SYNTHETIC COOLING AGENT IN ORAL NICOTINE POUCH PRODUCTS MARKETED AS “FLAVOR-BAN APPROVED”

Abstract

Background

US sales of oral nicotine pouches (ONPs) have rapidly increased, with cool/mint-flavoured ONPs the most popular flavour category. Restrictions on sales of flavoured tobacco products have either been implemented or proposed by several US states and localities. Zyn, the most popular ONP brand, is marketing Zyn Chill and Zyn Smooth as ‘Flavour-Ban Approved’ or ‘unflavoured’, probably to evade flavour bans and increase product appeal. At present, it is unclear whether these ONPs are indeed free of flavour additives that can impart pleasant sensations such as cooling.

Methods

Sensory cooling and irritant activities of ‘Flavour-Ban Approved’ Zyn ONPs, Chill and Smooth, along with minty varieties (Cool Mint, Peppermint, Spearmint, Menthol), were analysed by Ca²⁺ microfluorimetry in HEK293 cells expressing the cold/menthol (TRPM8) or menthol/irritant receptor (TRPA1). Flavour chemical content of these ONPs was analysed by gas chromatography/mass spectrometry.

Results

Zyn Chill ONP extracts robustly activated TRPM8, with much higher efficacy (39%–53%) than the mint-flavoured ONPs. In contrast, mint-flavoured ONP extracts elicited stronger TRPA1 irritant receptor responses than Chill extracts. Chemical analysis demonstrated that Chill exclusively contained WS-3, an odourless synthetic cooling agent, while mint-flavoured ONPs contained WS-3 together with mint flavourants.

Conclusions

ONP products marketed as ‘Flavour-Ban Approved’ or ‘unflavoured’ contain flavouring agents, proving that the manufacturer’s advertising is misleading. Synthetic coolants such as WS-3 can provide a robust cooling sensation with reduced sensory irritancy, thereby increasing product appeal and use. Regulators need to develop effective strategies for the control of odourless sensory additives used by the industry to bypass flavour bans.

Background

The sales of flavoured oral nicotine pouch (ONP) products have rapidly increased.(1,2) ONPs are currently marketed in a wide variety of fruit, candy, menthol/mint and concept flavours. The most popular ONP brand, Zyn (Swedish Match / Philip Morris International) is advertising two of their products, labelled ‘Chill’ and ‘Smooth’, as ‘Flavour-Ban Approved’ or ‘unflavoured’,(3) likely in response to bans targeting tobacco products with characterising flavours, as recently enacted in California, and in anticipation of regulatory interventions in other jurisdictions.(4) It remains unclear whether ONPs marketed as ‘Flavour-Ban Approved’ or ‘unflavoured’ are indeed free of flavour additives, including constituents imparting pleasant sensations such as cooling.

Since the name of one of the ONP products, Zyn Chill, implies a cooling effect, we hypothesized that the product may contain a synthetic cooling agent. Synthetic cooling agents such as WS-3 or WS-23 elicit cooling sensations but lack the characteristic minty odour of menthol.(5,6) These agents are also less irritating than menthol that causes burning and painful sensations at higher concentrations. Synthetic cooling agents are increasingly used in electronic cigarette products, but haven’t been reported as ingredients in ONPs.(6–9)

Similar to menthol, synthetic cooling agents activate the cold/menthol receptor, TRPM8, a chemosensory receptor expressed in trigeminal and vagal sensory neurons.(10) In addition to sensory cooling, activation of TRPM8 has analgesic effects, facilitating the inhalation of tobacco smoke and increasing the palatability of smokeless tobacco products.(11–13) Menthol’s irritant effects are mediated by TRPA1, a sensory irritant receptor. Compared to menthol, synthetic cooling agents (WS-3, WS-23) are only weak activators of TRPA1, explaining their lack of irritant effects.

The replacement of menthol with synthetic cooling agents is likely an attempt by the tobacco industry to circumvent flavoured tobacco product bans based on the concept of ‘characterising flavour’, as in California.(4) While odourless, these agents impart the desired cooling effect of menthol.

