Evaluation of the Tobacco Heating System 2.2. Part 3: Influence of the tobacco blend on the formation of harmful and potentially harmful constituents of the Tobacco Heating System 2.2 aerosol.

The Tobacco Heating System (THS2.2), which uses "heat-not-burn" technology, generates an aerosol from tobacco heated to a lower temperature than occurs when smoking a combustible cigarette. The concentrations of harmful and potentially harmful constituents (HPHCs) are significantly lower in THS2.2 mainstream aerosol than in smoke produced by combustible cigarettes. Different tobacco types and 43 tobacco blends were investigated to determine how the blend impacted the overall reductions of HPHCs in the THS2.2 mainstream aerosol. The blend composition had minimal effects on the yields of most HPHCs in the aerosol. Blends containing high proportions of nitrogen-rich tobacco, e.g., air-cured, and some Oriental tobaccos, produced higher acetamide, acrylamide, ammonia, and nitrogen oxide yields than did other blends. Most HPHCs were found to be released mainly through the distillation of HPHCs present in the tobacco plug or after being produced in simple thermal reactions. HPHC concentrations in the THS2.2 aerosol may therefore be further minimized by limiting the use of flue- and fire-cured tobaccos which may be contaminated by HPHCs during the curing process and carefully selecting nitrogen rich tobaccos with low concentrations of endogenous HPHCs for use in the tobacco plug blend.

Evaluation of the Tobacco Heating System 2.2. Part 1: Description of the system and the scientific assessment program.

This publication introduces a series of eight other publications describing the non-clinical assessment and initial clinical study of a candidate modified risk tobacco product (MRTP) - the Tobacco Heating System 2.2 (THS2.2). This paper presents background information on tobacco harm reduction, to complement the approaches aimed at increasing smoking cessation and reducing smoking initiation to reduce the morbidity and mortality caused by cigarette smoking. THS2.2 heats tobacco without combustion, and the resulting formation of harmful and potentially harmful constituents (HPHC) is greatly reduced compared with cigarette smoke. Assessment of the THS2.2 aerosol in vitro and in vivo reveals reduced toxicity and no new hazards. Additional mechanistic endpoints, measured as part of in vivo studies, confirmed reduced impact on smoking-related disease networks. The clinical study confirmed the reduced exposure to HPHCs in smokers switching to THS2.2, and the associated transcriptomic study confirmed the utility of a gene expression signature, consisting of only 11 genes tested in the blood transcriptome of subjects enrolled in the clinical study, as a complementary measure of exposure response. The potential of THS2.2 as an MRTP is demonstrated by the assessment and additional publications cited in this series.

Evaluation of the Tobacco Heating System 2.2. Part 2: Chemical composition, genotoxicity, cytotoxicity, and physical properties of the aerosol.

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.

A framework for in vitro systems toxicology assessment of e-liquids.

Various electronic nicotine delivery systems (ENDS), of which electronic cigarettes (e-cigs) are the most recognized prototype, have been quickly gaining ground on conventional cigarettes because they are perceived as less harmful. Research assessing the potential effects of ENDS exposure in humans is currently limited and inconclusive. New products are emerging with numerous variations in designs and performance parameters within and across brands. Acknowledging these challenges, we present here a proposed framework for an in vitro systems toxicology assessment of e-liquids and their aerosols, intended to complement the battery of assays for standard toxicity assessments. The proposed framework utilizes high-throughput toxicity assessments of e-liquids and their aerosols, in which the device-to-device variability is minimized, and a systems-level investigation of the cellular mechanisms of toxicity is an integral part. An analytical chemistry investigation is also included as a part of the framework to provide accurate and reliable chemistry data solidifying the toxicological assessment. In its simplest form, the framework comprises of three main layers: (1) high-throughput toxicity screening of e-liquids using primary human cell culture systems; (2) toxicity-related mechanistic assessment of selected e-liquids, and (3) toxicity-related mechanistic assessment of their aerosols using organotypic air-liquid interface airway culture systems. A systems toxicology assessment approach is leveraged to enable in-depth analyses of the toxicity-related cellular mechanisms of e-liquids and their aerosols. We present example use cases to demonstrate the suitability of the framework for a robust in vitro assessment of e-liquids and their aerosols.

