Results

Degree of oral mucosal lesions

Oral mucosal lesions were assessed, and clinical photographs were taken at each treatment visit (Fig. 1). Normal mucosa, with no observable alterations, was assigned a score of 0. Oral mucosal lesions were graded from 1 to 4 according to Axéll et al. [13]. During Visits 2 and 3, when participants were using tobacco-based snus, 30–50% had oral mucosal lesions graded as 4. No statistically significant difference in lesion severity was observed between Visit 2 and Visit 3 during continued snus use (p = 0.2869), supporting the use of Visit 3 as the baseline before nicotine pouch substitution. Following exclusive use of either the dry or moist formulation, noticeable reductions in the proportion of participants with lesions scored as 4 were observed at Visit 4 (14 days) and Visit 5 (28 days). For the dry formulation, the proportions decreased to 17% and 13%, respectively, and for the moist formulation to 36% and 14%, respectively. Additionally, at Visits 4 and 5, an increase in the proportion of participants with oral mucosal lesions scores of 0 or 1 was observed, particularly among those using the dry formulation, increasing from 8.7% at Visit 3 to 13% at Visit 4 and 34.3% at Visit 5.

Fig. 1: Oral mucosal lesions before (A) and after (B) switching to non-tobacco-based nicotine pouches. Clinical photographs of four participants taken during visit 3 (while using regular snus) and visit 5 (after exclusively using nicotine pouches for four weeks). The oral mucosal lesions at the pouch placement site were assessed using a four-point clinical scale. n = 45 participants

Both groups showed a significant reduction in lesion severity between Visit 3 and Visit 5 (p < 0.0001), with a large effect size (r ≈ 0.60). In the moist formulation group, lesion grade decreased significantly from Visit 3 to Visit 5 (p < 0.0001). The dry formulation group demonstrated a similar significant improvement over the same period (p < 0.0001) (Fig. 2).

Fig. 2: Comparison of mucosal lesion severity at pouch placement site. The severity of mucosal lesions at the pouch placement site was assessed at visit 3 and visit 5 using a four-point clinical scale by Axéll et al. Statistical analysis was performed with Wilcoxon matched-pairs signed-rank test. **** p < 0.0001, ** p < 0.01. n = 45 participants

Epithelial thickness and presence of inflammatory cells

Following microscopic evaluation, some samples were excluded due to alignment issues that could result in inaccurate measurements. Specifically, samples were excluded if papillary structures were not clearly visible or if no tissue was present directly adjacent to the epithelial layer. Only slides that were properly embedded, correctly sectioned, and exhibited continuous tissue in direct contact with the epithelium were included in the epithelial thickness analysis. This approach ensured that only precise and reliable data were included in the final evaluation.

Fig. 3: Inflammatory cell presence and epithelial thickness. Representative histological images of snus-induced lesions at visit 3 (A) and visit 5 (B), stained with hematoxylin and eosin, illustrating inflammatory cell infiltration and epithelial thickness

The thickness of the epithelial layer and the percentage of inflammatory cells were assessed in oral mucosal biopsies collected at Visit 3 and Visit 5 (Fig. 3). The analysis showed that neither the percentage of inflammatory cells (Fig. 4) nor the epithelial thickness (Fig. 5) changed significantly over this period in either the dry or moist formulation groups.

Fig. 4: Inflammatory cell presence in snus-induced lesions. The presence of inflammatory cells in biopsies from snus-induced lesions at visit 3 and visit 5 was quantified as the percentage of positively stained cells per selected tissue area. Statistical analysis was conducted using the Wilcoxon matched-pairs signed-rank test. The notation ‘ns’ indicates no statistically significant differences. n = 45 participants

Fig. 5: Epithelial thickness in snus-induced lesions at visit 3 and visit 5. Epithelial thickness (µm) was measured in biopsies from snus-induced lesions at visit 3 and visit 5. Statistical comparisons between visits were made using the Wilcoxon matched-pairs signed-rank test to assess changes in epithelial thickness over time. The notation ‘ns’ indicates no statistically significant differences. n = 45 participants

Measurement of total protein concentration in saliva

Total protein concentration in saliva was measured, and no statistically significant differences were observed between Visit 3 and Visit 5 in either group (Fig. 6).

Fig. 6: Total protein levels in saliva. Total protein levels were measured in saliva samples collected at visit 3 and visit 5. Statistical analysis was performed using the Wilcoxon matched-pairs signed-rank test to evaluate changes in protein concentration between visits. The notation ‘ns’ indicates no statistically significant differences. n = 45 participants

Measurement of cytokines in saliva

Cytokine concentrations in saliva were measured using a multiplex assay. Only mean cytokine levels within the detectable range of the standard curve are presented. Values below the lower limit of the standard curve were assigned the lowest standard value, while values above the upper limit were assigned the highest standard value. Cytokine concentrations were normalized to total protein concentration, and the results are shown in Fig. 7. No significant differences were observed in the levels of the pro-inflammatory cytokines interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), and the chemokine monocyte chemoattractant protein-1 (MCP-1) in saliva samples between Visit 3 and Visit 5. In contrast, levels of the chemokine interleukin-8 beta (IL-8) were significantly increased (p < 0.01) from Visit 3 to Visit 5, but only in the dry formulation group.

Fig. 7: Cytokine levels in saliva. Cytokine levels in saliva samples collected at visit 3 and visit 5 were measured. The concentrations of pro-inflammatory cytokines IL-1β, IL-8, TNF-α, and MCP-1 were quantified using a multiplexed bead-based immunoassay. Cytokine concentrations were normalized to total protein levels. Statistical analysis was conducted using the Wilcoxon matched-pairs signed-rank test. The notation ‘ns’ indicates no statistically significant differences. ** p < 0.01. n = 45 participants

Analysis of pattern of use

In the dry formulation group, the cumulative exposure time was 10.4 h at Visit 3 and decreased slightly to 10.1 h at Visit 5. In the moist formulation group, the cumulative exposure time was 10.7 h at Visit 3 and remained essentially unchanged at 10.6 h at Visit 5.

Safety and tolerability

Ad libitum use of dry formulation NPs and moist formulation NPs was found to be safe and well tolerated among current daily users of tobacco-based snus in this study. No deaths, serious adverse events, or withdrawals due to AEs were reported. Two participants (4.4%) reported a total of two AEs, both cases of the common cold. Both AEs were assessed as mild and unlikely to be related to the IPs, and they resolved within 4–5 days. No additional AEs were reported, and no differences in AE frequency were observed between the dry and moist formulation groups.