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.