It is debated whether synthetic cooling agents can be considered flavours. However, the Flavour Extracts Manufacturers Association (FEMA), the tobacco-industry supported flavour industry association tasked with flavour safety evaluations for US Food and Drug Administration (USFDA), classifies synthetic cooling agents as flavour chemicals.(14,15) FEMA defines flavour as an entire range of sensations that encompasses taste, smell and physical traits such as hotness and cooling.(16)

The USFDA’s new proposed product standard for menthol in combustible cigarettes contains similar language, defining ‘characterising flavour’ to incorporate the ‘multisensory experience’ of a flavour chemical, including cooling sensations.(17) However, the proposed rule does not apply to other tobacco products such as ONPs.

Here, we examined those ONPs marketed as ‘Flavour-Ban Approved’ for sensory cooling activity and irritant activity, and for the presence of synthetic odourless cooling agents and minty flavourants. We further compared them with ONPs named for their mint characterising flavours.

Methods

ONP extract preparation:

Zyn ONPs marketed as ‘Flavour-Ban Approved’ (Chill, Smooth) and Zyn ONPs labelled for cool/mint characterising flavours (Cool Mint, Peppermint, Spearmint and Menthol) were purchased at 3 and 6 mg nicotine/pouch strength between July and October 2022, either directly from Zyn’s official website (www.us.zyn.com) or from local gas stations. For calcium microfluorimetry, ONP contents were stirred overnight in calcium assay buffer (Hank’s Balanced Salt Solution with 10 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid)) and dilutions of these extracts (diluted 1X-100X in assay buffer) were prepared to test for receptor activity. The 1X dilution is defined as the extract of one ONP pouch in 10 mL assay buffer, and 100X the 100-fold dilution thereof. For chemical analysis, ONP contents were weighed and extracted in 2 mL of methanol for 7 days in the dark with occasional stirring. This protocol was adapted from an established protocol for the quantification of flavourants in pouched snus.(18)

Testing for sensory cooling and irritant activity by calcium microfluorimetry

Buffer ONP extracts (diluted 1X-100X) were tested for activity on the human cold/menthol receptor, TRPM8, and the menthol irritant receptor, TRPA1, by intracellular calcium microfluorimetry in HEK-293t cells (RRID:CVCL1926) as described.(19,20) Responses to maximally activating L-menthol (1 mM), WS-3 (100 μM), cinnamaldehyde (1 mM), and a nicotine dilution series (1X: 6 mg/10 mL, 1X-100X)) were measured for comparison. To control for any variations in receptor expression levels and loading of Ca²⁺indicator across experiments, Ca²⁺- influx data was normalised to the Ca²⁺-response elicited by a maximally activating concentration of agonist L-menthol (1 mM; TRPM8) or cinnamaldehyde (1 mM; TRPA1). Dose-response curves for receptor activity and associated calcium influx changes were plotted using non-linear regression analysis with a 4-parameter logistic equation (GraphPad Prism V.9.0, San Diego, California, USA). To further characterise receptor activation by ONP extracts, we determined and compared their efficacies (maximal receptor activation) and potencies (the concentration that produces 50% of its maximal response at a receptor) of activation. Experiments were repeated no less than three times (n ≥ 3) and each conducted at least in triplicates (N ≥ 3) with independent ONP extractions.

Chemical Analysis

Methanolic ONP extracts (N=3) were filtered (0.22μm) and 1 uL was injected directly into a GC/MS (Perkin-Elmer Clarus 580-SQ8S) using previously established methods.(7,20) Extracts were analyzed for the presence of synthetic cooling agents commonly used in food products and other tobacco product categories, including WS-3 (CAS No. 39711–79-0), WS-5 (68489–14-5), WS-12 (68489–09-8), WS-23 (51115–67-4), and Frescolat MGA (63187–91-7), ML (17162–29-7), and XCool (1122460–01-8), (5,7) as well as for the minty flavourants menthol (89–78-1), menthone (10458–14-7), menthyl acetate (89–48-5), and carvone (2244–16-8). Identified synthetic cooling agents or minty flavourants were quantified using a GC/FID (Shimadzu 2010) using commercially available standards by previously established methods.(7,8)

