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A study to investigate changes in the levels of biomarkers of exposure to selected cigarette smoke constituents in Japanese adult male smokers who switched to a non-combustion inhaler type of tobacco product
Regulatory Toxicology and Pharmacology 71 (2015) 498–506
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A study to investigate changes in the levels of biomarkers of exposure to selected cigarette smoke constituents in Japanese adult male smokers who switched to a non-combustion inhaler type of tobacco product Naoki Miura ⇑, Dai Yuki, Naoki Minami, Aoi Kakehi, Yasuyuki Futamura Product Science Division, R&D Group, Japan Tobacco Inc., Yokohama, Kanagawa, Japan
a r t i c l e
i n f o
Article history: Received 28 July 2014 Available online 12 February 2015 Keywords: Biomarkers Exposure Cigarette Smoking Smokeless tobacco Clinical study
a b s t r a c t In a clinical study, changes in 14 biomarkers of exposures (BOEs) from 10 tobacco smoke constituents and mutagens detected by the urine mutagenicity test were investigated using a non-combustion inhaler type of tobacco product (NCIT) by switching from a conventional cigarette. This study was conducted in 80 Japanese healthy adult males with a 4-week residential, controlled, open-label, parallel group design. After randomization, 40 smokers used NCIT with approximately 750 aspirations, other 20 smokers smoked approximately 20 pieces of an assigned 1-mg ISO tar conventional cigarette (CC1) every day. Twenty non-smokers (NS) did not use any tobacco product. Under this study condition, switching from cigarette to NCIT showed significant reduction in all BOEs measured. On day 29, the levels of these BOEs were almost the same as those in the NS group, except BOEs of nicotine and 4-(methylnitrosamino)-1-(3pyridyl)-1-butanone (NNK). This suggested that the exposure to 8 constituents and mutagens in the NCIT group was similar to that in the NS group, while the exposure to nicotine was higher. Although the precise exposure level to NNK was not estimated because of the long half-life of its BOE, it would be substantially lower in the NCIT group than in the CC1 group. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction Many previous studies reported that cigarette smoking is a risk factor for serious diseases including lung cancer, coronary heart disease, emphysema, and chronic bronchitis. There are many types of tobacco products other than cigarette in the world, such as cigars, pipes, and nasal or oral snuff, and it has been reported that the users of such products are at a risk for serious diseases (International Agency for Research on Cancer, 2004, 2007). In addition to the various traditional tobacco products available, some non-traditional forms of tobacco products, such as electrically heated cigarettes (Buchhalter and Eissenberg, 2000) and dissolvable tobacco tablets (Rainey et al., 2011), have been recently introduced. Some of these were designed for reducing exposure to a series of selected harmful and potentially harmful constituents (Schorp et al., 2012). For the comprehensive evaluation of such emerging products, it is important to measure the users’ exposure to chemical constituents derived from the products. ⇑ Corresponding author at: Corporate, Scientific and Regulatory Affairs Group, Japan Tobacco Inc., 2-2-1 Toranomon, Minato-ku, Tokyo 105-8422, Japan. Fax: +81 3 5572 1477. E-mail address: [email protected] (N. Miura). http://dx.doi.org/10.1016/j.yrtph.2015.02.007 0273-2300/Ó 2015 Elsevier Inc. All rights reserved.
The non-combustion inhaler type of tobacco product (NCIT) is a new form of smokeless tobacco product that consists of a tapered mouthpiece and cartridge filled with finely cut tobacco leaves. The flavor components can be delivered from the tobacco leaves not by burning or heating but just by the air flow when users aspirate through its mouthpiece. We previously reported the results of a clinical study investigating the pharmacokinetics of nicotine when using prototype NCIT (Miura et al., 2013). That study results revealed that the bioavailability of nicotine delivered from prototype NCIT was similar to that delivered by conventional cigarette smoking, regardless of the form of the tobacco product. However, the degree of user’s exposure to chemical constituents derived from the product, except nicotine, is unclear. The measurement of several chemical constituents delivered from NCIT by machine aspiration showed that many of these constituents were below the limit of detection (LOD), even when approximately 2000 aspirations were integrated (described in detail in this article). This result indicates a prospect of significant reduction in exposure to those constituents in NCIT users. The measurement of biomarkers of exposure (BOE) is one of the effective methods to evaluate human exposure to smoke constituents. BOEs for tobacco smoke constituents are mainly the chemical constituents themselves and/or their metabolites, and at
N. Miura et al. / Regulatory Toxicology and Pharmacology 71 (2015) 498–506
present, some BOEs are validated and recommended to use for measuring the exposure of smoke constituents (Institute of Medicine, 2012). The advantage of monitoring BOEs is that they could reflect intra- and inter-individual differences in the tobacco smoking or using behavior. Moreover, the BOE monitoring method is applicable not only to the conventional cigarette but to the various other types of tobacco products, including the emerging products. The biomarker level must be background level if the exposure is zero. However, many of the validated BOEs are well known to be influenced by the other source of their corresponding chemicals existing in our living environment (Scherer, 2005). Therefore, for precise exposure evaluation, it is important to determine the degree of the influence of environmental factors other than tobacco on BOE levels. The objective of this study was to evaluate changes in the selected BOEs with the use of NCIT in Japanese healthy adult male smokers, using a 1 mg ISO tar conventional cigarette (CC1) as a control. In addition, the changes in BOEs in Japanese healthy male non-smokers (NS) were evaluated for identifying the influence of factors other than tobacco on BOE levels in the same study environment. The BOEs monitored in this study were selected with reference to previously published information on harm reduction studies (Scherer, 2005) and the results of our preliminary BOEs exploratory study with smokers and non-smokers and our recent publication (Sakaguchi et al., 2014).
2. Materials and methods 2.1. Study design This study was conducted at 4 medical institutions in Japan using a permuted-block, randomized (for smokers), controlled, forced-switching, open-label, parallel group design. All subjects who passed the screening test stayed at one of the medical institutions throughout the study period, except several times of outings accompanied by the clinical staff. The study period of 31 consecutive days was set in consideration of the elimination half-lives of some BOEs. The outing, such as bowling, seeing a movie, and art appreciation, was about 2 h, and was set once a week. All smoking subjects smoked their usual brand of a 1 mg ISO tar cigarette within ±10% of their usual daily consumption for the first 2 days (day 1 and 0, baseline period). Following the baseline
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period, the smokers were randomly assigned to 2 groups: NCIT and CC1, as described in Fig. 1. Subjects in the NCIT group used 2 NCIT cartridges per day, and those in the CC1 group smoked an assigned CC1 within ±10% of their usual daily consumption from day 1 to day 28. All non-smokers were automatically assigned to the NS group and did not smoke throughout the study period. All four medical institutions had approximately 20–30 subjects each, and the number of subjects assigned to each group were set as equal (CC1–NCIT–NS, 1:2:1) in order to avoid the institutionspecific effect on the particular group. The clinical staff observed and recorded any adverse events. They also ensured that the subjects abstained from using nicotine-containing products except for the tobacco products specified for the study. The study was conducted at the Maruyama hospital (Hamamatsu, Japan), OCROM clinic (Osaka, Japan), Sumida hospital (Tokyo, Japan), and Hakata clinic (Fukuoka, Japan) in accordance with Good Clinical Practice (GCP) and the principles that have their origin in the Declaration of Helsinki. This study protocol was approved by the Institutional Review Board of Japan Tobacco Inc. and the medical institutions. 2.2. Subjects Eighty Japanese healthy males, 60 smokers and 20 non-smokers, aged 21–49 years and having a body mass index (BMI) in the range of 18.5–25.0 kg/m2 were enrolled. The health of the subjects was checked before their entry into the trial by physical examination, examination of medical history and vital signs, 12-lead electrocardiography and laboratory tests. Eligible smokers for the study had smoked a 1 mg ISO tar conventional cigarette without regard to menthol or non-menthol with a daily consumption of at least 20 cigarettes for at least 1 year prior to screening and their serum cotinine levels had exceeded 14 ng/ml at the screening (Pérez-Stable et al., 1992). Eligible nonsmokers had no experience of routinely smoking the conventional cigarette and had not used any tobacco products including the conventional cigarette for at least 1 year before screening. Non-smokers with their serum cotinine levels lower than 14 ng/ml at the screening were enrolled. All volunteers were paid for participating and provided written informed consent before the enrollment. 2.3. Study procedure
Day −1
Day 1
Day 8
Day 15
Day 29
Blood sampling
forced-switching
24hr Urine sampling
1 mg tar cigarette smokers (N = 60) Non-smokers (N = 20)
CC1 group (N = 20) NCIT group (N = 40) NS group (N = 20)
Baseline period = for COHb
Investigation period = for all BOE in blood except COHb
Fig. 1. Study design. All smoking subjects smoked their usual brand of 1 mg ISO tar cigarette on day 1 and 0, and then the smokers were randomly assigned to the NCIT and CC1 groups. All non-smokers were automatically assigned to the NS group and did not smoke throughout the study period. NCIT: non-combustion inhaler type of tobacco product. CC1: 1 mg ISO tar conventional cigarette. NS: non-smokers. BOE: biomarker of exposure. COHb: carboxyhemoglobin.
All smoking subjects smoked or used their assigned test tobacco product whenever they wanted from 7 to 23 o’clock throughout the study period, except for some constraints on the timing of tobacco smoking or usage; the subjects were not allowed to use NCIT or smoke the conventional cigarette from the time of awakening to the completion of clinical examination in the morning on day 1, 1, 8, 15, 22, and 29. During outings, the subjects were not allowed to use test tobacco because accurate checking of each subjects’ tobacco use was difficult for the clinical staff. In addition, the subjects used NCIT with 15 aspirations or smoked 1 piece of the conventional cigarette at approximately 16 o’clock, and then 30– 45 min after use or smoking, blood sampling was performed on day 0, 7, 14, and 28 to measure carboxyhemoglobin (COHb) levels. The subjects abstained from using or smoking the test tobacco product until the completion of this blood sampling. Subjects who smoked the conventional cigarette (smoking subjects on day 1 and 0 and the CC1 group subjects from day 1 to day 28) received the cigarette from the clinical staff in each case and were allowed to smoke only in the designated smoking room. Subjects in the NCIT group received 2 pieces of NCIT cartridge daily from the clinical staff at a set time and were forbidden to enter
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the smoking room since they were allowed to bring NCIT and use it in their living space in the institution. Regarding the total daily aspiration number of NCIT use, 180 aspirations were set as the minimal number per day and were confirmed using a Puff Profiler (CReSSmicro™, Plowshare/BorgwaldtKC), and the other voluntary aspiration number of the day was investigated by a self-administered questionnaire. No upper limit was set for the daily aspiration number of NCIT use. For the CC1 group, the daily number of cigarettes smoked by each subject was recorded. To avoid the potential effects on BOEs, clinical institutions served meals without smoked food and baked or fried processed food for 2 days prior to the day of blood sampling, day 8, 15 and 29. The health of the subjects was checked during this study period by physical examination and examination of vital signs on day 1, 8, 15, 22, and 29 and by 12-lead electrocardiography and laboratory tests on day 1 and 29. 2.4. Test tobacco products NCIT consists of a cartridge filled with finely cut tobacco leaves and a tapered mouthpiece. Volatile substances are delivered from the cartridge not by burning or heating but just by the air flow of users’ aspiration. Water, flavorings, potassium carbonate, and propylene glycol were added to the tobacco leaves. NCIT was mint flavored, unlike the prototype NCIT used in our previous study (Miura et al., 2013), in which flavorings were not added. For measuring the amount of chemical constituents derived from NCIT by machine aspiration, the following regimen was set referring to the Health Canada Intense method for cigarette machine smoking conditions (Health Canada, 1999): 55 ml aspiration volume, 2-s duration, and 30-s interval. The number of aspirations, 1920, was set according to the assumption of continual aspiration for 16 h with 30-s intervals. The CC1 chosen for the investigation period (from day 1 to day 28) is one of the most popular 1 mg ISO tar brands in Japan. The mainstream smoke delivered from CC1 was analyzed using the smoking machine protocol standardized by the International Organization for Standardization (ISO, 2000). The brand of 1 mg ISO tar conventional cigarette for the baseline period (day 1 and 0) was conformed to the each subjects’ usual brand which they declared. For this study, NCIT (it was marketed under the name ‘‘Zerostyle Mint’’ in Japan at that time), subjects’ usual brand of cigarette and assigned CC1 were purchased from the market. 2.5. Blood and urine sampling To measure plasma BOE levels, venous blood samples were obtained from the forearm vein. Blood samples (7 ml) were drawn for cotinine and thiocyanate (SCN) measurement before breakfast and first use or smoking of the test tobacco product of the day on day 1, 8, 15, and 29. Moreover, 2 ml of blood were obtained for COHb measurement at approximately 16 o’clock on day 0, 7, 14, and 28, because the half-life of COHb has been reported be 1–4 h (Scherer, 2006) and COHb was expected to largely disappear prior to the next morning. Blood samples were collected in blood collection tubes containing EDTA-2Na and centrifuged at 3000 rpm for 10 min at 4 °C. Plasma was transferred into plastic tubes and stored at 80 °C until further analysis. The 24-h urine pools for measuring urine mutagenicity and BOEs were sampled from the morning on day 0, 7, 14, and 28 to the next morning (in this article, the study day for each 24-h urine sample data were expressed as the end of the sampling). Approximately 300 ml of 24-h collected urine was divided into plastic tubes or bottles and stored at 80 °C until further analysis.
