Defined Approaches for EPA Testing

The US Environmental Protection Agency (US EPA) continues to drive significant efforts in the United States to modernize the battery of acute toxicity tests classically known as the “6-pack.” Known for many years as an “all in vivo” testing strategy for products registered under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), the 6-pack was revised in 2015 to include non-animal (in vitro/ex vivo) test methods for classification and labeling for Eye Irritation, both for commonly used household cleaning products with anti-microbial claims and more conventional pesticides products.

In 2018, EPA has continued the modernization of the 6-pack through its Draft Interim Science Policy to allow the Skin Sensitization endpoint to be performed in in vitro models as well. Currently, only pure substances are permitted to be used as test materials for submission purposes and one of the Integrated Testing Strategies, either the “2 out of 3 Defined Approach” or the “Key Event 3/1 Sequential Testing Strategy” must be followed (see figures 1a and 1b below). It is expected that EPA will release further information regarding the acceptance of mixtures and formulations in the coming months.

Schematic of the AOP "2 out of 3" defined approach

Figure 1a:  Schematic of the AOP “2 out of 3” defined approach. OECD TG methods for Key Events (KEs) 1-2 are run in an undefined order until least two of the three methods show consensus. (, 2018)

Schematic of the Key Event (KE) 3/1 Sequential Testing Strategy defined Approach

Figure 1b:  Schematic of the Key Event (KE) 3/1 Sequential Testing Strategy defined approach. (, 2018)

Currently, assays that can be used in this approach are:

IIVS has considerable knowledge and hands-on expertise in test methods representing all 3 Key Events and can assist with US EPA regulatory submission needs. For more information, please contact Brent Gilbert, Business Development Manager at 813-598-5570.