A framework for the systematic evaluation of a novel cigarette filter technology.

Any functional change in cigarette filter design warrants a rigorous assessment to ensure comparability to existing filter functionality. This study compares the functionality of a standard CA filter with a novel cellulose-based alternative using a combination of emissions, in silico approaches, pre-clinical assessments and behavioural studies. We assess the challenges faced with a significant filtration change, the substantiation of this change and the limitations of such assessments. We explore cigarette emission chemical profiles; assess the potential toxicological impacts (in vitro and statistical modelling) of the differing chemical profiles of cigarette smoke aerosol resulting from the respective filter types; and, finally investigate the behavioural aspects associated with use of the novel filter as compared to the traditional one. The aim of the study was to establish a weight of evidence assessment framework for the comprehensive evaluation of a novel cigarette filter design as part of robust stewardship approach. The data show comparability to a standard CA filter across all assessments and highlight potential areas of investigation for future novel filter product iterations. The approach demonstrates the applicability of a comprehensive step-wise assessment framework to identify any potential increased toxicant emissions and exposures associated with using the novel filter.

Key Challenges for In Vitro Testing of Tobacco Products for Regulatory Applications: Recommendations for the In Vitro Mouse Lymphoma Assay.

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.

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.

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. The test matrices assessed were as follows, total particulate matter (TPM), gas vapour phase (GVP), a combination of TPM + GVP and whole aerosol (WA). Here we use an example assay, the mouse lymphoma assay (MLA) to demonstrate that data generated across four cigarette smoke test matrices can be compared. The results show that all test matrices were able to induce positive mutational events, but with clear differences in the biological activity (both potency and toxicity) between them. TPM was deemed the most potent test article and by extension, the particulate phase is interpreted as the main driver of genotoxic induced responses in the MLA. However, the results highlight that the vapour phase is also active. MLA appeared responsive to WA, with potentially lower potency, compared to TPM approaches. However, this observation is caveated in that the WA approaches used for comparison were made on a newly developed experimental method using dose calculations. The TPM + GVP matrix had comparable activity to TPM alone, but interestingly induced a greater number of mutational events at comparable relative total growth (RTG) and TPM-equivalent doses when compared to other test matrices. In conclusion, this case study highlights the importance of understanding test matrices in response to the biological assay being assessed and we note that not all test matrices are equal.

In Silico Approaches In Carcinogenicity Hazard Assessment: Current Status and Future Needs.

Historically, identifying carcinogens has relied primarily on tumor studies in rodents, which require enormous resources in both money and time. In silico models have been developed for predicting rodent carcinogens but have not yet found general regulatory acceptance, in part due to the lack of a generally accepted protocol for performing such an assessment as well as limitations in predictive performance and scope. There remains a need for additional, improved in silico carcinogenicity models, especially ones that are more human-relevant, for use in research and regulatory decision-making. As part of an international effort to develop in silico toxicological protocols, a consortium of toxicologists, computational scientists, and regulatory scientists across several industries and governmental agencies evaluated the extent to which in silico models exist for each of the recently defined 10 key characteristics (KCs) of carcinogens. This position paper summarizes the current status of in silico tools for the assessment of each KC and identifies the data gaps that need to be addressed before a comprehensive in silico carcinogenicity protocol can be developed for regulatory use.

Skin sensitization in silico protocol.

The assessment of skin sensitization has evolved over the past few years to include in vitro assessments of key events along the adverse outcome pathway and opportunistically capitalize on the strengths of in silico methods to support a weight of evidence assessment without conducting a test in animals. While in silico methods vary greatly in their purpose and format; there is a need to standardize the underlying principles on which such models are developed and to make transparent the implications for the uncertainty in the overall assessment. In this contribution, the relationship between skin sensitization relevant effects, mechanisms, and endpoints are built into a hazard assessment framework. Based on the relevance of the mechanisms and effects as well as the strengths and limitations of the experimental systems used to identify them, rules and principles are defined for deriving skin sensitization in silico assessments. Further, the assignments of reliability and confidence scores that reflect the overall strength of the assessment are discussed. This skin sensitization protocol supports the implementation and acceptance of in silico approaches for the prediction of skin sensitization.

