Toxicological Assessment of Highly Mentholated Reduced-Risk Tobacco Products in Sprague Dawley Rats Following Sub-Chronic Inhalation Exposure

      Ho, J.; Oviedo, A.; Patrice, L.; Garcia, L.; Lebrun, S.; Tung, C. K.; Yeo, D.; Chng, Y. X.; Phillips, B. W.; Peitsch, M.; Hoeng, J.; Vanscheeuwijck, P.

      Conference date
      Oct 1, 2021
      Conference name
      EUROTOX 2021

      Menthol is a flavor that is commonly used additive in many products, including conventional cigarettes and other inhaled products (e.g. e-cigarettes and heated tobacco products). It is therefore important to assess potential toxicological effects of inhaled menthol in the context of heated tobacco products. This study characterized the toxicity related to highly mentholated (ranging from 1.3 to 4.2 mg menthol/stick) Tobacco Heating System (THS) compared with fresh air control, non-mentholated THS as well as two conventional reference cigarettes (3R4F and 1R6F) in a sub-chronic inhalation exposure study using Sprague Dawley rats following the OECD Test Guideline 413. Three concentrations of ‘THS high menthol’ aerosols (15, 23 and 50 μg nicotine/L) were delivered by nose-only exposure (6 hours/day, 5 days/week for 13 weeks) to the rats, while additional rats of selected groups were included to observe reversibility of any effects after 42 days post-exposure period. All test atmospheres were well tolerated by the rats. Respiratory minute volume and breathing frequency (both sexes) increased in a concentration-dependent manner in response to menthol levels up to a certain level (THS high menthol), after which the values for these parameters started to decrease. Pulmonary inflammation, determined by quantification of free-lung cells, and of inflammatory cytokines in cell-free bronchoalveolar lavage fluid, were lower in the THS (mentholated or non-mentholated) exposure groups compared to the conventional reference cigarettes smoke, and without any effects of menthol, which was confirmed following histopathological evaluation of the lungs, showing minimal inflammation in the THS-exposed rats. Histopathology of respiratory organs paralleled these observations and further confirmed minimal sub-chronic effects for THS groups, which is consistent with lower content of irritants in the aerosols. Microscopic findings in respiratory tract organs including epithelial cell hyperplasia and squamous metaplasia were reduced in THS-exposed groups as compared with conventional reference cigarettes groups at equivalent nicotine concentration. Findings in the non-respiratory tract organs were considered generally low incidence and severity, while exposure-related effects were not observed in the THS-exposed groups. Clinical pathological changes following exposure to aerosols of THS, such as higher blood neutrophil counts, elevated liver enzymes and lower energy metabolism parameters were observed, all of which reversible following recovery period, and likely represent a nicotine-effect. In general, only minimal biological effects were seen following exposure to the mentholated THS aerosols. Despite the high levels of menthol in the aerosols (equivalent up to 116 mg nicotine and 360 mg menthol per day based on human body surface area), there were no additional toxicity effects compared with conventional reference cigarettes or non-mentholated THS.