2.6. Bioanalysis All measurements, except for the urine mutagenicity test, were conducted by Mitsubishi Chemical Medience Corporation (Tokyo, Japan) using validated methods. The BOEs in urine except urine mutagenicity were measured based on a liquid chromatography with tandem mass spectrometry (LC–MS/MS) (Sakaguchi et al., 2014). Urinary nicotine equivalent (NE) was calculated as the molar sum of nicotine and its 5 major metabolites (cotinine, trans-3-hydroxycotinine, nicotine-N-glucuronide, cotinine-N-glucuronide, and trans-3-hydroxycotinine-O-glucuronide). NE in mg/ 24 h was calculated by multiplying molar NE by the molecular weight of nicotine. Urinary total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a major metabolite of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), was calculated as the molar sum of its free and glucuronide form. COHb levels in whole blood were measured by the cyanmethemoglobin method using a microplate reader (Okuzono et al., 1976), and plasma SCN levels were assayed spectrophotometrically using the microplate reader (Vesey et al., 1999). Plasma cotinine levels were measured by liquid chromatography–tandem mass spectrometry (LC–MS/MS) (Miura et al., 2013). Mutagenicity of 24-h urine samples was measured by a modified quantitative Salmonella typhimurium reverse mutation assay at Labstat International (Ontario, Canada). The urine sample was concentrated using XAD-2 resin and diluted with dimethylsulfoxide to intended dose levels. The samples were incubated at 37 ± 1 °C with the metabolic activating system and YG1024 (tester strain), which was derived from strain TA98 with a high sensitivity for detecting the mutagenicity of aromatic amine compounds (Kado et al., 1983; Yamasaki and Ames, 1977). To evaluate urine mutagenicity, the activity indicator value per day (revertants/24h urine) was calculated by determining the slope (revertants/ml of urine) on the basis of the number of revertant colonies including 3 levels or more and by multiplying the slope by the 24-h urine pool volume of each subject. A slope of the regression line whose correlation coefficient was the highest was chosen among all the slopes of the regression lines calculated as above. If the slope was negative, the activity indicator value was 0.
2.7. Data analysis Changes in BOE levels by switching from the conventional cigarette to NCIT were evaluated by comparison of BOE levels between the NCIT and CC1 groups. The value of day 29, when the effect of switching tobacco products on the BOE level was believed to be the largest during the investigation period, was used as the evaluation value (the value of day 28 was used for COHb). Because homoscedasticity and normality of all evaluation values for BOEs were not observed, statistical analyses were performed using a non-parametric Wilcoxon rank sum test. Statistical significance was based on a 2-sided significance level of 0.05. When the BOE level in blood or urine was less than the lower limit of quantification (LLOQ), data analysis was performed by substituting one-half of LLOQ for the value. Because the levels of COHb, nicotine-glucuronide, cotinine-glucuronide, trans-30 -hydroxycotinine glucuronide, and NNAL glucuronide, and urine mutagenicity values were calculated using math formulae and there is no quantification limit, if these calculated values were negative, ‘‘0’’ was used for analysis. Statistical analysis was performed using SAS for Windows (version 9.2, SAS Institute). This study result is based on the per-protocol population.
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N. Miura et al. / Regulatory Toxicology and Pharmacology 71 (2015) 498–506 Table 2 Demographic and baseline characteristics of subjects.
3. Results 3.1. Measurement of tobacco smoke constituents by machine aspiration/smoking Table 1 shows the measurement results of tar and 10 constituents, which were reported to be corresponding to each BOE measured in this study (Scherer, 2005), by machine aspiration/ smoking of the test product NCIT and CC1. Among these constituents, the level of them, excluding nicotine, were below LOD for NCIT. A total of 1.1 mg of nicotine was detected in the aspirated air with 1920 machine aspirations (correspond to approximately 0.6 lg of nicotine per aspiration). The measurement of nicotine delivered by each of the 160 machine aspirations showed that the amount of nicotine delivered was almost constant by approximately 320–480 times of aspiration and decreased gradually to approximately half of the maximum at the last 160 of the 1920 aspirations (data not shown).
Subject group NCIT
CC1
NS
Number of subjects (n) Per protocol population (Enrolled)
40 (40)
20 (20)
18 (20)
Age (years) Mean ± SD Range
28.3 ± 6.12 21–43
28.6 ± 7.07 21–49
32.1 ± 4.55 22–39
Body mass index (kg/m2) Mean ± SD Range
20.7 ± 1.75 18.5–24.6
20.9 ± 1.33 19.0–24.1
21.6 ± 1.81 18.8–24.9
Duration of Smoking (year) Mean ± SD Range
8.20 ± 6.12 1–23
8.10 ± 7.17 1–29
–
Daily cigarette consumption (cig.) Mean ± SD Range
21.0 ± 2.24 20–30
21.1 ± 2.61 20–30
–
NCIT; non-combustion inhaler type of tobacco product. CC1; 1 mg ISO tar conventional cigarette, NS; non-smokers.
3.2. Study population A total of 97 smokers and 44 non-smokers who gave written informed consent were screened. Of these, 60 smokers were enrolled and randomly assigned to the NCIT and CC1 groups (40 and 20 subjects, respectively) and 20 non-smokers were assigned to the NS group. Two subjects in the NS group were discontinued prematurely according to the investigator’s judgment because a clinically significant incident or disease occurred on day 6 and 11, respectively. A total of 78 subjects completed the study. Demographic and some baseline characteristics are shown in Table 2 by subject group. With regard to age and BMI, no remarkable bias was observed among the subject groups, and smoking duration and daily cigarette consumption for smoking subjects were not remarkable bias as well between the NCIT and the CC1 groups,
3.3. Test tobacco product consumption The number of cigarettes smoked per day by the CC1 group and the daily number of aspirations for the NCIT group (total aspiration number recorded on the Puff Profiler and the self-recorded numbers in the questionnaire) are shown in Fig. 2.
Subjects in the CC1 group smoked approximately 21 cigarettes per day and were confirmed to keep their daily smoked number of CC1 within ±10% of their usual daily consumption throughout the study period. In the NCIT group, the mean of the total daily aspiration number ranged from 632 to 801 times throughout the study period. 3.4. Biomarkers Changes in BOE levels are shown in Table 3 and Fig. 3. The data of BOEs for 2 subjects in the NS group, who withdrew from the study prematurely, were removed from analysis. Moreover, because of failure of 24-h urine sampling for 1 subject in the NCIT group from day 7 to day 8, the urine data for 39 subjects were used for data analysis of day 8. In the baseline period (day 1 and 0), all smoking subjects (NCIT and CC1 groups) smoked their usual brands of 1 mg ISO tar cigarettes. At that time, the levels of all BOEs measured in this study were higher in the smoking subjects than in the non-smoking subjects (the NS group). On the other hand, no remarkable difference was observed between the NCIT and CC1 groups in the levels of all BOEs measured (Table 3, day 1).
Table 1 Yields of tar and cigarette smoke constituents that correspond to biomarkers of exposure measured in this study by machine aspiration/smoking. Cigarette smoke constituents
Tar Nicotine CO NNK Acrolein Crotonaldehyde 1,3-Butadiene Benzene HCN Benzo(a)pyrene 4-ABP
NCIT
mg mg mg ng lg lg lg lg lg ng ng
CC1
Total of 1920 aspirations
Per each aspirationa
Per cigarette
Per each puffb
n.d.c 1.1 n.d. (0.064) n.d. (4.93) n.d. (1.992) n.d. (3.138) n.d. (1.32) n.d. (0.263) n.d. (0.221) n.d. (0.03) n.d. (0.124)
n/a 0.0006 n/a n/a n/a n/a n/a n/a n/a n/a n/a
1d 0.1d 1.6 6.14 5.12 n.d. (0.718) 5.60 3.85 3.56 1.98 0.321
0.2 0.02 0.25 0.92 0.81 n/a 0.89 0.61 0.58 0.31 0.048
A machine smoking regimen was set as follows: 55 ml aspiration volume, 2-s duration, and 30-s interval for NCIT, and 35 ml aspiration volume, 2-s duration, and 60-s interval for CC1. Lower LOD is shown in parenthesis when a chemical constituent was not detected. NCIT: non-combustion inhaler type of tobacco product. CC1: 1 mg ISO tar conventional cigarette. NNK: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. HCN: Hydrogen cyanide. 4-ABP: 4-aminobiphenyl. n.d.: not detected. n/a: not applicable. a Values for ‘‘per aspiration’’ mean the value for the ‘‘A total of 1920 aspirations’’ divided by 1920. b Values for ‘‘per puff’’ mean the value for ‘‘per cigarette’’ divided by the puff count average for each measurement. c No increase was observed in the weight of a Cambridge filter pad used for trapping particulate matter. d Values of tar and nicotine printed on the package.
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NCIT: aspiration count (times/day)
CC1: cigarette smoked (n/day)
1000
30 (mean± SEM)
25
750
20
500
CC1 group NCIT group
15
250
0
10
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 study day
Fig. 2. Daily consumption of the test tobacco product. Plots show the mean number of cigarettes smoked by the CC1 group and aspiration number for the NCIT group. NCIT: non-combustion inhaler type of tobacco product. CC1: 1 mg ISO tar conventional cigarette.