In vitro mutagenicity of gas-vapour phase extracts from flavoured and unflavoured heated tobacco products.

The in vitro mutagenic and genotoxic potential of Heated Tobacco Products (HTPs) has already been studied with the particulate phase and reported previously. This study has been designed to complement the in vitro assessment of the HTP and to determine whether the inclusion of potential flavourings would alter the in vitro response by testing the other phase of the aerosol, the gas-vapour phase (GVP). Both flavoured and unflavoured Neostik GVP samples did not show any sign of mutagenic activity in the Ames test but induced a mutagenic response in the mouse lymphoma assay (MLA), however, these responses were significantly less than those of the reference cigarette, 3R4F. The results demonstrated that GVP emissions of this HTP did not induce either new qualitative or quantitative mutagenic hazards compared to 3R4F, as assessed by the Ames test (no new responsive strains) and MLA (a lower mutagenic response), respectively. A statistical comparative analysis of the responses showed that the addition of flavourings that may thermally decompose under the conditions of use did not add to the in vitro baseline responses of the unflavoured Neostik.

Genetic toxicology in silico protocol.

In silico toxicology (IST) approaches to rapidly assess chemical hazard, and usage of such methods is increasing in all applications but especially for regulatory submissions, such as for assessing chemicals under REACH as well as the ICH M7 guideline for drug impurities. There are a number of obstacles to performing an IST assessment, including uncertainty in how such an assessment and associated expert review should be performed or what is fit for purpose, as well as a lack of confidence that the results will be accepted by colleagues, collaborators and regulatory authorities. To address this, a project to develop a series of IST protocols for different hazard endpoints has been initiated and this paper describes the genetic toxicity in silico (GIST) protocol. The protocol outlines a hazard assessment framework including key effects/mechanisms and their relationships to endpoints such as gene mutation and clastogenicity. IST models and data are reviewed that support the assessment of these effects/mechanisms along with defined approaches for combining the information and evaluating the confidence in the assessment. This protocol has been developed through a consortium of toxicologists, computational scientists, and regulatory scientists across several industries to support the implementation and acceptance of in silico approaches.

Assessment of priority tobacco additives per the requirements in the EU Tobacco Products Directive (2014/40/EU): Part 2: Smoke chemistry and in vitro toxicology.

This publication is part of a series of three publications and describes the non-clinical assessment performed to fulfill the regulatory requirement per Art. 6 (2) of the EU Tobacco Products Directive 2014/40/EU under which Member States shall require manufacturers and importers of cigarettes and Roll Your Own tobacco containing an additive that is included in the priority list established by Commission Implementing Decision (EU) 2016/787 to carry out comprehensive studies (European Comission, 2016). This publication contains the results of a literature search, comprehensive smoke chemistry, additive transfer, and in vitro toxicity studies for the 13 priority additives (carob bean extract, cocoa powder, fenugreek extract, fig juice concentrate, geraniol, glycerol, guaiacol, guar gum, liquorice extract powder, maltol, l-menthol (synthetic), propylene glycol, and sorbitol) commissioned by the members of the Priority Additives Tobacco Consortium to independent Contract Research Organizations. Comparisons of the 39 World Health Organisation smoke emissions in smoke from cigarettes with and without priority additives identified some differences that, with few exceptions, were minor and well within the inherent variability of the analytical method observed for the 3R4F monitor cigarette. Most differences were not statistically significant and did not show consistent additive-related increases or decreases. However, test cigarettes with guar gum showed a statistically significant, additive-related increase in formaldehyde and cadmium; test cigarettes with sorbitol showed a statistically significant, additive-related increase in formaldehyde and acrolein; test cigarettes with glycerol showed a statistically significant, additive-related decrease in phenols, benzo[a]pyrene and N-nitrosoanabasine; and test cigarettes with propylene glycol showed a statistically significant, additive-related decrease in phenol and m + p-cresols. These changes were not observed when the additives were tested as a mixture. None of the increases or decreases in smoke chemistry translated into changes in the in vitro toxicity. Comparisons of the in vitro toxicity of smoke from cigarettes with and without priority additives gave some differences that were minor, well within the inherent variability of the assays, not statistically significant, and did not show consistent additive-related increases or decreases. Thus, it can be concluded that the addition of priority additives had no effect on the in vitro toxicity of the cigarette smoke. The results obtained in our studies are consistent with those in scientific literature.

