1. Introduction
It is generally acknowledged that use of tobacco products is associated with risks. In an effort to combat these risks, tobacco science and production have refocused their efforts to provide consumers with products that may limit their potential exposure to harmful or potentially harmful constituents (HPHCs). In 2009, the Family Smoking Prevention and Tobacco Control Act (‘Tobacco Control Act’) was passed in which control over regulatory oversight was given to the US Food and Drug Administration (FDA) [
1,
2]. Included in this act were specific requirements for the language to be included on warning labels for various tobacco products and the need for scientific rigor when making claims for any modified risk profile a product may offer [
3]. To be able to claim a modified risk profile, manufacturers must submit scientific evidence to support the claim as part of a Modified-Risk Tobacco Product (MRTP) application, and FDA permission must be received. The Tobacco Control Act further required the FDA to establish a list of harmful or potentially harmful constituents to human health found in mainstream smoke and tobacco products (referred to as the ‘HPHC list’) [
4].
Reducing the consumer’s exposure to compounds on the HPHC list is one way that risk can conceivably be lowered. Given that combustion is the main source for many of the HPHC compounds, alternative means of delivering nicotine are being promoted and developed. Examples of such products are electronic nicotine delivery systems (ENDS) and heated tobacco products (HTP), which both produce aerosolized nicotine for inhalation. An alternative to smoking altogether is the use of traditional smokeless tobacco products (STPs), such as chewing tobacco and snuff. STPs have seen a 23.1% increase in total usage over the same period that cigarette consumption has declined by 38.7%, though whether the two are linked is unclear given that there is a perception among some US smokers that STPs do not provide any reduction in toxicant exposure [
5,
6,
7]. Indeed, STPs are known to contain a number of HPHCs categorized by the IARC (International Agency for Research on Cancer) as being Group 1 carcinogens (e.g., formaldehyde,
N-Nitrosonornicotine (NNN), 4-(
N-Methylnitroamino)-1-(3-pyridyl)-1-butanone (NNK), and cadmium) [
8,
9,
10,
11]. The production techniques for STPs vary considerably and can include curing, fermentation, and pasteurization processes that will influence the HPHC profile of the resulting product, creating a broad spectrum of potential risk among this product class. For instance, typical US moist snuff (including snus) uses a fermentation process that leads to high levels of tobacco-specific nitrosamines (TSNAs) whereas Swedish-style moist snuff is heat-treated (pasteurized) and contains lower TSNA levels [
12]. Of the few tobacco products that have been granted MRTP designation by the FDA, eight are Swedish-style snus products manufactured by Swedish Match USA, Inc., which can state they present a reduced risk for certain cancers and diseases when compared with cigarettes [
13,
14]. Similar to cigarettes, there has been an emergence of alternative products that replicate the STP usage experience with a lowered HPHC exposure risk, namely modern oral nicotine products.
Modern oral nicotine products (MONPs), also known as tobacco-free nicotine products (TFNPs), are a novel class of nicotine-containing products aimed at further reducing toxicant exposure while still delivering the desired nicotine dosage. These products are intended to be consumed in a similar way as STPs, with placement between the gum and the lip/cheek. MONPs are produced in two main formulations—white granular powders (WGP) and plant-based versions. The white granular powder MONPs are pre-portioned and composed of a number of ingredients that include a stabilized form of nicotine (e.g., nicotine salt, nicotine-polacrilex), pH-adjusting agents (e.g., sodium carbonates), filler materials (e.g., modified cellulose, microcrystalline cellulose), sweeteners, and flavorings. Plant-based MONPs more closely mimic traditional STPs in that they are moist products produced using many of the same techniques and are packaged as either long-cut (loose) or pre-portioned pouches. Plant-based MONPs differ from STPs in that they are made from non-tobacco plant-based materials with pharmaceutical-grade nicotine added during the production process. Both forms of MONPs are typically sold in a variety of flavors with some also having the option of multiple nicotine strengths, granting the consumer a variety of options to choose from. At the time of publication, no MRTP applications for any MONPs have been made public on the FDA website (as is required by the Tobacco Control Act).
With the requirements set forth by the FDA pertaining to the classification of modified-risk products, the challenges of accurately assessing potential HPHC exposure comes to the forefront of any analytical testing laboratory. Currently, there are a number of published, standardized methods for the analysis of HPHCs found in smokeless tobacco products. However, as potential HPHC levels are expected to be low, the question of whether these standardized methods are “fit for purpose” must be addressed. A 2021 collaborative study undertaken by the Tobacco and Tobacco Products Analytes (TTPA) sub-group of CORESTA (Cooperation Centre for Scientific Research Relative to Tobacco) examined the suitability of existing CORESTA recommended methods (CRMs) for the analysis of select HPHCs (nicotine, TSNAs, carbonyls, benzo[
a]pyrene (B[
a]P), and metals (arsenic and cadmium)) in nicotine pouches [
15]. This study found that the methods were suitable and the nicotine pouch matrix was subsequently added to the scope of the respective CRMs in December 2021.
