Transparency in scientific research promotes the use of best practices and methods, leading to robust and reproducible results. In this article, we share some of our methodology and protocols for the scientific assessment of heated tobacco aerosols.
One of the best choices a person can make for their health is to never smoke, or, when they do, to quit as early as possible. Increasingly, new smoke-free products are recognized as an opportunity to accelerate the decline in smoking prevalence by encouraging people who would otherwise continue smoking cigarettes to switch. To properly understand their potential impact on public health, the aerosols of these smoke-free products must be compared with cigarette smoke in order to assess their relative risk-reduction potential.
We have dedicated substantial efforts to the development, characterization, and, when possible, validation of methods and technologies to study and compare these aerosols. We shared detailed information on these efforts in an extensive review as well as on our online sharing platform INTERVALS.science, because we believe that transparency in science promotes the use of best practices in research.
Reproducibly generating, collecting, and using cigarette smoke and heated tobacco aerosols is essential to the scientific assessment and comparison of these products. Heated tobacco product aerosols are very different from cigarette smoke. As a result, comparing their toxicities can be difficult, and methods that were developed to study cigarette smoke often can’t be used as-is to study heated tobacco product aerosols. In 2020, we published an extensive review of our efforts to adapt methods developed for cigarette smoke to be used for heated tobacco product aerosols. That review article includes a discussion on the development of scientifically substantiated smoke-free products, the verification by others of existing studies and recommendations for further research. For a more-detailed dive into the study, you can refer to this handout on INTERVALS.science.
Cigarette smoke is an aerosol generated by burning tobacco. It is made of more than 6000 different components. Aerosols generated by heating tobacco are made of a significantly lower number of components, mostly water, glycerin, nicotine, and tobacco flavors. These two aerosols have very different properties and behave differently when studied. For example, the aerosol of our leading Heated Tobacco product, the Tobacco Heating System or THS, has a very high water content. Because of this, the aerosol can condense to liquid under certain conditions, which is not the case with cigarette smoke.
Diagram showing some of the biggest differences in the components found in cigarette smoke and Tobacco Heating System (THS) aerosol. Both cigarette smoke (left) and the aerosol of THS (right) are comprised of water, nicotine, glycerin, and other compounds. However, the amounts of each of those constituents are significantly different between the two products. Further, the number and levels of the “other chemicals” are significantly lower in THS aerosol compared to cigarette smoke. Cigarette smoke and THS aerosol were collected on laboratory filter pads, with 5 sticks used for each product. The numbers shown are on a per-stick basis.
We have created solutions to limit the impact of these differences in our studies. In our review and on INTERVALS.science, we describe in detail the protocols and instruments we have developed or adapted in order to generate, collect and use cigarette smoke and THS aerosol in a manner that allows the collection of comparable data.
We use linear and rotary smoking machines for the automatized smoking of cigarettes. These machines have been modified to accommodate the specific features ofTHS, including a tobacco stick holder and a rechargeable electronic device.
Image of a rotary smoking machine that has been modified for use in studies on THS.
Standardized machine puffing regimes enable meaningful comparisons between different products. For example, a machine puffing regime defines the number of puffs and puff duration for a cigarette or a tobacco stick. The World Health Organization Study Group on Tobacco Product Regulation recommends using the Health Canada Intense regime, also defined by the International Organization for Standardization (ISO intense) for the study of heated tobacco products. THS aerosol can be generated according to standard regimes like the ISO intense, but some of the requirements can’t apply to the product for technical reasons. For example, there is no ignition of THS tobacco sticks by an external lighter, there is a fixed heating time of six minutes, and the sticks remain the same size as they’re used. All unlike smoking a cigarette.
For many applications, such as the determination of aerosol compounds or in vitro testing in submersed cell cultures, it is necessary to collect aerosol fractions. Standard trapping and extraction procedures have been developed for cigarette smoke. We have developed and published methods to capture comparable samples from cigarette smoke and THS aerosol. We have optimized such parameters as the number of tobacco sticks, solvent volume, or trapping temperature.
Standard toxicity assessment of cigarette smoke has traditionally relied on a series of long-established in vitro assays in submersed cell cultures to measure cytotoxicity, mutagenicity, and genotoxicity. We conduct these studies as well, and we have also developed many new in vitro assays to test the progress toward toxicity reduction. These tests use cell cultures derived from the animal and human respiratory tract and cardiovascular system. We measure cellular endpoints that are known to be linked to smoking-related chronic diseases. This document on INTERVALS.science goes into more detail on some of those results.
Image of a human bronchial cell culture. These cells were cultured partly in solution with their top surface exposed to air in order to form a 3D structure with different cell types. This 3D structure allows the cell culture to more closely mimic the behavior and structure of cells in the human body versus more simple cell cultures.
Submersed cell cultures are fundamentally different from the human lung, as they are exposed to a liquid, not to air or an aerosol. Therefore, exposure systems have been developed in which cell cultures have their upper side exposed to air and can be exposed to cigarette smoke or THS aerosol. These exposure systems are called exposure systems at the air-liquid interface. The cell cultures are complex, three-dimensional cell cultures that mimic the organization and physiology of cells in the human tissue of interest. A complete exposure system is made of an aerosol-generating machine delivering the aerosol to a pipe circulating on top of the cell cultures housed in an exposure chamber.
Schematic representation of the Vitrocell® 24/48 exposure system. The cells are placed in a 48 well plate that allows them to be partially submerged in liquid, exposed to both liquid and air, like in the human body. The Vitrocell® 24/48 can simultaneously expose up to 48 cell cultures.
We are using the VITROCELL® 24/48 exposure system that allows the simultaneous exposure of up to 48 cell cultures. We have determined, for both cigarette smoke and THS aerosol, the dose that could be delivered to each cell culture, its composition, the uniformity of aerosol delivery to the different cell cultures, and whether these parameters are stable over time. More details on the characterization of the Vitrocell system is provided on INTERVALS.science. Overall, we found that the aerosol that is delivered to the cells is not necessarily representative of the applied aerosol. Hence, it is critical to characterize the aerosol delivered to the cells in order to interpret and compare results. We have developed several methods to that effect, by determining key aerosol compounds, such as nicotine or carbonyl compounds, either during or after the experiment.
These are some of the methods we use in our research, studying the aerosol chemistry and physics, and in vitro toxicity. In December of 2020, we also published a similar paper sharing some of our best methods and practices for generating aerosols from e-cigarettes. As part of our smoke-free product assessment program, these are the kinds of studies that are mostly carried out in the Cube, PMI’s research center in Neuchâtel, Switzerland.
Publishing scientific papers and presenting our methods and data on INTERVALS.science isn’t the only way we share our work openly and transparently. As of June 2020, we started hosting a series of virtual conferences where our scientists speak openly about our latest research, our methods, and the fundamentals behind our smoke-free product research. People interested in learning more about our research are welcome to take a virtual tour of the Cube, to learn more about our findings on our portfolio of smoke-free products, or to read through a booklet summarizing those findings.