Introduction

The use of tobacco products contributes to the development of a number of pre-ventable diseases, however, there are still many smokers who are not or unable to quit [1]. All tobacco products use can become an addiction for some users. The effects of nicotine intake and exposures to harmful substances from tobacco products on the human body are complex, and the assessment of such effects needs to be conducted on different aspects of the product use [2].

The risks of cigarette smoking to health are well known, in recent years many to-bacco companies have developed alternative tobacco and nicotine products, such like electronic cigarettes (e-cigarettes) and heated tobacco products (HTPs) [3]. HTPs were first launched in Japan and then European markets in 2014 [4], with their claimed benefits of harm reduction and aiding smoke cessation their use have increased in re-cent years [5].

HTPs are designed to heat tobacco below the ignition temperature and deliver aerosolized nicotine and other tobacco constituents for inhalation [6]. Based on con-trolled heating technologies, different HTPs are now available for producing aerosols containing nicotine without burning tobacco [7].

As a new form of nicotine delivery, HTPs have significantly different smoke gen-eration principles and smoke components from CCs [8]. According to data from the tobacco industry, the toxicant contents in the aerosols of some commercial HTPs are about 90-95% lower than that of conventional cigarette smoke, the levels of harmful and potentially harmful components (HPHCs) such as aldehydes and volatile organic compounds in heated cigarette smoke are also significantly reduced [9]. A review including 25 relevant studies published in English between 2015 and 2021 suggests that exclusive HTP use over smoking may reduce the risk of some chronic diseases, includ-ing respiratory disease, cardiovascular diseases and cancer, although they may pose a risk for those that are non-smokers, so HTPs have been intended to be an alternative to CCs for heavy smokers who had failed comprehensive treatment for nicotine addiction [8]. In addition, HTPs do not emit 2nd hand smoke and may reduce environmental pol-lution [3].

Several types of HTPs have been introduced into the market so far, including brands such as iQOS, Glo, Ploom, Pax and iFuse and are sold in more than 40 countries [8].  A recent market research found that a large number of Chinese smokers are in-terested in HTPs [10], either in combination with other tobacco products or as quitting aids. However, some studies found that these types of novel tobacco products can also attract young generation users who never smoke [11].

As a newly developed alternative nicotine products, emission analysis of the chemical composition of HTPs aerosol is an important step in determining whether HTPs offer both individual and population-level harm reduction potential. In fact, some studies reported the levels of harmful and potentially harmful constituents (HPHCs) in the emissions from HTPs are significantly reduced as compared to CCs [1, 12].  Emission data from chemical analysis are typically collected under standard machine-smoking regimens defined by the International Organization for Standardization (ISO) or the Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) [13]. However, the standardized regimens are intended for product com-parison purposes and do not necessarily reflect human puffing behavior. To estimate the real emissions or the levels of chemicals inhaled by their users from HTPs, which are more relevant to assess these products’ true harm reduction potential, puffing topographic parameters close to realworld usage have to be gathered.

Puffing topography of a tobacco product is influenced by the design and specifica-tions of the product, and the smoking habits of the user. There is also a correlation with user preferences, etc. Puffing topography data not only reflect personal preferences and smoking habits of smokers to a considerable extent, but also significantly affects the toxicant intake by the tobacco users [14, 15]. Harsher puffing conditions such as more, longer, and larger puffs yield higher levels for most toxicant emissions [16]. For instance, a lot of researches have reported puffing topographic data among smokers using different generations of e-cigarettes [13, 17, 18], most of them showed that puff duration, inter-puff interval and puff volume among e-cigarette users varied from conventional cigarettes. In addition, different flavors of e-cigarettes could also result in different puff durations and different levels of plasma nicotine as measured in clinical studies [14]. There is evidence that both the levels and forms of nicotine affects the user’s puffing topography.

With the rapid development of HTPs, it is important to study the puffing topog-raphy of HTPs for smokers of different demographics. So far few studies on puffing topographies of HTPs compared to CCs have been reported for Chinese smokers [19]. Cigarettes available in China are predominantly a local Virginia blends, which are quite different to the dominant tobacco varieties outside China. Transition patterns by Chinese smokers to use HTPs may be different to those seen in other parts of the world where HTPs are available. We therefore conducted a randomized experiment to meas-ure the puffing topographic behavior by using two differently flavored HTPs (one to-bacco and one menthol) and compared those to CCs under similar standardized conditions, in order to further understand the nicotine exposure and metabolism of heated cigarettes on the human body. The results of this study were expected to provide in-sights on potential risk in the switching of Chinese smokers from conventional ciga-rettes to HTPs. An understanding of any product characteristics that pertain to HTPs use may also facilitate the development of a standard protocol for HTPs testing and clinical studies [13]. In addition, it can also provide a basis for the experimental design of evaluating the exposure of heated cigarette smoke.