A survey of the current literature turns up few studies that focus on the assessment of MONPs and their potential HPHC exposure risk [
16,
17,
18,
19,
20]. Further, these studies tend to have a narrow focus on a single product brand, limiting their overall scope. It is the intention of this publication to address this deficiency. Herein we describe the screening of seven brands of modern oral nicotine products. Within each brand, where possible, multiple flavors were analyzed. In addition to these products, two CORESTA smokeless tobacco reference products were screened, along with two smokeless tobacco products that are currently on the US market. In all, 25 unique products were assessed for a select list of HPHCs including TSNAs, carbonyls, nitrite, benzo[
a]pyrene, and metals.
3. Results and Discussion
3.1. Study Design
Current product innovation is focused on reducing consumer exposure to HPHCs through the development of modified-risk products. As these products aim to reduce the consumer’s exposure to harmful or potentially harmful constituents, the expected levels of each analyte examined is expected to be lower than those observed in traditional smokeless tobacco products. As MONPs are a relatively new product class, most of the literature published are by the product manufacturers and only focus on their own products, which can make cross product comparisons difficult. This study sought to address this by providing a direct comparison within a single study.
For this work, twenty-five (25) different products from nine individual manufacturers were selected for evaluation (
Table 5). Of these twenty-five, four were traditional smokeless tobacco products chosen for comparative purposes. The goal was to obtain products from a range of manufacturers that also varied in flavor and product type (pouch vs. long cut). An effort was made to diversify the flavor profiles, but also to ensure flavor overlap between manufacturers when possible. It must be noted that given the variety of nicotine strengths, not only from brand to brand, but even within a manufacturer’s own flavor, the products selected for analysis were at the maximum nicotine strength available. As this is a relatively new product classification, consideration of overall market share was not a top priority in the selection of brands. As mentioned previously, MONP are produced in two main forms: white granular powders (pouches) and plant-based (pouched or long cut). Based on these forms of consumption (pouch/long cut), four traditional smokeless tobacco products, two commercial brands and two CORESTA reference products (CRP1.1 and CRP2.1), were selected as the comparators. The commercial pouch product, General Wintergreen White Portion, was chosen as this product has received permission from the FDA to be marketed as a modified-risk tobacco product [
13]. Each of these products was screened for several HPHCs that are shown in
Table 6, along with their IARC designations.
Our own in-house methods (see
Section 2.3) for the analysis of the selected HPHCs are fully validated and based upon the corresponding CORESTA recommended methods, with the exception of B[
a]P, where a UHPLC method with fluorescence detection is used, and nitrite, which is based on Astoria-Pacific Method A181 (itself based on EPA method 353.2) [
40]. The challenge presented by MONPs is that most HPHCs should be found in levels lower than what are considered typical in tobacco products. As a result, the limits of detection (LOD) and quantitation (LOQ) become more critical for each analytical method and may not be appropriate for the analysis of MONPs should the analytes be present. The 2021 CORESTA collaborative study [
15] did not assess the suitability of the respective CRM LOQs, which were set with regard to the typical native levels found in tobacco products, and so this was an additional consideration in our study. The LOQs for our in-house methods are listed in
Table 7, along with the LOQ values from the analogous CRM. Except for the metals and nitrite analysis methods, all of the LOQs for the validated methods are comparable to those listed in the analogous CRM. It is worth noting that CRMs are consensus methods that are evaluated by multiple laboratories before approval and publishing. As such, the method LOQs can be much higher than a particular laboratory may be capable of achieving in order to allow for differences in instrumentation and expertise across laboratories.
Using our in-house methods (see
Section 2.3), we analyzed the selected products for the 17 HPHCs listed in
Table 7 and, where necessary, nicotine. For ease of comparison, the results were initially calculated on a per-gram basis without correction for water content, a full summary of which can be found in
Table S1 of the Supplementary Materials. The results were not corrected for any moisture content since our aim was to examine the consumer’s potential exposure during use. Overall, the general trend observed followed what was expected, with the average levels measured in MONPs being lower than those measured in selected smokeless tobacco products. The trends for each analyte class are discussed in the following sections.
3.2. Nicotine
Since MONPs do not contain tobacco, nicotine is added during their manufacture. For the vast majority of the products, including most of those used in this study, the nicotine levels are reported on the packaging and range from 4 mg to 12 mg per portion (
Table 5). This is comparable to the levels observed in the traditional smokeless products used as comparators (7 or 8 mg per portion for the snus products and 10 or 12 mg per portion for the long-cut products). Since some of the products selected for this study did not list their nicotine content on their packaging, they were assessed experimentally, and were found to exhibit comparable nicotine content to the other products being tested, ranging from 3.1 to 8.0 mg/portion.
