Multi-endpoint in vitro toxicological assessment of snus and tobacco-free nicotine pouch extracts.

'Modern' oral tobacco-free nicotine pouches (NPs) are a nicotine containing product similar in appearance and concept to Swedish snus. A three-step approach was taken to analyse the biological effects of NPs and snus extracts in vitro. ToxTracker was used to screen for biomarkers for oxidative stress, cell stress, protein damage and DNA damage. Cytotoxicity, mutagenicity, and genotoxicity were assessed in the following respective assays: Neutral Red Uptake (NRU), Ames and Mouse Lymphoma Assay (MLA). Targeted analysis of phosphorylation signalling and inflammatory markers under non-toxic conditions was used to investigate any potential signalling pathways or inflammatory response. A reference snus (CRP1.1) and four NPs with various flavours and nicotine strengths were assessed. Test article extracts was generated by incubating one pouch in 20 mL of media (specific to each assay) with the inclusion of the pouch material. NP extracts did not induce any cytotoxicity or mutagenic response, genotoxic response was minimal and limited signalling or inflammatory markers were induced. In contrast, CRP1.1 induced a positive response in four toxicological endpoints in the absence of S9: Srxn1 (oxidative stress), Btg2 (cell stress), Ddit3 (protein damage) and Rtkn (DNA damage), and three endpoints in presence of S9: Srxn1, Ddit3 and Rtkn. CRP1.1 was genotoxic when assessed in MLA and activated signalling pathways involved in proliferation and cellular stress and specifically induced phosphorylation of c-JUN, CREB1, p53, p38 MAPK and to a lesser extent AKT1S1, GSK3α/ß, ERK1/2 and RSK1 in a dose-dependent manner. CRP 1.1 extracts resulted in the release of several inflammatory mediators including cytokines IL-1α, IL5, IL6, IL8, IL-1RA, MIF and TNF-ß, receptor IL-2RA, and growth factors FGF-basic, VEGF and M-CSF. In conclusion these assays contribute to the weight of evidence assessment of the potential comparative health risks of NPs and snus.

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.

Advances in whole aerosol approaches for in vitro e-cigarette testing.

Electronic-cigarette regulation and risk assessment is a prominent and developing field, as the popularity and prevalence of this product category increases. Over the last 10 years since their emergence, there have been many advances and adaptations to current in vitro testing techniques to better assess and predict absolute consumer risk. However, there are still requirements to create a cross-field harmonised approach to appropriate exposure and experimental design. With many assessments still being carried out using methods developed and optimised for cigarette smoke, there must first be an acknowledgement regarding the differences between cigarette smoke and tobacco-free e-cigarette aerosols before we can accurately assess these distinct products. Here, we discuss five published studies from within our own research to demonstrate how in vitro testing techniques have evolved to improve determination of risk by considering appropriate dosimetry and exposure for both e-cigarette and cigarette aerosols and how we can contextualise the data through human consumption and dose extrapolation, ultimately giving more relevance to in vitro data. Furthermore, we have demonstrated the evolution of techniques, which has allowed us to bridge between platforms, simplify exposure set-up, experimental design and demonstrate technology evolution within our products, thus fulfilling a responsible duty of care to consumers via an appropriate and robust in vitro product assessment.

Key Challenges and Recommendations for In Vitro 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 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.

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.

Evaluation of behavioural, chemical, toxicological and clinical studies of a tobacco heated product glo™ and the potential for bridging from a foundational dataset to new product iterations.

Background: Tobacco Heating Products (THPs) are tobacco products that heat rather than burn tobacco with temperatures less than 350 °C. Because of this operating principle, they produce substantially fewer and lower levels of tobacco smoke toxicants than combustible cigarette smoke produced when tobacco is burnt, which occurs at much higher temperatures of around 900 °C. This paper analyses data on a THP, glo™, and assesses whether its use would result in reduced health risks compared to the health risks of smoking cigarettes. It also looks at the possibility of bridging datasets across the different variants of the glo™ product. Methods: The approach is to consider whether datasets from behavioural, chemical, toxicological and clinical studies provide consistent findings of reductions in toxicant exposure with glo™ use by subjects who switch completely from smoking cigarettes to using glo™ and whether these reductions are similar to those who stop smoking cigarettes without switching to glo™ or any other tobacco or nicotine product. We also examine the similarities and differences of different versions of the glo™ product and benchmark it against a THP from another manufacturer. Results: The studies indicate that the use of the glo™ results in substantial and prolonged reductions in toxicant exposure for smokers who switch to glo™ completely. A long-term clinical study shows substantial reductions in toxicant exposure over a period of time, similar to reduction of some biomarkers of exposure found following smoking cessation without switching to glo™ or any other tobacco product, and biomarkers of potential harm trending in a favourable manner for both groups that switch to glo™ and that quit all tobacco and nicotine use. Data suggests that all iterations of glo™ result in substantial reductions in toxicant exposure compared to smoking cigarettes and that bridging across datasets is feasible. Conclusions: Given the accumulated scientific data summarised in this paper, and particularly the findings from a long-term clinical study, the data demonstrate that glo™ is a reduced exposure product compared to combustible cigarettes and is reasonably deemed to reduce the risk of smoking-related diseases and supports the conclusion that smokers who would have otherwise continued to smoke and instead switch entirely to THP glo™ use, will reduce their relative risk of developing smoking-related diseases as compared to continued smoking. The extent of reduction in risk compared to continuing to smoke is likely to vary by smoking-related disease and by an individuals' smoking history, other risk factors and an individual's susceptibility to disease. Use of the THP will present some level of increased health risk as compared to cessation of tobacco and nicotine products and will cause dependence. As long as the principles of heat-not-burn are maintained, THP use will result in substantially reduced exposure to smoke toxicants as compared to continued conventional cigarette smoking. It is possible to use bridging or read across to apply these conclusions to new iterations of the glo™ product, extending the utility and validity of the evidence generated through study of prior iterations.