Comparing the preclinical risk profile of inhalable candidate and potential candidate modified risk tobacco products: A bridging use case.

Cigarette smoking causes major preventable diseases, morbidity, and mortality worldwide. Smoking cessation and prevention of smoking initiation are the preferred means for reducing these risks. Less harmful tobacco products, termed modified-risk tobacco products (MRTP), are being developed as a potential alternative for current adult smokers who would otherwise continue smoking. According to a regulatory framework issued by the US Food and Drug Administration, a manufacturer must provide comprehensive scientific evidence that the product significantly reduces harm and the risk of tobacco-related diseases, in order to obtain marketing authorization for a new MRTP. For new tobacco products similar to an already approved predicate product, the FDA has foreseen a simplified procedure for assessing "substantial equivalence". In this article, we present a use case that bridges the nonclinical evidence from previous studies demonstrating the relatively reduced harm potential of two heat-not-burn products based on different tobacco heating principles. The nonclinical evidence was collected along a "causal chain of events leading to disease" (CELSD) to systematically follow the consequences of reduced exposure to toxicants (relative to cigarette smoke) through increasing levels of biological complexity up to disease manifestation in animal models of human disease. This approach leverages the principles of systems biology and toxicology as a basis for further extrapolation to human studies. The experimental results demonstrate a similarly reduced impact of both products on apical and molecular endpoints, no novel effects not seen with cigarette smoke exposure, and an effect of switching from cigarettes to either MRTP that is comparable to that of complete smoking cessation. Ideally, a subset of representative assays from the presented sequence along the CELSD could be sufficient for predicting similarity or substantial equivalence in the nonclinical impact of novel products; this would require further validation, for which the present use case could serve as a starting point.

Characterizing the precision of mass-spectrometry-based metabolic profiling platforms.

In conventional analytical chemistry it is customary to report figures of merit - such as precision, analysis time, limit of detection - for the chemical analysis performed. Usually, such figures of merit are reported for each analyte separately, generating a list of figures of merit. In metabolomics such a listing is not informative, since very many compounds are measured. An ANOVA-based strategy is proposed for a global measure of precision of the whole experiment, broken down in components of variation contributing to the total variation. This strategy uses well established statistical techniques and can be used easily. It was implemented to study the reproducibility of different comprehensive GC- and LC-MS methods for the analysis of tobacco aerosols. The results give insight into different sources of variation contributing to the total variation, such as biological variability, sampling variability and repeatability. For the specific example, median CV values ranged from 4.6% to 12.5% for repeatability; from 14.7% to 18.0% for sampling variability and, finally, from 24.2% to 26.8% for biological variability. Such a breakdown of sources of variability gives clues for improving the methods.

Attitudes of families affected by adrenoleukodystrophy toward prenatal diagnosis, presymptomatic and carrier testing, and newborn screening.

Families affected by adrenoleukodystrophy (ALD) and adrenomyeloneuropathy (AMN) were surveyed to elicit attitudes toward prenatal, presymptomatic and carrier testing, and newborn screening in order to determine the level of support that these families have for current and future genetic testing protocols. Identifying attitudes toward genetic testing, including newborn screening, is especially important because of new data regarding therapeutic options and the possible addition of ALD to newborn screening regimens. The Kennedy Krieger Institute (KKI) database identified 327 prospective participants. Families that were willing to participate in the study received an anonymous questionnaire for completion. Frequencies were generated using SPSS software for Windows. Questionnaires were returned from 128 families for a response rate of 39%. Sons who were at risk for inheriting the ALD gene would be tested by 93% of respondents, and 89.3% would ideally have this testing performed prenatally or in the newborn period. Eighty-nine percent would test an at-risk daughter and 51.2% would ideally have this testing performed prenatally or shortly after birth. ALD newborn screening for males and females was supported by 90% of respondents. If newborn screening for ALD/AMN commences, or there is a new diagnosis of ALD, genetic professionals need to be prepared to have extensive conversations with families regarding the benefits and limitations of current therapeutic and genetic testing options.