In vitro exposure systems and dosimetry assessment tools for inhaled tobacco products: Workshop proceedings, conclusions and paths forward for in vitro model use

Abstract

In 2009, the passing of the Family Smoking Prevention and Tobacco Control Act facilitated the establishment of the FDA Center for Tobacco Products (CTP), and gave it regulatory authority over the marketing, manufacture and distribution of tobacco products, including those termed 'modified risk'. On 4-6 April 2016, the Institute for In Vitro Sciences, Inc. (IIVS) convened a workshop conference entitled, In Vitro Exposure Systems and Dosimetry Assessment Tools for Inhaled Tobacco Products, to bring together stakeholders representing regulatory agencies, academia and industry to address the research priorities articulated by the FDA CTP. Specific topics were covered to assess the status of current in vitro smoke and aerosol/vapour exposure systems, as well as the various approaches and challenges to quantifying the complex exposures in in vitro pulmonary models developed for evaluating adverse pulmonary events resulting from tobacco product exposures. The four core topics covered were: a) Tobacco Smoke and E-Cigarette Aerosols; b) Air-Liquid Interface-In Vitro Exposure Systems; c) Dosimetry Approaches for Particles and Vapours/In Vitro Dosimetry Determinations; and d) Exposure Microenvironment/Physiology of Cells. The 2.5-day workshop included presentations from 20 expert speakers, poster sessions, networking discussions, and breakout sessions which identified key findings and provided recommendations to advance these technologies. Here, we will report on the proceedings, recommendations, and outcome of the April 2016 technical workshop, including paths forward for developing and validating non-animal test methods for tobacco product smoke and next generation tobacco product aerosol/vapour exposures. With the recent FDA publication of the final deeming rule for the governance of tobacco products, there is an unprecedented necessity to evaluate a very large number of tobacco-based products and ingredients. The questionable relevance, high cost, and ethical considerations for the use of in vivo testing methods highlight the necessity of robust in vitro approaches to elucidate tobacco-based exposures and how they may lead to pulmonary diseases that contribute to lung exposure-induced mortality worldwide.

Figure 1.

Summary of experimental design

Figure 2.

Allometric plot of alveolar surface to body mass Source: Pinkerton KE et al. Architecture and Cellular Composition of the Air-Blood Tissue Barrier, Chapter 9. In: Parent RA, ed. Comparative biology of the normal lung. Second edition. ed. New York: Elsevier Academic Press; 2015:105–17.

Figure 3.

Factors involved in respiratory tract dosimetry

Figure 4.

Chemical complexity – smoke vs. e-aerosol GC x GC – TOFMS analysis of a single 55 ml puff

Figure 5.

SEIVS – Smoke flow

Figure 6.

Experimental setup

Figure 7.

The complete exposure system

Figure 8.

The Vitrocell 24/48 Exposure System

Figure 9.

Imaging-based CFD Model Development Corley RA et al. Toxicol Sci 2015;146:65–88. Corley RA et al. Toxicol Sci 2012;128:500–16.

Figure 10.

Processes and system characteristics affecting particle transport rates in liquid containing in vitro systems Source: Hinderliter PM et al. Part Fibre Toxicol 2010;7:36.

Figure 11.

Brief revisit of the respiratory tract. Airway tissue transitions: cell types and functionality https://upload.wikimedia.org/wikipedia/commons/c/c8/Respiratory_Tract_Histological_Differences.png

Figure 12.

In vitro/ex vivo models: RHuA

Figure 13.

The “dripping” technique

Figure 14.

(A) Histology cross section of normal human bronchus from a lung transplant donor (hematoxylin and eosin stain). Asterisks mark capillaries. (B) Schematic of the airway mucosa model including three vertically stacked compartments with three different cell types separated by two nanoporous membranes, arrows indicate channels for fluid or air. From Sellgren et al. Lab Chip 2014;14:3349–58.

Figure 15.

Microfluidic device configuration. (A) and (B) scanning electron microscope images of the polytetrafluoroethylene (PTFE) and polyester (PET) membranes; (C) exploded view and schematic (D) photograph of a 10 × 1 mm device with dyes in the three fluidic channels; (E) optical microscope image of a 10 × 1 mm device cross section. From Sellgren et al. Lab Chip 2014;14:3349–58.

Figure 16.

Turbo RASL-seq procedure Method modified from Li et, al. 2012, PNAS, 109(12):4609–4614

Figure 17.

Seed and soil model for the epigenetic basis of interindividual variability in exposure responses. Note: Dr. McCullough is chairing a Society of Toxicology Contemporary Concepts of Toxicology meeting with Dana Dolinoy, PhD from the University of Michigan on “Toxicoepigenetics: The Interface of Epigenetics and Risk Assessment” (www.toxicology.org/teg) this coming November in Tyson’s Corner, Virginia.