Impact of Factors Related to Loose and Pouched Moist Smokeless Tobacco Product Use Behavior on Biomarkers of Exposure to Nicotine and N-Nitrosonornicotine

Although moist smokeless tobacco (MST) use is not as prevalent as cigarette smoking, approximately 5.2 million adults in the United States (2.1%) reported they were currently using MST in a 2021 survey (1). The two primary forms of MST found in the United States (U.S.) market are loose and pouched; like loose MST, pouched MST is made from ground or cut tobacco. These are different to the rapidly emerging, newer oral tobacco-derived nicotine pouches that do not contain any cut, ground, or leaf tobacco, accounting for the low to non-quantifiable levels of tobacco-specific nitrosamines (TSNAs) and other harmful and potentially harmful components (HPHCs) and resulting exposure (2, 3). Users of loose MST self-select a pinch of the product and place it between the cheek and gum, whereas users of pouched MST place pre-portioned MST, encased in pouch material, between the cheek and gum.

Earlier studies (summarized in Supplemental Table 1) indicated that the average pinch mass among adults who use loose MST products was approximately 2 g used for ~ 45–60 min. Hatsukami et al. surveyed a local population of young U.S. college males using Copenhagen^® MST (N = 56) and observed an average pinch mass of 1.97 g (range 0.62–5.91 g) that was kept in the mouth for approximately 39.9 min (4). A second study of MST use in undergraduate males (N = 30) by Hatsukami et al. found that the average dry weight of tobacco after use was 0.75 g per dip (range 0.13–3.12 g) and was kept in the mouth for an average of 39.6 min (range 19.1–106 min) (5). A small 1992 study by Baldini et al. (N = 12) reported use patterns in both “light” and “heavy” users, and found that “light” users were using an average of 1.8 cans per week and 6 dips per day, which at 34 g per can of MST can be extrapolated to approximately 1.5 g per pinch (assuming that they were daily users) (6). Lemmonds et al. reported that a population of Upper Midwestern males (N = 54) were using an average of 3.4 cans per week and 6.8 dips per day for 71.2 min, extrapolating to an average pinch mass of 2.4 g (7). Severson et al. described that a population of U.S. males (N = 94) used on average a 1.2 g pinch for 59 min (8). Gritz et al. found that a small population of undergraduate males (N = 12) used an average of 10.8 g/day for 24.2 min per pinch (9). Thomas et al. reported an average time in the mouth of 82.9 min in a population of predominantly male users (N = 68) (10).

Most recently, Nighbor et al. investigated the use topography and nicotine pharmacokinetics in loose and pouched MST products and reported an average use of a single pinch of loose MST as 2.99 g used for 53.2 min, and the average amount for portioned (pouched) MST use as 1.52 g for 45.5 min (11). The wide-ranging reported use patterns may be driven by differences in sample sizes, study specific populations, different tobacco use criteria and length of use, or study designs. Moreover, very little information exists on pouched MST users, therefore additional systematic characterization is needed to better understand the use topography of MST.

Understanding MST use topography is essential as it can directly impact exposure to harmful and potentially harmful constituents (HPHCs). Two key HPHCs in MST are nicotine and tobacco-specific nitrosamines (e.g., N-nitrosonornicotine (NNN)). Both nicotine and NNN are naturally occurring constituents found in tobacco. Nicotine, while addictive and not benign is not directly responsible for tobacco-caused cancer, lung disease, and heart diseases (12). NNN is classified as a human carcinogen by the International Agency for Research on Cancer (IARC) (13, 14). Exposure to both constituents can be measured non-invasively with biomarkers in urine.

The use behavior of MST products, such as how much (pinch mass/number of pouches), how often (number of use occasions) and how long (duration of use), have not been systematically characterized for a full day of use. Moreover, the relationship between patterns of use and the resulting exposure to HPHCs, like nicotine and NNN, have not been reported in detail. In the current study, we characterized the MST use topography and daily exposure to nicotine and NNN over an entire day. In addition, we examined the relationship between the measured use parameters and the resulting exposure to gain insights regarding the role of different product use behavior parameters on biomarkers of HPHCs linked to potential disease outcomes (15).

A total of 230 eligible people were enrolled; one participant was excluded from this analysis due to use of both pouch and loose MST on Day -1. The demographic characteristics are shown in Table 1.

Overall, the mean age was 37.4 years, and the mean BMI was 29.3 kg/m². The majority of participants were male (93.5%), White (85.2%) or Black or African American (10.9%); only 2.6% were Hispanic or Latino. Only one participant reported using MST some days/occasionally, all others reported daily MST use.

