Effects of Sweeteners and Cinnamon Flavor on Oral Nicotine Choice Behaviors

Abstract

Introduction

Oral nicotine products (ONPs) are emerging as a new nicotine delivery method, with varied types and flavors such as sweeteners and cinnamon. This study evaluates how sucrose, saccharin, and cinnamaldehyde influence nicotine preference, shedding light on the potential appeal of ONPs and how they may impact on harm reduction.

Methods

For oral choice behavior studies, we utilized a four-bottle choice (BC) test in male and female adult Sprague-Dawley rats. We first examined most common sucrose (1%) and saccharin (0.32%) concentrations as sweet solutions, and quinine (0.01%) as a bitter solution, to determine 4BC sensitivity and ability to distinguish between sweet and bitter tastes. We then performed dose–response analyses with sucrose (0.01%, 0.1%, and 1%), saccharin (0.032%, 0.1%, and 0.32%), and cinnamaldehyde (0.0005%, 0.005%, and 0.05%), in comparison to water in 4BC. Lastly, we tested nicotine (10 µg/mL) choice behaviors in the presence of sweeteners and/or cinnamaldehyde.

Results

Female and male rats significantly preferred sucrose (1%) and saccharin (0.1% and 0.32%) but not cinnamaldehyde. Moreover, rats differentiated sweet and bitter solutions with the highest preference for saccharin. Sucrose increased nicotine preference in females, but cinnamaldehyde increased nicotine preference in males. Saccharin increased nicotine preference in females, but not in males. Additionally, the combination of cinnamaldehyde and saccharin increased nicotine preference in females.

Conclusions

We found differential preferences among the test solution concentrations with the highest sweetener concentrations being most preferred. Sweetness value of the nicotine solution played a major role on nicotine preference in females but not in males.

Implications

Understanding how sweeteners and flavor additives affect oral nicotine choice behavior and nicotine preference in ONPs can guide the development of targeted harm reduction strategies and regulatory policies. By identifying which additives enhance product appeal and potentially influence addiction, this research can inform the creation of safer ONP formulations. This research also supports the utility of evidence-based guidelines for ONP use.

Introduction

According to the World Health Organization, tobacco use remains a major global health threat, with 1.3 billion tobacco users, and 8 million tobacco-related deaths annually.¹ While cigarette smoking is the most prevalent form of worldwide tobacco use, individuals also use other tobacco products including waterpipe, cigars, electronic delivery systems, smokeless tobacco, and non-medicinal oral nicotine products (ONPs). Overall, all forms of nicotine use are harmful, with no safe level of exposure.^1,2 The rise of ONPs, including gums, pouches, and other novel formats, represents a significant shift in nicotine consumption patterns, particularly as a harm reduction strategy.³ For example, there is noted interest and use of nicotine pouches among adult smokers planning to quit.⁴ Youth also perceive nicotine pouches as less addictive, due to their noncombustible form and due to the influence of youth-oriented social media advertising.^5,6 Given that ONPs are gaining traction among youth and adult tobacco product users, investigations of ONP constituents, product choice, and use patterns are urgently needed.

As the ONP market expands, with an increasing diversity in flavors, nicotine concentration, and sources, there is a critical need to understand the implications of these products on consumer behavior and public health. Along with nicotine lozenges and gum, pouches are part of the modern ONPs and they are available with sweetener and flavor additives.^7–9 A national online survey in youth revealed that flavored ONP use is favored among youth.⁵ A Swedish research preprint showed that although flavors do not significantly impact nicotine pharmacokinetics, they significantly influence user satisfaction and increase the likelihood of continued use for oral nicotine pouches.¹⁰ Indeed, oral nicotine pouches contain sweeteners such as acesulfame-K and sucralose, which are associated with higher nicotine concentrations.⁹ Preclinical research shows that increasing sweetener content in oral nicotine pouch extracts enhances consumption in mice, particularly with higher nicotine concentrations.⁹ This association suggests that these sweeteners could be included to mask the harshness of nicotine and enhance its appeal. Furthermore, intraoral sweetener saccharin enhanced intravenous nicotine self-administration and seeking behaviors in rats.¹¹ Because sweetness and flavoring influence both nicotine’s orosensory properties and addiction potential,^11–13 it is important to determine the impact of sweeteners and flavors on oral nicotine choice behaviors. However, prior studies have isolated these components to directly examine oral choice comparisons between non-flavored, flavored, sweetened, and unsweetened ONPs.

