Interest in the toxicological assessment of iterations of e-cigarette devices, e-liquid formulations and flavour use is increasing. Here, we describe a multiple test matrix and in vitro approach to assess the biological impact of differing e-cigarette activation mechanism (button vs. puff-activated) and heating technology (cotton vs. ceramic wick). The e-liquids selected for each device contained the same nicotine concentration and flavourings. We tested both e-liquid and aqueous extract of e-liquid aerosol using a high throughput cytotoxicity and genotoxicity screen. We also conducted whole aerosol assessment both in a reconstituted human airway lung tissue (MucilAir) with associated endpoint assessment (cytotoxicity, TEER, cilia beat frequency and active area) and an Ames whole aerosol assay with up to 900 consecutive undiluted puffs. Following this testing it is shown that the biological impact of these devices is similar, taking into consideration the limitations and capturing efficiencies of the different testing matrices. We have contextualised these responses against previous published reference cigarette data to establish the comparative reduction in response consistent with reduced risk potential of the e-cigarette products tested in this study as compared to conventional cigarettes.

The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to develop recommendations for optimal scientific and technical approaches for conducting in vitro assays to assess potential toxicity within and across traditional tobacco and various tobacco and nicotine next-generation products (NGPs), including Heated Tobacco Products (HTPs) and Electronic Nicotine Delivery Systems (ENDS). This report was developed by a working group composed of attendees of the seventh IIVS workshop, 'Approaches and recommendations for conducting the mouse lymphoma gene mutation assay (MLA) and introduction to in vitro disease models', which was held virtually on 21-23 June 2022. This publication provides a background overview of the MLA, and includes the description of assay conduct and data interpretation, key challenges and recommended best practices for evaluating tobacco and nicotine products, with a focus on the evaluation of NGPs, and a summary of how the assay has been used to evaluate and compare tobacco and nicotine products.

The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to 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 products (NGPs) including heated tobacco products (HTPs) and electronic nicotine delivery systems (ENDSs). This publication was developed by a working group of the workshop members in conjunction with the sixth workshop in that series entitled “Dosimetry for conducting in vitro evaluations” and focuses on aerosol dosimetry for aerosol exposure to combustible cigarettes, HTP, and ENDS aerosolized tobacco products and summarizes the key challenges as well as documenting areas for future research.

Electronic nicotine delivery products (ENDPs) emit lower levels of toxicants than cigarettes, and, consequently, ENPD aerosols have the potential to be less toxic than cigarette smoke (CS). Toxicological assessment of ENDPs relies heavily on in vitro studies to verify this assertion. First, a battery of standardized and validated regulatory toxicology assays is used to verify that ENDP aerosols are less cytotoxic and genotoxic than CS. Second, a systems toxicology approach is employed to gain a deeper understanding of the biological mechanisms affected by CS. This knowledge then allows quantification of the effects of ENDP aerosols on the mechanisms perturbed by CS and identification of potential new effects of ENDP aerosols. While regulatory toxicology assays rely on standardized prokaryotic and mammalian cells, systems toxicology aims to employ in vitro systems derived from primary human cells and more closely recapitulate the key features of human tissues and mechanisms linked to smoking-related diseases. This chapter presents the experimental requirements, summarizes the results, and discusses the challenges of in vitro assessment of ENDPs.

Smokers are exposed to the harmful and potentially harmful constituents generated by cigarette combustion. Discriminating the independent effects of nicotine from those of other chemicals present in cigarette smoke is difficult. Nevertheless, investigating circumstances in which individuals are exposed to nicotine without combustion—as with the use of smokeless tobacco products, nicotine replacement therapies, and, to some extent, e-vapor products—provides evidence on the possible health effects of nicotine. Where epidemiological data are lacking, nonclinical studies may provide mechanistic insights into nicotine's ability to modulate molecular and cellular processes. This chapter summarizes the current knowledge about the effects of nicotine exposure on the circulatory system, lungs, female and male reproductive organs, and developing fetus and critically reviews nicotine's potential involvement in cardiovascular diseases, lung cancer, and developmental defects. This chapter also highlights key gaps in our current understanding of the biological effects of nicotine.

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 (R) 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.