We report the most current and systematic characterization of use behavior in a relatively large sample of 229 participants using their own brand loose or pouched MST products. The observations were carefully documented by clinic staff from ad libitum use over the study day period of 16 h. Our findings indicate that for loose MST products, the average pinch mass is larger than previously reported for loose MST (~ 4 g vs ~ 2–3 g), and these products are kept in use for a longer duration (~ 74 min) and are used for ~ 6 occasions per day. We also observe that, on average, adults who use pouched MST products use ~ 2 pouches per use occasion, which lasts for ~ 70 min. The exposure to nicotine (NE) resulting from this use behavior pattern indicates that NE levels were significantly lower among pouched MST users than loose MST users, but NNN levels were not significantly different. Our analyses indicate that ~ 50% and ~ 33% of the variability in daily exposure to nicotine and NNN, respectively, can be explained by use behavior, with the number of use occasions exhibiting the highest impact. The findings reported from our analysis are unique and add to the current body of literature on how MST products are used. Most studies report consumption of cans per day or cans per week and rely on self-reported observations. Additionally, there is limited evidence regarding ad libitum use behavior over an entire day, especially for pouched products. Earlier studies have suggested that the average pinch mass among adults who use commercial MST products is approximately 2 g and used for ~ 40 min (4, 5, 8). In a recent study, Nighbor et al. (9) observed an inclinic measurement of pinch mass as 2.99 g among a relatively small sample size of 29 participants using loose MST products for 53 min and pouch users using 1.5 pouches at a time for 45 min (11). Of note was an outlier participant who used 74.7 g over the course of 8 h. Additionally, in the Nighbor et al. study, the participants took a pinch of loose or pouch for the first hour, and blood samples were collected to measure plasma nicotine for the entire hour, followed by ad libitum use for an additional seven hours with blood samples collected every hour; the interventions for blood sampling may have disrupted true ad libitum use. In comparison, in our study, the participants were using their own brand loose or pouch products under ad libitum use settings for a period of 16 h, thus reflecting a full day of product use. Overall, our study suggests that MST product use behavior (amount and duration), when systematically characterized, is higher than typically reported in published literature. Our observations generally align with those reported by Nighbor et al., where they report a higher pinch mass and longer duration of use than earlier publications. Total number of pouches may be comparable as well; participants in this study used on average a total of 13.3 (SD = 7.38) pouches over 16 h, whereas Nighbor et al. reported participants using an average total of 7.4 (SD = 3.2) pouches over 8 h (11).

Our study provides insights regarding the relationship between BOEs to nicotine and NNN and actual use behaviors. Direct comparison of BOE levels observed in our study with those reported in the literature should take into consideration differences in sampling methods (e.g., 24-h urine vs spot urine) or units of measurements (e.g., mg/24 h, mg/g creatinine, μmol/g creatinine) or differences in descriptive statistics (geometric means vs arithmetic means) for the BOE. Although we utilized analytical methods generally consistent with methods described in the literature, minor differences in the bioanalytical methods may further complicate head-to-head comparisons. Cheng et al. reported geometric mean values for NNN in spot urine collections among exclusive daily smokeless tobacco users at a population level based on spot urine collection from the PATH Wave 1 dataset as 33.9 ng/g creatinine (95% CI: 29.6–38.8) (20). In our study population, the geometric mean levels for NNN, pooled across loose and pouched MST products, were 25.0 ng/g creatinine (95% CI: 27.69–30.68). Our observations, while trending lower than those reported by Cheng et al. (CI: 29.6–38.8), have comparable and overlapping 95% confidence intervals (20). Moreover, the differences could be attributed to differences in study populations and study design. In our study, we report exposure to NNN from use of a subset of smokeless tobacco products, i.e., own-brand loose and pouched MST products, from in-clinic measurements, whereas Cheng et al. report observations based on a cross-sectional assessment of a larger set of smokeless tobacco products (20). However, the arithmetic mean values for NNN (78.0 ± 91.71 mg/24 h) were higher in our study compared to those reported by Prasad et al. (48.9 ± 34.7 mg/24 h) in 40 MST users (21). The differences in NNN levels may be attributed to the reasons described above. A potential confounder could be the large interindividual variability observed with urinary measurements of NNN. In some cases, NNN excretion in urine can be influenced by NNN formation from nitrosation of nornicotine under acidic conditions endogenously and exogenously; hence, this additional variability in urinary NNN levels may complicate product-specific exposure assessments (22, 23).

