Acute and subacute inhalation toxicity assessment of WS‐23 in Sprague–Dawley rats

Abstract

2‐isopropyl‐N,2,3‐trimethylbutyramide (WS‐23) is a well‐known artificial synthesis cooling agent widely used in foods, medicines, and tobaccos. As a commonly cooling agent in e‐cigarette liquids, WS‐23 has led to concerns about the inhalation toxicity with the prosperous of e‐cigarettes in recent years. Thus, the aim of this study is to assess the acute and subacute inhalation toxicity of WS‐23 in Sprague–Dawley (SD) rats according to the Organization for Economic Cooperation and Development (OECD) guidelines. In the acute toxicity study, there was no mortality and behavioral signs of toxicity at the limit test dose level (340.0 mg/m3) in the exposure period and the following 14‐day observation period. In the subacute inhalation toxicity study, there was no significant difference observed in the body weights, feed consumption, and relative organ weights. Haematological, serum biochemical, urine, and bronchoalveolar lavage fluid (BALF) analysis revealed the non‐adverse effects after 28‐day repeated WS‐23 inhalation (342.85 mg/m3), accompanied by slight changes in few parameters which returned to normal during the 28‐day recovery period. The histopathologic examination also did not show any differences in vital organs. In conclusion, the maximum tolerated dose for WS‐23 acute inhalation is not less than 340.0 mg/m3, and the No Observed Adverse Effect Level (NOAEL) of WS‐23 subacute inhalation was determined to be over 342.85 mg/m3.

Ice flavours and non-menthol synthetic cooling agents in e-cigarette products: a review

“…Cellular genotoxic assays observed clastogenic effects of WS-23, inducing breaks in chromosomes on metabolic activation 53. A 4-week WS-23 rat inhalation toxicity study conducted by an e-cigarette company (RLX Technology, China) that administered 19 mg/kg body weight/day and 29 mg/kg body weight/day detected exposure-related changes for several lung function parameters 58. Across these studies, no-observed-adverse-effect levels for WS-23 and WS-3 were determined to be 5 mg/kg body weight/day and 8 mg/kg body weight/day, respectively.…”

Synthetic Cooling Agents in US-marketed E-cigarette Refill Liquids and Popular Disposable E-cigarettes: Chemical Analysis and Risk Assessment

“…Recently, an e-vapor product company (RLX technology, China) has conducted a nose-only acute and sub-acute inhalation study for WS-23 (28-day exposure at doses of 19 and 29 mg/kg-bw/day) 24 . Whole-body plethysmography was conducted after 28 days of repeated exposure to measure various parameters of pulmonary function.…”

“…Exposure of airway-epithelial cells in-vitro to various concentrations of synthetic coolants and WS-23 containing e-liquids also demonstrated a dose-dependent cellular toxicity 15 . A recent inhalation exposure study in rodents by an E-liquid manufacturer for WS-23 determined a NOAEL of 29 mg/kg-bw (342 mg/m3) in rodents 24 . Due to their evident toxicity and increased use in E-cigarettes, it is critical to evaluate the potential health risks associated with synthetic coolant exposure from E-cigarette use.…”

“…. A recent inhalation exposure study in rodents by an E-liquid manufacturer for WS-23 determined a NOAEL of 29 mg/kg-bw (342 mg/m3) in rodents 24 . Due to their evident toxicity and increased use in E-cigarettes, it is critical to evaluate the potential health risks associated with synthetic coolant exposure from E-cigarette use.…”

Synthetic Coolant WS-23 increases E-Cigarette Generated Aerosolized Acellular Reactive Oxygen Species (ROS) Levels

“…In rodent studies, rats exposed to aerosolized e-liquid containing WS-23 at tested doses (via acute and subacute exposures) found no substantial changes in histopathologic analyses of vital organs nor relative organ weights(Wu, Liu et al 2021). This same study, via a bronchioalveolar lavage fluid (BALF) analysis, found no significant difference in neutrophil concentration between rats which had undergone repeated 28-day WS-23 exposure and those apart of the respective control group(Wu, Liu et al 2021).Neutrophils are major sources of endogenous ROS production.Future studies aimed at understanding the role of WS-23 in modulating e-cig-induced oxidative stress should involve measurements of intracellular and extracellular ROS using isolated Polymorphonuclear Neutrophils (PMNs)(Kuhns, Priel et al 2015). More specifically, PMNs isolated from blood collected from mice exposed to aerosolized e-liquids of varying WS-23 concentrations can be analyzed via luminol enhanced chemiluminescence exposure(Kuhns, Priel et al 2015).…”