In the CC1 and NS groups, levels of all BOEs, except the urine mutagenicity values, were not changed markedly throughout the study period. On the other hand, levels of all BOEs measured dropped to a lower value in the NCIT group after switching from their usual brand of 1 mg ISO tar conventional cigarette smoking to NCIT use. The urine mutagenic activities in all groups decreased on day 8 in comparison to the respective baseline levels. The levels of BOEs, except NE, cotinine, SCN, and total NNAL, in the NCIT group were almost same as those in the NS group on day 8, and these levels were maintained throughout the study period. The levels of total NNAL and SCN in the NCIT group declined gradually throughout the study period. On day 29, levels of all BOEs measured in this study were significantly (p < 0.05) lower in the NCIT group than in the CC1 group. The levels of BOEs, except cotinine, NE, and total NNAL in the NS and NCIT groups were almost the same. The levels of cotinine and NE in the NCIT group were almost constant from day 8 to day 29 and were slightly but definitely higher than those in the NS group on day 29. 3.5. Safety Nine adverse events developed in 8 of the 80 subjects (10.0%) enrolled in this study. Of these, colonic diverticulitis, which developed in 1 subject in the NS group, was rated serious, and other 8 events were rated non-serious by the investigator. Study participation for two subjects in the NS group, one had the adverse event rated as serious as described above and the other had enterocolitis rated non-serious and mild severity, was terminated prematurely according to the investigator’s judgment. For the causal relationship with the test product, 1 event, increased creatine phosphokinase, in CC1 group was classified into ‘‘probably related’’, 1 event, diarrhoea, in NCIT group was into ‘‘unlikely related’’ and other 7 events were into ‘‘not related’’ by the investigator. All adverse events recovered. 4. Discussion No smoke is generated when subjects use NCIT. The amount of chemical constituents, corresponding to BOEs measured in this
study, delivered from the product by machine aspiration was below LOD, except nicotine, in this analytical setting (Table 1). Accordingly, the levels of selected BOEs were markedly reduced after the subjects switched from their usual brand of 1 mg ISO tar conventional cigarette smoking to a new form of smokeless tobacco product in this clinical study setting. The mean number of daily cigarette consumption in the CC1 group during the entire study period was stable (Fig. 2), and this was believed to be the result of this study setting; all subjects in the CC1 group were allowed to smoke CC1 within ±10% of their usual daily consumption. Regarding NCIT use, the mean of the total daily aspiration number of NCIT was almost the same; the range was from 632 to 801, with an average of approximately 750 aspirations (Fig. 2). Some amount of inter- and intra-subject differences were observed among the study days. This may be due to the study setting for free use of NCIT except for 180 aspirations counted by the Puff Profiler. Furthermore, relatively lower values in the mean of the total daily aspiration number were observed almost weekly, and this was believed to be the result of the outings; all subjects were not allowed to use their assigned test tobacco product during outings. In the baseline period (day 1 and 0), the levels of all BOEs measured showed no remarkable difference between the CC1 and NCIT groups, and these levels were higher than those in the NS group (Table 3 and Fig. 3, day 1). The ratios of the each BOE level in the smoking subjects to that in the non-smoking subjects calculated from the baseline data (Table 3, day 1) were mostly consistent with the ratios reported (Scherer, 2005). In the NCIT group, levels of all BOEs measured dropped to a lower value after switching from their usual brand of cigarette to NCIT (Table 3 and Fig. 3). On day 29, levels of all BOE measured were significantly (p < 0.05) lower in the NCIT group than in the CC1 group and were almost the same as those in the NS group except cotinine, NE, and NNAL. These reductions in BOE levels indicate that the exposure to cigarette smoke constituents corresponding to each BOE were quite lower in the NCIT group than in the CC1 group, and suggested that the exposure levels in the NCIT group, except of nicotine and NNK, were similar to those in the NS group in this study setting. The levels of NE and cotinine in the NCIT group were higher than those in the NS group from day 8 to day 29 (Table 3). This
Table 3 Levels of biomarkers of exposure measured. Cigarette smoke constituents
Biomarker
Study day
Median (IQR)
Mean ± SD
Median (IQR)
Mean ± SD
Median (IQR)
Mean ± SD
Median (IQR)
Nicotine
Plasma cotinine (ng/ml) CC1 (n = 20) 200 ± 115 NCIT (n = 40) 173 ± 103 NS (n = 18) 0.25 ± 0.0
168 (103–274) 133 (105–268) 0.25 (0.25–0.25)
186 ± 98.6 24.9 ± 21.5 0.25 ± 0.0
172 (107–242) 18.4 (8.2–34) 0.25 (0.25–0.25)
204 ± 108 23.1 ± 23.5 0.25 ± 0.0
181 (133–262) 12.1 (4.8–34.1) 0.25 (0.25–0.25)
218 ± 102 26.2 ± 33.7 0.25 ± 0.0
227 (161–278) 10.2 (4.6–37.2)* 0.25 (0.25–0.25)
NEs (mg/24 h) CC1 NCIT NS
12.8 ± 7.87 12.0 ± 6.49 0.009 ± 0.008
11.9 (7.9–13.7) 10.5 (7.6–17.3) 0.007 (0.004–0.010)
11.9 ± 7.36 1.49 ± 1.36 0.007 ± 0.011
10.0 (7.5–13.3) 1.0 (0.53–2.0) 0.004 (0.003–0.006)
13.3 ± 8.52 1.64 ± 1.9 0.006 ± 0.008
11.3 (8.9–14.3) 0.83 (0.33–2.4) 0.004 (0.003–0.006)
12.5 ± 8.18 1.51 ± 1.81 0.007 ± 0.012
10.4 (7.8–13.2) 0.68 (0.26–2.1)* 0.004 (0.003–0.005)
COHb (%)à CC1 NCIT NS
5.61 ± 2.61 4.75 ± 1.66 2.12 ± 0.95
5.06 (4.15–6.74) 4.93 (3.72–5.77) 2.39 (1.43–2.84)
5.50 ± 1.87 2.11 ± 0.72 2.25 ± 0.73
5.06 (4.12–6.83) 2.14 (1.52–2.60) 2.16 (1.67–2.80)
5.61 ± 1.79 2.20 ± 0.69 1.81 ± 0.47
5.58 (4.38–6.28) 2.32 (1.80–2.72) 1.69 (1.58–2.03)
5.54 ± 1.67 2.04 ± 0.73 1.74 ± 0.61
5.16 (4.45–6.72) 1.94 (1.55–2.51)* 1.72 (1.24–2.11)
Total NNAL (ng/24 h) CC1 187 ± 100 NCIT 157 ± 63.5 NS 3.40 ± 3.62
178 (119–219) 155 (118–199) 2.32 (1.32–3.58)
170 ± 90.9 60.5 ± 25.5 2.39 ± 3.03
155 (100–212) 60.8 (43.6–79.2) 1.60 (0.63–2.66)
161 ± 77.3 41.0 ± 21.1 1.68 ± 1.48
160 (93.3–218) 38.6 (24.8–53.0) 1.43 (0.54–2.22)
151 ± 78.4 24.2 ± 19.8 0.99 ± 0.85
142 (80.8–204) 19.4 (13.4–24.3)* 0.56 (0.48–1.17)
3-HPMA (mg/24 h) CC1 3.97 ± 2.45 NCIT 3.35 ± 1.62 NS 0.95 ± 0.22
3.00 (2.36–4.62) 2.92 (2.22–4.24) 0.93 (0.82–1.12)
3.23 ± 1.23 0.81 ± 0.30 0.96 ± 0.30
3.08 (2.26–3.67) 0.81 (0.62–0.94) 0.93 (0.80–1.03)
3.64 ± 2.48 0.79 ± 0.38 0.80 ± 0.31
2.89 (2.25–3.50) 0.72 (0.48–0.97) 0.76 (0.62–1.06)
2.85 ± 2.19 0.67 ± 0.29 0.66 ± 0.20
2.45 (1.86–2.75) 0.66 (0.43–0.82)* 0.69 (0.48–0.82)
HMPMA (lg/24 h) CC1 1616 ± 793 NCIT 1503 ± 684 NS 257 ± 58.1
1394 (1061–2200) 1504 (936–2002) 246 (215–308)
1622 ± 792 244 ± 79.4 242 ± 63.0
1388 (1094–1791) 243 (188–285) 238 (216–292)
1630 ± 873 213 ± 65.8 226 ± 60.5
1509 (1138–1832) 211 (158–258) 229 (184–250)
1575 ± 850 191 ± 81.7 198 ± 75.0
1212 (934–2115) 185 (128–228)* 178 (144–232)
MHBMA (lg/24 h) CC1 7.15 ± 8.41 NCIT 9.16 ± 10.6 NS 0.85 ± 1.11
3.85 (1.63–8.57) 4.57 (1.60–12.5) 0.45 (0.31–0.76)
4.58 ± 5.39 0.32 ± 0.21 0.35 ± 0.18
2.66 (1.38–5.09) 0.25 (0.22–0.33) 0.31 (0.22–0.43)
6.77 ± 7.04 0.70 ± 0.87 0.46 ± 0.23
3.88 (1.32–9.62) 0.42 (0.24–0.68) 0.38 (0.33–0.51)
5.73 ± 6.55 0.44 ± 0.64 0.84 ± 1.34
3.22 (1.89–6.19) 0.26 (0.18–0.38)* 0.26 (0.20–0.50)
DHBMA (lg/24 h) CC1 496 ± 144 NCIT 458 ± 128 NS 346 ± 87.8 Mean ± SD
474 (393–561) 441 (357–536) 344 (287–405) Median (IQR)
451 ± 107 281 ± 82.4 358 ± 94.2 Mean ± SD
442 (376–501) 288 (217–326) 342 (280–441) Median (IQR)
486 ± 140 305 ± 65.6 329 ± 63.6 Mean ± SD
459 (407–523) 305 (257–338) 340 (280–357) Median (IQR)
403 ± 177 288 ± 85.7 304 ± 89.5 Mean ± SD
382 (322–459) 290 (216–349)* 290 (240–367) Median (IQR)
TMA (lg/24 h) CC1 NCIT NS
155 ± 68.8 123 ± 54.0 49.0 ± 25.1
138 (100–191) 109 (89.8–160) 43.2 (30.1–59.6)
130 ± 46.7 39.5 ± 15.3 53.9 ± 24.0
129 (94.1–163) 36.9 (28.6–51.8) 48.9 (35–68.8)
147 ± 54.0 47.1 ± 18.8 49.9 ± 20.2
143 (113–165) 44.8 (36.6–54.5) 45.9 (34.5–64.6)
161 ± 82.3 72.6 ± 49.9 77.4 ± 38.9
156 (117–200) 54.1 (38.3–92.3)* 66.7 (52–99.7)
S-PMA (ng/24 h) CC1 NCIT NS
420 ± 276 445 ± 238 156 ± 90.7
383 (238–514) 430 (245–614) 140 (101–181)
496 ± 273 156 ± 104 190 ± 98.8
427 (292–620) 117 (93.7–183) 162 (118–263)
501 ± 286 180 ± 113 238 ± 165
526 (274–607) 133 (82.7–272) 197 (128–288)
491 ± 312 203 ± 118 231 ± 161
432 (351–543) 194 (111–264)* 160 (117–255)
1-OHP (ng/24 h) CC1 NCIT NS
346 ± 178 321 ± 170 129 ± 63.9
349 (247–412) 265 (226–407) 108 (81.8–176)
340 ± 134 130 ± 61.2 123 ± 53.2
318 (234–439) 119 (82.4–173) 113 (86.5–152)
309 ± 97.5 133 ± 75.1 122 ± 58.0
291 (259–372) 128 (77.9–153) 101 (73.8–167)
264 ± 119 61.2 ± 32.5 70.1 ± 23.4
230 (194–309) 55.5 (41.4–73.5)* 71.3 (61.2–79.9)
Day 1 Mean ± SD
Nicotine
NNK
Acrolein
Crotonaldehyde
1,3-Butadiene
1,3-Butadiene
Benzene
Benzene
Pyrene
Day 15
Day 29
503
(continued on next page)
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CO
Day 8
13,522 4449 4040 15,112 4406 2933
69.1 (55.2–86.2) 37.8 (32.3–43.7) 28.0 (26.8–29.6)
Mutagenicity (revertants/24 h) CC1 34,459 NCIT 24,292 NS 10,479 Mutagen
75.4 ± 34.1 68.9 ± 25.4 29.7 ± 6.52 SCN (lmol/l) CC1 NCIT NS HCN
22,522 21,574 8054
71.1 (52.5–92.0) 67.3 (51.9–84.4) 28.5 (24.7–32.1)
16,142 5531 5752
70.7 ± 27.6 46.4 ± 14.7 26.0 ± 8.67
14,939 5005 3668
63.7 (52.8–82.6) 44.4 (36.0–55.5) 27.6 (23.9–29.3)
19,134 6823 4768
73.7 ± 28.1 39.3 ± 9.24 28.5 ± 7.19
9.65 (5.25–13.1) 1.03 (0.82–1.36) 1.02 (0.86–1.26) 9.21 ± 4.81 1.22 ± 0.65 1.11 ± 0.45 8.37 (4.40–11.8) 1.10 (0.92–1.26) 1.17 (1.02–1.28) 8.89 ± 6.17 1.15 ± 0.52 1.19 ± 0.48 8.28 (6.63–12.8) 8.96 (5.66–11.6) 1.13 (0.80–1.41) 11.0 ± 7.37 9.30 ± 5.29 1.43 ± 1.39 4-ABP (ng/24 h) CC1 NCIT NS 4-ABP
15,679 5519 6830
74.6 ± 28.5 31.2 ± 7.01 26.7 ± 6.35
7.80 (3.89–10.8) 0.89 (0.73–1.13)* 0.98 (0.75–1.13) 7.74 ± 4.49 1.02 ± 0.54 1.02 ± 0.49
Median (IQR) Mean ± SD
Day 29
Median (IQR) Mean ± SD
Day 15
Mean ± SD
Median (IQR) Day 8
Mean ± SD
Study day
Median (IQR) Day 1
Biomarker Cigarette smoke constituents
Table 3 (continued)
In the baseline period (day 1 and 0), all subjects in the CC1 and NCIT groups smoked their usual brand of 1 mg ISO tar cigarette, and the values of day 1 reflected the baseline values. NCIT: non-combustion inhaler type of tobacco product. CC1: 1 mg ISO tar conventional cigarette. NS: non-smoker. NEs: nicotine equivalents. COHb: carboxyhemoglobin. NNK: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Total NNAL: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and glucuronide. 3-HPMA: 3-hydroxypropylmercapturic acid. HMPMA: 3-hydroxy-1-methylpropyl-mercapturic acid. MHBMA: monohydroxybutenylmercapturic acid. DHBMA: dihydroxybutenylmercapturic acid. TMA: trans, trans-muconic acid. S-PMA: S-phenylmercapturic acid. 1-OHP: 1-hydroxypyrene. 4-ABP: 4-aminobiphenyl. HCN: hydrogen cyanide. SCN: thiocyanate. IQR: interquartile range. * Significant difference in BOE levels in the NCIT group vs. the CC1 group on day 29 (day 28 for COHb). p < 0.05. à Blood samplings for COHb measurement were conducted between 16 to 17 o’clock on day 0, 7, 14, and 28. Data of BOE in urine for day 8 were for 39 subjects because of failure of 24-h urine collection for 1 subject.