Materials and Methods

Devices and Products

PuffmanTM puff behavior recorders (Puffman automation and instruments com-pany, China) were used to measure the parameters of puffing topography of every participant in this study. The technical specifications are shown Table 1.

Table 1: The technical parameters of puff behavior recorder used in this research.

Before using each puff behavior recorder in this test, trial runs were performed to compare the recorded data and to ensure they were within the specified ranges. Dur-ing the experiment, care was taken to wipe any aerosol residuals within the product adaptation hole every half hour of usage.

All of the HTP devices and consumable tobacco sticks, and test cigarettes in the experiment were provided by Shanghai New Tobacco Product Research Institute Co., Ltd. As shown in Figure 1, the HTP device had a maximum heating temperature of 350?. The nicotine content of the HTP consumable sticks (both original tobacco and menthol flavor) was 1.0 mg per stick. CCs were commercially available with a nicotine content of 1.0 mg/stick and a tar content of 11 mg/stick.

Figure 1: The schematic of the tested HTP device with a tobacco stick inserted.

Human Subjects and Study Groups

Subjects were selected from four cities, including Shanghai and Suzhou in Eastern China, Hefei in Central China, and Guiyang in Western China. We used the simple random sampling method to select 50 participants in each city totaling 200, and then divided them randomly into three groups. Tests were run from July to August 2022.

The inclusion criteria of participants included: aged 19–65 years, without cardio-vascular, respiratory or reproductive diseases, no psychiatric and other serious psy-chiatric disorders, no pregnancy or breastfeeding, not taking any medication in the past week that could be considered interference to the test purpose. Volunteers smoked 5–20 cigarettes with ISO tar yield between 10–12 mg, nicotine level of 1.0 mg daily for at least 2 years (current brand for >6 months) [20]. The exclusion criteria included: ev-er or known illicit drug abuse, current or former mental disorders, diagnosed cardio-vascular diseases, chronic obstructive pulmonary disease, other respiratory diseases or cancer of any kind, or any other reasons that could be considered noncompatible with the purpose of the study. The investigators of the study also evaluated the participants’ adherence of the experimental protocol. Informed consent was obtained from all the subjects after explaining the purpose of the study. The Biomedical Ethics Review Committee of Tongren Traditional Chinese Medicine Hospital approved the study (Approval number: TRSZYY-2022-02).

Experimental Methods

All the subjects were allowed to familiarize themselves with the usage of the HTP products and also the puff behavior recorder. Efforts were made to encourage them puff naturally and to reproduce their usual puff behaviors as needed. The adaptation period lasted six days, during which each participants were provided with an average of 5 cigarettes and HTP sticks. Instructors were on hand when needed to guide the par-ticipants to custom their stable puffing behaviors.

In the first round of the experiment starting from July 2022, the three groups of participants were randomly assigned to puff CCs (group 1, n = 72), the Original- HTPs (group 2, n = 68), and the Menthol- HTPs (group 3, n = 60) respectively. On the seventh day, they were required to smoke one single cigarette sample which they had been as-signed within 5 minutes in very similar experimental environments including atmos-pheric pressure, temperature and indoor ventilation. Throughout the entire process of puffing, we recorded their smoking behavioral parameters including puff number, puff volume, puff duration, inter-puff interval, flow velocity, peak flow rate, peak time us-ing the puff behavior recorder as well as atmospheric pressure at the same time. The process was video recorded in order to verify each participant’s compliance and en-sure that all of data had been correctly recorded.

In the second round beginning at the eighth day, the participants in group 1, group 2 and group 3 were switched to puff Original- HTPs, Menthol- HTPs and CCs respectively. In the last round beginning at the fifteenth day, participants in group 1, group 2 and group 3 were switched to puff Menthol- HTPs, CCs and Original- HTPs respectively. Similar to the first round, we also set a six-day adaptation period for the second and the third round, as well as required each participant to smoke a cigarette sample assigned to them within 5 minutes and recorded the above smoking behavioral parameters at the same time (Figure 3).

Figure 2: The flowchart of the experiment.