In this study, we aimed to determine how flavors influence flavor appeal, as reflected in preference and choice behavior. Specifically, we investigated the effects of the sweeteners sucrose and saccharin, as well as the commonly used flavor additive cinnamaldehyde,^7,8 on nicotine preference and oral choice behavior. Cinnamaldehyde, the principal chemical of cinnamon, acts as a transient receptor potential ankyrin 1 (TRPA1) channel agonist.¹⁴ Therefore, cinnamon provides a distinct sweet-smelling flavor accompanied by a burning sensation through TRPA1 activation, which contrasts with sweetness itself, allowing us to examine its unique impact on nicotine choice behaviors. By comparing the effects of sucrose, saccharin, and cinnamaldehyde, we aimed to elucidate how different sweeteners and flavor additives uniquely influence nicotine preference and oral choice behavior.

Materials and Methods

Animals

Female and male adult Sprague-Dawley rats (weighing 250–275 g and 300–350 g, respectively; Charles River Laboratories, Wilmington, MA) were housed in a temperature- and humidity-controlled husbandry room. Rats were single-housed and provided with ad libitum food and water on a standard 12-hour light/dark cycle (from 7 am to 7 pm). All experiments were approved by the Yale University Institutional Animal Care and Use Committee and conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Drugs and Chemicals

Freebase nicotine (#N3876), sucrose (#S0389), saccharin sodium salt hydrate (#S1002), and cinnamaldehyde (#W228613) were purchased from Sigma-Aldrich (St. Louis, MO). Quinine monohydrochloride (#163720050) was obtained from Acros Organics (Geel, Belgium). Solutions were prepared in tap water with pH adjusted to 7.0, using NaOH and HCl. The solutions were prepared 1 day prior to the experiment, stored in the refrigerator, and then moved to room temperature 1 hour before the start of the experiment. Each solution was used a single time on the test day. The selected nicotine concentration (10 µg/mL, which is equivalent to 0.001%) used in this study was chosen based on established concentrations from our previous oral nicotine administration studies in rats.^12,15–17 At 10 µg/mL, rats maintained stable nicotine drinking behavior, and nicotine was less aversive than at other concentrations, making this concentration suitable for evaluating the effects of tobacco product constituents on nicotine intake.^15,17 Dose–response analyses were performed, and preference was characterized, for the other chemicals.

Four-Bottle Free Choice Test

Rats were individually housed after their arrival at the facility, and they were habituated to their housing cages for 5 days. To assess preferences, a four-bottle free choice test (4BC) was employed, adapted from the 2BC test we previously described.¹⁸ This modified test provides the subjects with a choice between four different bottles, allowing for a more comprehensive evaluation of their preferences compared to the original two-bottle setup. Two water bottles were always present in the home cage, except during the 4BC test. On test days, animals underwent a 4-hour period without water, immediately prior to the experiment, to induce a thirsty state. They were then tested in the custom-built 4BC cages. The testing cages were identical to their home cages, but the lids were built from stainless steel to allow for the placement of four 25-mL plastic tubes with rubber stoppers and stainless-steel drinking spouts (Supplementary Figure S1).

During the final 10 minutes of the water deprivation period, the animals were transferred to the empty testing cages, without bedding or food, for a 10-minute habituation period. Following the habituation period, tubes containing test solutions were filled and placed in the cages in a randomized order, to prevent positional bias for each rat. The volume of each solution was measured in milliliters at the start of the experiment and again after 1 hour, at the end of the experiment. The amount of each solution consumed by the animals was recorded as fluid intake (mL) per solution. Data were reported as the percent preference for each solution, which was calculated based on these fluid intake measurements. Preference for the test solution was determined by dividing the volume of test solution consumed by the total volume consumed. Additionally, the body weight of all the rats was measured after each test. To account for any spillage that might naturally occur, an empty cage was used every test session, equipped with four bottles of test solutions. If any spillage occurred in the empty cage, the corresponding data in the test cage were excluded from the study. A between-subjects design was used for the experiments.