Evaluation of flavourings potentially used in a heated tobacco product: Chemical analysis, in vitro mutagenicity, genotoxicity, cytotoxicity and in vitro tumour promoting activity.

We designed a novel tobacco-heating product (THP) that heats tobacco to release nicotine and aerosolised components, such as glycerol and tobacco volatiles from a tobacco rod (Neostik). Heating tobacco significantly reduces levels of combustion-derived toxicants in the aerosol compared to cigarette smoke. This study was conducted to determine whether the inclusion of potential flavourings in the THP would add to the levels of toxicants in the emissions or alter in vitro responses. Levels of measured toxicants were similar in the flavoured and unflavoured Neostik emissions and significantly less than emissions from the reference cigarette, 3R4F. No mutagenicity was observed with the Neostiks in the Ames test or in the mouse lymphoma assay. There was evidence of a weak genotoxic response in the in vitro micronucleus test using V79 cells from both Neostiks and these responses were less than 3R4F. They did not show tumour-promoting potential in the Bhas 42 cell transformation assay and were not cytotoxic in the Neutral Red uptake assay. 3R4F elicited toxic responses in all assays at significantly lower concentrations. The addition of flavourings to the Neostik tested did not alter the chemical profile of THP emissions or change in vitro responses relative to the unflavoured Neostik.

A novel hybrid tobacco product that delivers a tobacco flavour note with vapour aerosol (Part 2): In vitro biological assessment and comparison with different tobacco-heating products.

This study assessed the toxicological and biological responses of aerosols from a novel hybrid tobacco product. Toxicological responses from the hybrid tobacco product were compared to those from a commercially available Tobacco Heating Product (c-THP), a prototype THP (p-THP) and a 3R4F reference cigarette, using in vitro test methods which were outlined as part of a framework to substantiate the risk reduction potential of novel tobacco and nicotine products. Exposure matrices used included total particulate matter (TPM), whole aerosol (WA), and aqueous aerosol extracts (AqE) obtained after machine-puffing the test products under the Health Canada Intense smoking regime. Levels of carbonyls and nicotine in these matrices were measured to understand the aerosol dosimetry of the products. The hybrid tobacco product tested negative across the in vitro assays including mutagenicity, genotoxicity, cytotoxicity, tumour promotion, oxidative stress and endothelial dysfunction. All the THPs tested demonstrated significantly reduced responses in these in vitro assays when compared to 3R4F. The findings suggest these products have the potential for reduced health risks. Further pre-clinical and clinical assessments are required to substantiate the risk reduction of these novel products at individual and population levels.

The combination of two novel tobacco blends and filter technologies to reduce the in vitro genotoxicity and cytotoxicity of prototype cigarettes.

Tobacco smoke from a combustible cigarette contains more than 6000 constituents; approximately 150 of these are identified as toxicants. Technologies that modify the tobacco blend to reduce toxicant emissions have been developed. These include tobacco sheet substitute to dilute toxicants in smoke and blend treated tobacco to reduce the levels of nitrogenous precursors and some polyphenols. Filter additives to reduce gas (vapour) phase constituents have also been developed. In this study, both tobacco blend and filter technologies were combined into an experimental cigarette and smoked to International Organisation on Standardisation and Health Canada puffing parameters. The resulting particulate matter was subjected to a battery of in vitro genotoxicity and cytotoxicity assays - the Ames test, mouse lymphoma assay, the in vitro micronucleus test and the Neutral Red Uptake assay. The results indicate that cigarettes containing toxicant reducing technologies may be developed without observing new additional genotoxic hazards as assessed by the assays specified. In addition, reductions in bacterial mutagenicity and mammalian genotoxicity of the experimental cigarette were observed relative to the control cigarettes. There were no significant differences in cytotoxicity relative to the control cigarettes.