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69.1 (53.5–89.3) 29.5 (27.3–35.1)* 26.2 (25.0–27.9)
504
result showed frequent aspiration of nicotine form NCIT by the subjects during the study period. The levels of total NNAL and SCN in the NCIT group declined gradually throughout the study period. The elimination half-lives of these two BOEs are relatively longer than those of the other BOEs measured in this study. The half-lives of total NNAL were reported as 1–4 and 10–45 days for initial (t1/2a) and terminal (t1/2b) phases, respectively (Goniewicz et al., 2009; Hecht et al., 1999). The total NNAL level in urine for the NCIT group was higher than that for the NS group on day 29, therefore it remains unclear whether the exposure level to NNK in the NCIT group would be almost the same as that in the NS group. However, the continuous decrease in the total NNAL level indicates that the exposure to NNK in the NCIT group would be substantially lower than that in the CC1 group. Because the reported elimination half-life of SCN was 6.4 days (Junge, 1985), almost the same SCN level observed in the NCIT and NS groups on day 29 suggested that exposure of hydrogen cyanide (HCN) in the NCIT group was almost the same as in the NS group. In the CC1 and NS groups, levels of all BOEs were relatively constant throughout the entire study period, except the urine mutagenicity values. Although the subjects in the CC1 group switched from their usual brand of 1 mg ISO tar cigarette to the assigned CC1 on day 1, the constant BOE levels in the CC1 group suggested that the levels were not considerably affected in this study if the smoking topography of the smoker changed by switching the cigarette brand. The urine mutagenicity values showed a decrease on day 8 compared with each baseline value in the CC1, NS, and NCIT groups. Because the half-life of the smoking-related urine mutagenic activity was reported to be approximately 7–23 h (Kado et al., 1985), the effect of smoking the usual brand of cigarette on the urine mutagenic activity is considered to be disappeared on day 8. This result showed that the use of NCIT decreased exposure to mutagens detected in the urine mutagenicity test, as much the exposure level of the NS group. It has been reported that the urine mutagenic activity increased with an intake of fried or boiled meal (Doolittle et al., 1989). In this study, the clinical institutions served daily meal excluding such heat-processed food for 2 days prior to the day of 24-h urine sampling completion, day 8, 15 and 29. The decrease in the urine mutagenic activity in the NS group on day 8, by approximately half of the activity of day 1, would be mainly due to this diet. Considering the constant levels of other BOEs in the CC1 group, the decrease in the urine mutagenic activity in the CC1 group would be due to the change in this diet as well. In summary, under the conditions of the present study, switching the tobacco product used from a conventional cigarette to NCIT, the new form of smokeless tobacco product, showed significant reduction in all BOEs measured in this study. Environmental factors such as daily food might have affected BOE levels in the NS group. On day 29, the exposure levels to 8 tobacco smoke constituents and mutagens detected by the urine mutagenicity test in the NCIT group were believed to be similar to those in the NS group, while the exposure levels to nicotine would be higher than those in the NS group. Although in this study, it is unclear whether the exposure level to NNK in the NCIT group is similar to that in the NS group because of the relatively long half-life of the corresponding BOE, it would be substantially lower than that in the CC1 group.
Conflict of interest This work was funded by Japan Tobacco Inc., and all authors are employees of the company.
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4 2
250 200 150
CC1
100
NCIT NS
50
0
80 60 40 20
0 day0
day7
day14
0 day1
day28
CC1 NCIT NS
100 SCN in blood (µmol/l)
COHb in blood (%)
6
300 Plasma cotinine (ng/ml)
CC1 NCIT NS
8
day8
Study day
day15
day29
day1
Study day
200
day8
day15
day29
Study day
600
6
500
5
CC1 NCIT NS
100 50
400
MHBMA (µg/24hr)
150
S-PMA (ng/24hr)
TMA (µg/24hr)
CC1
CC1 NCIT NS
300 200 100
day1
day8
day15
2 1 0
day1
day29
NS
3
0
0
NCIT
4
day8
Study day
day15
day29
day1
Study day
day15
500 CC1 NCIT NS
300 200
CC1 NCIT NS
100
NCIT NS
3 2 1
day8
day15
300 200 100 0
day1
day29
day8
Study day
day1
day29
Study day
CC1 NCIT NS
2000
day15
15
1000 500
10
5
0
0 day1
day8
day15
day1
day29
day8
Study day
CC1 NCIT NS
200
day15
day29
Study day
150 100 50 0
day29
CC1 NCIT
15
NS
10 5 0 day1
day8
day15
day29
Study day
CC1 NCIT NS
30000 Urine mutagenicity (revartants/24hr urine)
250
day15
20
CC1 NCIT NS
4-ABP (ng/24hr)
1500
day8
Study day
Nicotine equivalent (mg/24hr)
day1
HMPMA (µg/24hr)
400
0
0
Total NNAL (ng/24hr)
1-OHP (ng/24hr)
400
4
3-HPMA (mg/24hr)
DHBMA (µg/24hr)
CC1 500
day29
Study day
5
600
day8
20000
10000
0 day1
day8
day15
Study day
day29
day1
day8
day15
day29
Study day
Fig. 3. Biomarker of exposure (BOE) measurement results for all study groups. Data are shown as median. Blood samplings for COHb measurement were performed between 16 and 17 o’clock on day 0, 7, 14, and 28. Data of the NCIT group’s BOE in urine for day 8 were for 39 subjects because of failure of 24-h urine collection for 1 subject. In the baseline period (day 1 and 0), all subjects in the CC1 and NCIT groups smoked their usual brand of 1 mg ISO tar cigarette, and the values of day 1 reflected the baseline values. NCIT: non-combustion inhaler type of tobacco product. CC1: 1 mg ISO tar conventional cigarette. NS: non-smokers. COHb: carboxyhemoglobin. Total NNAL: 4(methylnitrosamino)-1-(3-pyridyl)-1-butanol and glucuronide. 3-HPMA: 3-hydroxypropylmercapturic acid. HMPMA: 3-hydroxy-1-methylpropyl-mercapturic acid. MHBMA: monohydroxybutenylmercapturic acid. DHBMA: dihydroxybutenylmercapturic acid. TMA: trans, trans-muconic acid. S-PMA: S-phenylmercapturic acid. 1-OHP: 1hydroxypyrene. 4-ABP: 4-aminobiphenyl. SCN: thiocyanate.
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Acknowledgments We would like to thank Drs. Kunio Iwata and Yasufumi Nagata for their invaluable input. References Buchhalter, A.R., Eissenberg, T., 2000. Preliminary evaluation of a novel smoking system: effects on subjective and physiological measures and on smoking behavior. Nicotine Tob. Res. 2, 39–43. Doolittle, D.J., Rahn, C.A., Burger, G.T., Lee, C.K., Reed, B., Riccio, E., Howard, G., Passananti, G.T., Vesell, E.S., Hayes, A.W., 1989. Effect of cooking methods on the mutagenicity of food and on urinary mutagenicity of human consumers. Food Chem. Toxicol. 27, 657–666. Goniewicz, M.L., Havel, C.M., Peng, M.W., Jacob III, P., Dempsey, D., Yu, L., ZielinskaDanch, W., Koszowski, B., Czogala, J., Sobczak, A., Benowitz, N.L., 2009. Elimination kinetics of the tobacco-specific biomarker and lung carcinogen 4(methylnitrosamino)-1-(3-pyridyl)-1-butanol. Cancer Epidemiol. Biomarkers Prev. 18, 3421–3425. Health Canada, 1999. Determination of ’’Tar’’, Nicotine and Carbon Monoxide in Mainstream Tobacco Smoke-Official Method. Health Canada, Ottawa (SOR2000-272). Hecht, S.S., Carmella, S.G., Chen, M., Dor. Koch, J.F., Miller, A.T., Murphy, S.E., Jensen, J.A., Zimmerman, C.L., Hatsukami, D.K., 1999. Quantitation of urinary metabolites of a tobacco-specific lung carcinogen after smoking cessation. Cancer Res. 59, 590–596. International Agency for Research on Cancer, 2004. Tobacco smoke and involuntary smoking. IARC Monogr. Eval. Carcinog. Risks Hum. 83, 1–1438. International Agency for Research on Cancer, 2007. Smokeless tobacco and some tobacco-specific N-nitrosamines. IARC Monogr. Eval. Carcinog. Risks Hum. 89, 1–592. International Organization for Standardization, 2000. Routine analytical cigarettesmoking machine, ISO3308. Institute of Medicine, 2012. Scientific Standards for Studies on Modified Risk Tobacco Products. The National Academy Press, Washington DC.
Junge, B., 1985. Changes in serum thiocyanate concentration on stopping smoking. Br. Med. J. (Clin. Res. Ed.) 291, 22. Kado, N.Y., Langley, D., Eisenstadt, E., 1983. A simple modification of the Salmonella liquid-incubation assay. Increased sensitivity for detecting mutagens in human urine. Mutat. Res. 121, 25–32. Kado, N.Y., Manson, C., Eisenstadt, E., Hsieh, D.P., 1985. The kinetics of mutagen excretion in the urine of cigarette smokers. Mutat. Res. 157, 227–233. Miura, N., Yuki, D., Minami, N., Kakehi, A., Onozawa, M., 2013. Pharmacokinetic analysis of nicotine when using non-combustion inhaler type of tobacco product in Japanese adult male smokers. Regul. Toxicol. Pharmacol. 67, 198– 205. Okuzono, H., Tamagawa, S., Hattori, S., 1976. A spectrophotometric method for the determination of carboxyhemoglobin in blood, and some clinical reports. Rinsho Byori. 24, 616–621. Pérez-Stable, E.J., Marín, G., Marín, B.V., Benowitz, N.L., 1992. Misclassification of smoking status by self-reported cigarette consumption. Am. Rev. Respir. Dis. 145, 53–57. Rainey, C.L., Conder, P.A., Goodpaster, J.V., 2011. Chemical characterization of dissolvable tobacco products promoted to reduce harm. J. Agric. Food Chem. 59, 2745–2751. Sakaguchi, C., Kakehi, A., Minami, N., Kikuchi, A., Futamura, Y., 2014. Exposure evaluation of adult male Japanese smokers switched to a heated cigarette in a controlled clinical setting. Regul. Toxicol. Pharmacol. 69, 338–347. Scherer, G., 2005. Biomonitoring of inhaled complex mixtures-ambient air, diesel exhaust and cigarette smoke. Exp. Toxicol. Pathol. 57 (Suppl. 1), 75–110. Scherer, G., 2006. Carboxyhemoglobin and thiocyanate as biomarkers of Exposure to carbon monoxide and hydrogen cyanide in tobacco smoke. Exp. Toxicol. Pathol. 58, 101–124. Schorp, M.K., Tricker, A.R., Dempsey, R., 2012. Reduced exposure evaluation of an electrically heated cigarette smoking system. Part 1: non-clinical and clinical insights. Regul. Toxicol. Pharmacol. 64 (2 Suppl.), S1–10. Vesey, C.J., McAllister, H., Langford, R.M., 1999. A safer method for the measurement of plasma thiocyanate. J. Anal. Toxicol. 23, 134–136. Yamasaki, E., Ames, B.N., 1977. Concentration of mutagens from urine by absorption with the nonpolar resin XAD-2: cigarette smokers have mutagenic urine. Proc. Natl. Acad. Sci. U.S.A. 74, 3555–3559.