Introduction: The Institute for In Vitro Sciences is sponsoring a workshop series to identify, discuss, and develop recommendations for optimal scientific and technical approaches for conducting in vitro assays—focusing first on genotoxicity assays—used for assessing potential toxicity within and across tobacco and electronic nicotine delivery product categories (hereafter referred to as tobacco products). Materials and Methods: Workshops provide a unique opportunity for invited expert stakeholders to share experiences and to develop recommendations that may serve as a resource for developing optimal testing approaches and data interpretation. It is envisioned that some recommendations would form the basis for the generation of guidance documents and/or serve as authoritative reference publications to support regulatory submissions. Results and Discussion: During the first workshop (November 27–28, 2018), workgroup members identified important issues for using in vitro genotoxicity assays for evaluating tobacco products. These issues were triaged into three priority categories that will provide the basis for selecting high-priority topics for subsequent workshops. To provide background for future workshops and to serve as a scientific community resource, the workgroup developed a tabulated referenced summary of the types of tobacco product test samples that have been evaluated using the regulatory genotoxicity assays and the types of regulatory questions that have been addressed. A touch-base meeting was held March 7, 2019, and a second workshop June 4–5, 2019 to discuss ongoing issues and to further organize workgroup activities. Conclusion: The current publication lists priority topics and background summary information for using regulatory genotoxicity assays to evaluate tobacco products.

Tobacco-free 'modern' oral nicotine pouches (MOPs), are similar in appearance and use to Swedish-style snus, but without tobacco. There are few identified methods to create test samples for toxicologically assessment of MOPs in vitro. In this study we present a simple method for the extraction of pouch material in cell culture media, providing consistent nicotine concentration and easy in vitro assessment. A series of contemporary in vitro screening assays (viability, cell health markers, oxidative stress and genotoxicity) using human oral fibroblasts (HGF) and human lung epithelial cells (H292) were employed. Extracts were generated from LYFT and compared to snus (CRP1.1) and cigarette (1R6F) reference products. MOP and CRP1.1 extracts were generated by incubating one pouch in 20 ml of cell culture media, while 1R6F AqE was prepared by smoking 1 cigarette into 20 ml of cell culture media. 1R6F demonstrated toxicological responses in most assays; CRP1.1 had minimal to moderate effects while MOP demonstrated little or no response in all assays. This study demonstrated the generation of MOPs extracts and their toxicological evaluation using in vitro screening approaches. Future product usage, pharmacokinetics and clinical studies will further substantiate the reduced risk potential of MOPs.

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.

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) trimethylammonium 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 gamma H2AX, 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-a-vis nature may facilitate further comprehension of the range of effects that can manifest in human cells in response to DNA-damaging agents.

Electronic nicotine delivery systems (ENDSs; e.g. e-cigarettes) are being developed as potentially reduced-risk alternatives to the continued use of conventional tobacco products. They typically comprise a device that heats an e-liquid to generate inhalable vapor. E-liquids and ENDS-derived vapor have been the focus of toxicological assessment; in particular, their DNA-damaging potential has been investigated with varying outcomes and conclusions. In vitro genetic toxicology assays have formed a part of these assessments. However, they are susceptible to producing misleading or false positive results, especially under extreme conditions. In the present study, we evaluated a series of six neat (non-vaporized) non-flavored e-liquids (NFEL-A to F) in a flow cytometry version of the in vitro micronucleus assay in order to characterize their baseline effects on Chinese hamster ovary cells under hazard identification conditions. The NFELs induced cytotoxicity universally despite differing in propylene glycol (PG), vegetable glycerin (VG), and nicotine content. In addition, significant genotoxic responses were also detected with the PG-predominant e-liquids NFEL-A, D, and F but not with NFEL-B, C, or E, which contained higher proportions of VG. All six NFELs induced extreme cell culture conditions (i.e. increases in pH and osmolality) at the concentrations assessed. They also exhibited nonbiologically relevant effects on the mechanistic endpoints (i.e. cell cycle and phosphorylated histones H2AX and H3). In conclusion, although the PG component of the NFELs drove micronucleus formation in the assay, data on the complementary mechanistic endpoints suggest that this apparent DNA damage is potentially misleading and of negligible biological relevance as a risk for DNA integrity. In future assessments, any adverse changes (such as signatures of micronuclei induction, G 2 M arrest, and increases in γH2AX) relative to this reference data set might indicate a possible genotoxic hazard and would prompt further investigations for exploring the extent of risk.

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.

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.

Emergent diseases caused by viral and bacterial infections have proven to be a current and future challenge. The occurrence of these diseases is usually accompanied by the lack of vaccines and dedicated therapies leaving prevention as the best strategy to adopt. In that context, and apart from confinement and physical distancing measures, an increase in hygiene actions, namely hand and surface cleaning and disinfection can reduce the infection spread originated from our day-to-day routines. However, during crisis situations the high disinfectants demand can very likely lead to having them running out of stock. This impels many individuals and companies to produce their own disinfectants. Here, we explore the main components of a disinfection formulation, both for hand-rub and surface cleaning. Alcohol and non-alcohol based formulations are described, including the possibility to fine tune the properties of the final product in order to increase public acceptance while maintaining product efficacy. The action mechanisms of the main active principles are also described conjugating information from experimental and theoretical data. Overall, the main aspects to develop a disinfectant formulation are addressed, as well as their function, helping formulation developers to better understand the impact of their choices.