Before testing the effects of ONP additives on nicotine preference, we assessed the 4BC model’s sensitivity to distinguish between sweet and bitter tastes. Additionally, we performed dose–response analyses for sweeteners and cinnamon flavor (sucrose, saccharin, cinnamaldehyde). Sucrose (caloric table sugar) is a disaccharide composed of glucose and fructose, commonly found in sugar cane and sugar beets. Saccharin, a noncaloric artificial sweetener, is 200 to 700 times sweeter than sucrose.¹⁹ Quinine is a bitter alkaloid derived from the bark of the cinchona tree, used both medicinally and in research analyses of taste perception. To test the effectiveness and functionality of the 4BC model, we included both sweeteners (sucrose and saccharin), and we compared sweetener preference to quinine and water. This setup allowed us to evaluate whether the 4BC model accurately reflects the expected preference hierarchy, with sweet solutions (sucrose at 1% and saccharin at 0.32%) anticipated to be more preferred over the bitter solution (quinine at 0.01%), and over water. By including these distinct taste profiles, we aimed to verify that the 4BC model effectively reflects taste preferences and differentiates between rewarding and aversive stimuli.

Statistical Analyses

We used GraphPad Prism software (version 9; GraphPad Software, Inc., San Diego, CA) for create graphs and to perform all statistical analyses. Prior to statistical analysis, Brown–Forsythe and Barlett’s tests were used. Data were checked for adherence to a normal distribution. All data met the necessary assumptions. For statistical analysis, preferences within each sex were analyzed using a one-way ANOVA, followed by Tukey’s post hoc test. Comparisons across sexes were conducted using a two-way ANOVA with Bonferroni’s post hoc test to correct for multiple comparisons. Additionally, solution-specific analyses were performed using a two-way ANOVA with Sidak’s post hoc test to further refine sex comparisons. Significance was determined at p < .05.

Results

Evaluation of 4BC Utility in Distinguishing Between Sweet and Bitter Preferences

To test the utility of 4BC model in differentiating between sweet and bitter preferences, we performed a comparative test of both sweeteners and a bitter solution. We hypothesized that rats would prefer sweet solutions over water and over the bitter solution. This setup allowed us to assess whether the 4BC model could accurately test the expected preference, with sweet solutions (sucrose at 1% and saccharin at 0.32%) anticipated to be favored over the bitter solution (quinine at 0.01%) and water. We found significant main effect of test solution in both females (F_3,36 = 33.31; p < .0001, Figure 1A) and in males (F_3,36 = 22.69; p < .0001, Figure 1B). The preference was the highest for saccharin, followed by sucrose, water, and quinine. Although saccharin was the only solution to show a significant increase in preference compared to water (p < .05), the order of preference remained consistent between female and male rats. Quinine and water preferences were not significantly different from each other (p > .05). In addition, statistical analysis across sex revealed no sex differences (F_1,72 = 4.697e-019; p > .9999), but a significant effect of solution (F_3,72 = 54.96; p < .0001).

Figure 1.

Four-bottle choice test of water, quinine, sucrose, and saccharin in (A) female and (B) male rats. Rats were provided water, quinine (0.01%), sucrose (1%), and saccharin (0.32%) for 1 hour following 4 hours of water deprivation. Data were expressed as mean ± standard error of the mean of n = 10/sex. ***p < .001, ****p < .0001.

Sucrose, Saccharin, and Cinnamaldehyde Preferences Across Varied Concentrations in 4BC

Figure 1 shows the average preference per test concentration and solution in females and males in 4BC. To assess the impact of sucrose concentration on oral choice, we characterized sucrose preference across three different concentrations (0.01%, 0.1%, and 1%). We found significant main effects of sucrose in females (F_3,36 = 13.34; p < .0001, Figure 2A) and in males (F_3,36 = 6.08; p < .01, Figure 2B). While all concentrations resulted in some preference, both female and male rats significantly preferred the highest concentration (1%) over the others (p < .05). In addition, statistical analysis across sex revealed no sex differences (F_1,72 = 2.820e-021; p > .9999), but revealed a significant effect of sucrose (F_3,72 = 18.57; p < .0001).

Figure 2.

Dose–response curve of sucrose, saccharin, and cinnamaldehyde in the four-bottle choice test. Average preferences of sucrose solutions (0.01%, 0.1%, and 1%) and water in (A) female and (B) male rats. Average preferences of saccharin solutions (0.032%, 0.1%, solutions, and 0.32%) and water in (C) female and (D) male rats. Average preferences of cinnamaldehyde solutions (0.0005%, 0.005%, and 0.05%) and water in (E) female and (F) male rats. Rats were provided water and three different concentrations of the test chemical for 1 hour following 4 hours of water deprivation. Data were expressed as mean ± standard error of the mean of n = 10/sex/chemical. *p < .05, **p < .01, ***p < .001, ****p < .0001.