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Regulatory Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/yrtph
A study to investigate changes in the levels of biomarkers of exposure to selected cigarette smoke constituents in Japanese adult male smokers who switched to a non-combustion inhaler type of tobacco product Naoki Miura ⇑, Dai Yuki, Naoki Minami, Aoi Kakehi, Yasuyuki Futamura Product Science Division, R&D Group, Japan Tobacco Inc., Yokohama, Kanagawa, Japan
a r t i c l e
i n f o
Article history: Received 28 July 2014 Available online 12 February 2015 Keywords: Biomarkers Exposure Cigarette Smoking Smokeless tobacco Clinical study
a b s t r a c t In a clinical study, changes in 14 biomarkers of exposures (BOEs) from 10 tobacco smoke constituents and mutagens detected by the urine mutagenicity test were investigated using a non-combustion inhaler type of tobacco product (NCIT) by switching from a conventional cigarette. This study was conducted in 80 Japanese healthy adult males with a 4-week residential, controlled, open-label, parallel group design. After randomization, 40 smokers used NCIT with approximately 750 aspirations, other 20 smokers smoked approximately 20 pieces of an assigned 1-mg ISO tar conventional cigarette (CC1) every day. Twenty non-smokers (NS) did not use any tobacco product. Under this study condition, switching from cigarette to NCIT showed significant reduction in all BOEs measured. On day 29, the levels of these BOEs were almost the same as those in the NS group, except BOEs of nicotine and 4-(methylnitrosamino)-1-(3pyridyl)-1-butanone (NNK). This suggested that the exposure to 8 constituents and mutagens in the NCIT group was similar to that in the NS group, while the exposure to nicotine was higher. Although the precise exposure level to NNK was not estimated because of the long half-life of its BOE, it would be substantially lower in the NCIT group than in the CC1 group. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction Many previous studies reported that cigarette smoking is a risk factor for serious diseases including lung cancer, coronary heart disease, emphysema, and chronic bronchitis. There are many types of tobacco products other than cigarette in the world, such as cigars, pipes, and nasal or oral snuff, and it has been reported that the users of such products are at a risk for serious diseases (International Agency for Research on Cancer, 2004, 2007). In addition to the various traditional tobacco products available, some non-traditional forms of tobacco products, such as electrically heated cigarettes (Buchhalter and Eissenberg, 2000) and dissolvable tobacco tablets (Rainey et al., 2011), have been recently introduced. Some of these were designed for reducing exposure to a series of selected harmful and potentially harmful constituents (Schorp et al., 2012). For the comprehensive evaluation of such emerging products, it is important to measure the users’ exposure to chemical constituents derived from the products. ⇑ Corresponding author at: Corporate, Scientific and Regulatory Affairs Group, Japan Tobacco Inc., 2-2-1 Toranomon, Minato-ku, Tokyo 105-8422, Japan. Fax: +81 3 5572 1477. E-mail address: [email protected] (N. Miura). http://dx.doi.org/10.1016/j.yrtph.2015.02.007 0273-2300/Ó 2015 Elsevier Inc. All rights reserved.
The non-combustion inhaler type of tobacco product (NCIT) is a new form of smokeless tobacco product that consists of a tapered mouthpiece and cartridge filled with finely cut tobacco leaves. The flavor components can be delivered from the tobacco leaves not by burning or heating but just by the air flow when users aspirate through its mouthpiece. We previously reported the results of a clinical study investigating the pharmacokinetics of nicotine when using prototype NCIT (Miura et al., 2013). That study results revealed that the bioavailability of nicotine delivered from prototype NCIT was similar to that delivered by conventional cigarette smoking, regardless of the form of the tobacco product. However, the degree of user’s exposure to chemical constituents derived from the product, except nicotine, is unclear. The measurement of several chemical constituents delivered from NCIT by machine aspiration showed that many of these constituents were below the limit of detection (LOD), even when approximately 2000 aspirations were integrated (described in detail in this article). This result indicates a prospect of significant reduction in exposure to those constituents in NCIT users. The measurement of biomarkers of exposure (BOE) is one of the effective methods to evaluate human exposure to smoke constituents. BOEs for tobacco smoke constituents are mainly the chemical constituents themselves and/or their metabolites, and at
N. Miura et al. / Regulatory Toxicology and Pharmacology 71 (2015) 498–506
present, some BOEs are validated and recommended to use for measuring the exposure of smoke constituents (Institute of Medicine, 2012). The advantage of monitoring BOEs is that they could reflect intra- and inter-individual differences in the tobacco smoking or using behavior. Moreover, the BOE monitoring method is applicable not only to the conventional cigarette but to the various other types of tobacco products, including the emerging products. The biomarker level must be background level if the exposure is zero. However, many of the validated BOEs are well known to be influenced by the other source of their corresponding chemicals existing in our living environment (Scherer, 2005). Therefore, for precise exposure evaluation, it is important to determine the degree of the influence of environmental factors other than tobacco on BOE levels. The objective of this study was to evaluate changes in the selected BOEs with the use of NCIT in Japanese healthy adult male smokers, using a 1 mg ISO tar conventional cigarette (CC1) as a control. In addition, the changes in BOEs in Japanese healthy male non-smokers (NS) were evaluated for identifying the influence of factors other than tobacco on BOE levels in the same study environment. The BOEs monitored in this study were selected with reference to previously published information on harm reduction studies (Scherer, 2005) and the results of our preliminary BOEs exploratory study with smokers and non-smokers and our recent publication (Sakaguchi et al., 2014).
2. Materials and methods 2.1. Study design This study was conducted at 4 medical institutions in Japan using a permuted-block, randomized (for smokers), controlled, forced-switching, open-label, parallel group design. All subjects who passed the screening test stayed at one of the medical institutions throughout the study period, except several times of outings accompanied by the clinical staff. The study period of 31 consecutive days was set in consideration of the elimination half-lives of some BOEs. The outing, such as bowling, seeing a movie, and art appreciation, was about 2 h, and was set once a week. All smoking subjects smoked their usual brand of a 1 mg ISO tar cigarette within ±10% of their usual daily consumption for the first 2 days (day 1 and 0, baseline period). Following the baseline
499
period, the smokers were randomly assigned to 2 groups: NCIT and CC1, as described in Fig. 1. Subjects in the NCIT group used 2 NCIT cartridges per day, and those in the CC1 group smoked an assigned CC1 within ±10% of their usual daily consumption from day 1 to day 28. All non-smokers were automatically assigned to the NS group and did not smoke throughout the study period. All four medical institutions had approximately 20–30 subjects each, and the number of subjects assigned to each group were set as equal (CC1–NCIT–NS, 1:2:1) in order to avoid the institutionspecific effect on the particular group. The clinical staff observed and recorded any adverse events. They also ensured that the subjects abstained from using nicotine-containing products except for the tobacco products specified for the study. The study was conducted at the Maruyama hospital (Hamamatsu, Japan), OCROM clinic (Osaka, Japan), Sumida hospital (Tokyo, Japan), and Hakata clinic (Fukuoka, Japan) in accordance with Good Clinical Practice (GCP) and the principles that have their origin in the Declaration of Helsinki. This study protocol was approved by the Institutional Review Board of Japan Tobacco Inc. and the medical institutions. 2.2. Subjects Eighty Japanese healthy males, 60 smokers and 20 non-smokers, aged 21–49 years and having a body mass index (BMI) in the range of 18.5–25.0 kg/m2 were enrolled. The health of the subjects was checked before their entry into the trial by physical examination, examination of medical history and vital signs, 12-lead electrocardiography and laboratory tests. Eligible smokers for the study had smoked a 1 mg ISO tar conventional cigarette without regard to menthol or non-menthol with a daily consumption of at least 20 cigarettes for at least 1 year prior to screening and their serum cotinine levels had exceeded 14 ng/ml at the screening (Pérez-Stable et al., 1992). Eligible nonsmokers had no experience of routinely smoking the conventional cigarette and had not used any tobacco products including the conventional cigarette for at least 1 year before screening. Non-smokers with their serum cotinine levels lower than 14 ng/ml at the screening were enrolled. All volunteers were paid for participating and provided written informed consent before the enrollment. 2.3. Study procedure
Day −1
Day 1
Day 8
Day 15
Day 29
Blood sampling
forced-switching
24hr Urine sampling
1 mg tar cigarette smokers (N = 60) Non-smokers (N = 20)
CC1 group (N = 20) NCIT group (N = 40) NS group (N = 20)
Baseline period = for COHb
Investigation period = for all BOE in blood except COHb
Fig. 1. Study design. All smoking subjects smoked their usual brand of 1 mg ISO tar cigarette on day 1 and 0, and then the smokers were randomly assigned to the NCIT and CC1 groups. All non-smokers were automatically assigned to the NS group and did not smoke throughout the study period. NCIT: non-combustion inhaler type of tobacco product. CC1: 1 mg ISO tar conventional cigarette. NS: non-smokers. BOE: biomarker of exposure. COHb: carboxyhemoglobin.
