Pathway-based predictive approaches for non-animal assessment of acute inhalation toxicity

Abstract

New approaches are needed to assess the effects of inhaled substances on human health. These approaches will be based on mechanisms of toxicity, an understanding of dosimetry, and the use of in silico modeling and in vitro test methods. In order to accelerate wider implementation of such approaches, development of adverse outcome pathways (AOPs) can help identify and address gaps in our understanding of relevant parameters for model input and mechanisms, and optimize non-animal approaches that can be used to investigate key events of toxicity. This paper describes the AOPs and the toolbox of in vitro and in silico models that can be used to assess the key events leading to toxicity following inhalation exposure. Because the optimal testing strategy will vary depending on the substance of interest, here we present a decision tree approach to identify an appropriate non-animal integrated testing strategy that incorporates consideration of a substance's physicochemical properties, relevant mechanisms of toxicity, and available in silico models and in vitro test methods. This decision tree can facilitate standardization of the testing approaches. Case study examples are presented to provide a basis for proof-of-concept testing to illustrate the utility of non-animal approaches to inform hazard identification and risk assessment of humans exposed to inhaled substances.

Keywords: Acute inhalation toxicity; Adverse outcome pathway; Aggregate exposure pathway; Dosimetry; Ex vivo; In silico; In vitro; Integrated approach to testing and assessment (IATA); Quantitative structure-activity relationships (QSAR); Risk assessment.

Fig. 1.

Integration of exposure information into an aggregate exposure pathway:adverse outcome pathway (AEP:AOP) framework. Key events (KE) in the AOP are indicated by the pink-colored boxes. The arrows between the source and the target site exposure (TSE) indicate analogous exposure KEs in the AEP. The molecular initiating event (MIE) links the AEP and AOP frameworks.

Fig. 2.

Selected key events and adverse outcomes that may occur systemically or in the respiratory tract as the portal of entry following acute exposures to an inhaled material. The TSE is a function of dosimetry including absorption, distribution, elimination and metabolism (ADME) processes that dictate disposition of the parent chemical or its metabolite(s). Chronic lung effects may occur as a result of acute toxicity that does not cause death or with repeated acute exposures notably when either the chemical or its damage persists; exposure concentration, frequency, and duration also impact the trajectories of different pathways. Tissue remodeling would not be evident after a single acute exposure, and protective cellular effects that could mitigate the listed adverse responses are not listed here. Additionally, while respiratory sensitization may occur following acute exposure to an inhaled substance, this endpoint is not included as it is not generally considered in the current acute systemic toxicity animal tests.

Fig. 3:

Decision tree that can be used to guide the exposure parameters and design of an integrated strategy for inhalation testing. Results of proof-of-concept testing based on this decision tree, the AOPs in Table 1, and the considerations in Table 2 can be evaluated to develop guidance on classification and other inhalation toxicity applications. The decision tree does not supplant expert judgement but is intended as conceptual structure to help identify considerations for experimental design. (1) Evaluate target exposure scenario and type of inhaled agent: Target scenario includes parameters that characterize specific population considerations (e.g., general population, children, or occupational), regimen (e.g., number of days per week), and duration (e.g., hours per day and work week or lifetime). Evaluating the type of inhaled agent requires assessment of exposure generation and characterization. Target scenario may also include consideration of problem formulation or objective of the testing, including the information requirement (e.g., identification of intrinsic hazard properties of chemicals or end-use products; hazard communication (i.e., classification; product labeling statements/signal words); industrial hygiene (e.g., short-term exposure levels or time-weighted averages); and emergency response (e.g., egress or re-entry levels). (2) Determine particle size distribution and density: a) Conversion of diameter to aerodynamic diameter (d_ae) or mass median aerodynamic diameter (MMAD) may be required (US EPA 1994). b) Additional physicochemical properties will help determine likely dosimetry, such as molecular diffusivity; blood:air partition coefficients; ability to be reactive including hydrolysis or to serve as an enzymatic substrate for ADME in respiratory tract tissue; or contains structural alerts indicative of inherent reactivity (e.g., if the chemical contains an electrophilic center such as a carbonyl carbon in an aldehyde). (3) Determine if human inhalation is likely: Human is presumed target population; if evaluating or translating an in vivo animal study would require consideration of that species’ anatomy and ventilation parameters. It may be possible to avoid testing if the aerodynamic diameter of the particle is greater than 100 μM (Vincent 2005). (4) Potential to be corrosive: All available existing information that may be informative should be evaluated, including QSAR, read-across, and data from in vitro assays. Evidence of skin or eye irritation or severe pH could be used to justify waiving an LC₅₀ study (OECD 2014; OECD 2017); however, this generalization does not apply to all chemistries as some agents with a high/low pH are not corrosive, and not all corrosive agents will possess a high acute toxicity. (5) Electrophile, reactive or specific toxicity or metabolism alert: If molecular structure (e.g., chemical contains an electrophilic center such as the case with an activated ester or aldehyde) indicates likelihood of specific reactions such as hydrolysis or specific metabolism by cells in respiratory tract, then characterization of POE toxicity is necessary in addition to systemic toxicity. (6) Calculate HEC for effects in portal of entry (POE): Human equivalent concentration (HEC) is calculated with different models for particles and gases; and for respiratory versus remote or systemic effects. The choice of model also depends on data availability and ranges from default to sophisticated model structures. The HEC calculations should use input parameters relevant to target exposure scenario and potential mode of action in the respiratory tract. a) For particles, the MPPD model is recommended and can predict regional (extrathoracic, tracheobronchial, or pulmonary) deposition to aid study design. b) For gases likely to react with tissues in the respiratory tract, these are designated as gas Category 1 (described in section 4b above and Table 5 of (Clippinger, Allen et al. 2018)) and default algorithms are applied or models such as CFD or hybrid PBPK-CFD models can refine dose predictions. If potential for both POE and systemic toxicity exists, these are gas Category 2 and both types of toxicity must be addressed (8a and 8b). Guidance on the choice of model structure and necessary data are provided in (US EPA 1994; US EPA 2012; Clippinger, Allen et al. 2018). (7) Calculate HEC for systemic toxicity: Human equivalent concentration (HEC) is calculated with different models for particles and gases; and for respiratory versus remote or systemic effects. The choice of model also depends on data availability and ranges from default to sophisticated model structures. The HEC calculations should use input parameters relevant to target exposure scenario and potential mode of action for systemic toxicity, or possibly both POE and systemic toxicity. a) For particles, the MPPD model is recommended and can predict regional (extrathoracic, tracheobronchial, or pulmonary) deposition to aid study design. b) For gases likely to cause systemic toxicity, these are designated as gas Category 3 and default algorithms are applied, or PBPK-CFD models can refine dose predictions. If potential for both POE and systemic toxicity exists, these are gas Category 2 and both types of toxicity must be addressed (8a and 8b). Guidance on the choice of model structure and necessary data are provided in (US EPA 1994; US EPA 2012; Clippinger, Allen et al. 2018). (8) Assess in in vitro system for POE effects or systemic toxicity: a) Assess in in vitro system, such as a three-dimensional human reconstructed human lung tissue model, for POE effects including respiratory irritation. See Supplementary Table 1 for potential cell systems of interest. b) Assess in in vitro system, such as cardiac, liver, or neuronal cells, for systemic toxicity.