

Last updated: November 20, 2016

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

National/ Regional2

Acute mammalian toxicity (oral)Acute oral toxicityIn vivoESAC (2000)OECD TG 401; Deleted in 2002
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)

JaCVAM (2011)

OECD GD 129 (2010)
3T3 NRU Assay supporting identification of substances not requiring classification for acute oral toxicityIn vitroEURL ECVAM (2013)
Waiving or bridging of mammalian acute toxicity tests30Draft OECD GD (2015)
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)

Fixed concentration procedureIn vivoDraft OECD TG 433 (2015)
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 (2009)

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 (2003)

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

Deletion of target-animal batch safety test (TABST) for batch safety testing of veterinary vaccines after consistency in 10 consecutive batchesNAESAC (2002)VICH GL50 (2013)European Pharmacopeia (2012)
Vaccines for Veterinary Use (0062): around 80 vaccine-specific monographs with VICH Guidelines; around 40 inactivated vaccine-specific monographsVICH 41; VICH 44European Pharmacopeia (2012)

European Pharmacopoeia, 2015 (effective January 1, 2017)

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 products15aIn vitroICCVAM agency: FDA

ICCVAM Workshop (2006)

US FDA: Allergan, Inc., method accepted (2011)
Cell-based assay for stability and potency of botulinum neurotoxin type A products15bIn vitroUS FDA (2015)

German Federal Institute for Drugs and Medical Devices and all other EU drug regulatory authorities (2015)

Alternative test procedure for tuberculin, PPD Bovis, intradermicIn vivoICCVAM agency: USDAUSDA: 9 CFR 113.409(c), Memorandum 800.114 (2012)
CarcinogenicityCell Transformation Assays (CTAs) in: Syrian Hamster Embryo cells (SHE) performed at pH 6.7 and at pH 7.04In vitroEURL ECVAM (2012)

OECD Detailed Review Paper No. 31

OECD GD No. 214 (2015)

Bhas 42 Cell Transformation Assay (Bhas 42 CTA)4In vitroEURL ECVAM (2013)

OECD Validation study report no. 208 (2014)

OECD GD No. 231 (2016)

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)JaCVAM (2013)

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)EU Biocides Regulation and REACH Guidance
Acute avian toxicity (oral): Sequential testing procedure to minimize numbers of birds usedIn vivoOECD TG 223 (2010; updated 2016)
Zebrafish embryo acute toxicity test (ZFET)18In vivoEURL ECVAM (2014)OECD TG 236 (2013)
Honey bee larval toxicity test, repeated dose29In vivoDraft OECD GD (2015)
Endocrine active substancesOECD Detailed Review Paper No. 97 (2008)
Androgen receptor binding assay (rat prostate cytosol)Ex vivoOPPTS TG 890.1150 (EPA, 2009)
Aromatase inhibition assay (human recombinant)In vitroOPPTS TG 890.1200 (EPA, 2009)
Performance-based Test Guideline for stably transfected human receptor-alpha transcriptional activation assays for detection of estrogenic agonist-activity of chemicalsIn vitroOECD/US EPAOECD TG 455 (2012; updated 2015; updated 2016)5OPPTS TG 890.1300
Estrogen Receptor binding assay Rat Uterine Cytosol (ER-RUC)Ex vivoOPPTS TG 890.1250 (EPA, 2009)
H295R steroidogenesis assay5In vitroOECD/US EPAOECD TG 456 (2011)OPPTS TG 890.1550 (EPA, 2009)
US EPA Endocrine Disruptor Screening ProgramIn vitro / In vivo

 

In vitro

US EPA Tier 1 Screening Battery (2009)5

US EPA Toxcast™ ER Model for Bioactivity (2015)

BG1Luc ER TA test method for estrogen agonists and antagonists6In vitroICCVAM (2012)JaCVAM (2013)

OECD TG 457 (2012)

Draft Performance Standards for TG 457 (2012)

Performance-based Test Guideline for human recombinant estrogen receptor (hrER) in vitro assays to detect chemicals with ER binding affinity

