traditional animal testing to new approach methodologies (NAMs), aiming to reduce, refine, or replace (known as the "3R") animal-based methods typically used for toxicity and risk evaluations (European Parliament resolution, 2010). Overall, NAMs are designed intended to ease the transitionreplace from animal studies due to the ethical concerns of using animals, alongside the and challenges in animals providing su icient clinically more relevant risk assessment, such due to as the necessity for large safety factors and the limited predictive value of animal models for in relation to human diseases (Monticello et al. , 2017;Clark and Steger-Hartmann et al. , 2018). NAMs o er several advantages, including the use of relevant human cells, investigation of humanspecific disease mechanisms, provision of mechanistic insights to improve predictive capabilities, development of microphysiological systems that mimic physiological conditions, and integration with Artificial Intelligence (AI) to enhance analysis and interpretation (Schmeisser et al. , 2023). NAMs, which include in vitro and computational (in silico) methods, are becoming valuable tools for safety and risk assessment for novel tobacco and nicotine products (Lee et al. , 2022;Thorne et al. , 2024). Although these tools are growing in use, many are not truly "new"; what is novel is their broader applications beyond traditional hazard identification allowing regulatory decisions based on NAMs without requiring new animal tests. In these issuesthis Research Topic, we have presented a total of 6six peer-reviewed original studies aimed at advancing NAM approaches especially which highlight the use of in vitro tools for evaluating next-generation tobacco and nicotine products (NGPs), such as heated tobacco products (HTPs), nicotine pouches (NPs), and electronic nicotine delivery systems (ENDS). Researchers from both academic institutions and the tobacco industryfoot₀ contributed their testing data and perspectives about the use of selected NAM tools, highlighting strengths, limitations, and potential opportunities in evaluating NGPs. The studies (Cao et al. , 2025;Chapman et al. , 2024;Hayashida et al. , 2025;Ichikawa et al. , 2025;Ito et al. , 2025;Wong et al. , 2025) cover a wide range of novel nicotine and tobacco products, including oral tobaccoderived nicotine (OTDN) products (Cao et al. , 2025), HTPs (Chapman et al. , 2024;Hayashida et al. , 2025), and ENDS (Chapman et al. , 2024;Wong et al. , 2025). These publications utilized various NAM platforms, such as cell-based assays (Cao et al. , 2025;Wong et al. , 2025;Ichikawa et al. , 2025), organ-on-a-chip systems (Chapman et al. , 2024;Hayashida et al. , 2025), omics technologies (Wong et al. , 2025), adverse outcome pathways (Ichikawa et al. , 2025), and in-silico models (Ito et al. , 2025). Below is a summary of the NAMs used in these studies; readers are encouraged to review the six relevant publications for more detail. Cell-based assays employing human-derived cells and tissues allows investigation of chemical e ects within a controlled laboratory setting, enhancing the clinical relevance to toxicity outcomes. Cao et al. (2025) compared the biological impact of OTDN productsextracted using artificial saliva and complete artificial saliva-on cytotoxicity, oxidative stress, and inflammatory responses across four in vitro oral models. These included monolayer cultures of normal human gingival fibroblasts (NHGFs) and oral epithelial cells (NHOEs), as well as three-dimensional organotypic models (EpiOral™ and EpiOral™ Full Thickness). For inhalable products, Wong et al. (2025) conducted an integrated study exposing three-dimensional (3D) human bronchial epithelial tissues (EpiAirway™) at the airliquid interface (ALI) to aerosols containing aldehydes generated through the thermal decomposition of electronic cigarette (EC) solvents, followed by proteomic analyses. Ichikawa et al. (2025) combined an Adverse Outcome Pathway (AOP) framework with an in vitro approach, subjecting 3D cultured human bronchial epithelial cells (HBECs) to whole cigarette smoke (WCS). Organ-on-a-chip (OoC) systems are microphysiological models cultured on microfluidic chips that supply medium and signals through microchannels. Two studies introduced pathogenesis of atherosclerosis models to evaluate ENDS (Chapman et al. , 2024) and HTPs (Chapman et al. , 2024;Hayashidae et al. , 2025). Atherogenic endpoints (oxidative stress, monocyte adhesion, ICAM-1 expression, and inflammatory markers) were assessed on a Human Coronary Artery Endothelial Cells (HCAECs) -chip combined with THP-1 monocytes under flow conditions (Chapman et al. , 2024). Endothelial barrier impairment, monocyte adhesion, and monocyte migration were measured on HCAECs-chip. Macrophages were exposed to HTP aerosol extracts, and conditioned medium was collected. HCAECs cultured on chips were exposed to these conditioned media to mimic the e ects on the vascular system caused by inflammatory responses (Hayashidae et al. , 2025). As supplementary to standardized in