Roadmap Implementation: Acute Systemic Toxicity

The ATWG published a scoping document (Strickland et al. 2018) that identifies U.S. agency information requirements, needs, and decision contexts for acute systemic toxicity testing. A paper by NICEATM scientists and international collaborators (Strickland et al. 2023) describes international information requirements, needs, and decision contexts for acute systemic toxicity testing.

ICCVAM and NICEATM facilitate public-private partnerships to share data and information on the development and evaluation of alternative methods for acute systemic toxicity assessment. For example, EPA established a stakeholder group to discuss the development, evaluation, and implementation of alternatives for the “six-pack” of acute toxicity tests required for pesticide registration, including the three systemic toxicity tests. Activities within this group have informed EPA efforts toward adoption of alternative methods, such as guidance on waivers for dermal toxicity testing (described below).

NICEATM and ICCVAM partner with other organizations to hold workshops to discuss the state of the science for non-animal alternatives to current in vivo acute systemic toxicity tests and implementation progress. These include:

• A 2015 workshop (summarized in Hamm et al. 2017) identified resources necessary to advance identification and implementation of alternative methods for acute systemic toxicity testing, including high-quality reference data, training on use and interpretation of computational approaches, and global harmonization of testing requirements.
• A 2016 webinar series and workshop (summarized in Clippinger et al. 2018a) focused on alternatives for inhalation toxicity testing. The workshop identified activities needed to develop alternative testing strategies that will meet the information needs of regulatory agencies. Workgroups were established to coordinate these activities; some outcomes are described below under “Developing IATA and Defined Approaches."
• A 2018 workshop (summarized in Kleinstreuer et al. 2018) presented the results of a global project (described below) to develop in silico models for acute oral systemic toxicity. Insights on the reproducibility of in vivo studies were also discussed.
• A 2019 workshop (summarized in Sullivan et al. 2021) considered approaches available to assess acute lethality associated with chemicals and chemical mixtures, with the purpose of designing comprehensive strategies to predict toxicity while avoiding animal tests. Follow-up actions include an analysis of variability in the in vivo LD50 test to establish confidence in toxicity predictions (Karmaus et al. 2022), a comprehensive assessment of an additivity equation for predicting mixtures toxicity (Hamm et al. 2021), and exploring the addition of biological and mechanistic information to complement in silico predictions.

Development of new testing approaches requires high-quality data. One of the follow-up activities from the inhalation toxicity workshop was to gather acute inhalation toxicity data, and these efforts are underway. NICEATM and the EPA National Center for Computational Toxicology (NCCT; now part of the EPA Center for Computational Toxicology & Exposure) compiled acute oral LD50 data from multiple curated databases, and NICEATM has also compiled EPA data on pesticide formulations. These data sets are available through the NICEATM Integrated Chemical Environment (ICE).

NICEATM continues to work with the ATWG to quantify and define the variability of in vivo endpoints from acute systemic toxicity bioassays, such as oral LD50 values. These analyses provide important insight into the reproducibility of the in vivo tests and add context for appropriately evaluating the performance of non-animal approaches that are under development.

• Analyses of curated acute oral toxicity data are described in a paper by Karmaus et al. (2022).
• Curated acute inhalation toxicity data were released via the NICEATM Integrated Chemical Environment in 2023. This was a follow-up activity from the 2016 workshop described above.

Efforts are ongoing to establish public-private partnerships to share acute systemic toxicity data. Such partnerships could provide data for evaluating alternative methods for a broader range of substances than is available from the scientific literature.

NICEATM and ICCVAM agencies are considering a number of different approaches to reduce animal use for required testing. These include using integrated approaches to testing and assessment (IATA) and defined approaches, which enable prediction of acute systemic toxicity through high-throughput screening assays and in silico models.

One product from the 2016 inhalation toxicity workshop was a state-of-the-science manuscript (Clippinger et al. 2018b), which describes relevant adverse outcome pathways that could be used to guide development of non-animal testing strategies for inhalation toxicity. The paper also summarizes the toolbox of in vitro and in silico models that can be used to assess key events from inhalation exposure to toxic outcomes.

In November 2017, the ATWG initiated a global project to develop in silico models of acute oral systemic toxicity that predict five specific endpoints identified by ATWG members as being relevant to U.S. regulatory agencies (Mansouri et al. 2021). Predictive models were developed and evaluated in both a qualitative and quantitative manner, using a large body of rat oral acute toxicity data collected by NICEATM and NCCT. Models developed for the project that met criteria defined by the project organizing committee were used to generate consensus predictions for the acute oral toxicity endpoints of interest. Models were presented at an 2018 workshop (summarized in Kleinstreuer et al. 2018) and the consensus predictions generated for the project are available via the Open (Quantitative) Structure-activity/property Relationship App (OPERA), a free and open-source QSAR tool. The predictions are available via ICE and the EPA’s CompTox Dashboard.

Other approaches being used by ICCVAM agencies to reduce animal use for required testing include non-testing approaches such as waivers. For example, the EPA Office of Pesticide Products issued guidance in 2016 for waiving the acute dermal toxicity test for pesticide formulations, based on a NICEATM analysis that indicated that oral acute toxicity tests were often more informative and sensitive. Similarly, in February 2021, EPA published guidance to reduce testing on birds when registering conventional outdoor pesticides. Read more about U.S. regulatory applications of 3Rs approaches.

A common theme that has emerged from past NICEATM workshops is the need for training opportunities to inform industry and regulatory staff about the use and interpretation of in vitro and in silico data and defined approaches. Thus, efforts to facilitate regulatory acceptance and use of defined approaches and other alternative methods will include the development of training materials and technical support to assist agencies in issuing guidance.