In vitro Alternatives to Acute Inhalation Toxicity Studies in Animal Models—A Perspective

Abstract

When assessing the risk and hazard of a non-pharmaceutical compound, the first step is determining acute toxicity, including toxicity following inhalation. Inhalation is a major exposure route for humans, and the respiratory epithelium is the first tissue that inhaled substances directly interact with. Acute inhalation toxicity testing for regulatory purposes is currently performed only in rats and/or mice according to OECD TG403, TG436, and TG433 test guidelines. Such tests are biased by the differences in the respiratory tract architecture and function across species, making it difficult to draw conclusions on the potential hazard of inhaled compounds in humans. Research efforts have been therefore focused on developing alternative, human-relevant models, with emphasis on the creation of advanced In vitro models. To date, there is no In vitro model that has been accepted by regulatory agencies as a stand-alone replacement for inhalation toxicity testing in animals. Here, we provide a brief introduction to current OECD test guidelines for acute inhalation toxicity, the interspecies differences affecting the predictive value of such tests, and the current regulatory efforts to advance alternative approaches to animal-based inhalation toxicity studies. We then list the steps that should allow overcoming the current challenges in validating In vitro alternatives for the successful replacement of animal-based inhalation toxicity studies. These steps are inclusive and descriptive, and should be detailed when adopting in house-produced 3D cell models for inhalation tests. Hence, we provide a checklist of key parameters that should be reported in any future scientific publications for reproducibility and transparency.

A Human-Relevant 3D In Vitro Platform for an Effective and Rapid Simulation of Workplace Exposure to Nanoparticles: Silica Nanoparticles as Case Study

“…In this complex scenario, the development of three-dimensional (3D) in vitro models coupled with a nebulizer system (as explained above) that can mimic tissue structure and in vivo functionalities could provide great benefits to answer, although partially, the above questions raised by in vivo and epidemiological studies, especially regarding toxicity testing [ 17 , 18 ]. Advanced in vitro 3D models resembling the lung are currently available, as two-dimensional (2D) models lack the complexity of physiological systems and long-term exposure studies [ 18 , 19 ]. Pulmonary interface is mimicked by using a 3D (airway/lung) model culture at the air–liquid interface (ALI), which more closely resembles the in vivo lung epithelium, where the apical surface is exposed to air and the basal surface of the cells is in contact with the liquid culture medium.…”

Preclinical Development of Orally Inhaled Drugs (OIDs)—Are Animal Models Predictive or Shall We Move Towards In Vitro Non-Animal Models?

“…Although in vitro, cell-based NAMs still have limitations, the advantages associated with their use is evident and future efforts should aim at validating these systems for regulatory acceptance [59]. In the development of OIDs, we should therefore invest in moving away from animal studies.…”

“…Removal mechanisms constitute the third feature to keep into account for developing an effective inhalation therapy. As described in detail in Section 2.1.1, this feature is species-specific [59] and, therefore, human-specific removal mechanisms are not replicated by animal models. Notably, human-specific removal mechanisms can be reproduced by in vitro, cell-based NAMs [60][61][62][63], as discussed in detail in Section 2.2.…”

“…For example, rodents are obligate nose breathers; this strongly influences how inhaled compounds deposit in the respiratory tract. This and other interspecies differences have been extensively discussed by the authors in a recent perspective [59]. Preclinical studies during OID development requires a clear understanding of such interspecies differences and their impact on the screening outcomes in terms of OID efficacy, toxicity and recovery from adverse effects.…”

“…The next section of this commentary focuses on this specific aspect, complementing the authors' previous publication [59] and further discussing if and how new approach methodologies (NAMs) could become useful in the attempt to overcome the limitations of current animal models and increase OID translation rate. For completeness, it should be mentioned here that the abbreviation "NAMs" is often used in toxicology to refer broadly to any non-animal technology, methodology, approach, or combination thereof that can be used to provide information on chemical hazard and risk assessment.…”