Ciliary Beat Frequency: Proceedings and Recommendations from a Multi-laboratory Ring Trial Using 3-D Reconstituted Human Airway Epithelium to Model Mucociliary Clearance.

The use of reconstituted human airway (RHuA) epithelial tissues to assess functional endpoints is highly relevant in respiratory toxicology, but standardised methods are lacking. In June 2015, the Institute for In Vitro Sciences (IIVS) held a technical workshop to evaluate the potential for standardisation of methods, including ciliary beat frequency (CBF). The applicability of a protocol suggested in the workshop was assessed in a multi-laboratory ring study. This report summarises the findings, and uses the similarities and differences identified between the laboratories to make recommendations for researchers in the absence of a validated method. Two software platforms for the assessment of CBF were used - Sisson-Ammons Video Analysis (SAVA; Ammons Engineering, Clio, MI, USA) and ciliaFA (National Institutes of Health, Bethesda, MD, USA). Both were utilised for multiple read temperatures, one objective strength (10×) and up to four video captures per tissue, to assess their utility. Two commercial RHuA tissue cultures were used: MucilAir™ (Epithelix, Geneva, Switzerland) and EpiAirway™ (MatTek, Ashland, MA, USA). IL-13 and procaterol were used to induce CBF-specific responses as positive controls. Further testing addressed the impact of tissue acclimation duration, the number of capture fields and objective strengths on baseline CBF readings. Both SAVA and ciliaFA reliably collected CBF data. However, ciliaFA failed to generate accurate CBF measurements above ∼10 Hz. The positive controls were effective, but were subject to inter-laboratory variability. CBF endpoints were generally uniform across replicate tissues, objective strengths and laboratories. Longer tissue acclimation increased the percentage active area, but had minimal impact on CBF. Taken together, these findings support the development and validation of a standardised CBF measurement protocol.

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.

Application of ToxTracker for the toxicological assessment of tobacco and nicotine delivery products.

Consumer demands and innovation have led to an increasingly diverse range of nicotine delivery systems, driven by a desire to reduce risk associated with traditional combustible cigarettes. This speed of change provides a mandate for rapid new product assessment. We have used the validated technology ToxTracker®, to assess biomarkers of DNA damage, protein misfolding, oxidative and cellular stress, across the categories of cigarette (1R6F), tobacco heating product (THP 1.4) and electronic cigarette (ePen 3). In addition, we compared the commonly used test matrices for tobacco and nicotine products; whole aerosol aqueous extracts (AqE) and gas vapour phase (GVP), determining their suitability across the product categories. We demonstrated a significant reduction in oxidative stress and cytotoxicity for THP 1.4 over cigarette, further reduced for ePen 3, when assessed by both dilution and nicotine dosimetry. We also identified that while the extraction matrices AqE and GVP from combustible products were equivalent in the induced responses, this was not true of the other category examples, moreover THP 1.4 GVP demonstrates a >50 % reduction in both toxicity and cytotoxicity endpoints over AqE. This indicates that unlike cigarette, the active components or toxicants for THP and electronic cigarette are associated with the aerosol fraction of these categories.

Geometrical Scaling Effects in the Mechanical Properties of 3D-Printed Body-Centered Cubic (BCC) Lattice Structures.