All smoking subjects smoked or used their assigned test tobacco product whenever they wanted from 7 to 23 o’clock throughout the study period, except for some constraints on the timing of tobacco smoking or usage; the subjects were not allowed to use NCIT or smoke the conventional cigarette from the time of awakening to the completion of clinical examination in the morning on day 1, 1, 8, 15, 22, and 29. During outings, the subjects were not allowed to use test tobacco because accurate checking of each subjects’ tobacco use was difficult for the clinical staff. In addition, the subjects used NCIT with 15 aspirations or smoked 1 piece of the conventional cigarette at approximately 16 o’clock, and then 30– 45 min after use or smoking, blood sampling was performed on day 0, 7, 14, and 28 to measure carboxyhemoglobin (COHb) levels. The subjects abstained from using or smoking the test tobacco product until the completion of this blood sampling. Subjects who smoked the conventional cigarette (smoking subjects on day 1 and 0 and the CC1 group subjects from day 1 to day 28) received the cigarette from the clinical staff in each case and were allowed to smoke only in the designated smoking room. Subjects in the NCIT group received 2 pieces of NCIT cartridge daily from the clinical staff at a set time and were forbidden to enter
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the smoking room since they were allowed to bring NCIT and use it in their living space in the institution. Regarding the total daily aspiration number of NCIT use, 180 aspirations were set as the minimal number per day and were confirmed using a Puff Profiler (CReSSmicro™, Plowshare/BorgwaldtKC), and the other voluntary aspiration number of the day was investigated by a self-administered questionnaire. No upper limit was set for the daily aspiration number of NCIT use. For the CC1 group, the daily number of cigarettes smoked by each subject was recorded. To avoid the potential effects on BOEs, clinical institutions served meals without smoked food and baked or fried processed food for 2 days prior to the day of blood sampling, day 8, 15 and 29. The health of the subjects was checked during this study period by physical examination and examination of vital signs on day 1, 8, 15, 22, and 29 and by 12-lead electrocardiography and laboratory tests on day 1 and 29. 2.4. Test tobacco products NCIT consists of a cartridge filled with finely cut tobacco leaves and a tapered mouthpiece. Volatile substances are delivered from the cartridge not by burning or heating but just by the air flow of users’ aspiration. Water, flavorings, potassium carbonate, and propylene glycol were added to the tobacco leaves. NCIT was mint flavored, unlike the prototype NCIT used in our previous study (Miura et al., 2013), in which flavorings were not added. For measuring the amount of chemical constituents derived from NCIT by machine aspiration, the following regimen was set referring to the Health Canada Intense method for cigarette machine smoking conditions (Health Canada, 1999): 55 ml aspiration volume, 2-s duration, and 30-s interval. The number of aspirations, 1920, was set according to the assumption of continual aspiration for 16 h with 30-s intervals. The CC1 chosen for the investigation period (from day 1 to day 28) is one of the most popular 1 mg ISO tar brands in Japan. The mainstream smoke delivered from CC1 was analyzed using the smoking machine protocol standardized by the International Organization for Standardization (ISO, 2000). The brand of 1 mg ISO tar conventional cigarette for the baseline period (day 1 and 0) was conformed to the each subjects’ usual brand which they declared. For this study, NCIT (it was marketed under the name ‘‘Zerostyle Mint’’ in Japan at that time), subjects’ usual brand of cigarette and assigned CC1 were purchased from the market. 2.5. Blood and urine sampling To measure plasma BOE levels, venous blood samples were obtained from the forearm vein. Blood samples (7 ml) were drawn for cotinine and thiocyanate (SCN) measurement before breakfast and first use or smoking of the test tobacco product of the day on day 1, 8, 15, and 29. Moreover, 2 ml of blood were obtained for COHb measurement at approximately 16 o’clock on day 0, 7, 14, and 28, because the half-life of COHb has been reported be 1–4 h (Scherer, 2006) and COHb was expected to largely disappear prior to the next morning. Blood samples were collected in blood collection tubes containing EDTA-2Na and centrifuged at 3000 rpm for 10 min at 4 °C. Plasma was transferred into plastic tubes and stored at 80 °C until further analysis. The 24-h urine pools for measuring urine mutagenicity and BOEs were sampled from the morning on day 0, 7, 14, and 28 to the next morning (in this article, the study day for each 24-h urine sample data were expressed as the end of the sampling). Approximately 300 ml of 24-h collected urine was divided into plastic tubes or bottles and stored at 80 °C until further analysis.
2.6. Bioanalysis All measurements, except for the urine mutagenicity test, were conducted by Mitsubishi Chemical Medience Corporation (Tokyo, Japan) using validated methods. The BOEs in urine except urine mutagenicity were measured based on a liquid chromatography with tandem mass spectrometry (LC–MS/MS) (Sakaguchi et al., 2014). Urinary nicotine equivalent (NE) was calculated as the molar sum of nicotine and its 5 major metabolites (cotinine, trans-3-hydroxycotinine, nicotine-N-glucuronide, cotinine-N-glucuronide, and trans-3-hydroxycotinine-O-glucuronide). NE in mg/ 24 h was calculated by multiplying molar NE by the molecular weight of nicotine. Urinary total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a major metabolite of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), was calculated as the molar sum of its free and glucuronide form. COHb levels in whole blood were measured by the cyanmethemoglobin method using a microplate reader (Okuzono et al., 1976), and plasma SCN levels were assayed spectrophotometrically using the microplate reader (Vesey et al., 1999). Plasma cotinine levels were measured by liquid chromatography–tandem mass spectrometry (LC–MS/MS) (Miura et al., 2013). Mutagenicity of 24-h urine samples was measured by a modified quantitative Salmonella typhimurium reverse mutation assay at Labstat International (Ontario, Canada). The urine sample was concentrated using XAD-2 resin and diluted with dimethylsulfoxide to intended dose levels. The samples were incubated at 37 ± 1 °C with the metabolic activating system and YG1024 (tester strain), which was derived from strain TA98 with a high sensitivity for detecting the mutagenicity of aromatic amine compounds (Kado et al., 1983; Yamasaki and Ames, 1977). To evaluate urine mutagenicity, the activity indicator value per day (revertants/24h urine) was calculated by determining the slope (revertants/ml of urine) on the basis of the number of revertant colonies including 3 levels or more and by multiplying the slope by the 24-h urine pool volume of each subject. A slope of the regression line whose correlation coefficient was the highest was chosen among all the slopes of the regression lines calculated as above. If the slope was negative, the activity indicator value was 0.
2.7. Data analysis Changes in BOE levels by switching from the conventional cigarette to NCIT were evaluated by comparison of BOE levels between the NCIT and CC1 groups. The value of day 29, when the effect of switching tobacco products on the BOE level was believed to be the largest during the investigation period, was used as the evaluation value (the value of day 28 was used for COHb). Because homoscedasticity and normality of all evaluation values for BOEs were not observed, statistical analyses were performed using a non-parametric Wilcoxon rank sum test. Statistical significance was based on a 2-sided significance level of 0.05. When the BOE level in blood or urine was less than the lower limit of quantification (LLOQ), data analysis was performed by substituting one-half of LLOQ for the value. Because the levels of COHb, nicotine-glucuronide, cotinine-glucuronide, trans-30 -hydroxycotinine glucuronide, and NNAL glucuronide, and urine mutagenicity values were calculated using math formulae and there is no quantification limit, if these calculated values were negative, ‘‘0’’ was used for analysis. Statistical analysis was performed using SAS for Windows (version 9.2, SAS Institute). This study result is based on the per-protocol population.
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N. Miura et al. / Regulatory Toxicology and Pharmacology 71 (2015) 498–506 Table 2 Demographic and baseline characteristics of subjects.
3. Results 3.1. Measurement of tobacco smoke constituents by machine aspiration/smoking Table 1 shows the measurement results of tar and 10 constituents, which were reported to be corresponding to each BOE measured in this study (Scherer, 2005), by machine aspiration/ smoking of the test product NCIT and CC1. Among these constituents, the level of them, excluding nicotine, were below LOD for NCIT. A total of 1.1 mg of nicotine was detected in the aspirated air with 1920 machine aspirations (correspond to approximately 0.6 lg of nicotine per aspiration). The measurement of nicotine delivered by each of the 160 machine aspirations showed that the amount of nicotine delivered was almost constant by approximately 320–480 times of aspiration and decreased gradually to approximately half of the maximum at the last 160 of the 1920 aspirations (data not shown).
Subject group NCIT
CC1
NS
Number of subjects (n) Per protocol population (Enrolled)
40 (40)
20 (20)
18 (20)
Age (years) Mean ± SD Range
28.3 ± 6.12 21–43
28.6 ± 7.07 21–49
32.1 ± 4.55 22–39
Body mass index (kg/m2) Mean ± SD Range
20.7 ± 1.75 18.5–24.6
20.9 ± 1.33 19.0–24.1
21.6 ± 1.81 18.8–24.9
Duration of Smoking (year) Mean ± SD Range
8.20 ± 6.12 1–23
8.10 ± 7.17 1–29
–
Daily cigarette consumption (cig.) Mean ± SD Range
21.0 ± 2.24 20–30
21.1 ± 2.61 20–30
–
NCIT; non-combustion inhaler type of tobacco product. CC1; 1 mg ISO tar conventional cigarette, NS; non-smokers.
3.2. Study population A total of 97 smokers and 44 non-smokers who gave written informed consent were screened. Of these, 60 smokers were enrolled and randomly assigned to the NCIT and CC1 groups (40 and 20 subjects, respectively) and 20 non-smokers were assigned to the NS group. Two subjects in the NS group were discontinued prematurely according to the investigator’s judgment because a clinically significant incident or disease occurred on day 6 and 11, respectively. A total of 78 subjects completed the study. Demographic and some baseline characteristics are shown in Table 2 by subject group. With regard to age and BMI, no remarkable bias was observed among the subject groups, and smoking duration and daily cigarette consumption for smoking subjects were not remarkable bias as well between the NCIT and the CC1 groups,
3.3. Test tobacco product consumption The number of cigarettes smoked per day by the CC1 group and the daily number of aspirations for the NCIT group (total aspiration number recorded on the Puff Profiler and the self-recorded numbers in the questionnaire) are shown in Fig. 2.
Subjects in the CC1 group smoked approximately 21 cigarettes per day and were confirmed to keep their daily smoked number of CC1 within ±10% of their usual daily consumption throughout the study period. In the NCIT group, the mean of the total daily aspiration number ranged from 632 to 801 times throughout the study period. 3.4. Biomarkers Changes in BOE levels are shown in Table 3 and Fig. 3. The data of BOEs for 2 subjects in the NS group, who withdrew from the study prematurely, were removed from analysis. Moreover, because of failure of 24-h urine sampling for 1 subject in the NCIT group from day 7 to day 8, the urine data for 39 subjects were used for data analysis of day 8. In the baseline period (day 1 and 0), all smoking subjects (NCIT and CC1 groups) smoked their usual brands of 1 mg ISO tar cigarettes. At that time, the levels of all BOEs measured in this study were higher in the smoking subjects than in the non-smoking subjects (the NS group). On the other hand, no remarkable difference was observed between the NCIT and CC1 groups in the levels of all BOEs measured (Table 3, day 1).
Table 1 Yields of tar and cigarette smoke constituents that correspond to biomarkers of exposure measured in this study by machine aspiration/smoking. Cigarette smoke constituents
Tar Nicotine CO NNK Acrolein Crotonaldehyde 1,3-Butadiene Benzene HCN Benzo(a)pyrene 4-ABP
NCIT
mg mg mg ng lg lg lg lg lg ng ng
CC1
Total of 1920 aspirations
Per each aspirationa
Per cigarette
Per each puffb
n.d.c 1.1 n.d. (0.064) n.d. (4.93) n.d. (1.992) n.d. (3.138) n.d. (1.32) n.d. (0.263) n.d. (0.221) n.d. (0.03) n.d. (0.124)
n/a 0.0006 n/a n/a n/a n/a n/a n/a n/a n/a n/a
1d 0.1d 1.6 6.14 5.12 n.d. (0.718) 5.60 3.85 3.56 1.98 0.321
0.2 0.02 0.25 0.92 0.81 n/a 0.89 0.61 0.58 0.31 0.048
A machine smoking regimen was set as follows: 55 ml aspiration volume, 2-s duration, and 30-s interval for NCIT, and 35 ml aspiration volume, 2-s duration, and 60-s interval for CC1. Lower LOD is shown in parenthesis when a chemical constituent was not detected. NCIT: non-combustion inhaler type of tobacco product. CC1: 1 mg ISO tar conventional cigarette. NNK: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. HCN: Hydrogen cyanide. 4-ABP: 4-aminobiphenyl. n.d.: not detected. n/a: not applicable. a Values for ‘‘per aspiration’’ mean the value for the ‘‘A total of 1920 aspirations’’ divided by 1920. b Values for ‘‘per puff’’ mean the value for ‘‘per cigarette’’ divided by the puff count average for each measurement. c No increase was observed in the weight of a Cambridge filter pad used for trapping particulate matter. d Values of tar and nicotine printed on the package.
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NCIT: aspiration count (times/day)
CC1: cigarette smoked (n/day)
1000
30 (mean± SEM)
25
750
20
500
CC1 group NCIT group
15
250
0
10
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 study day
Fig. 2. Daily consumption of the test tobacco product. Plots show the mean number of cigarettes smoked by the CC1 group and aspiration number for the NCIT group. NCIT: non-combustion inhaler type of tobacco product. CC1: 1 mg ISO tar conventional cigarette.