Results

Average total pouch mass for the Zyn pouches was 405±18 mg (n=12). Zyn Chill extract robustly activated the cold/menthol receptor TRPM8, even at 20-fold dilution, indicating the presence of a cooling agent (figure 1A). The Zyn Chill extract dilution series activated TRPM8 with a potency similar to mint-flavoured/menthol-flavoured ONP extracts (Zyn Cool Mint, and Zyn Peppermint), but with higher efficacy than these extracts (Chill 39±5% higher vs. Cool Mint; 53±4% higher vs. Peppermint) and a maximally activating menthol concentration (1 mM, 84±10% higher) (n=3–4) (figure 1A). The sensory irritant receptor, TRPA1 was robustly activated by mint-flavoured ONP extracts (Zyn Cool Mint and Zyn Peppermint), yet TRPA1 activation was significantly weaker for Zyn Chill extracts (Chill 48±13% lower vs. Cool Mint and 52±8% lower vs. Peppermint; n=3–4) (figure 1B) indicating the presence of a non-irritating cooling ingredient in Zyn Chill. Extracts from Zyn Smooth showed weak activity on TRPM8 and TRPA1, and only at 1X dilution (ie, no dilution of the extract).

Figure 1: Sensory cooling and irritant activity of Zyn “Flavor-Ban Approved” and mint-flavored ONPs, measured by Ca²⁺ microfluorimetry in HEK293 cells.

A. Dose-response analysis of human TRPM8 cold/menthol receptor-mediated Ca²⁺-influx into HEK293 cells following superfusion with a dilution series of Zyn ONP extracts (“Chill”, “Smooth”, “Cool Mint”, “Peppermint”) and nicotine (1X: 6mg/10ml). The increase in fluorescence units (F_max-F₀) of the fluorescent Ca²⁺ indicator (Calcium 6), was normalized to the Ca²⁺-response elicited by a saturating concentration of agonist L-menthol (1 mM). Response to a saturating concentration of WS-3 (100 μM) is shown for comparison.

B. Dose-response analysis of human TRPA1 irritant receptor-mediated Ca²⁺-influx into HEK293 cells following superfusion with the same dilution series of ONP extracts and nicotine as in A. Ca²⁺-response to a saturating concentration of TRPA1 agonist cinnamaldehyde (1 mM) was used for normalization.

Experiments were performed 3–4 times in triplicates with independent ONP extractions. Error bars for each data point show standard error of the mean.

Chemical analysis of the ONPs demonstrated the presence of WS-3, an odourless menthol-derived synthetic cooling agent in Zyn Chill (234±7 μg/pouch) as the only major flavourant and the only observable peak besides nicotine. WS-3 was also present in Zyn Peppermint (201±11 μg/pouch) and in Zyn Spearmint (209±15 μg/pouch). Other minty flavourants were detected in Zyn Peppermint (menthol: 1,925±140 μg; menthone: 791±55 μg; and menthyl acetate: 105±9 μg) and in Zyn Spearmint (carvone: 1180±185 μg and menthol: 470±44 μg). Menthol was the only flavourant present in Zyn Cool Mint (5406±259 μg/pouch) and Zyn Menthol (1390 μg/pouch; online supplemental table 1). For Zyn Smooth, no cooling and/or minty flavourants were detected, nor any other flavour chemical detectable with our method. For WS-3 analysis, recovery was 99.5% (n=7), precision 1.8 % relative SD, limit of detection 5 μg/mL and limit of quantification 15 μg/mL.

Discussion:

Our findings demonstrate that WS-3, an odourless synthetic cooling agent, is added to several of the tested cool-/mint-flavoured Zyn ONPs, and to Zyn Chill, an ONP advertised as ‘Flavour-Ban Approved’ or ‘unflavoured’. Extracts from the cool-flavoured/mint-flavoured ONPs also contained menthol and robustly activated the cold/menthol receptor, TRPM8. Extract from Zyn Chill, containing only WS-3, displayed higher efficacy for TRPM8 activation, exceeding the efficacy of a maximally activating menthol concentration. While extracts of cool-flavoured/mint-flavoured ONPs strongly activated the sensory irritant receptor, TRPA1, Zyn Chill extracts showed only weak efficacy and potency at TRPA1, suggesting reduced irritancy. Containing WS-3 exclusively, ‘Flavour-Ban Approved’ Zyn Chill likely provides a robust oral cooling sensation by strongly activating TRPM8, in combination with lower sensory irritation (ie. weaker TRPA1 activation) than menthol would cause, thereby likely increasing the appeal of the product.

