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Advanced In Vitro Methodologies Demonstrate New Paradigms for Toxicological Testing

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Scientists from Philip Morris International (PMI) have presented details of a series of advanced techniques for the in vitro toxicological assessment of complex mixtures.1 For the scientific assessment of heated tobacco and e-cigarette aerosols, PMI has developed and tested methodologies using human cells grown in three-dimensional cultures, networks-based systems toxicological analysis, and organ-on-a-chip models combining cells from multiple human organs. These techniques have significant ramifications for the widely recognized 3Rs of animal research: the replacement, reduction and refinement of animal use in scientific research. The presentation of these techniques and the results obtained was made at a dedicated session on the risk assessment of chemical mixtures at the recent TOXCON 2018 interdisciplinary toxicology conference in Stará Lesná, Slovakia.

“Humans are routinely exposed to many different chemicals, in the air we breathe, the water we drink, and the food and products we consume,” said Dr A. Wallace Hayes, University of South Florida and Michigan State University, and Chairperson of the session at TOXCON. “We need new approaches to testing to assess the health impacts of these complex chemical mixtures and a variety of models, from in silico to non-mammalian in vivo, are proving promising. PMI has demonstrated the significant potential of advanced in vitro methodologies in this area, which not only reduce the need for animal models but also offer more timely, robust, and human-specific scientific findings.”

Human cells grown in three-dimensional cultures were used in a series of studies comparing the effects of exposure to cigarette smoke with the aerosol of the Tobacco Heating System (THS 2.2), one of PMI’s Reduced-Risk Products. Using THS 2.2, tobacco is heated rather than burned, resulting in significantly reduced levels of harmful chemicals emitted and inhaled as compared with cigarette smoke.2 The three-dimensional configuration allowed primary epithelial cells taken from nasal, oral, and bronchial cavities to develop ‘organotypic’ tissues with complexity closely resembling that found in the human body. Cultures were then exposed to either cigarette smoke or THS 2.2 aerosol. Following these exposures, biological effects were assessed at various time-points and using an array of endpoints. Multiple experimental repetitions ensured results were robust, reliable and reproducible.

The biological effects of exposures in the nasal, oral, and bronchial studies were assessed using a combination of well-established in vitro testing endpoints such as deposition of toxic carbonyls, morphological alterations, and secretion of inflammatory mediators. Furthermore, collected data was also processed through a set of manually curated biological network models that are known to relate to respiratory disease.3 Biological impacts were then quantified, compared, and complemented by standard gene-set analysis. Combined results indicated that the biological impact of THS 2.2 aerosol is significantly reduced when compared with cigarette smoke and comparable to that of fresh-air exposure.

Three-dimensional in vitro models can be further enhanced through the incorporation of small devices that reproduce the complex microenvironments of specific organs. These are often referred to as ‘organs-on-chips’. While they typically only represent a single organ, and therefore cannot be used to study the organ-to-organ interactions observed in the human body, PMI has developed a combined lung-liver-on-a-chip. This is an important development because while airborne compounds are absorbed by the lung, their true toxicity may only be fully realized following their metabolization by the liver. PMI has demonstrated the ability of the lung-liver-on-a-chip to hold both lung and liver tissues in a stable state for at least four weeks and has also confirmed its metabolic capacity.

“Testing the biological effects of exposure to heated tobacco aerosols requires the use of specialized test systems and exposure models,” said Dr Anita Iskandar, Senior Scientist, PMI. “The systems biology approaches that we have presented at TOXCON allow us to uncover biological effects at cellular and molecular levels which are undetectable using standard methods of toxicity testing. In collaboration with the wider scientific community, industry, and regulators, we are delighted to be developing the field of toxicological risk assessment and bringing it in line with the vision for 21st century toxicology set out by the US National Research Council.”

To facilitate the reproducible and independent assessment of heated tobacco products, as well as other nicotine delivery systems such as e-cigarettes, PMI is making all their study data available on a dedicated database and searchable web portal called INTERVALS. Datasets are annotated for ease of use and full details of study designs and protocols are also included. The INTERVALS platform is a collaborative initiative that aims to develop a robust methodology for verifying scientific methods and results.

This article has been republished from materials provided by Philip Morris International (PMI). Note: material may have been edited for length and content. For further information, please contact the cited source.


1. Iskandar, A. et al. Advanced in vitro approaches for comparative assessment of heated tobacco products. TOXCON 2018, Stará Lesná, Slovakia.

2. Smith, MR. et al. Evaluation of the Tobacco Heating System 2.2. Part 1: description of the system and the scientific assessment program. Regulatory Toxicology and Pharmacology 81. 2016: S17-S26. https://doi.org/10.1016/j.yrtph.2016.07.006.

3. Boué, S. et al. Causal biological network database: a comprehensive platform of causal biological network models focused on the pulmonary and vascular systems. Database. 2015. http://dx.doi.org/10.1093/database/bav030.