3.3. Benzo[a]pyrene
Benzo[
a]pyrene (B[
a]P) is typically produced during combustion or any manufacturing process that may require heat (curing) [
44]. Even if some MONPs are exposed to these types of processes, levels are still expected to be low. Of all the MONPs examined, only Fully Loaded, Berry Long Cut (F4) had detectable levels of B[
a]P (1.27 ± 0.04 ng/g). This was far below those levels seen in the two long-cut STP comparators, which were 77.2 and 151 ng/g.
3.4. Nitrite and TSNAs
Nitrite is a precursor to TSNA formation [
45,
46] and is readily found in smokeless tobacco products [
47]. Generally, the nitrite observed in MONPs was lower than those levels seen in STPs, except for one product (G2) that contained three-to-four times more nitrite than observed in the STPs. Given the low nitrite observed, it is not surprising that the levels of TSNAs observed for almost all of the MONPs in this study were below the LOQ or non-detectability threshold. The exceptions to this were the two Black Buffalo long-cut products (G3 and G4) that were found to contain NAT (18.5 ± 0.3 and 14.8 ± 1.3 ng/g, respectively), NNK (13.7 ± 0.4 ng/g in G3 only), and NNN (39.4 ± 1.3 and 32.4 ± 0.8 ng/g, respectively), shown in
Figure 1. Both the NAT and NNK amounts were close to the respective LOQ values, while the NNN was close to three times the LOQ. In contrast to the MONPs, all four STPs were found to contain the four TSNAs at higher levels, particularly the CRP2.1 reference product (NAB: 9 to 274 ng/g; NAT: 125 to 4168 ng/g; NNK: 40 to 2104 ng/g; NNN: 177 to 3380 ng/g). While our analysis of MONPs shows that the LOQs associated with the method used are suitable for comparison to STPs, lower LOQs may be more beneficial when analyzing for regulatory reporting purposes. Adapting methods for trace TSNA analysis in ENDS e-liquid and aerosol may be a means for achieving this [
48].
3.5. Carbonyls
One of the more interesting classes of compounds assessed in this study was carbonyls, for which there were distinct trends observed based solely on the filler composition of the MONP (
Figure 2). The formaldehyde levels measured in all MONPs were comparable to those measured in the smokeless tobacco products tested (0.33 to 3.33 µg/g for MONPs and 0.78 to 3.64 µg/g for STPs). Two WGP-based MONPs, E1 and E2 (Zyn Nicotine Pouches), exhibited levels that were approximately three-to-four times those seen in STPs, being in the 10 to 14 µg/g range.
Although the levels of formaldehyde were fairly consistent among all products screened, acetaldehyde appeared to be composition specific. The acetaldehyde levels measured in WGP-based products were typically below the LOQ or non-detectability except for one product, B3, that contained 0.56 ± 0.06 µg/g. Conversely, the acetaldehyde measured in the plant-based products ranged from lower to substantially higher than the levels observed in STPs, topping out at 23.5 µg/g versus 5.7 µg/g measured in the commercial snus product (H1).
Interestingly, the highest levels of acetaldehyde measured in MONP were observed in products F3 and G4, both of which happen to be peach flavored. It is also noteworthy that one of these products (G4) was the only product tested to have detectable crotonaldehyde, albeit just above the LOQ. Given that manufacturers will likely use the same base composition across their respective products, it is indicative of the elevated acetaldehyde levels observed being due to this particular flavorant.
3.6. Metals
Similar to carbonyls, there are clear differences in the metal analyte profiles of the two MONP types (
Figure 3). WGP-based MONPs generally show much lower levels of metals, if present at all, than the plant- and tobacco-based products. Arsenic, beryllium, cadmium, and selenium were all either below the LOD or below the LOQ. Cobalt (4.5 to 10.4 ng/g) and lead (3.9 to 19.6) were all within six-times the LOQ (both 3.7 ng/g), except for lead in the Rogue Pouch products, C1 to C3 (77 to 83 ng/g), which were closer to the levels seen in the pouched STPs (approximately 107 ng/g). The more prevalent metals in WGP-MONPs were chromium (22.5 to 274 ng/g) and nickel (39.4 to 138 ng/g), though the levels were, for the most part, much lower than in the pouched STPS (Cr: 242 to 336 ng/g; Ni: 644 to 676 ng/g). The highest levels were seen for the Rogue Pouch products (Cr: 215 to 274 ng/g; Ni: 92.5 to 138 ng/g), with two of the on! Nicotine Pouch products (B1 and B2) and Fré Lush also showing elevated Cr levels relative to the majority of the WGP-MONPs tested.
