A Comparative In Vitro Toxicological Screening of a Closed-End Heated Tobacco Product *

Smoking is one of the main preventable causes of respiratory and other chronical diseases, including COPD and lung cancers (1). The best cause of action for smokers is to quit. However, despite significant public health efforts and the availability of pharmaceutical nicotine replacement therapy products (NRTs), many smokers remain unwilling or unable to give up smoking. As a harm reduction intervention, validated low-risk tobacco products have been made available to current smokers. In the U.S., under the auspice of the FDA’s comprehensive tobacco regulation program, modified risk tobacco products (MRTPs) are an authorized category of alternatives that can be marketed to adult smokers (2).

Heated tobacco products (HTPs) are among the classes of novel tobacco products that are available in some markets. They are designed to heat tobacco through a battery-powered heating system to generate nicotine-containing aerosol for inhalation (3, 4). It has been shown that most of the toxicants found in cigarette smoke are formed as a result of incomplete tobacco combustion (5). In a typical HTP device, its heating temperature program and the aerosol formation can be accurately controlled, resulting in the elimination of tobacco combustion and therefore the aerosol formed is found to contain significantly less toxicants than cigarette smoke (6, 7). Most commercial HTPs heat the tobacco below 350 °C using a varied but controlled temperature profile. This contrasts to a burning cigarette which typically runs between 600 and 900 °C (5). However, commercial HTPs come with different heating technologies such as e.g., IQOS^TM and glo^TM (3, 4), where the heating energy is either transmitted from inside a tobacco rod outwardly or inwardly. More innovations are coming onto the market in HTP designs, some using resistive while others using radiative/non-contact means for heat transfer. This may lead to different degrees of tobacco heating at different locations, and therefore some differences in the chemicals formed in the aerosol. Hence it is required by regulators that each HTP should be chemically and biologically assessed under rigorous preclinical and clinical programs before it is considered for market assess and potentially reduced risk claims.

In commercial HTPs, aerosol is formed by heating reconstituted tobacco material containing aerosol-forming agents such as vegetable glycerol and propylene glycol (3). During heating, the volatile compounds and the aerosol-forming agents are vaporized from the tobacco substrate and reach supersaturation; upon cooling by the puffing air flow condense to form the aerosol. In most HTP designs, puffing airflow enters from the open end of a tobacco rod and exits through its mouth end - for comparison purpose, this design is called an open-end HTP. In this work, a novel HTP design that prevents the air flow from entering the tobacco end is described - this is called a closed-end HTP. The key design features and its working principle have been described before (8, 9), more details will be given in the experimental section.

In this work, we aimed to compare the aerosol chemistry, first using a limited number of toxicants measured on this close-ended HTP and a commercial HTP (IQOS^TM). Afterwards biological properties of the aerosol produced were assessed using the standardized regulatory in vitro assays (Ames, Micronucleus and Neutral Red Uptake). The results were discussed and presented as the first step towards building a comprehensive preclinical dataset for this novel HTP before any clinical evaluation.

In our previous studies, the aerosol release behaviors of the main TPM components of these two HTP systems were compared, such as glycerol, nicotine and water, on a paired closed-end vs. open-end product comparison. On an identical tobacco stick and device design (apart from the open vs. closed air flow) basis, closed-end HTP-A was found to be more efficient in releasing these agents (8). The reason was that the HTP-A system removed the convective heat transfer of the tobacco bed better when compared with existing open-ended HTP-B. And the HTP-A’s mainstream aerosol was formed without the repeated interruption of incoming air and its cooling effects (8, 9). The formation process and mechanism of the two aerosols also directly determined the differences in the content of various components in aerosols, especially for those harmful and potentially harmful constituents.

