toxicological evaluation of pouched portioned oral nicotine products.

Introduction: Modern oral nicotine pouch products (ONPs) are a category of oral nicotine products which contain pharmaceutical-grade nicotine, flavors, and other food-grade ingredients but no tobacco leaf. Recent reports indicate that ONPs in general do not contain (or only at minimal levels) the harmful and potentially harmful constituents (HPHCs) identified in cigarette smoke, suggesting their potential as alternative products for reducing harm from cigarette smoking.

Methods: We assessed toxicological effects of eight ONPs, designated as modern oral (MO) 1 to 8 along with an ONP, an oral tobacco (snus), and a combustible cigarette market comparator using established regulatory toxicological assays including Ames, micronucleus (ivMN), and neutral red uptake (NRU) assays.

Evaluation of Cytotoxicity and Oxidative Stress of Whole Aerosol from Vuse Alto ENDS Products.

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.

Key Challenges and Recommendations for Testing of Tobacco Products for Regulatory Applications: Consideration of Test Materials and Exposure Parameters.

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 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.

Characterization of smoke and aerosol deliveries from combustible cigarettes, heated tobacco products and electronic nicotine delivery systems in the Vitrocell® Mammalian 6/48 exposure module.

The rapid development associated with Next Generation Tobacco Products (NGTP) has necessitated the development of high throughput methodologies to test their genotoxic potential in vitro when compared to conventional cigarette smoke (CS). An assessment of two Vitrocell® Mammalian 6/48 exposure modules in three independent experiments was made by comparing results from multiple dosimetric techniques applied to aerosol generated from 3R4F Kentucky Reference cigarettes, commercially available electronically heated tobacco product (eHTP) and Electronic Nicotine Delivery System (ENDS) using the Vitrocell® VC10®. Real-time aerosol particle concentration was assessed by means of light scattering photometers and expressed as area under the curve (∑AUC).

A comparison of cigarette smoke test matrices and their responsiveness in the mouse lymphoma assay: A case study.

No cigarette smoke test matrix is without limitation, due to the complexity of the starting aerosol and phase to phase dynamics. It is impossible to capture all chemicals at the same level of efficiency, therefore, any test matrix will inadvertently or by design fractionate the test aerosol. This case study examines how four different test matrices derived from cigarette smoke can be directly compared.

Key challenges for in vitro testing of tobacco products for regulatory applications: Recommendations for dosimetry.

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.

Investigation of multiple whole smoke dosimetry techniques using a VITROCELL®VC10® smoke exposure system.

The Vitrocell® VC10® smoke exposure system offers multiple platforms for air liquid interface (ALI) and air agar interface (AAI) exposure that mimic conditions for assessing toxicological impact of whole smoke using assays. The aim of this study was to investigate and compare multiple dosimetry techniques that may be employed during combustible cigarette whole smoke exposure using the Vitrocell® VC10® smoking robot. The following techniques were assessed: (1) quartz crystal microbalances (QCMs), (2) aerosol photometers (using area under curve, AUC), and (3) fluorescence of anhydrous dimethyl sulfoxide (DMSO)-captured smoke constituents.

Genotoxicity evaluation of tobacco and nicotine delivery products: Part One. Mouse lymphoma assay.

Conduct of the mouse lymphoma assay (MLA) is underpinned by Organisation for Economic Co-operation and Development (OECD) Test Guideline 490 and International Conference on Harmonisation S2(R1) guidance and is a recognised in vitro genotoxicity test battery assay. It has been used on a limited number of occasions for the assessment of some tobacco and nicotine products, such as e-cigarettes and tobacco heating products (THP). The aim of this study was to assess the suitability of the MLA for genotoxicity testing with a variety of tobacco and nicotine products.

Genotoxicity evaluation of tobacco and nicotine delivery products: Part Two. In vitro micronucleus assay.