OECD TG 493 (2015)21

Stably Transfected Human Androgen Receptor Transcriptional Activation Assay for detection of androgenic agonist and antagonist activity of chemicalsOECD TG 458 (2016)
Eye corrosion/Serious eye damageBovine Corneal Opacity Permeability (BCOP) test34Ex vivoICCVAM (2007)

ESAC (2007)

JaCVAM (2009)

OECD TG 437 (2009; updated 2013)

OECD Summary Document 189 (2013)10

REACH regulation
Isolated Chicken Eye (ICE) testEx vivoICCVAM (2007)ESAC (2007)

JaCVAM (2009)

OECD TG 438 (2009; updated 2013; draft revised 2015)10

OECD Summary Document 188 Part 1 (2013); 188 Part 2 (2013)

REACH regulation
Cytosensor MicrophysiometerIn vitroESAC (2009)7ICCVAM (2010)8Draft OECD TG (2012)
Fluorescein LeakageIn vitroESAC (2009)9ICCVAM

 

JaCVAM (2012)

OECD TG 460 (2012)

OECD Summary Document No. 180

REACH regulation
Hen’s Egg Test-ChorioAllantoic Membrane (HET-CAM)In vitroEU Competent Authorities for Dangerous Substances Directive
Isolated rabbit eye test

Ex vivo

EU Competent Authorities for Dangerous Substances Directive

Short Time Exposure (STE) in vitro test method for identifying chemicals inducing serious eye damageIn vitroJaCVAM ICCVAM (2013)OECD TG 491 (2015)REACH regulation
Routine use of topical anesthetics, systemic analgesics, and humane endpointsIn vivoICCVAM (2010) JaCVAM (2014)

OECD TG 405 (updated 2012)

OECD GD No. 19

Sequential testing strategy for eye irritation and corrosionIn vitro/Ex vivo/In vivoICCVAM16 JaCVAM (2014)OECD TG 405 (updated 2012)
Alternate testing framework for classification of eye irritation potential of EPA pesticide products28In vitroEPA OPP (2015)
Eye irritationCytosensor MicrophysiometerIn vitroESAC (2009)7ICCVAM (2010)8Draft OECD TG (2012)
Short Time Exposure (STE) in vitro test method for identifying chemicals not requiring classification for eye irritation or serious eye damageIn vitroJaCVAM ICCVAM (2013)OECD TG 491 (2015)REACH regulation
Reconstructed Human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage22In vitroEURL ECVAMDraft Revised OECD TG 492 (2016)

Draft updated performance standards for validation of similar methods (2016)

REACH regulation
Bovine Corneal Opacity Permeability (BCOP) TestEx vivoICCVAM (2007)

ESAC (2007)

JaCVAM (2013)

OECD TG 437 (2009; updated 2013)10

OECD Summary Document 189 (2013)

OECD GD 160

REACH regulation
Isolated Chicken Eye (ICE) testEx vivoICCVAM (2007)

ESAC (2007)

JaCVAM (2014)

OECD TG 438 (2009; updated 2013; draft revised 2015)10

Draft Position Paper on ICE for UN GHS No Category

OECD Summary Document 188 Part 1 (2013); 188 Part 2 (2013)

OECD GD 160

REACH regulation
Routine use of topical anesthetics, systemic analgesics, and humane endpointsIn vivoICCVAM (2010) JaCVAM (2014)

OECD TG 405 (updated 2012)

OECD GD No. 19

Sequential testing strategy for eye irritation and corrosionIn vitro/Ex vivo/In vivoICCVAM16 JaCVAM (2014)Updated OECD TG 405 (2012)
Alternate testing framework for classification of eye irritation potential of EPA pesticide products28In vitroEPA OPP (2015)
Genotoxicity25Bacterial reverse mutation (Ames) testIn vitroOECD TG 471 (1997)
In vitro mammalian cell gene mutation tests using the Hprt and xprt genesIn vitroOECD TG 476 (1997; updated 2015; updated 2016)
In vitro mammalian cell gene mutation test using the thymidine kinase geneIn vitroOECD TG 490 (2015; updated 201619
In vitro mammalian chromosomal aberration testIn vitroOECD TG 473 (1983; updated 2014; updated 2016)