To assess the impact of saccharin concentration on oral choice behavior, we characterized saccharin preference across three different concentrations (0.032%, 0.1%, and 0.32%). Statistical analyses revealed significant main effects of saccharin in females (F_3,36 = 30.84; p < .0001, Figure 2C) and in males (F_3,36 = 7.19; p < .001, Figure 2D). Both 0.1% and 0.32% saccharin concentrations resulted in significantly higher preferences compared to water in females and males (p < .05). While female rats significantly preferred the highest saccharin concentration (0.32%) over the other concentrations (p < .05), male rats preferred saccharin at 0.1% and 0.32% concentrations similarly (p > .05) but more than others (p < .05). In addition, no main effect of sex (F_1,72 = 2.590e-018; p > .9999), but a significant effect of solution (F_3,72 = 28.41; p < .0001) was found.

To assess the impact of cinnamaldehyde concentration on oral choice, we then characterized cinnamaldehyde preference across three different concentrations (0.0005%, 0.001%, and 0.005%). Statistical analyses showed no significant main effect of cinnamaldehyde (F_3,36 = 0.17; p = .9098, Figure 2E, and F_3,36 = 0.78; p = .5113, Figure 2F), with all three cinnamaldehyde concentrations resulting in preferences that were not significantly different from water in both females and males (Figure 2E and Figure 2F, respectively). In addition, statistical analysis across sex revealed no sex differences (F_1,72 = 1.914e-007; p = .9997) and no effect of solution (F_3,72 = 0.025; p = .9945).

After completing the dose–response curve analyses, we selected the sweetener and flavor concentrations with the highest preference, if possible, for use in subsequent experiments. The concentrations selected were sucrose at 1%, saccharin at 0.1% and 0.32%, and cinnamaldehyde at 0.005%.

Impact of Sucrose and Cinnamaldehyde on Nicotine Choice Behavior in 4BC

To investigate whether the sweetener sucrose and the flavor cinnamaldehyde alter nicotine choice behaviors, sucrose and cinnamaldehyde were added to nicotine, either alone or in combination. The selected concentrations of sucrose and cinnamaldehyde (Figure 3) were based on the most preferred concentrations, from our dose–response analyses (Figure 2). Each animal was presented with the following four-bottle choices: water + nicotine, cinnamaldehyde (0.005%) + nicotine, sucrose (1%) + nicotine, and a combination of cinnamaldehyde (0.005%) and sucrose (1%) + nicotine. The data revealed a significant main effect of test solutions in females (F_3,36 = 11.44; p < .0001, Figure 3A). Female rats preferred sucrose + nicotine solution significantly more than both the water + nicotine and cinnamaldehyde + nicotine solutions (p < .05). The combination of cinnamaldehyde, sucrose, and nicotine showed a trend toward increase in preference compared to water; however, it did not reach significance (p > .05). Additionally, male rats showed a significant main effect across all solutions (F_3,36 = 4.563; p < .01, Figure 3B). Overall, cinnamaldehyde-containing solutions showed higher preference; however, only the cinnamaldehyde + nicotine solution showed significantly higher preference compared to nicotine alone (p < .05). Although the combination of cinnamaldehyde, sucrose, and nicotine solution showed a trend toward an increase in preference, no significant differences were found, compared to the other solutions (p > .05). In addition, statistical analysis revealed no effect of sex (F_1,72 = 2.401e-018; p > .9999), but a significant effect of solution (F_3,72 = 4.746; p = .0045).

Figure 3.

Effects of sucrose and cinnamaldehyde on nicotine preference in the four-bottle choice test. (A) Female and (B) male rats were given four bottles of test solutions: nicotine (10 µg/mL), cinnamaldehyde (0.005%) + nicotine, sucrose (1%) + nicotine, and a combination of cinnamaldehyde, sucrose, and nicotine. Rats were provided these test solutions for 1 hour following 4 hours of water deprivation. Data were expressed as mean ± standard error of the mean of n = 10/sex. *p < .05, ***p < .001, ****p < .0001.

