Differences in the anatomy and physiology of the human and rat respiratory tracts and impact on toxicological assessments.

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.

Use of Cause-and-Effect Analysis to Optimize the Reliability of In Vitro Inhalation Toxicity Measurements Using an Air-Liquid Interface.

In vitro inhalation toxicology methods are increasingly being used for research and regulatory purposes. Although the opportunity for increased human relevance of in vitro inhalation methods compared to in vivo tests has been established and discussed, how to systematically account for variability and maximize the reliability of these in vitro methods, especially for assays that use cells cultured at an air-liquid interface (ALI), has received less attention. One tool that has been used to evaluate the robustness of in vitro test methods is cause-and-effect (C&E) analysis, a conceptual approach to analyze key sources of potential variability in a test method. These sources of variability can then be evaluated using robustness testing and potentially incorporated into in-process control measurements in the assay protocol. There are many differences among in vitro inhalation test methods including the use of different types of biological test systems, exposure platforms/conditions, substances tested, and end points, which represent a major challenge for use in regulatory testing. In this manuscript, we describe how C&E analysis can be applied using a modular approach based on the idea that shared components of different test methods (e.g., the same exposure system is used) have similar sources of variability even though other components may differ. C&E analyses of different in vitro inhalation methods revealed a common set of recommended exposure systems and biological in-process control measurements. The approach described here, when applied in conjunction with Good Laboratory Practices (GLP) criteria, should help improve the inter- and intralaboratory agreement of in vitro inhalation test results, leading to increased confidence in these methods for regulatory and research purposes.

Cryopreserved human precision-cut lung slices provide an immune competent pulmonary test system for "on-demand" use and long-term cultures.

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 1 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 (4-6 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.

A multiplex inhalation platform to model in situ like aerosol delivery in a breathing lung-on-chip.

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.

Human cell-based in vitro systems to assess respiratory toxicity: a case study using silanes.

Inhalation is a major route by which human exposure to substances can occur. Resources have therefore been dedicated to optimize human-relevant in vitro approaches that can accurately and efficiently predict the toxicity of inhaled chemicals for robust risk assessment and management. In this study-the IN vitro Systems to PredIct REspiratory toxicity Initiative-2 cell-based systems were used to predict the ability of chemicals to cause portal-of-entry effects on the human respiratory tract. A human bronchial epithelial cell line (BEAS-2B) and a reconstructed human tissue model (MucilAir, Epithelix) were exposed to triethoxysilane (TES) and trimethoxysilane (TMS) as vapor (mixed with N2 gas) at the air-liquid interface. Cell viability, cytotoxicity, and secretion of inflammatory markers were assessed in both cell systems and, for MucilAir tissues, morphology, barrier integrity, cilia beating frequency, and recovery after 7 days were also examined. The results show that both cell systems provide valuable information; the BEAS-2B cells were more sensitive in terms of cell viability and inflammatory markers, whereas MucilAir tissues allowed for the assessment of additional cellular effects and time points. As a proof of concept, the data were also used to calculate human equivalent concentrations. As expected, based on chemical properties and existing data, the silanes demonstrated toxicity in both systems with TMS being generally more toxic than TES. Overall, the results demonstrate that these in vitro test systems can provide valuable information relevant to predicting the likelihood of toxicity following inhalation exposure to chemicals in humans.

Maximizing the relevance and reproducibility of A549 cell culture using FBS-free media.

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.

Use of new approach methodologies (NAMs) to meet regulatory requirements for the assessment of tobacco and other nicotine-containing products.

Regulatory frameworks on tobacco and other nicotine-containing products (TNCP) continue to evolve as novel products emerge, including electronic nicotine delivery systems (e.g., electronic cigarettes or vaping products), heated tobacco products, or certain smokeless products (e.g., nicotine pouches). This article focuses on selected regulations for TNCPs that do not make health claims, and on the opportunities to use new approach methodologies (NAMs) to meet regulatory requirements for toxicological information. The manuscript presents a brief overview of regulations and examples of feedback from regulatory agencies whilst highlighting NAMs that have been successfully applied, or could be used, in a regulatory setting, either as stand-alone methods or as part of a weight-of-evidence approach to address selected endpoints. The authors highlight the need for agencies and stakeholders to collaborate and communicate on the development and application of NAMs to address specific regulatory toxicological endpoints. Collaboration across sectors and geographies will facilitate harmonized use of robust testing approaches to evaluate TNCPs without animal testing.