LIPE is an intracellular neutral lipase, which is capable of hydrolyzing a variety of esters and plays a key role in the mobilization of fatty acids from diacylglycerols. The objectives of this study were to characterize the genetic polymorphism of bovine LIPE gene and to evaluate the possible association between three SNPs in the coding regions of this gene with the fatty acid composition of meat in a cattle population. Forty-three unrelated animals from different cattle breeds were re-sequenced and 21 SNPs were detected over approximately 2600 bp, five of these SNPs were novel. Three SNPs were selected, on the basis of evolutionary conservation, to perform validation and association studies in a crossbred cattle population. Our results may suggest a possible association of SNP1 with contents of oleic acid and total monounsaturated fatty acids (p < 0.01), and SNP2 and SNP3 with Heneicosylic acid content (p < 0.01), may be helpful to improve the quality of meat and improve health.

The chemical composition, in vitro genotoxicity, and cytotoxicity of the mainstream aerosol from the Tobacco Heating System 2.2 (THS2.2) were compared with those of the mainstream smoke from the 3R4F reference cigarette. In contrast to the 3R4F, the tobacco plug in the THS2.2 is not burnt. The low operating temperature of THS2.2 caused distinct shifts in the aerosol composition compared with 3R4F. This resulted in a reduction of more than 90% for the majority of the analyzed harmful and potentially harmful constituents (HPHCs), while the mass median aerodynamic diameter of the aerosol remained similar. A reduction of about 90% was also observed when comparing the cytotoxicity determined by the neutral red uptake assay and the mutagenic potency in the mouse lymphoma assay. The THS2.2 aerosol was not mutagenic in the Ames assay. The chemical composition of the THS2.2 aerosol was also evaluated under extreme climatic and puffing conditions. When generating the THS2.2 aerosol under "desert" or "tropical" conditions, the generation of HPHCs was not significantly modified. When using puffing regimens that were more intense than the standard Health Canada Intense (HCI) machine-smoking conditions, the HPHC yields remained lower than when smoking the 3R4F reference cigarette with the HCI regimen.

Mutagenesis in S. typhimurium and in vitro induction of DNA single-strand breaks in primary rat hepatocytes (DNA-SSB) have been investigated for two new N-nitroso compounds, mononitrosocaffeidine (MNC) and dinitrosocaffeidine (DNC). Mononitrosamidocaffeidine (MNAC) and tert.-(butyloxy)carbonyl-mononitrosamidocaffeidine (t-BOC-MNAC), both nitrosated derivatives of caffeidine with nitrosation at methylcarboxamide-N only, were also similarly studied. MNC, an asymmetric nitrosamine, failed to show mutagenicity in any of the tester strains used, and also did not induce DNA-SSB in rat hepatocytes. DNC, having both N-nitrosamide and N-nitrosamine groups in the molecule, showed direct mutagenicity in TA100, TA1535 and TA102. The mutagenic potential of the compound was found to increase on S9 activation. However, it was non-mutagenic in TA98 and TA1537. DNC also exhibited a high potential for inducing alkali-labile DNA-SSB in rat hepatocytes (70–78% C-T value) and was cytotoxic at concentrations over 0.1 μmole/ml. Both MNC and DNC were found to produce formaldehyde on S9 activation. MNAC was not mutagenic directly but showed weak mutagenicity on metabolic activation, whereas t-BOC-MNAC was mutagenic both with and without S9 activation in TA100, TA1535 and TA102. t-BOC-MNAC was more cytotoxic to hepatocytes than MNAC, though both caused DNA-SSB to the same extent (62% C-T value). On the basis of the presented data it is inferred that while DNC is a direct-acting mutagen in TA100, TA1535 and TA102 due to the presence of a reactive N-methylnitrosamido group, its mutagenic potential is greatly enhanced in the presence of S9 possibly due to the synergistic influence of an activated N-methylnitrosamino group in the molecule. Additionally, the study shows a qualitative consistency between Salmonella mutagenicity, genotoxicity in hepatocytes and the reactivity of the methyl group at the nitrosamido-N in nitrosated caffeidine compounds.