Impact of Saccharin and Cinnamaldehyde on Nicotine Choice Behavior in 4BC

To investigate whether the sweetener saccharin and the flavor cinnamaldehyde alter nicotine choice behaviors, saccharin and cinnamaldehyde were added to nicotine solutions, either alone or in combination. The concentrations were based on the most preferred concentrations, as determined in our prior dose–response experiments (Figure 1). Because saccharin induced significantly higher preference at both 0.1% and 0.32% concentrations in the dose–response curve study, we first examined naïve rats with 0.1% saccharin. Five days later, the same animals were tested with 0.32% saccharin. This allowed us to investigate the animals’ responses to both low and high saccharin concentrations.

First, each animal was presented with four bottles containing either: water + nicotine, cinnamaldehyde (0.005%) + nicotine, saccharin (0.1%) + nicotine, or a combination of cinnamaldehyde (0.005%) and saccharin (0.1%) + nicotine. We found significant main effect of test solutions in females (F_3,36 = 21.14; p < .0001, Figure 4A). Specifically, female rats preferred saccharin + nicotine, and cinnamaldehyde + saccharin + nicotine solutions significantly more than water + nicotine or cinnamaldehyde + nicotine (p < .05). Additionally, female rats showed significantly increased preference for saccharin + nicotine compared to all other tested solutions (p < .05). Preference for cinnamaldehyde + nicotine preference was similar to preference for nicotine alone (p > .05). In contrast, male rats showed no significant differences in preference, across all tested solutions (F_3,36 = 0.7907; p = .5070, Figure 4B). However, statistical analysis revealed no sex differences (F_1,72 = 4.260e-018; p > .9999), but a significant effect of solution (F_3,72 = 9.699; p < .0001).

Figure 4.

Effects of saccharin and cinnamaldehyde on nicotine preference in the four-bottle choice test. (A) Female and (B) male rats were given four bottles of test solutions: nicotine (10 µg/mL), cinnamaldehyde (0.005%) + nicotine, saccharin (0.1%) + nicotine, and a combination of cinnamaldehyde, saccharin, and nicotine. Five days later, same (C) female and (D) male rats were given four bottles of test solutions: nicotine (10 µg/mL), cinnamaldehyde (0.005%) + nicotine, saccharin (0.32%) + nicotine, and a combination of cinnamaldehyde, saccharin, and nicotine. Rats were provided these test solutions for 1 hour following 4 hours of water deprivation. Data were expressed as mean ± standard error of the mean of n = 7–10/sex. *p < .05, **p < .01, ****p < .0001.

Five days after the first experiment, each animal underwent a second experiment where they were presented with four bottles in a counterbalanced fashion. Animals receive one of the following test solutions: water + nicotine, cinnamaldehyde (0.005%) + nicotine, saccharin (0.32%) + nicotine, and a combination of cinnamaldehyde (0.005%) and saccharin (0.32%) + nicotine. We found a similar pattern with this higher saccharin concentration (0.32%) in females and males, as we observed in the prior experiment with 0.1% saccharin. Statistical analysis revealed a significant main effect of test solutions in females (F_3,24 = 7.612; p < .001, Figure 4C). Specifically, female rats significantly preferred saccharin + nicotine solution over all other tested solutions (p < .05). When cinnamaldehyde was added to nicotine, either alone or in combination with saccharin, the preference was similar to that of nicotine alone. (p > .05). In contrast, male rats did not show any statistically significant main effect to test solutions (F_3,32 = 1.613; p = .2058, Figure 4D). In addition, statistical analysis revealed no sex differences (F_1,56 = 0.6366; p = .4283), but a significant effect of solution (F_3,56 = 7.927; p = .0002).

Discussion

Understanding choice behaviors and the interactions between flavors and sweeteners is crucial for determining their impact on nicotine use behaviors. Here, we used a 4BC paradigm to examine the impact of cinnamon flavor (cinnamaldehyde) and sweeteners (sucrose and saccharin) on nicotine choice and preference. The choice preference comparisons between quinine, sucrose, and saccharin demonstrated that 4BC effectively distinguishes preferences between the test solutions and water. Both sucrose and saccharin increased nicotine preference in female rats but not in male rats. Cinnamaldehyde alone increased nicotine preference only in male rats. The combination of cinnamaldehyde and saccharin increased nicotine preference in females in a concentration-dependent manner. Together, our findings provide new understanding of whether cinnamon flavor or sweetener additives enhance ONP appeal.

