New approach methodologies (NAMs) are increasingly being used for regulatory decision making by agencies worldwide because of their potential to reliably and efficiently produce information that is fit for purpose while reducing animal use. This article summarizes the ability to use NAMs for the assessment of human health effects of industrial chemicals and pesticides within the United States, Canada, and European Union regulatory frameworks. While all regulations include some flexibility to allow for the use of NAMs, the implementation of this flexibility varies across product type and regulatory scheme. This article provides an overview of various agencies’ guidelines and strategic plans on the use of NAMs, and specific examples of the successful application of NAMs to meet regulatory requirements. It also summarizes intra- and inter-agency collaborations that strengthen scientific, regulatory, and public confidence in NAMs, thereby fostering their global use as reliable and relevant tools for toxicological evaluations. Ultimately, understanding the current regulatory landscape helps inform the scientific community on the steps needed to further advance timely uptake of approaches that best protect human health and the environment.

Inhalation is a critical route through which substances can exert adverse effects in humans; therefore, it is important to characterize the potential effects that inhaled substances may have on the human respiratory tract by using fit for purpose, reliable, and human relevant testing tools. In regulatory toxicology testing, rats have primarily been used to assess the effects of inhaled substances as they-being mammals-share similarities in structure and function of the respiratory tract with humans. However, questions about inter-species differences impacting the predictability of human effects have surfaced. Disparities in macroscopic anatomy, microscopic anatomy, or physiology, such as breathing mode (e.g., nose-only versus oronasal breathing), airway structure (e.g., complexity of the nasal turbinates), cell types and location within the respiratory tract, and local metabolism may impact inhalation toxicity testing results. This review shows that these key differences describe uncertainty in the use of rat data to predict human effects and supports an opportunity to harness modern toxicology tools and a detailed understanding of the human respiratory tract to develop testing approaches grounded in human biology. Ultimately, as the regulatory purpose is protecting human health, there is a need for testing approaches based on human biology and mechanisms of toxicity.

Prolonged exposure to environmental respirable toxicants can lead to the development and worsening of severe respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD) and fibrosis. The limited number of FDA-approved inhaled drugs for these serious lung conditions has led to a shift from in vivo towards the use of alternative in vitro human-relevant models to better predict the toxicity of inhaled particles in preclinical research. While there are several inhalation exposure models for the upper airways, the fragile and dynamic nature of the alveolar microenvironment has limited the development of reproducible exposure models for the distal lung. Here, we present a mechanistic approach using a new generation of exposure systems, the Cloud α AX12. This novel in vitro inhalation tool consists of a cloud-based exposure chamber (VITROCELL) that integrates the breathing ^AXLung-on-chip system (AlveoliX). The ultrathin and porous membrane of the AX12 plate was used to create a complex multicellular model that enables key physiological culture conditions: the air-liquid interface (ALI) and the three-dimensional cyclic stretch (CS). Human-relevant cellular models were established for a) the distal alveolar-capillary interface using primary cell-derived immortalized alveolar epithelial cells (^AXiAECs), macrophages (THP-1) and endothelial (HLMVEC) cells, and b) the upper-airways using Calu3 cells. Primary human alveolar epithelial cells (^AXhAEpCs) were used to validate the toxicity results obtained from the immortalized cell lines. To mimic in vivo relevant aerosol exposures with the Cloud α AX12, three different models were established using: a) titanium dioxide (TiO2) and zinc oxide nanoparticles b) polyhexamethylene guanidine a toxic chemical and c) an anti-inflammatory inhaled corticosteroid, fluticasone propionate (FL). Our results suggest an important synergistic effect on the air-blood barrier sensitivity, cytotoxicity and inflammation, when air-liquid interface and cyclic stretch culture conditions are combined. To the best of our knowledge, this is the first time that an in vitro inhalation exposure system for the distal lung has been described with a breathing lung-on-chip technology. The Cloud α AX12 model thus represents a state-of-the-art pre-clinical tool to study inhalation toxicity risks, drug safety and efficacy.

Scientists are using in vitro methods to answer important research questions and implementing strategies to maximize the reliability and human relevance of these methods. One strategy is to replace the use of fetal bovine serum (FBS)-an undefined and variable mixture of biomolecules-in cell culture media with chemically defined or xeno-free medium. In this study, A549 cells, a human lung alveolar-like cell line commonly used in respiratory research, were transitioned from a culture medium containing FBS to media without FBS. A successful transition was determined based on analysis of cell morphology and functionality. Following transition to commercially available CnT-Prime Airway (CELLnTEC) or X-VIVO™ 10 (Lonza) medium, the cells were characterized by microscopic evaluation and calculation of doubling time. Their genotype, morphology, and functionality were assessed by monitoring the expression of gene markers for lung cell types, surfactant production, cytokine release, the presence of multilamellar bodies, and cell viability following sodium dodecyl sulphate exposure. Our results showed that A549 cells successfully transitioned to FBS-free media under submerged and air-liquid-interface conditions. Cells grown in X-VIVO™ 10 medium mimicked cellular characteristics of FBS-supplemented media while those grown in CnT-Prime Airway medium demonstrated characteristics possibly more reflective of normal human alveolar epithelial cells.

Human precision-cut lung slices (hPCLS), considered a highly relevant ex vivo model of the lung, offer native architecture and cells of the lung tissue including respiratory parenchyma, small airways, and immune competent cells. However, the irregular availability of donor lungs has limited the accessibility of this system. As described here, thousands of hPCLS can be created from one lung, cryopreserved, and used “on demand” by applying slicing and cryopreservation methodology improvements. Fresh and cryopreserved (∼7 and ∼34 weeks; F&C) hPCLS from 1 donor lung were cultured for up to 29 days and evaluated for biomass, viability, tissue integrity, and inflammatory markers in response to lipopolysaccharide (LPS; 5 µg/mL) and Triton™ X-100 (TX100; 0.1%) challenge (24 h) at days 1, 8, 15, 22, and 29 following culture initiation. The F&C hPCLS retained biomass, viability, and tissue integrity throughout the 29 days and demonstrated immune responsiveness with up to ∼30-fold LPS-induced cytokine increases. Histologically, more than 70% of normal cytomorphological features were preserved in all groups through day 29. Similar retention of tissue viability and immune responsiveness post cryopreservation (4 to 6 weeks) and culture (up to 14 days) was observed in hPCLS from additional 3 donor lungs. Banking cryopreserved hPCLS from various donors (and disease states) provides a critical element in researching human-derived pulmonary tissue. The retention of viability and functional responsiveness (≥ 4 weeks) allows evaluation of long-term, complex endpoints reflecting key events in Adverse Outcome Pathways and positions hPCLS as a valuable human-relevant model for use in regulatory applications.

Editorial: Chemical testing using new approach methodologies (NAMs)