This paper investigates size effects on the mechanical response of additively manufactured lattice structures based on a commercially available polylactic acid (PLA) polymer. Initial attention is focused on investigating geometrical effects in the mechanical properties of simple beams and cubes. Following this, a number of geometrically scaled lattice structures based on the body-centered cubic design were manufactured and tested in order to highlight size effects in their compression properties and failure modes. A finite element analysis was also conducted in order to compare the predicted modes of failure with those observed experimentally. Scaling effects were observed in the compression response of the PLA cubes, with the compression strength increasing by approximately 19% over the range of scale sizes investigated. Similar size-related effects were observed in the flexural samples, where a brittle mode of failure was observed at all scale sizes. Here, the flexural strength increased by approximately 18% when passing from the quarter size sample to its full-scale counterpart. Significant size effects were observed following the compression tests on the scaled lattice structures. Here, the compression strength increased by approximately 60% over the four sample sizes, in spite of the fact that similar failure modes were observed in all samples. Finally, reasonably good agreement was observed between the predicted failure modes and those observed experimentally. However, the FE models tended to over-estimate the mechanical properties of the lattice structures, probably as a result of the fact that the models were assumed to be defect free.

The development of an in vitro 3D model of goblet cell hyperplasia using MUC5AC expression and repeated whole aerosol exposures.

Goblet cell hyperplasia and overproduction of airway mucin are characteristic features of the lung epithelium of smokers and COPD patients. Tobacco heating products (THPs) are a potentially less risky alternative to combustible cigarettes, and through continued use solus THPs may reduce smoking-related disease risk. Using the MucilAir™ in vitro lung model, a 6-week feasibility study was conducted investigating the effect of repeated cigarette smoke (1R6F), THP aerosol and air exposure. Tissues were exposed to nicotine-matched whole aerosol doses 3 times/week. Endpoints assessed were dosimetry, tight-junction integrity, cilia beat frequency (CBF) and active area (AA), cytokine secretion and airway mucin MUC5AC expression. Comparison of incubator and air exposed controls indicated exposures did not have a significant effect on the transepithelial electrical resistance (TEER), CBF and AA of the tissues. Cytokine secretion indicated clear differences in secretion patterns in response to 1R6F and THP exposure. 1R6F exposure resulted in a significant decrease in the TEER and AA (p=0.000 and p=0.000, respectively), and an increase in MUC5AC positive cells (p=0.002). Repeated THP exposure did not result in a significant change in MUC5AC positive cells. This study demonstrates repeated cigarette smoke whole aerosol exposure can induce these morphological changes in vitro.

Optimization of aqueous aerosol extract (AqE) generation from e-cigarettes and tobacco heating products for in vitro cytotoxicity testing.

Electronic cigarettes (e-cigarettes) and tobacco heating products (THPs) have reduced yields of toxicants and have recently emerged as a potentially safer alternative to combustible cigarettes. To understand if reduced toxicant exposure is associated with reductions in biological responses, there is a need for high-quality pre-clinical in vitro studies. Here, we investigated the cytotoxic response of human umbilical vein endothelial cells to conventional cigarette aqueous aerosol extracts (AqE) and highly concentrated AqEs from e-cigarettes (two generations of atomisers) and THPs (two variants). All AqE samples were generated by a standardized methodology and characterized for nicotine, propylene glycol and vegetable glycerol. The cigarette AqE caused a maximum 100 ± 0.00 % reduction in cell viability at 35 % dose (2.80 puffs) as opposed to 96.63 ± 2.73 % at 50 % (20 puffs) and 99.85 ± 0.23 % at 75 % (30 puffs) for the two THP variants (glo Bright Tobacco, glo Rich Tobacco), and 99.07 ± 1.61 % at the neat ePen2.0 e-cigarette (200 puffs). The AqE of the remaining e-cigarettes either resulted in an incomplete dose-response or did not elicit any response. The methods utilized were suitably sensitive to not only differentiate between cigarette, THP and e-cigarette aerosols but also to distinguish between products within each product category.

In vitro biological assessment of the stability of cigarette smoke aqueous aerosol extracts.

OBJECTIVES: Cigarette smoke aqueous aerosol extracts (AqE) have been used for assessing tobacco products, particularly with in vitro models such as oxidative stress and inflammation. These test articles can be generated easily, but there are no standardised methods for the generation and characterisation or stability. We investigated the effects of pro-oxidant smoke-derived chemicals by using 3R4F AqE generated under standardised conditioning and smoking regimes and assessed the stability over 31-week timeframe. Twenty batches generated from ten puffs per cigarette bubbled through 20 ml cell culture media were used fresh and thawed from frozen aliquots stored at - 80 ºC. RESULTS: Nicotine levels quantified by gas chromatography/mass spectrometry and optical density at 260 nm showed chemical and physical stability from week 0 (fresh sample) to weeks 1, 4, 8 and 31 (frozen samples). No significant change in H292 human bronchial epithelial cell viability or oxidative stress were observed between fresh AqE at week 0 and frozen AqE at 31 weeks. AqEs generated by our protocol were stable for up to 31 weeks for all tested end points, suggesting that it may not be necessary to use freshly generated AqE for each study, thus reducing batch-to-batch variability.

