We advocate that a modern, non-animal testing paradigm is needed for these new product categories.
There is a growing consensus that there is a risk continuum for nicotine delivery products, and most products that do not combust tobacco are likely to be substantially less risky to use than smoking cigarettes. We have developed a range of New Category products such as vapour and tobacco heating products (THPs).
The gold standard to demonstrate that these products are less risky is epidemiology, but it would take 25 to 30 years to collect data in relation to many of the chronic diseases related to smoking. So, we are taking a weight of evidence approach to evaluate where these products sit on a risk continuum1.
The latest technologies adopting 21st century toxicological approaches2 include the advances in human tissue engineering and computational toxicology, along with harnessing molecular biology techniques. These advances are also allowing us to analyse the mechanistic processes that underlie many smoking-related diseases (in particular oxidative stress).
To ensure that our New Category products meet high quality standards, we extensively test their safety by looking for and measuring potentially harmful effects relative to cigarettes. As part of this, we run a wide range of in vitro toxicity tests – lab studies that don’t involve humans or animals.
Classical in vitro regulatory toxicology tests are used in other industries, including cosmetics and pharmaceuticals, and they have been adapted for use in testing cigarette smoke and, more recently, aerosols from New Category products. These types of tests are conducted on simple cell systems – using monolayers of cells grown in a dish. Under well-defined laboratory and experimental conditions, we expose the cells to cigarette smoke or aerosol from one of our New Category products and assess and compare them afterwards. We examine, for instance, whether the cells have died as a result of exposure (cytotoxicity), or whether they show signs of DNA damage, or any mutations or changes that could lead to the potential formation of tumours.
Results from a battery of these tests show that, in stark contrast to cigarette smoke which gives rise to a great deal of biological activity in most tests, vapour from our Vype ePen or aerosol from our THP glo is substantially less toxic to cells, doesn’t cause mutations, and doesn’t promote the formation of tumours in these laboratory test conditions1,3. Further studies are needed to determine whether these reductions are sustained and translate to a reduction in smoking-related health risks.
These tests are based on well-established methods, and regulators have issued clear test guidance on how to conduct them, ensuring standardisation across laboratories. There are drawbacks, however, as some tests don’t use human cells, and perhaps more importantly, human-derived tissues such as airway tissue are much more complicated than a single layer of cells – meaning that its behaviour and response to smoke or vapour, especially over the long term, could differ greatly from what we see in a dish.
Finally, many of the standardised tests use only part of the aerosol (the part that can be collected easily) and cell are exposed in submerged conditions. So, we have also been pioneering the use of the whole aerosol in a more physiologically relevant scenario – at the air-liquid interface.
For more meaningful assessment, we and others have been leading the way in the use of 3D human tissue models that more accurately and better reflect the structure and function of normal human organs and physiology. We have adapted smoking and vaping machines so that we can expose these 3D human airway models to aerosols at exposures that more closely resemble consumer use. And to be certain that the cells are being exposed to what we think they are, we measure the amount of nicotine in the aerosol as it is generated, and again on the cells after they’ve been exposed to it.
By analysing the integrity and function of the 3D human airway models after exposing them to vapour or smoke, we can better assess the biological impact it has had. "By using human cells and human 3D reconstructed tissues where possible, not only do we avoid animal testing, but we can be more confident in extrapolating the findings to what actually happens in humans," explains BAT senior scientist, Dr. Damien Breheny.
Results from these more sophisticated in vitro tests suggest that our vapour products and THPs have much reduced or no toxic effect compared to cigarette smoke. Further studies are needed to determine whether these reductions are sustained and translate to a reduction in smoking-related health risks. So, we are also investigating and developing test models that are even more realistic. And, in an effort to identify the more subtle changes that vapour might be causing but that other methods are too blunt to detect, we’re also taking a more holistic, systems biology approach to understand the impact that aerosols from our NGPs have on cells.
The human genome has over 25,000 genes, and the pattern of genes that are differentially expressed in response to cigarette smoke or aerosols from our New Category products can be studied to understand whether exposure has had an effect.
We have exposed a 3D lung tissue model to cigarette smoke, vapour from the Vype ePen or aerosol from glo for an hour and compared the effects on gene expression. The results clearly show that exposure to cigarette smoke has a strongly adverse effect on cells, triggering thousands of changes in expression of genes including those involved in oxidative stress, inflammation and fibrosis, which are cellular processes that underlie smoking-related diseases such as lung cancer and Chronic Obstructive Pulmonary Disease.
In contrast, exposure to vapour from Vype ePen4 or aerosol from glo5 induces very few changes in gene expression – it’s a striking difference, but again further studies are needed to determine whether these results translate to a reduction in smoking-related health risks.
Going forward, we are planning to conduct similar experiments over days or weeks to study the effect of more realistic long-term exposure to various aerosols. We are also trying to identify gene sets specific to tobacco-specific diseases to establish models that relate better to COPD, cardiovascular disease and lung cancer.
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A Strategic Roadmap for Establishing New Approaches to Evaluate the Safety of Chemicals and Medical Products in the United States.
Murphy JJ et al. (2017).
Assessment of tobacco heating product THP1.0. Part 9: The placement of a range of next-generation products on an emissions continuum relative to cigarettes via pre-clinical assessment studies. Regul Toxicol Pharmacol 93: 92–104.
Haswell L et al. (2017).
Reduced biological effect of e-cigarette aerosol compared to cigarette smoke evaluated in vitro using normalized nicotine dose and RNA-seq-based toxicogenomics. Sci Rep 7: 888.
Haswell L et al. (2018).
In vitro RNA-seq-based toxicogenomics assessment shows reduced biological effect of tobacco heating products when compared to cigarette smoke. Sci Rep 8: 1145.