Advancing New Approach Methodologies (NAMs) for Tobacco Harm Reduction: Synopsis from the 2021 CORESTA SSPT-NAMs Symposium.

New approach methodologies (NAMs) are emerging chemical safety assessment tools consisting of in vitro and in silico (computational) methodologies intended to reduce, refine, or replace (3R) various in vivo animal testing methods traditionally used for risk assessment. Significant progress has been made toward the adoption of NAMs for human health and environmental toxicity assessment. However, additional efforts are needed to expand their development and their use in regulatory decision making. A virtual symposium was held during the 2021 Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) Smoke Science and Product Technology (SSPT) conference (titled "Advancing New Alternative Methods for Tobacco Harm Reduction"), with the goals of introducing the concepts and potential application of NAMs in the evaluation of potentially reduced-risk (PRR) tobacco products. At the symposium, experts from regulatory agencies, research organizations, and NGOs shared insights on the status of available tools, strengths, limitations, and opportunities in the application of NAMs using case examples from safety assessments of chemicals and tobacco products. Following seven presentations providing background and application of NAMs, a discussion was held where the presenters and audience discussed the outlook for extending the NAMs toxicological applications for tobacco products. The symposium, endorsed by the CORESTA In Vitro Tox Subgroup, Biomarker Subgroup, and NextG Tox Task Force, illustrated common ground and interest in science-based engagement across the scientific community and stakeholders in support of tobacco regulatory science. Highlights of the symposium are summarized in this paper.

Use of new approach methodologies (NAMs) to meet regulatory requirements for the assessment of industrial chemicals and pesticides for effects on human health.

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.

Medium throughput breathing human primary cell alveolus-on-chip model.

Organs-on-chips have the potential to improve drug development efficiency and decrease the need for animal testing. For the successful integration of these devices in research and industry, they must reproduce in vivo contexts as closely as possible and be easy to use. Here, we describe a 'breathing' lung-on-chip array equipped with a passive medium exchange mechanism that provide an in vivo-like environment to primary human lung alveolar cells (hAEpCs) and primary lung endothelial cells. This configuration allows the preservation of the phenotype and the function of hAEpCs for several days, the conservation of the epithelial barrier functionality, while enabling simple sampling of the supernatant from the basal chamber. In addition, the chip design increases experimental throughput and enables trans-epithelial electrical resistance measurements using standard equipment. Biological validation revealed that human primary alveolar type I (ATI) and type II-like (ATII) epithelial cells could be successfully cultured on the chip over multiple days. Moreover, the effect of the physiological cyclic strain showed that the epithelial barrier permeability was significantly affected. Long-term co-culture of primary human lung epithelial and endothelial cells demonstrated the potential of the lung-on-chip array for reproducible cell culture under physiological conditions. Thus, this breathing lung-on-chip array, in combination with patients' primary ATI, ATII, and lung endothelial cells, has the potential to become a valuable tool for lung research, drug discovery and precision medicine.

A lung-on-a-chip array with an integrated bio-inspired respiration mechanism.

We report a lung-on-a-chip array that mimics the pulmonary parenchymal environment, including the thin alveolar barrier and the three-dimensional cyclic strain induced by breathing movements. The micro-diaphragm used to stretch the alveolar barrier is inspired by the in vivo diaphragm, the main muscle responsible for inspiration. The design of this device aims not only at best reproducing the in vivo conditions found in the lung parenchyma but also at making the device robust and its handling easy. An innovative concept, based on the reversible bonding of the device, is presented that enables accurate control of the concentration of cells cultured on the membrane by easily accessing both sides of the membranes. The functionality of the alveolar barrier could be restored by co-culturing epithelial and endothelial cells that form tight monolayers on each side of a thin, porous and stretchable membrane. We showed that cyclic stretch significantly affects the permeability properties of epithelial cell layers. Furthermore, we also demonstrated that the strain influences the metabolic activity and the cytokine secretion of primary human pulmonary alveolar epithelial cells obtained from patients. These results demonstrate the potential of this device and confirm the importance of the mechanical strain induced by breathing in pulmonary research.