In the CC1 and NS groups, levels of all BOEs, except the urine mutagenicity values, were not changed markedly throughout the study period. On the other hand, levels of all BOEs measured dropped to a lower value in the NCIT group after switching from their usual brand of 1 mg ISO tar conventional cigarette smoking to NCIT use. The urine mutagenic activities in all groups decreased on day 8 in comparison to the respective baseline levels. The levels of BOEs, except NE, cotinine, SCN, and total NNAL, in the NCIT group were almost same as those in the NS group on day 8, and these levels were maintained throughout the study period. The levels of total NNAL and SCN in the NCIT group declined gradually throughout the study period. On day 29, levels of all BOEs measured in this study were significantly (p < 0.05) lower in the NCIT group than in the CC1 group. The levels of BOEs, except cotinine, NE, and total NNAL in the NS and NCIT groups were almost the same. The levels of cotinine and NE in the NCIT group were almost constant from day 8 to day 29 and were slightly but definitely higher than those in the NS group on day 29. 3.5. Safety Nine adverse events developed in 8 of the 80 subjects (10.0%) enrolled in this study. Of these, colonic diverticulitis, which developed in 1 subject in the NS group, was rated serious, and other 8 events were rated non-serious by the investigator. Study participation for two subjects in the NS group, one had the adverse event rated as serious as described above and the other had enterocolitis rated non-serious and mild severity, was terminated prematurely according to the investigator’s judgment. For the causal relationship with the test product, 1 event, increased creatine phosphokinase, in CC1 group was classified into ‘‘probably related’’, 1 event, diarrhoea, in NCIT group was into ‘‘unlikely related’’ and other 7 events were into ‘‘not related’’ by the investigator. All adverse events recovered. 4. Discussion No smoke is generated when subjects use NCIT. The amount of chemical constituents, corresponding to BOEs measured in this
study, delivered from the product by machine aspiration was below LOD, except nicotine, in this analytical setting (Table 1). Accordingly, the levels of selected BOEs were markedly reduced after the subjects switched from their usual brand of 1 mg ISO tar conventional cigarette smoking to a new form of smokeless tobacco product in this clinical study setting. The mean number of daily cigarette consumption in the CC1 group during the entire study period was stable (Fig. 2), and this was believed to be the result of this study setting; all subjects in the CC1 group were allowed to smoke CC1 within ±10% of their usual daily consumption. Regarding NCIT use, the mean of the total daily aspiration number of NCIT was almost the same; the range was from 632 to 801, with an average of approximately 750 aspirations (Fig. 2). Some amount of inter- and intra-subject differences were observed among the study days. This may be due to the study setting for free use of NCIT except for 180 aspirations counted by the Puff Profiler. Furthermore, relatively lower values in the mean of the total daily aspiration number were observed almost weekly, and this was believed to be the result of the outings; all subjects were not allowed to use their assigned test tobacco product during outings. In the baseline period (day 1 and 0), the levels of all BOEs measured showed no remarkable difference between the CC1 and NCIT groups, and these levels were higher than those in the NS group (Table 3 and Fig. 3, day 1). The ratios of the each BOE level in the smoking subjects to that in the non-smoking subjects calculated from the baseline data (Table 3, day 1) were mostly consistent with the ratios reported (Scherer, 2005). In the NCIT group, levels of all BOEs measured dropped to a lower value after switching from their usual brand of cigarette to NCIT (Table 3 and Fig. 3). On day 29, levels of all BOE measured were significantly (p < 0.05) lower in the NCIT group than in the CC1 group and were almost the same as those in the NS group except cotinine, NE, and NNAL. These reductions in BOE levels indicate that the exposure to cigarette smoke constituents corresponding to each BOE were quite lower in the NCIT group than in the CC1 group, and suggested that the exposure levels in the NCIT group, except of nicotine and NNK, were similar to those in the NS group in this study setting. The levels of NE and cotinine in the NCIT group were higher than those in the NS group from day 8 to day 29 (Table 3). This
Table 3 Levels of biomarkers of exposure measured. Cigarette smoke constituents
Biomarker
Study day
Median (IQR)
Mean ± SD
Median (IQR)
Mean ± SD
Median (IQR)
Mean ± SD
Median (IQR)
Nicotine
Plasma cotinine (ng/ml) CC1 (n = 20) 200 ± 115 NCIT (n = 40) 173 ± 103 NS (n = 18) 0.25 ± 0.0
168 (103–274) 133 (105–268) 0.25 (0.25–0.25)
186 ± 98.6 24.9 ± 21.5 0.25 ± 0.0
172 (107–242) 18.4 (8.2–34) 0.25 (0.25–0.25)
204 ± 108 23.1 ± 23.5 0.25 ± 0.0
181 (133–262) 12.1 (4.8–34.1) 0.25 (0.25–0.25)
218 ± 102 26.2 ± 33.7 0.25 ± 0.0
227 (161–278) 10.2 (4.6–37.2)* 0.25 (0.25–0.25)
NEs (mg/24 h) CC1 NCIT NS
12.8 ± 7.87 12.0 ± 6.49 0.009 ± 0.008
11.9 (7.9–13.7) 10.5 (7.6–17.3) 0.007 (0.004–0.010)
11.9 ± 7.36 1.49 ± 1.36 0.007 ± 0.011
10.0 (7.5–13.3) 1.0 (0.53–2.0) 0.004 (0.003–0.006)
13.3 ± 8.52 1.64 ± 1.9 0.006 ± 0.008
11.3 (8.9–14.3) 0.83 (0.33–2.4) 0.004 (0.003–0.006)
12.5 ± 8.18 1.51 ± 1.81 0.007 ± 0.012
10.4 (7.8–13.2) 0.68 (0.26–2.1)* 0.004 (0.003–0.005)
COHb (%)à CC1 NCIT NS
5.61 ± 2.61 4.75 ± 1.66 2.12 ± 0.95
5.06 (4.15–6.74) 4.93 (3.72–5.77) 2.39 (1.43–2.84)
5.50 ± 1.87 2.11 ± 0.72 2.25 ± 0.73
5.06 (4.12–6.83) 2.14 (1.52–2.60) 2.16 (1.67–2.80)
5.61 ± 1.79 2.20 ± 0.69 1.81 ± 0.47
5.58 (4.38–6.28) 2.32 (1.80–2.72) 1.69 (1.58–2.03)
5.54 ± 1.67 2.04 ± 0.73 1.74 ± 0.61
5.16 (4.45–6.72) 1.94 (1.55–2.51)* 1.72 (1.24–2.11)
Total NNAL (ng/24 h) CC1 187 ± 100 NCIT 157 ± 63.5 NS 3.40 ± 3.62
178 (119–219) 155 (118–199) 2.32 (1.32–3.58)
170 ± 90.9 60.5 ± 25.5 2.39 ± 3.03
155 (100–212) 60.8 (43.6–79.2) 1.60 (0.63–2.66)
161 ± 77.3 41.0 ± 21.1 1.68 ± 1.48
160 (93.3–218) 38.6 (24.8–53.0) 1.43 (0.54–2.22)
151 ± 78.4 24.2 ± 19.8 0.99 ± 0.85
142 (80.8–204) 19.4 (13.4–24.3)* 0.56 (0.48–1.17)
3-HPMA (mg/24 h) CC1 3.97 ± 2.45 NCIT 3.35 ± 1.62 NS 0.95 ± 0.22
3.00 (2.36–4.62) 2.92 (2.22–4.24) 0.93 (0.82–1.12)
3.23 ± 1.23 0.81 ± 0.30 0.96 ± 0.30
3.08 (2.26–3.67) 0.81 (0.62–0.94) 0.93 (0.80–1.03)
3.64 ± 2.48 0.79 ± 0.38 0.80 ± 0.31
2.89 (2.25–3.50) 0.72 (0.48–0.97) 0.76 (0.62–1.06)
2.85 ± 2.19 0.67 ± 0.29 0.66 ± 0.20
2.45 (1.86–2.75) 0.66 (0.43–0.82)* 0.69 (0.48–0.82)
HMPMA (lg/24 h) CC1 1616 ± 793 NCIT 1503 ± 684 NS 257 ± 58.1
1394 (1061–2200) 1504 (936–2002) 246 (215–308)
1622 ± 792 244 ± 79.4 242 ± 63.0
1388 (1094–1791) 243 (188–285) 238 (216–292)
1630 ± 873 213 ± 65.8 226 ± 60.5
1509 (1138–1832) 211 (158–258) 229 (184–250)
1575 ± 850 191 ± 81.7 198 ± 75.0
1212 (934–2115) 185 (128–228)* 178 (144–232)
MHBMA (lg/24 h) CC1 7.15 ± 8.41 NCIT 9.16 ± 10.6 NS 0.85 ± 1.11
3.85 (1.63–8.57) 4.57 (1.60–12.5) 0.45 (0.31–0.76)
4.58 ± 5.39 0.32 ± 0.21 0.35 ± 0.18
2.66 (1.38–5.09) 0.25 (0.22–0.33) 0.31 (0.22–0.43)
6.77 ± 7.04 0.70 ± 0.87 0.46 ± 0.23
3.88 (1.32–9.62) 0.42 (0.24–0.68) 0.38 (0.33–0.51)
5.73 ± 6.55 0.44 ± 0.64 0.84 ± 1.34
3.22 (1.89–6.19) 0.26 (0.18–0.38)* 0.26 (0.20–0.50)
DHBMA (lg/24 h) CC1 496 ± 144 NCIT 458 ± 128 NS 346 ± 87.8 Mean ± SD
474 (393–561) 441 (357–536) 344 (287–405) Median (IQR)
451 ± 107 281 ± 82.4 358 ± 94.2 Mean ± SD
442 (376–501) 288 (217–326) 342 (280–441) Median (IQR)
486 ± 140 305 ± 65.6 329 ± 63.6 Mean ± SD
459 (407–523) 305 (257–338) 340 (280–357) Median (IQR)
403 ± 177 288 ± 85.7 304 ± 89.5 Mean ± SD
382 (322–459) 290 (216–349)* 290 (240–367) Median (IQR)
TMA (lg/24 h) CC1 NCIT NS
155 ± 68.8 123 ± 54.0 49.0 ± 25.1
138 (100–191) 109 (89.8–160) 43.2 (30.1–59.6)
130 ± 46.7 39.5 ± 15.3 53.9 ± 24.0
129 (94.1–163) 36.9 (28.6–51.8) 48.9 (35–68.8)
147 ± 54.0 47.1 ± 18.8 49.9 ± 20.2
143 (113–165) 44.8 (36.6–54.5) 45.9 (34.5–64.6)
161 ± 82.3 72.6 ± 49.9 77.4 ± 38.9
156 (117–200) 54.1 (38.3–92.3)* 66.7 (52–99.7)
S-PMA (ng/24 h) CC1 NCIT NS
420 ± 276 445 ± 238 156 ± 90.7
383 (238–514) 430 (245–614) 140 (101–181)
496 ± 273 156 ± 104 190 ± 98.8
427 (292–620) 117 (93.7–183) 162 (118–263)
501 ± 286 180 ± 113 238 ± 165
526 (274–607) 133 (82.7–272) 197 (128–288)
491 ± 312 203 ± 118 231 ± 161
432 (351–543) 194 (111–264)* 160 (117–255)
1-OHP (ng/24 h) CC1 NCIT NS
346 ± 178 321 ± 170 129 ± 63.9
349 (247–412) 265 (226–407) 108 (81.8–176)
340 ± 134 130 ± 61.2 123 ± 53.2
318 (234–439) 119 (82.4–173) 113 (86.5–152)
309 ± 97.5 133 ± 75.1 122 ± 58.0
291 (259–372) 128 (77.9–153) 101 (73.8–167)
264 ± 119 61.2 ± 32.5 70.1 ± 23.4
230 (194–309) 55.5 (41.4–73.5)* 71.3 (61.2–79.9)
Day 1 Mean ± SD
Nicotine
NNK
Acrolein
Crotonaldehyde
1,3-Butadiene
1,3-Butadiene
Benzene
Benzene
Pyrene
Day 15
Day 29
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CO
Day 8
13,522 4449 4040 15,112 4406 2933
69.1 (55.2–86.2) 37.8 (32.3–43.7) 28.0 (26.8–29.6)
Mutagenicity (revertants/24 h) CC1 34,459 NCIT 24,292 NS 10,479 Mutagen
75.4 ± 34.1 68.9 ± 25.4 29.7 ± 6.52 SCN (lmol/l) CC1 NCIT NS HCN
22,522 21,574 8054
71.1 (52.5–92.0) 67.3 (51.9–84.4) 28.5 (24.7–32.1)
16,142 5531 5752
70.7 ± 27.6 46.4 ± 14.7 26.0 ± 8.67
14,939 5005 3668
63.7 (52.8–82.6) 44.4 (36.0–55.5) 27.6 (23.9–29.3)
19,134 6823 4768
73.7 ± 28.1 39.3 ± 9.24 28.5 ± 7.19
9.65 (5.25–13.1) 1.03 (0.82–1.36) 1.02 (0.86–1.26) 9.21 ± 4.81 1.22 ± 0.65 1.11 ± 0.45 8.37 (4.40–11.8) 1.10 (0.92–1.26) 1.17 (1.02–1.28) 8.89 ± 6.17 1.15 ± 0.52 1.19 ± 0.48 8.28 (6.63–12.8) 8.96 (5.66–11.6) 1.13 (0.80–1.41) 11.0 ± 7.37 9.30 ± 5.29 1.43 ± 1.39 4-ABP (ng/24 h) CC1 NCIT NS 4-ABP
15,679 5519 6830
74.6 ± 28.5 31.2 ± 7.01 26.7 ± 6.35
7.80 (3.89–10.8) 0.89 (0.73–1.13)* 0.98 (0.75–1.13) 7.74 ± 4.49 1.02 ± 0.54 1.02 ± 0.49
Median (IQR) Mean ± SD
Day 29
Median (IQR) Mean ± SD
Day 15
Mean ± SD
Median (IQR) Day 8
Mean ± SD
Study day
Median (IQR) Day 1
Biomarker Cigarette smoke constituents
Table 3 (continued)
In the baseline period (day 1 and 0), all subjects in the CC1 and NCIT groups smoked their usual brand of 1 mg ISO tar cigarette, and the values of day 1 reflected the baseline values. NCIT: non-combustion inhaler type of tobacco product. CC1: 1 mg ISO tar conventional cigarette. NS: non-smoker. NEs: nicotine equivalents. COHb: carboxyhemoglobin. NNK: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Total NNAL: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and glucuronide. 3-HPMA: 3-hydroxypropylmercapturic acid. HMPMA: 3-hydroxy-1-methylpropyl-mercapturic acid. MHBMA: monohydroxybutenylmercapturic acid. DHBMA: dihydroxybutenylmercapturic acid. TMA: trans, trans-muconic acid. S-PMA: S-phenylmercapturic acid. 1-OHP: 1-hydroxypyrene. 4-ABP: 4-aminobiphenyl. HCN: hydrogen cyanide. SCN: thiocyanate. IQR: interquartile range. * Significant difference in BOE levels in the NCIT group vs. the CC1 group on day 29 (day 28 for COHb). p < 0.05. à Blood samplings for COHb measurement were conducted between 16 to 17 o’clock on day 0, 7, 14, and 28. Data of BOE in urine for day 8 were for 39 subjects because of failure of 24-h urine collection for 1 subject.
