The Institute for In Vitro Sciences (IIVS) has sponsored a series of workshops with the aim of developing recommendations for scientific and technical approaches to conducting in vitro assays to assess potential toxicity within and across tobacco and nicotine products. As well as providing a background overview of oral tobacco and nicotine products, this report focuses on the methods used to generate extracts from these products for in vitro testing, the recommended best testing practices, and suggested areas for future research. This publication was developed by a working group of workshop members, in conjunction with the ninth IIVS in vitro workshop entitled 'In Vitro Models for Testing Oral Tobacco and Nicotine Products and Continued Discussion of In Vitro Models of Toxicity and Disease', which was held in January 2024.

The aim of this study was to evaluate an in vitro method using the EpiOralTM model, a three-dimensional cultured human buccal epithelium, for assessing the oral irritation potential of various products. We evaluated different concentrations of nine chemicals commonly found in over-the-counter (OTC) products and medical devices, including chlorhexidine digluconate, sodium hypochlorite, phosphoric acid, hydrogen peroxide, lactic acid, ethanol, sodium dodecyl sulfate, 1-decanol and methyl methacrylate. The method was able to identify the irritants with a clear dose–response relationship between cell viability and an increasing concentration of the chemicals in the tested solutions. Using three exposure times (1, 4 and 18 h) and calculating the ET-50 (time required to induce a 50% reduction in cell viability), the solutions were classified according to their irritant potency (strong, moderate, mild or non-irritant). The results showed excellent correlation with historical in vivo data by matching the potency classifications in most cases. This study highlighted the importance of multiple exposure times for accurate assessment, as some solutions with irritant chemicals require longer exposure to produce effects. By providing information on both the irritant potential and potency, this method proved useful for toxicologists in the risk assessment of OTC products and medical devices that come into contact with the oral cavity.

Risk of lung damage from inhaled chemicals or substances has long been assessed using animal models. However, New Approach Methodologies (NAMs) that replace, reduce, and/or refine the use of animals in safety testing such as 2D and 3D cultures are increasingly being used to understand human-relevant toxicity responses and for the assessment of hazard identification. Here we review 2D and 3D lung models in terms of their application for inhalation toxicity assessment. We highlight a key case study for the Organization for Economic Cooperation and Development (OECD), in which a 3D model was used to assess human toxicity and replace the requirement for a 90-day inhalation toxicity study in rats. Finally, we consider the regulatory guidelines for the application of NAMs and potential use of different lung models for aerosol toxicity studies depending on the regulatory requirement/context of use.

In vitro reconstructed models of human tissues are physiologically relevant alternatives to animal tests in many areas. They have been shown to reliably predict toxicity, interaction with pathogens, absorption and metabolism of pharmaceuticals and other substances as well as other processes. The prediction of acute respiratory toxicity (ART) and irritation is currently largely dependent on the use of animal models, which is problematic on both physiological and ethical grounds. The goal of this work was to develop physiologically relevant ART in vitro tests utilizing the EpiAirway™ tracheobronchial tissue model, to demonstrate correlation to OECD-accepted GHS categorization. Test articles (n=53) were applied to tissues produced at two separate facilities with two ART protocols, the Direct Application Protocol (DAP) for exposure to mists/sprays, and the Vapor Cap Protocol (VCP) for exposure to vapors/volatile liquids. The effects on tissue viability (MTT assay) and barrier properties (TEER) were determined. The effective doses were interpolated for the DAP and VCP methods and correlated to the GHS categories. Using MTT, the Sensitivity/Specificity/Accuracy (S/S/A) of DAP method were 63.5/76.1/69.8% (lab1) and 63.8/76.1/70.0% (lab2) - R2 =0.91. The VCP showed S/S/A of 70.8/83.2/77.0 (lab1) and 71.9/83.2/77.5% (lab2) - R2 =0.93. Using the MTT assay, both VCP and DAP demonstrated good predictivity of GHS categories and high interlaboratory reproducibility. Both protocols provide robust, efficient, physiologically relevant, organ-specific in vitro tests that can improve the predictivity of human responses and reduce the number of animals used to assess respiratory toxicity.

An efficient method for the C-C bond formation via water soluble Na2PdCl4/sSPhos mediated Suzuki-Miyaura cross-coupling reaction of DNA-conjugated aryl iodide with (het)aryl boronic acids has been developed. This reaction proceeds at 37°C in water and acetonitrile (4:1) system. We also demonstrated that numerous aromatic and heteroaromatic boronic acids of different electronic natures, and harboring various functional groups, were highly compatible providing the desired coupling products in good to excellent yields. This DNA-compatible Suzuki-Miyaura cross-coupling reaction has strong potential to construct DNA-Encoded Libraries (DELs) in the context of drug discovery.