Alternative Approaches for Acute Inhalation Toxicity Testing

Building upon 2015 workshops on inhalation toxicity testing of nanomaterials (here) and acute systemic toxicity testing (here), the PETA Science Consortium International and the US NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) co-hosted a meeting and webinar series to address current practices for acute inhalation toxicity testing. The goal of this work is to develop and implement approaches that reduce and replace acute inhalation testing in mammals for both global regulatory agencies and non-regulatory purposes.

In follow-up to this workshop, the Science Consortium is currently funding testing to show the utility of in vitro approaches to assess the effects of inhaled substances on the respiratory tract. To further this work, the Consortium also donated five VITROCELL® in vitro inhalation exposure devices (here) and funded the development of a three-dimensional human tissue model of the lower respiratory tract (here).


Clippinger AJ, Allen D, Jarabek AM, Corvaro M, Gaça M, Gehen S, Hotchkiss JA, Patlewicz G, Melbourne J, Hinderliter P, Yoon M, Huh D, Lowit A, Buckley B, Bartels M, BéruBé K, Wilson DM, Indans I, Vinken M. Alternative Approaches for Acute Inhalation Toxicity Testing to Address Global Regulatory and Non-Regulatory Data Requirements:  An International Workshop Report. Toxicol In Vitro. 2018;48:53-70.

Clippinger AJ, Allen D, Behrsing H, BéruBé KA, Bolger MB, Casey W, DeLorme M, Gaça M, Gehen SC, Glover K, Hayden P, Hinderliter P, Hotchkiss JA, Iskandar A, Keyser B, Luettich K, Ma-Hock L, Maione A, Makena P, Melbourne J, Milchak L, Ng S, Paini A, Page K, Patlewicz G, Prieto P, Raabe H, Reinke E, Roper C, Rose J, Sharma M, Spoo W, Thorne PA, Wilson DM, Jarabek AM. Pathway-based predictive approaches for non-animal assessment of acute inhalation toxicity. Toxicol In Vitro. 2018; 52:131-145.

Workshop Recommendations

Recommendations were made in four areas: 1) Develop a database of existing acute systemic toxicity data; 2) prepare a state-of-the-science review on mechanisms and assays for acute inhalation toxicity; 3) develop an in silico decision tree; and 4) to optimize in vitro assays and standardized protocols that can be used across laboratories. Working groups were developed and tasked with follow-up actions directed towards implementing each of these recommendations.

1) Develop a database of existing acute systemic toxicity data. Central to implementing alternative approaches for acute inhalation toxicity testing will be consolidating existing databases and obtaining additional data that has not yet been shared publicly. This working group will be tasked with cataloging the multitude of acute toxicity databases and determining the most efficient and user-friendly way to consolidate relevant information.

2) Prepare a state-of-the-science review on mechanisms and in vitro / in silico assays for acute inhalation toxicity. This working group published an article detailing the numerous mechanisms of action associated with acute inhalation toxicity, with careful consideration of the impact of dosimetry including metabolism. Relevant adverse outcome pathways that can be used to inform the appropriate integrated testing approach were defined. Additionally, the group cataloged the available alternative approaches and defined their usefulness and limitations. This review, published in 2018, encompassed the mechanisms of acute systemic toxicity and portal-of-entry effects.

3) Identify and optimize in silico approaches. This working group will be charged with identifying in silico methods that can be used to predict toxicity that may result from general acute toxicity mechanisms (e.g., reactivity) or mechanisms specific to the inhalation route and to predict whether exposure via the inhalation route is feasible based on a substance’s physicochemical properties. This work will directly inform working groups 2 and 4.

4) Develop a decision tree/testing strategy and conduct an in silico and in vitro proof-of-concept study. This working group is tasked with designing a testing approach to show the utility of in vitro and in silico methods in assessing acute inhalation toxicity. Building off the research of groups 1, 2, and 3, this group will (1) establish a list of reference chemicals based on availability of curated in vivo data (rat, human, and/or other species) that can be used to interrogate available alternative approaches; (2) develop a decision tree that will inform the need to conduct testing following in silico modeling; (3) select the relevant in vitro assays to include in an integrated approach based on mechanisms of acute inhalation toxicity in humans; and (4) optimize the in vitro and in silico assays and standardize protocols that can be used across laboratories.

Coordination of the four working groups and progress on established milestones and timelines is being coordinated by the Consortium and NICEATM.

In addition, the Science Consortium and NICEATM co-hosted a meeting at the 55th annual Society of Toxicology conference to provide an update on the progress made since our workshop and ongoing projects (see here for details) and presented a poster on “A Mechanistic Approach Using Adverse Outcome Pathways to Aid in the Design of In Vitro Inhalation Testing.” A poster was also presented at the 10th World Congress on Alternative and Animal Use in the Life Sciences (see here) and a talk presented at the 6th annual American Society for Cellular and Computational and Toxicology meeting (see here).

September 22-23, 2016 meeting slides

The case for an integrated approach to acute inhalation toxicity testing and assessment Jon Hotchkiss, The Dow Chemical Company
An alternative framework for acute toxicity using mechanistic in silico and in vitro approaches Dan Wilson, The Dow Chemical Company
An alternative approach for evaluating the human health risk from exposure to an irritant aerosol Paul Hinderliter, Syngenta
Toxicokinetics in risk assessment: evaluation of in silico approaches Michael Bartels,, LLC
Assessing bioavailability and systemic delivery of inhaled compounds: current status and future directions Miyoung Yoon, Scitovation
US EPA OPP regulatory perspective on acute inhalation toxicity testing Anna Lowit, US EPA Office of Pesticide Programs
US EPA OPPT regulatory perspective on acute inhalation toxicity testing Iris Camacho, US EPA Office of Pollution Prevention and Toxics
ICCVAM’s vision and strategy for acute toxicity testing Grace Patlewicz, US EPA National Center for Computational Toxicology



Current testing practices: regulatory requirements and non-regulatory testing Dr. Jon Hotchkiss, The Dow Chemical Company
Mr. Ian Indans, Health and Safety Executive, UK
State-of-the-science, practical application, and dosimetry considerations for in vitro and ex vivo methods Dr. Marianna Gaca, British American Tobacco
Dr. Annie Jarabek, US Environmental Protection Agency (NCEA, ORD)
State-of-the-science and practical application of in silico methods Dr. Grace Patlewicz, US Environmental Protection Agency (NCCT, ORD)
Dr. Dan Wilson, The Dow Chemical Company
GHS additivity approach to classify mixtures based on ingredient toxicity Dr. Marco Corvaro, Dow Agrosciences
Adverse outcome pathways Dr. Mathieu Vinken, Free University of Brussels-Belgium
Dr. Barbara Buckley, US Environmental Protection Agency (NCEA, ORD)
21st century testing approaches Dr. Dan Huh, University of Pennsylvania
Dr. Kelly BéruBé, Cardiff University

As a prelude to this webinar series, we encourage you to watch an August 2015 webinar recorded for the workshop on “Alternative Approaches for Identifying Acute Systemic Toxicity: Moving from Research to Regulatory Testing.” This webinar summarizes the EPA OPP’s regulatory requirements for acute inhalation toxicology, and details two commercially available in vitro organotypic models.

Further reading

Detailed information about the 2015 acute systemic toxicity workshop can be found here, and information about the Consortium’s work to develop an in vitro system to assess subchronic inhalation toxicity can be found here.