OECD Report on Statistical Issues…(No. 198)

In vitro mammalian cell micronucleus testIn vitroESAC (2006)ICCVAM16

OECD TG 487 (2010; updated 2014; updated 2016)

ICH (2011)

OECD Report on Statistical Issues…(No. 198)

In vitro sister chromatid exchange testIn vitroOECD TG 479 (1986; Deleted in 2014)
In vitro unscheduled DNA synthesis testIn vitroOECD TG 482 (1986; Deleted in 2014)
Saccharomyces cerevisiae gene mutation assayIn vitroOECD TG 480 (1986; Deleted in 2014)
Saacharomyces cerevisiae mitotic recombination assayIn vitroOECD TG 481 (1986; Deleted in 2014)
In vivo mammalian alkaline comet assayIn vitroJaCVAMOECD TG 489 (2016)
MetabolismHuman cytochrome P450 (CYP) n-fold induction in vitro test methodIn vitro

Draft OECD TG xxx (2014)

Draft Performance Standards (2014)

Human drug-drug interactions: pathway identification; enzyme inhibition; enzyme induction; transporter substrate identification; transporter inhibitionIn vitroUS FDA (2012) EMA (2013)
The use of metabolizing systems for in
vitro testing of endocrine disruptors
OECD Detailed Review Paper No. 97 (2008)
Phototoxicity3T3 Neutral Red Uptake Phototoxicity Test33In vitroESAC (1997)OECD TG 432 (2004)
3T3 NRU Phototoxicity Test: Application to UV filter chemicalsIn vitroESAC (1998)OECD TG 432 (2004)
(ROS) Reactive Oxygen Species assay photosafety (non-biological method)23In vitroJaCVAM26 (2013)ICH S10 (2013)

Draft OECD TG (2016)

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

PyrogenicityFive human cell assays for detecting pyrogenicity produced by Gram-negative endotoxins27: Human whole blood IL-1; Human whole blood IL-6; Human cryopreserved whole blood IL-1; PBMC IL-6; PBMC IL-6In vitroESAC (2006)ICCVAM (2008)12

European Pharmacopeia, Monocyte activation test (2.6.30) (2010); revised (2.6.30) (July 2017)

FDA Guidance: Pyrogen and Endotoxins Testing (2012)

Japan, Reservation

Limulus amebocyte lysate (LAL) testIn vivo/In vitro

EU, 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 vivoICCVAM16OECD TG 443 (updated 2012)
Skin corrosionIn vitro skin corrosion: reconstructed human epidermis (RHE) test method: Episkin™ SCT, Epiderm™ SCT, SkinEthic™ RHE SCT, epiCS® SCT (formerly EST-1000); Vitrolife-Skin™In vitro

ESAC (1998; 2000; 2006; 2009)

JaCVAM (2008)

ICCVAM 2002

OECD TG 431 (2004; updated 2015; updated 2016)13a

OECD Summary Document 190 (2013)

REACH regulation
In vitro Membrane Barrier test method for skin corrosion: CORROSITEX™In vitroICCVAM (1999)ESAC (2000)OECD TG 435 (2006; updated 2015)REACH regulation
Rat skin transcutaneous electrical resistance (TER) test methodEx vivoESAC (1998)ICCVAM (2002)OECD TG 430 (2004; updated 2015)REACH regulation
Integrated approach on testing and assessment (IATA) for skin corrosion and irritationOECD GD 203 (2014)
Skin irritationReconstructed human epidermis test method: Episkin™ SIT, Epiderm™ SIT, SkinEthic™ RHE SIT, LabCyte EPI-MODEL24 SITIn vitro

ESAC (2007; 2008; 2009)

JaCVAM (2013)