On the last day of each round, urine samples in the morning and 8 hours after smoking cigarette sample were collected to assess the individual’s nicotine exposure level from the sample cigarette. During this period, all volunteers freely drunk water to facilitate the elimination of nicotine. The following eight nicotine metabolites in urine will be primarily tested: nicotine (NIC), cotinine (COT), cotinine-N-ß-D-glucuronide (COTG), (1’S, 2’S)nicotine-1’-oxide (NNO), (S)-cotinine N-oxide (CNO), trans-3hydroxycotinine (OHCOT), nornicotine (NNIC), and norcotinine (NCOT). The urine sample was labeled and once collected, it was stored in liquid nitrogen for testing as soon as possible using the method reported before [10].

Data collection and statistical analysis

Participants’ smoking history and demographic information were collected before the first round of experiment using a selfadministered questionnaire designed in the study (see later).

Puff number was defined as the total number of puffing one single cigarette or single HTP stick. Puff volume was continuously recorded in unit of mL during a single mouth puffing process. Puff duration time refers to the duration of a single mouth puffing process. Inter-puff interval was defined as the time interval between two ad-jacent mouth puffing behaviors. Flow velocity, peak flow rate and peak time were rec-orded or calculated based on one single cigarette which the participants puffed in the experiment.

We calculated the means of the above puffing parameters and atmospheric pres-sure in three repeats of the experiment to describe the puffing topographies after ex-cluding the influence of puff order.

Data were checked for consistency and any abnormality. Characteristics of the participants were described as mean and standard deviation (SD) for continuous var-iables if normally distributed or as median and interquartile range (IQR) if nonnormally distributed. Categorical variables were presented as number and per-centages (%). We compared continuous variables using One-way analysis of variance (ANOVA) among the three groups divided by different types of tobacco sticks includ-ing CCs, Original - HTPs and Menthol - HTPs if the data are normally distributed. In the case of a significant difference had been found between the groups, Tukey’s post-hoc test was applied to examine the source of the difference. For non-normally distributed data or the data with heterogeneity in variance, Kruskal-Wallis test was used to compare average differences between multiple groups. For categorical variable data, Chi-square test was used to compare proportions’ differences. Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) version 23.0.

Results

Throughout the entire experimental phase, all the participants reported no ad-verse reactions or physical discomfort during or 1-week after the study.

Demographic characteristics and smoking history

The average age of the participants was 34.04 ± 10.52 years old, ranging from 19 to 65 years old, 164 (82.0%) participants were male, all the participants were current smokers, of which 96 (48.0%) had been smoking for more than 10 years, the average duration of cigarette smoking was 10.65 ± 8.38 years. Participants’ average body mass index (BMI) was 22.95 ± 3.04 kg/m2, of which 14.0%, 54.5%, 25.0%, 6.5% were under-weight, normal weight, overweight and obesity respectively. The proportions of daily cigarette consumption, Intention to quit smoking and occupation among the partici-pants were presented in Table 2.

Table 2: Demographic characteristics of the participants.

Except for daily cigarette consumption, there were no statistically significant differences in the demographic characteristics of participants from the four representa-tive cities, including gender, age, weight status and duration of cigarette smoking (Table 3), indicating that there were few differences in the demographic composition be-tween the participants from the four cities.

Table 3: Demographic characteristics of the participants among the four cities.

Differences of puffing topography

We compared the means of puff number, puff volume, puff duration, inter-puff interval, flow velocity, peak flow rate, peak time among the three groups of partici-pants who smoked different types of tobacco sticks, there were significant differences on all of the above variables (Table 4).

During the experiment, no statistical differences were found in the average at-mospheric pressure of the three groups of participants, which indicates that the levels of puffing parameters would not be affected by atmospheric pressure (Table 4).

When we compared the above puffing parameters in each city, we found that the puffing characteristics in each city were not similar except for flow velocity (Figure 3). The differences of puffing topography in the four cities between the Original- HTPs and Menthol- HTPs was subtle. However, the overall trend in variances between HTPs and CCs remained consistent.

Table 4: Comparisons on the means of puffing parameters and atmospheric pressure among different types of cigarettes.

Figure 3: Comparison of puffing parameters differences among different types of cig-arettes divided by cities. A. Puff number. B. Puff volume. C. Puff duration. D. Inter-puff interval. E. Flow velocity. F. Peak flow rate. G. Peak time. (NS: no significance; * p<0.05; ** p<0.01, respectively)

Differences in urinary nicotine metabolites levels

There were statistically significant differences in the average levels of all kinds of urinary nicotine metabolites among CCs, Original- HTPs and Menthol- HTPs, whether in urine samples in the morning or 8 hours after smoking cigarette sample (Table 5). In all the HTPs group, the total urinary nicotine metabolites content were lower than that of the CCs group (Figure 4).

Table 5: Comparisons on 8 urinary nicotine metabolites among test products (ng/L).

Figure 4: Comparison of total urinary nicotine metabolites.