Evaluation of Inhalation Exposures and Potential Health Impacts of Ingredient Mixtures Using in vitro to in vivo Extrapolation

Abstract

In vitro methods offer opportunities to provide mechanistic insight into bioactivity as well as human-relevant toxicological assessments compared to animal testing. One of the challenges for this task is putting in vitro bioactivity data in an in vivo exposure context, for which in vitro to in vivo extrapolation (IVIVE) translates in vitro bioactivity to clinically relevant exposure metrics using reverse dosimetry. This study applies an IVIVE approach to the toxicity assessment of ingredients and their mixtures in e-cigarette (EC) aerosols as a case study. Reported in vitro cytotoxicity data of EC aerosols, as well as in vitro high-throughput screening (HTS) data for individual ingredients in EC liquids (e-liquids) are used. Open-source physiologically based pharmacokinetic (PBPK) models are used to calculate the plasma concentrations of individual ingredients, followed by reverse dosimetry to estimate the human equivalent administered doses (EADs) needed to obtain these plasma concentrations for the total e-liquids. Three approaches (single actor approach, additive effect approach, and outcome-oriented ingredient integration approach) are used to predict EADs of e-liquids considering differential contributions to the bioactivity from the ingredients (humectant carriers [propylene glycol and glycerol], flavors, benzoic acid, and nicotine). The results identified critical factors for the EAD estimation, including the ingredients of the mixture considered to be bioactive, in vitro assay selection, and the data integration approach for mixtures. Further, we introduced the outcome-oriented ingredient integration approach to consider e-liquid ingredients that may lead to a common toxicity outcome (e.g., cytotoxicity), facilitating a quantitative evaluation of in vitro toxicity data in support of human risk assessment.

Use of quantitative in vitro to in vivo extrapolation (QIVIVE) for the assessment of non-combustible next-generation product aerosols

“…While important, these factors were not taken into consideration as the goal was to compare the potency of HTPs versus cigarettes. However, as a range of nicotine delivery products such as e-vapor products are gaining popularity among adult smokers, IVIVE modeling for mixtures must be considered ( Zhang et al, 2021 ). Qualitatively, differences in irritant properties of the inhaled mixtures may be the primary cause of the c-jun responses being greater with cigarettes compared to the HTP.…”

Key challenges for in vitro testing of tobacco products for regulatory applications: Recommendations for dosimetry

“…Indeed, the biological relevance of the in vitro assays to in vivo endpoints impacts the ability of the IVIVE approach to provide an accurate estimation of an equivalent administered dose (EAD), that is, the estimated external dose in humans that may induce similar bioactivity reflected by the in vitro assay. [10][11][12] These methods that place in vitro results in an in vivo context are gaining increasing importance in scientific decision making 13 and in the context of NGP evaluations. Chang et al 11 and Zhang et al 12 used an IVIVE approach to assess the toxicity of ingredients and their mixtures in e-cigarette aerosols.…”

“…It should be noted that the validity of these methods relies heavily on the accuracy of the in vitro response measurements and how relevant the in vitro assay is to the in vivo mode of action. Indeed, the biological relevance of the in vitro assays to in vivo endpoints impacts the ability of the IVIVE approach to provide an accurate estimation of an equivalent administered dose (EAD), that is, the estimated external dose in humans that may induce similar bioactivity reflected by the in vitro assay 10–12 …”

“…These methods that place in vitro results in an in vivo context are gaining increasing importance in scientific decision making 13 and in the context of NGP evaluations. Chang et al 11 and Zhang et al 12 used an IVIVE approach to assess the toxicity of ingredients and their mixtures in e‐cigarette aerosols. The authors concluded that “IVIVE is a useful tool for interpreting in vitro data in the context of in vivo human exposure and can be applied to mixtures assessment for hypothesis generation and preliminary risk assessment.” 12 This underscores the necessity to conduct appropriate and, relevant, in vitro studies utilizing the most appropriate dosimetry measurements to facilitate subsequent data use in applications such as IVIVE extrapolations.…”

“…Chang et al 11 and Zhang et al 12 used an IVIVE approach to assess the toxicity of ingredients and their mixtures in e‐cigarette aerosols. The authors concluded that “IVIVE is a useful tool for interpreting in vitro data in the context of in vivo human exposure and can be applied to mixtures assessment for hypothesis generation and preliminary risk assessment.” 12 This underscores the necessity to conduct appropriate and, relevant, in vitro studies utilizing the most appropriate dosimetry measurements to facilitate subsequent data use in applications such as IVIVE extrapolations.…”