In contrast to the WGP-based MONPs, the plant-based MONPs showed greater comparability with the tobacco-based products, though overall contained lower metal levels than the STPs with some noted exceptions. Beryllium was at or below the LOQ in all eight plant-based MONPs, and cadmium (16.0 to 86.0 ng/g) and cobalt (29.0 to 139 ng/g) were well below the levels in the STPs (Cd: 233 to 735 ng/g; Co: 293 to 546 ng/g). Arsenic (17.9 to 78.7 ng/g), chromium (233 to 456 ng/g), lead (21.7 to 159 ng/g), and selenium (12.4 to 81.8 ng/g) were observed at levels that were below or comparable to those in the STPs (As: 46.8 to 90.4 ng/g; Cr: 242 to 401 ng/g; Pb: 107 to 182 ng/g; Se: 93.5 to 197 ng/g). Nickel levels ranged from well below (175 ng/g in G2) to well above (1115 ng/g in F4) those in STPs (644 to 792 ng/g). Furthermore, there were clear differences between the two product brands tested, with the Fully Loaded brand generally containing higher metals levels than the Black Buffalo products for all analytes except cadmium and, for select products, selenium. This difference in levels may reflect the plants used to create the products and their respective uptake of metals from the soil and air. The generally higher levels in the Fully Loaded products may have been due to the use of kudzu root [
49], compared with edible green leaves for Black Buffalo products [
50], since a plant’s root system is responsible for the absorption of nutrients from the surrounding soil. The kudzu plant was even shown to have utility for the lead phytoremediation of soils through rhizofiltration (root absorption) [
51]. Finally, the metal content of the long-cut MONP formulations of the products were, with few exceptions, higher than their pouched counterparts. This may have been due to differences in the ingredients—the Fully Loaded long-cut formulation, for instance, contains molasses whereas the pouch formulation does not [
49]—or it could simply have been due to batch variations in the base plant matrix used during manufacturing.
3.7. Exposure Assessment
One of the goals of reduced risk products is to be able to provide comparable nicotine delivery with reduced exposure to potential HPHCs. However, assessing potential exposure is not a trivial task. The frequency of use is directly associated with the potential exposure to HPHCs and can vary widely across the globe. For example, a study conducted in the United States examined adult usage of specific brands of snus and found the study subjects consumed an average of 3.3 pouches per day [
52]. This is a relatively small amount when compared to the consumption habits found in Sweden where a survey conducted found the average daily consumption to be 11–12 g and 29–32 g for pouched snus and loose snus, respectively [
53]. Due to this wide range in consumption habits, where available, the approximate portion size (pouch weight) was obtained and is provided in
Table 5, providing context to potential usage. The observed HPHC analyte levels on a per-portion basis can also be found in
Table S2 of the Supplementary Materials, and were calculated using the actual number of pouches analyzed per replicate (for long-cut products, a 1 g portion size was used). For the WGP-based MONPs, the HPHC amount per portion will be reduced relative to both the STP amounts and their own per-gram amount since they have portion weights below 0.7 g. The plant-based MONP pouches, on the other hand, are either comparable (Black Buffalo, G1 and G2) or higher (Fully Loaded, F1 and F2) than their per-gram amounts. Relative to the pouched STPs, there are few changes to the trends described above, the exceptions being for the heavier Fully Loaded pouch products in which acetaldehyde becomes comparable, and lead and nickel become higher per portion.
Overall, the MONPs appear to pose a much-reduced exposure risk compared to STPs, though the caveat here is that the sample size for STPs is very limited in this study and does not represent the full range of products available to the consumer. Ultimately, however, it will be the end user’s consumption habits that determine their particular potential HPHC exposure risk.
4. Conclusions
The purpose of this work was to expand upon previous studies examining the potential HPHC content of modern oral nicotine products. With the push towards reducing the consumer’s potential HPHC exposure, interest in the science surrounding modern oral nicotine products has increased. Previous work typically focused on assessing a single product brand, where this study sought to assess 25 unique products, which also includes two CORESTA reference products and two traditional smokeless tobacco products. Generally, products that are composed of powder-based materials displayed much lower levels of the HPHCs being assessed than those observed in the plant-based oral nicotine products. This trend was most evident in the metals and acetaldehyde analysis, and was likely due to a combination of higher native levels in the plants being used and any curing or manufacturing processes employed during production.
Although the levels reported here for MONPs are typically lower than the commercial and reference STPs analyzed, it should be noted that the STPs used in this study do not represent all smokeless tobacco products and these results should be viewed in that context. Further work is suggested to provide a more complete picture of the toxicant exposure risk to consumers with relation to all available marketed products. Potential areas for inclusion would be an examination into how the nicotine level may affect the HPHC amounts and a probe of potential batch-to-batch variability.