In this study, we used a four-bottle paradigm with a 1-hour testing window after 4 hours of water deprivation to evaluate preferences between nicotine, sweetened nicotine, cinnamon-flavored nicotine, and both sweetened and flavored nicotine. The 1-hour consumption window provides an acute response of preference during a short-term test. This short-term test is useful for detecting immediate effects or acute choice behaviors, as well as initiation behaviors. However, we acknowledge that a longer testing period, such as 1 week, could capture more stable consumption patterns and accommodate potential tolerance to bitter tastes as we previously reported.¹⁷ Future experiments incorporating longer testing windows could provide further understanding of chronic consumption behavior, building on the findings from our current study.

In the 2BC model, rodents show a high preference for sucrose and saccharin but a low preference for quinine.²⁰ In this study, rats preferred saccharin, sucrose, water, and quinine in that order, with saccharin being significantly preferred over the others. This experiment confirmed that the 4BC model reliably captures animals’ preference behaviors, establishing its effectiveness and functionality for our further studies on flavor, sweetener, and nicotine preference. We also note that this 4BC model is well-suited for investigating oral choice behaviors related to ONP additives. In the literature, the most common used concentrations are 1%–2% for sucrose and 0.32% for saccharin.^13,21 In dose–response studies of sweeteners or flavors alone, no statistical sex differences were observed, but females consistently showed a higher preference than males for the highest concentrations of sweeteners. Both female and male rats showed a concentration-dependent preference for sucrose, with a significant preference for 1% sucrose over all other concentrations and water. Saccharin also elicited a concentration-dependent preference, with 0.1% and 0.32% concentrations being significantly preferred over water at the lowest concentration in both sexes. Our findings with sucrose and saccharin are consistent with those in previous studies.^13,20,21 However, preference for cinnamaldehyde did not differ among the three concentrations or water in either sex. No sex differences were observed in any of the test solutions.

This study compared preferences for nicotine alone versus nicotine combined with cinnamaldehyde, sweeteners, or both. The current study did not reveal overall main effects of sex across all solutions; however, solution-specific sex differences were observed, with females showing greater preference for sweetened nicotine and males preferring flavored nicotine. When combined with nicotine, sucrose (1%) led to the highest nicotine preference in females (Figure 3A), while cinnamaldehyde–nicotine combinations were most preferred in males (Figure 3B). Similar patterns were observed in the saccharin experiments (Figure 4, A and C). These findings suggest that sweeteners may play a greater role in nicotine preference for females, while flavors are more influential for males. Consistent with this potential differential extent of flavor effects in females and males, menthol flavoring increases oral nicotine intake and preference in male rats but not in female rats.¹⁶ An early study found that adult Holtzman albino both female and male rats consumed more caloric or noncaloric sweetener-containing solutions than water, with females showing a significantly stronger preference. The study also demonstrated that female rats preferred significantly higher concentrations of saccharin compared to males, highlighting a pronounced sex difference in sweetener sensitivity.²² Additionally, female Long–Evans rats exhibit more ingestive responses and fewer aversive reactions than males in taste reactivity tests. Furthermore, these differences are modulated by the estrous cycle, suggesting inherent sex differences in taste perception that may also influence nicotine preference.²³ A recent study found that wild-type mice consumed more oral nicotine pouch extracts with higher levels of sweeteners and nicotine compared to Tas1r2^−/− mice, which lack the ability to detect sweet taste.⁹ Moreover, sweeteners reduced the aversive effects of nicotine in wild-type mice, facilitating initiation and continued use.⁹ These findings emphasize the importance of sweetness in enhancing nicotine appeal, particularly in females, and suggest that flavors may hold a stronger influence on nicotine consumption behaviors in males. The findings from human studies also suggest that flavors and sweetness may increase product-liking for ONP.^10,24 However, these studies did not provide a detailed analysis of how these preferences might differ by sex. A 2024 paper using a cross-sectional survey of ONP users showed that 66% of participants identified as female.²⁵ Of note, this study also lacked information on sex differences in ONP use, leaving a gap in understanding the role of sex in ONP consumption patterns in human. Further research is needed to investigate these gaps in understanding use patterns between sexes.