In this study, a variety of test matrices from tobacco and nicotine delivery products were assessed against a 3R4F Kentucky reference cigarette using the in vitro micronucleus assay. Testing was conducted using two Chinese hamster cell lines (CHO and V79), and a human lymphoblastoid cell line (TK6), in accordance with established guidelines. Total particulate matter (TPM) from a 3R4F Reference cigarette was compared to an electronic cigarette e-liquid, electronic cigarette TPM and TPM from a commercial tobacco heating product using a standard and an extended treatment condition with recovery period.

Development of a quantitative method for assessment of dose in in vitro evaluations using a VITROCELL® VC10® smoke exposure system.

The assessment of potential cytotoxicity or genotoxicity of combustible tobacco products has historically been performed using partitioned exposures (i.e. total particulate matter [TPM], gas vapor phase [GVP]) rather than whole smoke.

Comparative in vitro toxicity profile of electronic and tobacco cigarettes, smokeless tobacco and nicotine replacement therapy products: e-liquids, extracts and collected aerosols.

The use of electronic cigarettes (e-cigs) continues to increase worldwide in parallel with accumulating information on their potential toxicity and safety. In this study, an in vitro battery of established assays was used to examine the cytotoxicity, mutagenicity, genotoxicity and inflammatory responses of certain commercial e-cigs and compared to tobacco burning cigarettes, smokeless tobacco (SLT) products and a nicotine replacement therapy (NRT) product. The toxicity evaluation was performed on e-liquids and pad-collected aerosols of e-cigs, pad-collected smoke condensates of tobacco cigarettes and extracts of SLT and NRT products.

Human G(alpha q): cDNA and tissue distribution.

G(alpha q), a member of the Gq family of heterotrimeric G proteins, transduces signals from several G protein-coupled receptors that stimulate membrane phosphoinositide hydrolysis. In order to further define the role of G(alpha q) in the function of G protein-coupled receptors, we have cloned the cDNA encoding human G(alpha q) from a prostate cDNA library. Human G(alpha q) exhibits high homology with its mouse homolog - 94% similarity at the nucleotide level, and 99% similarity at the amino acid level.

Elements essential for processing intronic U14 snoRNA are located at the termini of the mature snoRNA sequence and include conserved nucleotide boxes C and D.

Essential elements for intronic U14 processing have been analyzed by microinjecting various mutant hsc70/Ul4 pre-mRNA precursors into Xenopus oocyte nuclei. Initial truncation experiments revealed that elements sufficient for U14 processing are located within the mature snoRNA sequence itself. Subsequent deletions within the U14 coding region demonstrated that only the terminal regions of the folded U14 molecule containing con- served nucleotide boxes C and D are required for processing.

Processing of U14 small nucleolar RNA from three different introns of the mouse 70-kDa-cognate-heat-shock-protein pre-messenger RNA.

U14 is a small nucleolar RNA required for the processing of eukaryotic rRNA precursors. The U14 genes of mouse as well as rat, hamster, human, Xenopus and trout are encoded within introns of the constitutively expressed 70-kDa-cognate-heat-shock protein gene (hsc70). We demonstrate here that U14.

Heme Inhibition of [delta]-Aminolevulinic Acid Synthesis Is Enhanced by Glutathione in Cell-Free Extracts of Chlorella.

In plants, algae, and many bacteria, the heme and chlorophyll precursor, [delta]-aminolevulinic acid (ALA), is synthesized from glutamate in a reaction involving a glutamyl-tRNA intermediate and requiring ATP and NADPH as cofactors. In particulate-free extracts of algae and chloroplasts, ALA synthesis is inhibited by heme. Inclusion of 1.

Mouse U14 snRNA is a processed intron of the cognate hsc70 heat shock pre-messenger RNA.

U14 snRNA is a small nucleolar RNA species essential for eukaryotic pre-rRNA processing. We have previously shown that the mouse U14 snRNA genes are positioned within introns 5, 6, and 8 on the coding strand of the constitutively expressed cognate hsc70 heat shock gene. This genomic organization suggested the possibility that U14 snRNAs are transcribed as part of the hsc70 pre-mRNA and then excised from the intron to yield mature U14 snRNA species.