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69.1 (53.5–89.3) 29.5 (27.3–35.1)* 26.2 (25.0–27.9)
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result showed frequent aspiration of nicotine form NCIT by the subjects during the study period. The levels of total NNAL and SCN in the NCIT group declined gradually throughout the study period. The elimination half-lives of these two BOEs are relatively longer than those of the other BOEs measured in this study. The half-lives of total NNAL were reported as 1–4 and 10–45 days for initial (t1/2a) and terminal (t1/2b) phases, respectively (Goniewicz et al., 2009; Hecht et al., 1999). The total NNAL level in urine for the NCIT group was higher than that for the NS group on day 29, therefore it remains unclear whether the exposure level to NNK in the NCIT group would be almost the same as that in the NS group. However, the continuous decrease in the total NNAL level indicates that the exposure to NNK in the NCIT group would be substantially lower than that in the CC1 group. Because the reported elimination half-life of SCN was 6.4 days (Junge, 1985), almost the same SCN level observed in the NCIT and NS groups on day 29 suggested that exposure of hydrogen cyanide (HCN) in the NCIT group was almost the same as in the NS group. In the CC1 and NS groups, levels of all BOEs were relatively constant throughout the entire study period, except the urine mutagenicity values. Although the subjects in the CC1 group switched from their usual brand of 1 mg ISO tar cigarette to the assigned CC1 on day 1, the constant BOE levels in the CC1 group suggested that the levels were not considerably affected in this study if the smoking topography of the smoker changed by switching the cigarette brand. The urine mutagenicity values showed a decrease on day 8 compared with each baseline value in the CC1, NS, and NCIT groups. Because the half-life of the smoking-related urine mutagenic activity was reported to be approximately 7–23 h (Kado et al., 1985), the effect of smoking the usual brand of cigarette on the urine mutagenic activity is considered to be disappeared on day 8. This result showed that the use of NCIT decreased exposure to mutagens detected in the urine mutagenicity test, as much the exposure level of the NS group. It has been reported that the urine mutagenic activity increased with an intake of fried or boiled meal (Doolittle et al., 1989). In this study, the clinical institutions served daily meal excluding such heat-processed food for 2 days prior to the day of 24-h urine sampling completion, day 8, 15 and 29. The decrease in the urine mutagenic activity in the NS group on day 8, by approximately half of the activity of day 1, would be mainly due to this diet. Considering the constant levels of other BOEs in the CC1 group, the decrease in the urine mutagenic activity in the CC1 group would be due to the change in this diet as well. In summary, under the conditions of the present study, switching the tobacco product used from a conventional cigarette to NCIT, the new form of smokeless tobacco product, showed significant reduction in all BOEs measured in this study. Environmental factors such as daily food might have affected BOE levels in the NS group. On day 29, the exposure levels to 8 tobacco smoke constituents and mutagens detected by the urine mutagenicity test in the NCIT group were believed to be similar to those in the NS group, while the exposure levels to nicotine would be higher than those in the NS group. Although in this study, it is unclear whether the exposure level to NNK in the NCIT group is similar to that in the NS group because of the relatively long half-life of the corresponding BOE, it would be substantially lower than that in the CC1 group.
Conflict of interest This work was funded by Japan Tobacco Inc., and all authors are employees of the company.
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4 2
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Fig. 3. Biomarker of exposure (BOE) measurement results for all study groups. Data are shown as median. Blood samplings for COHb measurement were performed between 16 and 17 o’clock on day 0, 7, 14, and 28. Data of the NCIT group’s BOE in urine for day 8 were for 39 subjects because of failure of 24-h urine collection for 1 subject. In the baseline period (day 1 and 0), all subjects in the CC1 and NCIT groups smoked their usual brand of 1 mg ISO tar cigarette, and the values of day 1 reflected the baseline values. NCIT: non-combustion inhaler type of tobacco product. CC1: 1 mg ISO tar conventional cigarette. NS: non-smokers. COHb: carboxyhemoglobin. Total NNAL: 4(methylnitrosamino)-1-(3-pyridyl)-1-butanol and glucuronide. 3-HPMA: 3-hydroxypropylmercapturic acid. HMPMA: 3-hydroxy-1-methylpropyl-mercapturic acid. MHBMA: monohydroxybutenylmercapturic acid. DHBMA: dihydroxybutenylmercapturic acid. TMA: trans, trans-muconic acid. S-PMA: S-phenylmercapturic acid. 1-OHP: 1hydroxypyrene. 4-ABP: 4-aminobiphenyl. SCN: thiocyanate.
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Acknowledgments We would like to thank Drs. Kunio Iwata and Yasufumi Nagata for their invaluable input. References Buchhalter, A.R., Eissenberg, T., 2000. Preliminary evaluation of a novel smoking system: effects on subjective and physiological measures and on smoking behavior. Nicotine Tob. Res. 2, 39–43. Doolittle, D.J., Rahn, C.A., Burger, G.T., Lee, C.K., Reed, B., Riccio, E., Howard, G., Passananti, G.T., Vesell, E.S., Hayes, A.W., 1989. Effect of cooking methods on the mutagenicity of food and on urinary mutagenicity of human consumers. Food Chem. Toxicol. 27, 657–666. Goniewicz, M.L., Havel, C.M., Peng, M.W., Jacob III, P., Dempsey, D., Yu, L., ZielinskaDanch, W., Koszowski, B., Czogala, J., Sobczak, A., Benowitz, N.L., 2009. Elimination kinetics of the tobacco-specific biomarker and lung carcinogen 4(methylnitrosamino)-1-(3-pyridyl)-1-butanol. Cancer Epidemiol. Biomarkers Prev. 18, 3421–3425. Health Canada, 1999. Determination of ’’Tar’’, Nicotine and Carbon Monoxide in Mainstream Tobacco Smoke-Official Method. Health Canada, Ottawa (SOR2000-272). Hecht, S.S., Carmella, S.G., Chen, M., Dor. Koch, J.F., Miller, A.T., Murphy, S.E., Jensen, J.A., Zimmerman, C.L., Hatsukami, D.K., 1999. Quantitation of urinary metabolites of a tobacco-specific lung carcinogen after smoking cessation. Cancer Res. 59, 590–596. International Agency for Research on Cancer, 2004. Tobacco smoke and involuntary smoking. IARC Monogr. Eval. Carcinog. Risks Hum. 83, 1–1438. International Agency for Research on Cancer, 2007. Smokeless tobacco and some tobacco-specific N-nitrosamines. IARC Monogr. Eval. Carcinog. Risks Hum. 89, 1–592. International Organization for Standardization, 2000. Routine analytical cigarettesmoking machine, ISO3308. Institute of Medicine, 2012. Scientific Standards for Studies on Modified Risk Tobacco Products. The National Academy Press, Washington DC.
Junge, B., 1985. Changes in serum thiocyanate concentration on stopping smoking. Br. Med. J. (Clin. Res. Ed.) 291, 22. Kado, N.Y., Langley, D., Eisenstadt, E., 1983. A simple modification of the Salmonella liquid-incubation assay. Increased sensitivity for detecting mutagens in human urine. Mutat. Res. 121, 25–32. Kado, N.Y., Manson, C., Eisenstadt, E., Hsieh, D.P., 1985. The kinetics of mutagen excretion in the urine of cigarette smokers. Mutat. Res. 157, 227–233. Miura, N., Yuki, D., Minami, N., Kakehi, A., Onozawa, M., 2013. Pharmacokinetic analysis of nicotine when using non-combustion inhaler type of tobacco product in Japanese adult male smokers. Regul. Toxicol. Pharmacol. 67, 198– 205. Okuzono, H., Tamagawa, S., Hattori, S., 1976. A spectrophotometric method for the determination of carboxyhemoglobin in blood, and some clinical reports. Rinsho Byori. 24, 616–621. Pérez-Stable, E.J., Marín, G., Marín, B.V., Benowitz, N.L., 1992. Misclassification of smoking status by self-reported cigarette consumption. Am. Rev. Respir. Dis. 145, 53–57. Rainey, C.L., Conder, P.A., Goodpaster, J.V., 2011. Chemical characterization of dissolvable tobacco products promoted to reduce harm. J. Agric. Food Chem. 59, 2745–2751. Sakaguchi, C., Kakehi, A., Minami, N., Kikuchi, A., Futamura, Y., 2014. Exposure evaluation of adult male Japanese smokers switched to a heated cigarette in a controlled clinical setting. Regul. Toxicol. Pharmacol. 69, 338–347. Scherer, G., 2005. Biomonitoring of inhaled complex mixtures-ambient air, diesel exhaust and cigarette smoke. Exp. Toxicol. Pathol. 57 (Suppl. 1), 75–110. Scherer, G., 2006. Carboxyhemoglobin and thiocyanate as biomarkers of Exposure to carbon monoxide and hydrogen cyanide in tobacco smoke. Exp. Toxicol. Pathol. 58, 101–124. Schorp, M.K., Tricker, A.R., Dempsey, R., 2012. Reduced exposure evaluation of an electrically heated cigarette smoking system. Part 1: non-clinical and clinical insights. Regul. Toxicol. Pharmacol. 64 (2 Suppl.), S1–10. Vesey, C.J., McAllister, H., Langford, R.M., 1999. A safer method for the measurement of plasma thiocyanate. J. Anal. Toxicol. 23, 134–136. Yamasaki, E., Ames, B.N., 1977. Concentration of mutagens from urine by absorption with the nonpolar resin XAD-2: cigarette smokers have mutagenic urine. Proc. Natl. Acad. Sci. U.S.A. 74, 3555–3559.