JaCVAM (2010; 2012)OECD TG 439 (2010; updated 2015)13bREACH regulation
Integrated approach on testing and assessment (IATA) for skin corrosion and irritationOECD GD 203 (2014)
Skin sensitizationLocal lymph node assay (LLNA)In vivoICCVAM (1999; 2009)ESAC (2000)OECD TG 429 (2002; updated 2010)
Reduced LLNA: rLLNA11In vivoESAC (2007; 2008)

ICCVAM (2009)

JaCVAM (2012)

OECD TG 429 (updated 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 method20a: KeratinoSens™In vitroEURL ECVAM (2014)JaCVAM (2015)OECD TG 442D (2015)REACH regulation
Direct Peptide Reactivity Assay (DPRA)20bIn chemico

EURL ECVAM (2013)

JaCVAM (2015)OECD TG 442C (2015)REACH regulation
Human Cell Line Activation Test (h-CLAT)20cIn vitroEURL ECVAM (2015)OECD TG 442E (2016)REACH regulation
IL-8 Luc Assay32In vitroJaCVAMDraft OECD TG (2016)
U937 Skin Sensitization Test (U-SENS™)31In vitroEURL ECVAM (2016)Draft OECD TG (2016)
P&G/NICEATM Open-source Integrated Testing Strategy to identify potential skin sensitizers without conducting animal testsIn vitroNICEATM (2015)
Reporting of Defined Approaches and Individual Information Sources to be used within Integrated Approaches to Testing and Assessment (IATA) for Skin Sensitisation- ENV/JM/HA(2016)11OECD GD 256 (2016)

Annex 1: Case Studies… (2016)

Annex II: Information used within the case studies…(2016)

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 weight-of-evidence approach for the assessment of carcinogenic potential, and may reduce the use of in vivo testing.
5 The two reference test methods that provide the basis for this guideline are: the Stably Transfected TA (STTA) assay using the (h) ERα-HeLa-9903 cell line, and the BG1Luc ER TA assay. These methods are proposed for screening and prioritization purposes, but also provide mechanistic information that can be used in a weight of evidence approach.
6 BG1Luc ER TA test method has been adapted to a high throughput screening (HTS) format and 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)
13a Four validated test methods using commercially available RhE models are included in TG 431: EpiSkin™ Standard Model (SM), EpiDerm™ Skin Corrosivity Test (SCT) (EPI-200), SkinEthic™ RHE, and epiCS® (previously named EST-1000).
13b Four validated test methods adhere to TG 439: EpiSkin™, EpiDerm™ SIT (EPI-200), SkinEthic™ RHE, and LabCyte EPI-MODEL24 SIT. “Depending on the regulatory framework and the classification system in use, this procedure may be used to determine the skin irritancy of test substances as a stand-alone replacement test for in vivo skin irritation testing, or as a partial replacement test, within a tiered testing strategy.”
14 Recommended as screening test or as part of sequential testing strategy; only positive test results accepted in the 2007 endorsement
15a Cell-based assay for botulinum toxin potency testing is limited to products produced using the Allergan method
15b Cell-based assay for botulinum toxin type A testing is limited to products produced by Merz
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.
20a In vitro method 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. The only ARE-Nrf2 luciferase test method covered currently by this TG, is the KeratinoSens™ test method.
20b In chemico method 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.
20c In vitro method 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 This guideline describes assays providing the methodology for human recombinant in vitro assays to detect substances with estrogen receptor binding affinity, and comprises two similar reference test methods: Freyberger-Wilson (FW) in vitro estrogen receptor binding assay…, and Chemical Evaluation and Research Institute (CERI) in vitro estrogen receptor binding assay…; proposed for screening and prioritization purposes, and provide mechanistic information that can be used in a weight of evidence approach.
22 Methods currently covered by this TG are the EpiOcular™ Eye Irritation Test (EIT) and the SkinEthic™ Human Corneal Epithelium (HCE) Eye Irritation Test (EIT).
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.
24 OECD Series on Testing and Assessment contains additional useful guidance and performance standards. For example, Guidance Document No. 211: Guidance for describing non-guideline in vitro test methods to facilitate their consideration in regulatory applications, and the draft Guidance Document on Revisions to OECD Genetic Toxicology Test Guidelines.
25 Also see: Draft Guidance Document on Revisions to OECD Genetic Toxicology Test Guidelines (2015) and Report on Statistical Issues Related to OECD Test Guidelines (TGs) on Genotoxicity (Series on Testing and Assessment, No. 198, 2014)
26 “The JaCVAM peer review panel concluded that the reproducibility and predictivity of the ROS assay is sufficient to support its use in an integrated photosafety testing and decision strategy for drug research and development…[where] negative results in the ROS assay would not require further testing in animals or other tests, while positive, weakly positive, and inconclusive results would proceed to the next level of testing in an in vitro test system such as the 3T3 Phototoxicity Assay (OECD Test Guideline 432).”
27 The in vitro pyrogenicity methods are not considered valid for use in Japan at this time.
28 Includes explanation of expanded use of BCOP for antimicrobial testing, and decision tree approach using 3 assays (see IIVS Decision Tree Approach):