Menthol’s cooling effect is known to reduce the harsh sensations elicited by tobacco smoke, thereby facilitating initiation and continued product use especially among young and naïve users.(21) Menthol cigarette use is highly prevalent (~85%) among African-Americans who smoke, due to decades-long aggressive marketing strategies aimed at the community that have promoted the cooling effects of menthol.(21) Surveys of smokeless tobacco users revealed that menthol flavour is among the top two popular flavours, both among users of traditional products (snuff, snus) and of ONPs.(22)

Recent rodent studies demonstrated that menthol and synthetic cooling agents result in similar behavioral effects. Oral sensory cooling stimuli, either by menthol, or a synthetic cooling agent or cold water, were shown to act as potent conditioned reinforcers of intravenous nicotine self-administration in rats.(23) Also, similar to menthol, synthetic cooling agents have analgesic activity in rodents, suggesting that WS-3 may soothe the sensory irritation caused by nicotine in ONPs.(24) These studies suggest that cooling sensations and analgesia induced by odourless synthetic cooling agents in ONPs (eg., Zyn Chill) may improve the sensory experience of the user, thereby facilitating product use initiation and more frequent consumption under the guise of a ‘Flavour-Ban-Approved’ product.

The moniker ‘Flavour-Ban Approved’ was likely introduced by Swedish Match to position these ONPs in markets where all flavoured tobacco products have been banned, such as in the State of California.(4) In regions without such bans, or bans that do not apply to ONPs, the products may appeal to consumers concerned about flavours associated with increased addictiveness of tobacco products. While the influence of the term ‘Flavour-ban approved’ on health perceptions remains to be examined, it is possible that consumers may perceive these products as healthier and less addictive. However, our results suggest that these products contain additives which have been shown to enhance the reinforcing potential of nicotine.

While the USFDA has not announced any intent to regulate ONPs and their flavours, any such regulation will have to rely on the concept of ‘characterising flavour’. Regulations in California and the other US states and municipalities banning certain flavoured tobacco products also rely on this approach. The ambiguity of this approach may provide a loophole for the tobacco industry to add odourless sensory chemicals such as synthetic cooling agents that impart comparable or stronger physiological cooling and analgesic effects than menthol, and that at lower amounts per product unit. The manufacturer of Zyn products, Swedish Match/PMI, seems to be confident that synthetic cooling agents don’t fall under the category of ‘characterising flavour’ and have therefore marketed ‘Flavour-Ban Approved’ or ‘unflavoured’.(17) Regulatory approaches to control the addition of synthetic cooling agents include the use of positive lists of permitted additives, such as in Canada and currently in preparation in The Netherlands, banning these agents in electronic cigarettes.(25,26) Germany banned chemical scaffolds in combustible cigarettes that facilitate inhalation and nicotine intake, such as menthol and most synthetic cooling agents.(27) Belgium implemented an alternative approach: instead of regulating individual compounds or compound classes, the country prohibits the use of any additive activating TRPM8 cold/menthol receptors.(28) The USFDA’s proposed product standard for combustible menthol cigarettes, in which menthol is banned as a characterising flavour, extends the definition of ‘characterising flavour’ by adding ‘multisensory experience’, including cooling sensations, as a factor to determine whether a tobacco products has a characterising flavour.(17) If approved as written, this extended definition will allow regulation of synthetic cooling agents in combustible cigarettes. Similar language, if applied to ONPs, might allow regulation of these additives in this product category in the future.

What this paper adds:

What is already known on this topic:

• The tobacco industry recently started marketing “Flavour-ban-approved” or “unflavoured” oral nicotine pouch products (ONPs). However, user reports suggest that these products are flavoured.

What this study adds:

• This study demonstrates that an ONP advertised as “Flavour-ban-approved” or “unflavoured” is not free of flavour chemicals, but contains WS-3, an odourless synthetic cooling agent that elicits intense cooling sensations.

• The manufacturer seems to have confidence that odourless flavour additives such as WS-3 do not fall under the category of a “characterising flavour”, thereby eluding flavour regulations.

How this study might affect research, practice or policy

• Regulators need to implement rules that prevent manufacturers from bypassing flavour bans by adding odourless flavourants such as synthetic cooling agents.