vitro tools, discovery-focused omics technologies, such as genomics, metabolomics, proteomics, and transcriptomics, are useful to uncover underlying molecular signatures related to adverse outcomes and/or disease. These approaches encourage a move toward mechanistic toxicology, emphasizing early molecular changes over traditional late-stage pathological e ects. Utilizing proteomics data, researchers performed in vitro experiments on BEAS-2B bronchial epithelial cells, assessing reactive oxygen species, mitochondrial activity, and cytoskeletal stability after short-term exposure to two aldehydes (acetaldehyde or methylglyoxal) at levels commonly found in EC aerosols (Wong et al. , 2025). The results from this proteomics research were further confirmed by the concentration-response 2D in vitro results, providing insight into the mechanisms by which thermal breakdown of EC solvents, propylene glycol and glycerin, may impact biological systems. Adverse Outcome Pathways (AOPs) outline how toxicants cause adverse e ects through key events (KEs) which track the progression toward an adverse outcome (AO). Ichikawa et al. , (2025) developed an AOP-based in vitro method to test disease-related states that can be reproduced by exposing 3D-HBECs (from six di erent donors) to WCS and observe both acute phase responses (oxidative stress, epidermal growth factor receptor activation, and SP1 activation) and chronic phase responses (intracellular mucus production, goblet cell metaplasia/hyperplasia, and mucus hypersecretion) along the AOP. In sSilico models employ computational methodologies to simulate biological responses or predict chemical properties using existing datasets. Ito et al. (2025) integrated the AOP of mucus hypersecretion, -a well-recognized indicator of chronic airway disease as reported by Ichikawa et al. (2025), -with probabilistic quantitative models based on Bayesian networks. The incorporation of an AOP-based in vitro-in silico approach represents a promising tool for evaluating the chronic inhalation e ects associated with consumer products and chemicals. As more individual NAMs platforms were introduced, integrating NAMs (Vuong et al. , 2025) as ana holistic alternative tool for toxicologists has become more common for evaluating products, ingredients, and mixtures. The examples include approaches such as cell-based assays combined with omics technologies (Wong et al. , 2025), cell-based assays integrated with AOP frameworks (Ichikawa et al. , 2025), or in silico-AOP-based cell assay methods (Ito et al. , 2025). Several challenges still remain in accepting NAMs for regulatory purposes, including issues related to validation, reproducibility, such as setting upand the need for robust performance standards and reproducibility. Currently, many NAMs are typicallystill used primarily at a developmental stage, with limited validation that is essential for regulatory acceptance. For instance, As noted ain recent FDA draft guidance, states that "Forregulatory confidence in NAM data depends on the reliability of the methodology and on scientifically accepted validation criteriato be useful for regulatory decision-making, however, the data must be reliable and there must be confidence in the methodology used. This confidence is typically obtained through scientific consensus generated by the application of validation criteria to the design, performance, and interpretation of the studies" (FDA 2026b). Additional considerations include the need for fit-for-purpose methods, the limited availability of models encompassing the breadth of human disease processes, and the recognition that NAMs may not always function as direct one-to-one replacement for traditional involve conceptual changes-for instance, acknowledging that NAMs may not serve as direct oneto-one replacements for 90-day in vivo studies-benchmarking and ensuring comprehensive coverage of potential adverse e ects, ensuring methods are fit-for-purpose and ready for practical use, accurately identifying ingredients with low toxicity, and addressing the limited availability of NAMs that encompass the full spectrum of human diseases and pathologies (German Federal Institute for Risk Assessment, 2022; Ouedraogo et al. , 2025;Schmeisser et al. , 2023). Furthermore, many currently implemented NAMs are predominantly focused on cytotoxicity and related molecular pathways, which may not fully capture the e ects of pharmacologically active constituents, including nicotine, or other compounds associated with systemicfunctional physiological or hemodynamic e ectsresponses. This highlights the need for further methodological development in this area to address a broader range of functional and organism-level responsesbiologically relevant endpoints (Schmeisser et al. , 2023;Ouedraogo et al. , 2025). Overall, the papers included in this research Research topic Topic have served to highlight the growing value example use of NAMs in the toxicity toxicological assessment of various tobacco and nicotine products.
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