  • Bovine Corneal Opacity and Permeability test (BCOP)
  • EpiOcular assay
  • Cytosensor Microphysiometer assay

29 Honey bee larval toxicity tests complement OECD TGs 213 and 214 on young adult honey bees and should be seen as a lower tier screening test.
30 “Generally, waivers are considered when there is little or no significant human exposure by a given route of exposure or when it is technically not possible to perform a study for a certain endpoint, such as not requiring an acute oral toxicity study when the test chemical exists as a vapour or gas. Waivers are also possible taking into account animal welfare considerations, such as when the test chemical is corrosive.”
31 U-SENS™ is proposed to address the third key event of the skin sensitization AOP; recommended by EURL ECVAM as part of an IATA to support discrimination between sensitizers and non-sensitizers for hazard classification and labelling.
32 “IL-8 Luc assay is proposed to address the third key event (dendritic cell activation) of the skin sensitisation AOP by quantifying changes in the expression of cytokine associated with the process of activation of DC (i.e. IL-8), in the human monocytic leukemia cell line THP-1-derived IL-8 reporter cell line, THP-G8, following exposure to sensitisers.”
33 EPAA-IIVS Video of 3T3 NRU Phototoxicity Assay.
34 EPAA-IIVS Video of BCOP Eye Irritation Assay.

Supplemental Information

A. EURL ECVAM: Test methods endorsed by EURL 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. OECD Guidance Document for Describing Non-Guideline In Vitro Test Methods (No.211): intended to harmonize the way non-guideline in vitro test methods are described.

F. OECD Guidance Document on Developing and Assessing Adverse Outcome Pathways (No. 184) (2013): intended to provide guidance on the development of Adverse Outcome Pathways (AOPs).

G. OECD Guidance Document on the Reporting of Defined Approaches to be used within Integrated Approaches to Testing and Assessment (No. 255) (2016): intended “to provide a set of principles for reporting defined approaches to testing and assessment to facilitate their evaluation. Templates are also provided to enable a structured and harmonised approach to their documentation.”

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

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

J. 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)

K. USDA APHIS Supplemental Assay Methods (SAMs) (updated March 17, 2016)

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

IATA: Integrated Approach to Testing and Assessment, “Approach based on multiple information sources that integrates and weights all relevant existing evidence and guides the targeted generation of new data, where required, to inform regulatory decision-making regarding potential hazard and/or risk.”

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

Author(s)/Contributor(s):
Sherry L. Ward, PhD, MBA
AltTox Contributing Editor

AltTox Editorial Board reviewer(s):
Ian Kimber, PhD
Professor, University of Manchester

João Barroso, PhD
Systems Toxicology Unit, EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)

Albert P. Li, PhD
President & CEO, In Vitro ADMET Laboratories LLC

Disclaimer
The information provided here is intended only as an overview, and is neither guidance or a comprehensive review of the laws, regulations, or toxicity tests that may be accepted by an individual agency. Countries/regions and their regulatory authorities usually provide specific guidance on hazard/toxicity testing requirements.

Endocrine active substances