Validation & Regulatory Acceptance Status of Alternative Test Methods & Testing Strategies

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