In vitro toxicological evaluation of pouched portioned oral nicotine products

Abstract

Introduction: Modern oral nicotine pouch products (ONPs) are a category of oral nicotine products which contain pharmaceutical-grade nicotine, flavors, and other food-grade ingredients but no tobacco leaf. Recent reports indicate that ONPs in general do not contain (or only at minimal levels) the harmful and potentially harmful constituents (HPHCs) identified in cigarette smoke, suggesting their potential as alternative products for reducing harm from cigarette smoking.

Methods: We assessed in vitro toxicological effects of eight ONPs, designated as modern oral (MO) 1 to 8 along with an ONP, an oral tobacco (snus), and a combustible cigarette market comparator using established regulatory toxicological assays including Ames, in vitro micronucleus (ivMN), and neutral red uptake (NRU) assays.

Results: The MO test products 1-7 ZYN wintergreen, and General Snus white mint were negative for mutagenicity (Ames assay), genotoxicity (ivMN), and cytotoxicity (NRU). The combustible cigarette was positive in all three assays. The MO-8 test product was negative for mutagenicity; however, it was positive in the ivMN and NRU assays at concentrations either 42 to 135-fold based on the ivMN i to iv treatment schedule or 60-fold higher, respectively, when compared to combustible cigarettes.

Discussion: Thus, the MO test products are likely to be less harmful than combustible cigarettes and are alternatives to cigarettes. However, understanding of long-term effects of ONPs in general requires additional research.

Keywords: cytotoxicity; genotoxicity; in vitro toxicity assays; modern oral nicotine pouches; mutagenicity.

FIGURE 1

Ames Assay results (preincubation method) in the presence (A, C, E) and absence (B, D) of metabolic activation (±S9). Methods based on HC T-501 and OECD 471 guidelines. Results from Salmonella tester strains TA98 (±S9) (A, B), TA100 (±S9) (C, D), and TA1537 (+S9) (E) are displayed. Data not shown for Salmonella tester strains TA102 (±S9), TA1535 (±S9), or TA1537 (-S9) because no mutagenic activity was observed from any of these test items. Combustible cigarette (TPM) was mutagenic in the three strains shown, with dose-related increases exceeding the historical spontaneous revertant count range (-- - -). ONP and snus (CAS) test items consistently showed no mutagenic activity in all five tester strains at nicotine equivalent concentrations that were considerably higher than the combustible cigarette. ONP and snus revertant counts consistently fell within the spontaneous revertant historical range for each tester strain. Tested doses are based on the amount of nicotine equivalents (µg/plate) from the different sample preparations (TPM, CAS). Results (mean ± SD) from three (3) independent experiments.

FIGURE 2

In vitro micronucleus (ivMN) assay results (A, C, E, G) observed under four exposure schedules (i–iv). ivMN methods (without cytochalasin B) based on HC T-503 and OECD 487 guidelines. Schedule iv, 24 h exposure (-S9) with a 24-h recovery prior to harvesting, referenced from Thorne et al. (2019). Graphs (B, D, F, H) display the observed cytotoxicity (based on the relative increase in cell count: RICC) over the same dose range. Combustible cigarette (TPM) displayed genotoxicity in all four exposure schedules, indicated by the dose-related increase in micronuclei (MN) induction. ONP and snus (CAS) exposures resulted in no overall genotoxicity, with the exception of MO-8, which induced MN in all four exposure schedules. ivMN results (mean ± SD) from three (3) independent experiments.

FIGURE 3

NRU results for Marlboro Gold (TPM) and MO 1–8, ZYN wintergreen, and General Snus white mint (CAS extraction) test items (A, C). Osmolality (B, D) was concurrently measured following the exposure, and the osmolality difference from vehicle control was calculated. Dotted lines at ±20% (C, D) were used to account for any potential cytotoxic effects induced by osmotic stress. Marlboro Gold TPM and MO-8 CAS extract resulted in cytotoxic responses with a calculated IC₅₀ value based on delivery of 3.3 µg nicotine equivalents/mL and 180 µg nicotine equivalents/mL, respectively. No indication of cytotoxicity was observed for Market Snus and MO 1–7 test items up to the maximum deliverable doses. Results (mean ± SD) are from three (3) independent experiments.