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Heated tobacco products (HTPs) are non-combustible, inhaled tobacco products that generate an aerosol with fewer and lower levels of toxicants, with a potential to reduce risk relative to cigarette smoking. Here, we assessed in vitro toxicological effects of three menthol (glo neo neoCLICK, neo Smooth Menthol and Fresh Menthol) and one non-menthol (neo Smooth Tobacco) variants of glo HTP, along with market comparators for cigarettes and HTPs. Limited chemical characterization of the study products revealed significantly lower levels of acetaldehyde, acrolein, crotanaldehyde and formaldehyde in test samples from HTPs than those from cigarettes. The glo HTPs were non-mutagenic in the bacterial reverse mutagenesis assay. Although, the whole aerosol exposures of glo HTPs were classified as genotoxic in the in vitro micronucleus assay, and cytotoxic in the NRU (monolayer) and MTT (3 dimensional EpiAirway™ tissues) assays, the cigarette comparators were the most toxic study products in each of these assessments. Further, glo HTPs elicited oxidative stress responses only at the highest dose tested, whereas the cigarette comparators were potent inducers of oxidative stress at substantially lower doses in the EpiAirway tissues. The comparator (non-glo) HTP results were similar to the glo HTPs in these assays. Thus, the glo HTPs exhibit substantially lower toxicity compared to cigarettes., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Brian M Keyser reports financial support was provided by Reynolds American Inc. I, RL, JW, KJ, KZ, and PM are full time employees of RAI Services Company which is part of Reynolds American Inc. Brian M Keyser reports a relationship with Reynolds American Inc that includes: employment. Brian Keyser, Robert Leverette, John Wertman, Kristen Jordan, Ken Szeliga, and Patrudu Makena are full time employees of RAI Services Company (RAIS). Thomas Shutsky and Reagan McRae are former employees of RAIS and were full time employees during the conduct of these studies. RAIS is a wholly owned subsidiary of Reynolds American, Inc., which is a wholly owned subsidiary of British American Tobacco plc (BAT). The data presented in this manuscript were generated and analyzed in studies commissioned by RAI Services Company and conducted under contract at LabCorp Early Development Laboratories Ltd or Labstat International Inc. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Assessment of in vitro cytotoxicity is an important component of tobacco product toxicological evaluations. However, current methods of regulatory testing involve exposing monolayer cell cultures to various preparations of aerosols from cigarettes or other emerging products such as electronic nicotine delivery systems (ENDS), which are not representative of human exposure. In the present study, a whole aerosol (WA) system was used to expose lung epithelial cultures (2D and 3D) to determine the potential of six Vuse Alto ENDS products that varied in nicotine content (1.8%, 2.4%, and 5%) and flavors (Golden Tobacco, Rich Tobacco, Menthol, and Mixed Berry), along with a marketed ENDS and a marked cigarette comparator to induce cytotoxicity and oxidative stress. The WA from the Vuse Alto ENDS products was not cytotoxic in the NRU and MTT assays, nor did it activate the Nrf2 reporter gene, a marker of oxidative stress. In summary, Vuse Alto ENDS products did not induce cytotoxic or oxidative stress responses in the in vitro models. The WA exposures used in the 3D in vitro models described herein may be better suited than 2D models for the determination of cytotoxicity and other in vitro functional endpoints and represent alternative models for regulatory evaluation of tobacco products.

N-Ethyl-N-nitrosourea (ENU) was evaluated as part of the Stage III trial for the rat Pig-a gene mutation assay. Groups of six- to eight-week-old male Sprague Dawley (SD) or Fischer 344 (F344) rats were given 28 daily doses of the phosphate buffered saline vehicle, or 2.5, 5, or 10 mg/kg ENU, and evaluated for a variety of genotoxicity endpoints in peripheral blood, spleen, liver, and colon. Blood was sampled predose (Day-1) and at various time points up to Day 57. Pig-a mutant frequencies were determined in total red blood cells (RBCs) and reticulocytes (RETs) as RBC(CD592-) and RET(CD592-) frequencies. Consistent with the results from a reference laboratory, RBC(CD592-) and RET(CD592-) frequencies increased in a dose and time-dependent manner, producing significant increases at all doses by Day 15, with similar frequencies seen in both rat strains. ENU also induced small but significant increases in % micronucleated RETs on Days 4 and 29. No significant increases in micronuclei were seen in the liver or colon of the ENU-treated SD rats. Hprt and Pig-a lymphocyte mutation assays conducted on splenocytes from Day 56 F344 rats detected two- to fourfold stronger responses for Hprt than Pig-a mutations. Results from the in vivo Comet assay in SD rats at Day 29 showed generally weak increases in DNA damage in all tissues evaluated. The results with ENU indicate that the Pig-a RET and RBC assays are reproducible, transferable, and complement other genotoxicity endpoints that could potentially be integrated into 28-day repeat dose rat studies.

In recent years, experimental evidence has accumulated that supports the existence of sublinear dose-response relationships at low doses of DNA reactive mutagens. However, creating the in vivo data necessary to allow for a more detailed dose-response modeling with the currently available tools might not always be practical. The purpose of the current work was to evaluate the utility of the Pig-a gene mutation assay to rapidly identify dose-response relationships for direct acting genotoxicants. The induction of mutations in the peripheral blood of rats was evaluated following 28 days of exposure down to low doses of the direct acting alkylating agents ethyl methane sulfonate (EMS) and ethylnitrosourea (ENU). Using statistical modeling based on the 28-day studies, a threshold for mutation induction for EMS was estimated to be 21.9mg/kg, whereas for the more potent ENU, the threshold was estimated to be 0.88mg/kg. Comparing mutation frequencies from acute and sub-chronic dosing indicated less than additive dose-response relationships, further confirming the possibility of a threshold dose-response relationship for both compounds. In conclusion, the work presented provides evidence that the Pig-a assay might be a practical alternative to other in vivo mutation assays when assessing dose-response relationships for direct acting mutagens and that an experimental approach using fractionated dosing could be used to substantiate a biological mechanism responsible for the observation of a sublinear dose-response relationship., (2011 Elsevier B.V. All rights reserved.)

The in vivo bone marrow (BM) micronucleus assay is one of the three tests in the standard test battery to assess the genotoxic potential of a pharmaceutical candidate. In some cases, depending on results of in vitro studies, the route of administration or the degree of systemic exposure, in vivo assessment of genotoxicity in the BM alone may not be sufficient. Based on the potential for high gut exposures to orally administered compounds with low systemic exposures as well as the potential susceptibility of rapidly dividing cells of the intestinal tissues, we have developed a modified technique for evaluating micronuclei formation in both the duodenum and colon of rats based on earlier publications. Adult male Sprague Dawley rats were treated once daily for 2 days with either vehicle control or with the test articles acetyl salicylic acid (ASA), carbendazim (CAR), cyclophosphamide (CP), dimethylhydrazine (DMH), mitomycin C (MMC) or vinblastine sulfate (VIN). The duodenum, colon, and BM were harvested, processed, and analyzed for micronucleus induction. Results from these studies demonstrated differences in the susceptibility for different test compounds in the three tissues tested. When MMC and VIN were dosed by different routes at the same dose levels both compounds produced positive results in all three tissues by intraperitoneal injection but not oral administration. These studies suggest that overall the GI micronucleus assay might be a useful tool for clastogenic and aneugenic compounds that are expected to produce high sustained concentrations in the gastrointestinal tract with little systemic exposure., (Published 2010 Wiley-Liss, Inc.)