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The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to identify, discuss and develop recommendations for optimal scientific and technical approaches for conducting in vitro assays, to assess potential toxicity within and across tobacco and various next generation nicotine and tobacco products (NGPs), including heated tobacco products (HTPs) and electronic nicotine delivery systems (ENDS). The third workshop (24-26 February 2020) summarised the key challenges and made recommendations concerning appropriate methods of test article generation and cell exposure from combustible cigarettes, HTPs and ENDS. Expert speakers provided their research, perspectives and recommendations for the three basic types of tobacco-related test articles: i) pad-collected material (PCM); ii) gas vapour phase (GVP); and iii) whole smoke/aerosol. These three types of samples can be tested individually, or the PCM and GVP can be combined. Whole smoke/aerosol can be bubbled through media or applied directly to cells at the air-liquid interface. Summaries of the speaker presentations and the recommendations developed by the workgroup are presented. Following discussion, the workshop concluded the following: that there needs to be greater standardisation in aerosol generation and collection processes; that methods for testing the NGPs need to be developed and/or optimised, since simply mirroring cigarette smoke testing approaches may be insufficient; that understanding and quantitating the applied dose is fundamental to the interpretation of data and conclusions from each study; and that whole smoke/aerosol approaches must be contextualised with regard to key information, including appropriate experimental controls, environmental conditioning, analytical monitoring, verification and performance criteria.

Electronic nicotine delivery systems (ENDS) are being developed as potentially reduced-risk alternatives to the continued use of combustible tobacco products. Because of the widespread uptake of ENDS-in particular, e-cigarettes-the biological effects, including the toxic potential, of their aerosols are under investigation. Preclinically, collection of such aerosols is a prerequisite for testing in submerged cell culture-based in vitro assays; however, despite the growth in this research area, there is no apparent standardized collection method for this application. To this end, through an Institute for in vitro Sciences, Inc. workshop initiative, we surveyed the biomedical literature catalogued in PubMed® to map the types of methods hitherto used and reported publicly. From the 47 relevant publications retrieved, we identified seven distinct collection methods. Bubble-through (with aqueous solvents) and Cambridge filter pad (CFP) (with polar solvents) collection were the most frequently cited methods (57% and 18%, respectively), while the five others (CFP + bubble-through; condensation; cotton filters; settle-upon; settle-upon + dry) were cited less often (2-10%). Critically, the collected aerosol fractions were generally found to be only minimally characterized chemically, if at all. Furthermore, there was large heterogeneity among other experimental parameters (e.g., vaping regimen). Consequently, we recommend that more comprehensive research be conducted to identify the method(s) that produce the fraction(s) most representative of the native aerosol. We also endorse standardization of the aerosol generation process. These should be regarded as opportunities for increasing the value of in vitro assessments in relation to predicting effects on human health., (© 2020 The Authors. Journal of Applied Toxicology published by John Wiley & Sons Ltd.)

In vitro genetic toxicology assays are used to assess the genotoxic potential of chemicals or mixtures. They measure chromosome damage (e.g., micronucleus [MN] formation) or gene mutation, and different combinations of data generated from such assays are evaluated in concert in order to identify genotoxic hazards. Mode-of-action (MoA) information is also fundamental to understanding any apparent genotoxic response. In view of the importance of these types of data for full characterization of genotoxic potential, we leveraged relevant endpoints already established in the human TK6 cell line to develop a single integrated assay that measures MN formation, gene mutation (at the thymidine kinase locus), and MoA (DNA damage response biomarkers). Several prototypical direct-acting genotoxins (methyl methanesulfonate, mitomycin C, and 4-nitroquinoline 1-oxide), pro-genotoxins (benzo[a]pyrene and cyclophosphamide monohydrate), and one non-DNA reactive genotoxin (vinblastine sulfate) were assessed in the approach and found to elicit genotoxic profiles that were generally consistent with their MoA. In contrast, the non-genotoxic agents D-mannitol and (2-chloroethyl) trimethyl-ammonium chloride induced negligible effects on all endpoints up to a top concentration of 10 mM. Sodium diclofenac, presumed to be non-genotoxic, provoked an induction in the phosphoserine 10 -H3-positive cell population within a small window of concentrations (0.157-0.314 mM), as well as increases in γH2AX, nuclear p53, and MN at higher concentrations, although it had no effect on the mutation frequency endpoint. G 2 M cell cycle arrest was also largely observed in cells that exhibited genotoxicity in the in vitro MN assay. The TK6 cell-based integrated assay represents an in vitro approach that permits comprehensive genotoxicity analysis in a human-relevant test system. Moreover, its vis-à-vis nature may facilitate further comprehension of the range of effects that can manifest in human cells in response to DNA-damaging agents., Competing Interests: Declaration of Competing Interest All authors are employees of Philip Morris International., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)