It is noteworthy that the overall mean NE values observed in our study (24.7 ± 16.84 mg/24 h) were similar to that reported by Prasad et al. (29.4 ± 20.9 mg/24 h) from a cross-sectional sample of MST users (N = 40) (21). On the other hand, our NE values were lower than those reported by Cheng et al., however, their estimates from PATH were based on TNE-2 (urinary cotinine and trans-3′-hydroxycotinine), whereas we measured total NE (nicotine + five metabolites) based on mg/24-h urine collection. Therefore, we calculated the TNE-2 levels for our sample by utilizing only the urinary cotinine and trans-3′-hydroxycotinine in μmol/g creatinine units. The geometric mean TNE-2 values ranged from 41.6 μmol/g for loose MST and 35.7 μmol/g for pouched MST. These levels were lower than those reported by Cheng et al., 68.6 μmol/g (CI: 60.2–78.2) among exclusive daily ST users. The reasons for these differences may be attributed to the factors described earlier as well as the fact that the PATH analysis was based on spot urine collections from users of ST products (at the category level) whereas our measurements were based on 24-h urine collection among exclusive MST users. Additionally likely differences in duration of product use may also account for these differences.

Our analyses of the relationships between product use behavior and BOEs indicate that overall, the individual components of product use behavior (Table 2), while statistically significant, were weak (r² < 0.3) to moderate (r² > 0.31). Of the different parameters related to product use behavior, total duration of use over the study period was a better predictor of BOEs (Figures 1 and 2). However, our multivariate analysis demonstrates that all three parameters related to use behavior – average duration in mouth, average weight/number of pouches, as well as total number of use occasions, were all statistically significant (p < 0.001) factors in the model for both NE and NNN regardless of pouch or loose product use. Given that the statistical model explains ~ 33–50% of the variability in BOE, this suggests that these parameters are some of the primary factors impacting exposure to nicotine and NNN. Taken together, these three parameters provide a composite measure of the overall use behavior measure. Examination of the “dose-response” relationship by Rostron et al. indicated that the BOEs to NNK (based on concentrations of NNAL, a metabolite of NNK) for chewing tobacco and snuff users consistently increased by the frequency of use in terms of the number of days that they had used the product in the past 5 (24). These observations support our findings that exposure to nicotine and NNN is associated with product use behavior, of which the number of use occasions appears to have a relatively higher contribution than other parameters.

The strengths and limitations of this study must be considered when interpreting the results. To the best of our knowledge, this is the first study with such a large sample of own brand loose and pouched MST product users, as well as the systematic characterization of each product use behavior over a period of 16 h. We also examined the relationship between product use to NE and NNN based on 24-h urinary excretion. One limitation could be that the own brand MST use behavior may not be representative of the general population of MST users. However, based on their own brands used by the participants in the study (Supplemental Table 2), we believe that the brands of MST reasonably represent the most popular brands used in the U.S. Our secondary analysis (unpublished data) of the PATH dataset between 2015–2021 indicates a typical MST use pattern of about 0.6 cans/day of loose MST, whereas in our study the participants used on average ~ 0.8 cans/day (ranging from 0.1–4 cans/day). Comparison of our observations with published literature values for MST consumption is challenging because we report actual measurements whereas daily product use in published literature is often estimated from self-reported cans/week. Additionally, although the study participants were using the MST products under ad libitum conditions in an in-clinic setting to ensure precise measurements of product use, this may be considered a limitation since participants may exhibit different consumption patterns under a real-world, naturalistic setting. Another limitation could be that the 24-h NE and NNN could potentially include carry-over from previous day product use. However, the in-clinic measurement of the 24-h urinary excretion reflects a direct measurement of the systematically characterized product use and minimizes potential confounding from possible use of other tobacco product use. In addition, the use of the participants' own brand MST product allows for the most natural use occasion; however, each product may have different levels of nicotine and NNN, and this may confound the relationship of product used to exposure to these constituents.

Nighbor et al. (11) investigated the relationship between total and free nicotine and nicotine pharmacokinetic (PK) parameters and found weak correlations for both loose and portioned MST; the correlations between total nicotine and PK parameters were negative for loose MST. Furthermore, the chemical characterization of the loose and portioned products in their study revealed a very wide range of values for total and free nicotine where product pH likely plays an important role. We did not retain samples of the products used in our study, so our data are limited by the lack of information on the specific levels of pH, total nicotine, and free nicotine. Finally, typical MST use behavior is accompanied by expectoration of the saliva while the product is being used. We did not measure expectoration (whether it occurred or how much was expectorated during use), which could provide an additional variable of use behavior that may further inform the relationship between BOEs and use behavior.

In conclusion, under the conditions of this study, loose and pouched MST use appears to be higher than reported in the literature. Importantly, and not surprisingly, as much as ~ 33–50% of the variability in daily exposure to nicotine and NNN can be explained by use behavior. Although scientific evidence, including epidemiological studies, clearly indicates that smokeless tobacco products available in the U.S. are less risky than cigarettes, they are not risk-free (26). Understanding consumption patterns and their relationship with BOEs can provide valuable insights for developing suitable alternatives.