In this work, the two HTPs under investigation were not matched in their design characteristics, and therefore it was difficult to deduce the deeper mechanistic reasons behind the toxicant yields observed in Table 5. These have also been reflected by the differences in puff numbers and released nicotine levels for the two HTPs: HTP-A produced less nicotine and most of the measurable toxicants as compared to those in HTA-B on a per-stick basis, but when the yields were normalized to per-nicotine basis, the differences were marginal or reversed.

Generally speaking, the TobReg 9 toxicants cover the four main categories of releasing behavior (11, 22):

carbon monoxide being biomass-pyrolysis- and combustion-driven;

Significant reductions across these four categories are a reasonable indication that this novel closed-end HTP (HTP-A) was maintaining the key emission performance that is typically found in the leading commercial HTP products (such as HTP-B), and therefore it was worthwhile to be investigated for its toxicological effects. The relative increases or decreases in these toxicants were most likely caused by the differences in tobacco blends, heating mechanisms, and not just by the fact that HTP-A had a reduced availability of oxygen during puffing (23).

In previous toxicological studies of differently designed HTPs (18, 24–26), standard regulatory genotoxicity and cytotoxicity assays were used as the first steps in screening in addition to toxicant emission analyses. Under the test conditions, it was impossible to determine the IC₅₀ for the two HTPs up to 3000 μg/mL of TPM. This contrasted with the IC₅₀ of 3R4F smoke which was determined to be 49.18 μg/mL of TPM (Figure 2). The exposure of the cells to TPMs from the two HTPs at 2000 μg TPM/plate failed to elicit cell viability responses (Table 7). These results are in agreement with the fact that the two HTPs emitted significantly lower toxicant levels as compared to those found in 3R4F smoke (Table 5). This is due to their mainly heated aerosol formation mechanism, and glycerol and water making up the main components of TPMs (6, 7). The Ames and MN results showed that the TPMs of the two HTPs were not genotoxic under these regulatory toxicological assays. These assays were not sensitive to distinguish any difference between the two HTPs, whether or not the comparisons were made on a per-unit mass of TPM and/or per-unit mass of nicotine in the aerosol. These findings are also in line with those published previously for the other HTP products (24–26). It appears that despite all the design differences in heating technology and tobacco blend compositions (U.S. blended vs. Chinese blended), the general aerosol emission chemistry using a subset of representative analytes, such as the WHO’s TobReg 9, as used in this study, was reasonably robust to predict the qualitative, if not quantitative, toxicological outcomes. More sensitive cell lines such as 3D cell lines that represent the epithelial functions of respiratory systems and/in combination with system toxicology may be required to delineate some of the residual toxicological effects. As previous studies on HTPs have pointed out, inhalation exposures to HTP aerosols are not without risks. Further preclinical and clinical assessments should be carried out before a novel HTP could be considered a reduced-exposure- or reduced-risk tobacco product (27). As a preliminary screening evaluation, this study is not without its limitations, the main one being only based on a subset of aerosol chemistry and standard regulatory toxicological assays. Numerous studies on different HTP designs have been performed and are available in the literature. Our study used a comparative approach to set the basis for further evaluations. Any conclusions or observations should be viewed as such.

This work described a preliminary aerosol chemistry and regulatory in vitro screening on a novel closed-end HTP, with comparison to a commercial open-end HTP and 3R4F research cigarette. The effects of the HTP product design differences on aerosol chemistry were mainly reflected in the number of puffs available by the two systems. The resultant nicotine emissions were not matched on a per tobacco stick basis. Comparisons were therefore made on both per nicotine mass and per tobacco stick basis in order to set the ground for in vitro toxicological comparison. Using a set of regulatory toxicological assays (Ames, MN and NRU), the two HTPs behaved indistinguishably under the conditions tested and showed no observable responses, in contrast to the clear toxicity induced by TPM from 3R4F mainstream smoke and coincided with their significant reduced levels of WHO TobReg 9 toxicants. Further pre-clinical and clinical evaluations are needed before the novel HTP could be considered as a reduced-exposure or reduced-risk tobacco product.