Nicotine's genotoxic potential has been extensively studied in vitro. While the results of mammalian cell-based studies have inferred that it can potentially damage chromosomes, in general and with few exceptions, adverse DNA effects have been observed primarily at supraphysiological concentrations in nonregulatory assays that provide little information on its mode-of-action (MoA). In this study, a modern-day regulatory genotoxicity assessment was conducted using a flow cytometry-based in vitro micronucleus (MN) assay, Good Laboratory Practice study conditions, Chinese hamster ovary cells of known provenance, and acceptance/evaluation criteria from the current OECD Test Guideline 487. Nicotine concentrations up to 3.95 mM had no effect on background levels of DNA damage; however, concentrations above the point-of-departure range of 3.94-4.54 mM induced increases in MN and hypodiploid nuclei, indicating a possible aneugenicity hazard. Follow-up experiments designed to elucidate nicotine's MoA revealed cellular vacuolization, accompanying distortions in microtubules, inhibition of tubulin polymerization, centromere-positive DNA, and multinucleate cells at MN-inducing concentrations. Vacuoles likely originated from acidic cellular compartments (e.g., lysosomes). Remarkably, genotoxicity was suppressed by chemicals that raised the luminal pH of these organelles. Other endpoints (e.g., changes in phosphorylated histones) measured in the study cast doubt on the biological relevance of this apparent genotoxicity. In addition, three major nicotine metabolites, including cotinine, had no MN effects but nornicotine induced a nicotine-like profile. It is possible that nicotine's lysosomotropic properties drive the genotoxicity observed in vitro; however, the potency and mechanistic insights revealed here indicate that it is likely of minimal physiological relevance for nicotine consumers. Environ. Mol. Mutagen. 2019. © 2019 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society., (© 2019 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.)

Cytotoxicity assays are used to quantify the cytotoxic potential of chemicals. The neutral red uptake (NRU) assay is one of these assays and is routinely used in the pharmaceutical, cosmetic, and tobacco industries. In the context of e-cigarette development, an NRU assay-based screen was implemented to evaluate the cytotoxic potential of e-liquids. E-liquids induced a biphasic response in the BALB/c 3T3-based assay. The NRU initially increased in a concentration-dependent manner before decreasing following treatment with higher concentrations until NRU was abolished. Experiments were performed to characterize the mechanism underlying this biphasic signal. Nicotine alone was found to induce the same biphasic effects, while inducing concentration-dependent decreases in relative cell counts (RCC). Imaging and flow cytometry data revealed that the increases in NRU likely resulted from nicotine-induced vacuolization via a lysosomotropic mechanism. In support of this, two lysosomotropic agents, chloroquine and lapatinib, induced similar profiles. Nicotine's effects were also translatable, as brain-, lung-, bone marrow-, and smooth muscle-derived mammalian cells responded with the biphasic NRU signal. However, like RCC, three other cytotoxicity endpoints, resazurin, adenosine triphosphate, and water soluble tetrazolium salt (WST)-8, were not subject to these effects. The WST-8 assay is proposed as an alternative to screen the cytotoxic potential of e-liquids., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This study investigated the relationship between vertical and horizontal measures in bilateral and unilateral countermovement jump, drop jump and squat jump (SJ), and sprinting speed and muscle architecture of both the vastus lateralis and gastrocnemius. Subjects (n = 17) completed a 30-m sprint test, muscle stiffness test; ultrasound measures, and a jump testing session. Measures of horizontal peak and mean force, in both bilateral and unilateral jumps, tended to have greater relationships to sprint speeds (R = 0.132-0.576) than peak and mean force in the vertical plane (R = 0.008-0.504). Vertical velocity variables also showed some large and very large correlations to sprint speed (R = 0.062-0.635). Unilateral measures of velocity tended to have larger correlations to sprint performance than their bilateral counterparts across all jump types and peak and mean velocity in SJ showed large and very large correlations to sprint speed (bilateral R = 0.227-0.635; unilateral 0.393-0.574). Few large correlations were shown between muscle stiffness measures of muscle architecture and kinetic and kinematic variables in either vertical or horizontal jumps. The present findings suggest that sport scientists and strength and conditioning practitioners concerned with the prognostic value of kinetic variables to functional movements such as sprint speed should also use horizontal jumps in addition to vertical jumps in testing and training.

The purpose of this study was to investigate the chronic effect of a contrast training program designed to elicit an acute short term enhancement (STE) effect during training. A matched pairs training study design was implemented with a contrast (STE affected) and complex (control) training group completing a seven week training intervention. Twenty subjects participated. The contrast training group completed training based on a preloading protocol that had previously been shown to induce an acute STE effect within the subject population. The control group completed the same volume and type of training in a complex training format. Changes in squat 4RM strength and kinetic and kinematic performance in vertical and horizontal countermovement jump (CMJ) and drop jump (DJ) were measured via a Force Plate. Differences between the experimental and control group in change of mean strength (effect size 0.03 ± 0.33), vertical DJ (effect size: contact time -0.22 ± 0.52; peak force 0.20 ± 0.30; mean force 0.30 ± 0.74) and horizontal DJ (effect size: contact time -0.47 ± 0.73; peak force 0.03 ± 0.36; mean force 0.13 ± 0.56) were not meaningful. However, differences in mean change of vertical and horizontal CMJ measures of force (effect size range 0.40 - 0.46 ± 0.37 - 0.63), vertical CMJ peak velocity (effect size 0.84 ±0.66) and mean velocity (0.62 ± 0.88) were meaningful. These findings demonstrate that eliciting an acute STE response in dynamic training movements through contrast training can produce a chronic improvement in dynamic movements as a training effect.