In nicotine experiments with cinnamaldehyde and sweeteners (Figures 3 and 4), males showed the highest preference for cinnamaldehyde and nicotine, when both were combined with sucrose (Figure 3). In contrast, this effect was not seen in the saccharin experiments (Figure 4). These findings, thus, revealed a shift in male preference dynamics between exposure to different sweeteners. This finding highlights the importance of considering the specific flavor and sweetener combinations that influence preference. We suggest that saccharin’s stronger sweetening properties compared with sucrose could contribute to this finding. To our knowledge, such an effect between different sweeteners has not been previously observed in the context of nicotine drinking. However, our results suggest that cinnamon flavor could be more effective in changing the nicotine preference when the sweetening value is low. Although our study did not involve taste analyses, taste can also mediate in the preference for nicotine-containing solutions. Such findings were previously reported in a 2BC model in rats using quinine, sucrose, saccharin, and nicotine. Specifically, female rats were shown to consume more liquid than males, when both were exposed to sweet tastants such as saccharin and sucrose. In contrast, these effect was not observed with exposure to a bitter tastant like quinine.²⁶ When nicotine was introduced, no sex differences were reported in average liquid intake. These results suggest that sweet tastants preferentially influence liquid intake behavior in females, while nicotine intake does not show sex-specific differences in overall consumption.²⁶

While our findings provide valuable insights, there are some limitations that should also be considered when interpreting the findings. Notably, the lack of water availability in some of the experiments has introduced a moderate stressor that would impact drinking behavior. While this could serve as a limitation, the experimental design was necessary to understand the relative preferences of ONP additives in comparison to a neutral nicotine preference. We also note that animals still showed robust preference behavior. In the sucrose, saccharin, and quinine study (Figure 2), we did not observe a significant decrease in quinine preference compared to water. This lack of effect may have resulted from a floor effect, given the observed ~10% to 15% preference for water. However, considering our primary focus on sweetener and flavors, we anticipate that this limitation did not have a major impact on our overall conclusions. Although we did not examine many concentrations in the flavor–sweetener interactions, we selected concentrations based on their dose–response curves. Therefore, our approach ensured that the concentrations were optimal selected for capturing assessing preferences and for capturing significant effects.

Conclusions

Sweeteners and cinnamon flavoring altered oral nicotine preferences and choice behaviors. Saccharin was the more preferred sweetener for both male and female rats, compared to sucrose. While both sucrose and saccharin increased nicotine preference in female rats, no such effect was observed in males. In contrast, cinnamaldehyde alone increased nicotine preference only in male rats. Notably, the combination of cinnamaldehyde and saccharin further increased nicotine preference in females in a concentration-dependent manner; the combination of cinnamaldehyde and saccharin at low concentration also increased nicotine preference. Understanding the role of sweeteners and flavorants in ONP appeal can inform regulatory policies and harm reduction strategies. Our study contributes to the growing body of evidence needed to characterize ONP constituents, assess their impact on consumer behavior, and support the development of evidence-based regulations to mitigate potential risks associated with ONP use.

Funding

This work was supported by grant number (U54DA036151) from the National Institute on Drug Abuse (NIDA) of the National Institutes of Health (NIH) and Food and Drug Administration (FDA) Center for Tobacco Products (CTP) and the State of Connecticut, Department of Mental Health and Addiction Services. The content is solely the responsibility of the authors and does not represent the official views of the NIH, the FDA, or the Department of Mental Health and Addiction Services or the State of Connecticut.

Declaration of Interests

NAA: Royalties—Tyndale House Publishers. Speakers Bureau/Consultation Fees—American Program Bureau.

Author Contributions

Deniz Bagdas (Conceptualization [lead], Formal analysis [lead], Funding acquisition [equal], Investigation [lead], Methodology [lead], Project administration [lead], Resources [equal], Supervision [lead], Validation [lead], Visualization [equal], Writing—original draft [lead]), Jennifer Sedaille (Data curation [supporting], Formal analysis [supporting], Investigation [supporting], Methodology [supporting]), Mariam Khan (Data curation [supporting], Investigation [supporting], Methodology [supporting]), Nnedinma Okpala (Data curation [supporting], Investigation [supporting], Methodology [supporting]), and Nii Addy (Funding acquisition [equal], Project administration [equal], Resources [equal], Supervision [equal], Visualization [equal], Writing—review & editing [equal])

Data Availability

The data underlying this study will be made available upon reasonable request to the corresponding author.