The genotoxicological assessment of a tobacco heating product relative to cigarette smoke using the in vitro micronucleus assay.

In vitro studies have supported the toxicological evaluation of chemicals and complex mixtures including cigarette smoke and novel tobacco and nicotine products which include tobacco heating products (THP). This new environment requires faster testing, higher throughput and appropriate in vitro studies, to support product innovation and development. In this study, total particulate matter (TPM) from a commercially available THP and a reference cigarette (3R4F) were assessed up to 500 µg/mL using two in vitro micronucleus techniques. V79 and TK6 cells were assessed using conventional OECD 487 manual scoring techniques, whereas, CHO cells were assessed using contemporary, automated high content screening approaches (Cellomics ArrayScan® VTI). V79 cells gave the most consistent response with all three treatment conditions producing a clear positive genotoxic response. Human TK6 cells only produced dose-dependent response, indicative of a weak-positive response. CHO cells demonstrated a positive response with TPM using long (24 h) -S9 conditions. All three cell lines equally demonstrated a negative response with THP TPM up to 500 µg/mL. In conclusion, THP TPM did not increase micronuclei formation above control levels even at doses far exceeding that tested with reference cigarette smoke, in most cases up to 10x the dose delivered compared to that of cigarette smoke. This study supports the growing belief that THPs are less risky than conventional cigarettes and that 21st century screening techniques can be employed to support product design and decision making, as a potential 1st screen prior to more traditional assessments.

Evaluation of a high-throughput in vitro endothelial cell migration assay for the assessment of nicotine and tobacco delivery products.

Endothelial cell migration is a critical process in the maintenance of healthy blood vessels. Impaired endothelial migration is reportedly associated with the development of cardiovascular diseases. Here, we report on the development of a 96-well in vitro endothelial migration assay for the purpose of comparative toxicological assessment of a novel THP relative to cigarette smoke, to be able to rapidly inform regulatory decision making. Uniform scratches were induced in confluent human umbilical vein endothelial cells using the 96-pin wound maker and exposed to 3R4F cigarette or THP aqueous extracts (AqE). Endothelial migration was recorded over 24 h, and the rate of wound closure calculated using mean relative wound density rather than migration rate as previously reported. This self-normalising parameter accounts for starting wound size, by comparing the density of the scratch to the outer region at each time-point. Furthermore, wound width acceptance criteria was defined to further increase the sensitivity of the assay. 3R4F and THP AqE samples were tested at comparable nicotine concentrations. 3R4F showed significant cytotoxicity and inhibition of wound healing whereas THP AqE did not show any response in either endpoint. This 96-well endothelial migration assay was suitably sensitive to distinguish combustible cigarette and THP test articles.

An inter-laboratory in vitro assessment of cigarettes and next generation nicotine delivery products.

In vitro testing can facilitate the rapid assessment of next generation nicotine delivery products (NGPs) with comparisons to combustible tobacco products. In vitro assays for cytotoxicity and oxidative stress were employed at BAT (UK) and JT (Japan) to test total particulate matter (TPM) of a scientific reference cigarette and aerosol collected mass (ACM) of a commercially available E-cigarette and two tobacco heating products (THP). 3R4F TPMs were generated using the Health Canada intense (HCI) regimen, a modified regime (mHCI) for the THP ACMs and the CORESTA recommended method no. 81 for the E-cigarette ACM. Human lung cells were exposed to the test product TPM/ACMs at concentrations between 0-200 µg/ml followed by the employment of commercially available assays for endpoint analysis that included reactive oxygen species (ROS) generation, the glutathione ratio (GSH:GSSG), activation of the antioxidant response elements (ARE) and cellular viability. TPM/ACM nicotine concentrations were quantified using a UPLC-PDA technique. At both laboratories the 3R4F TPM induced significant and dose-dependent responses in all in vitro assays, whereas no significant responses could be measured for the NGP ACMs. In conclusion, both laboratories obtained comparable results across all endpoints therefore demonstrating the utility of the in vitro techniques combined with standardised test products to support the assessment of NGPs.

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. Total particulate matter (TPM) from a 3R4F cigarette was compared against a commercial electronic cigarette liquid (e-liquid), electronic cigarette (e-cigarette) aerosol matter captured from the same e-liquid, and TPM from a commercial THP. Treatment conditions included 3 h exposures with and without metabolic activation and a longer 24 h exposure without metabolic activation (-S9) at concentrations up to 500 µg/mL. Under all treatment conditions, 3R4F produced a clear positive response with regard to induction of mutation. In contrast, no marked induction of mutation was observed for the e-liquid, e-cigarette aerosol or THP. Additionally, data are presented as a function of nicotine equivalents for comparisons between these different tobacco products and test matrices.

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. Cells were assessed with 3R4F TPM prior to assessment of the other tobacco and nicotine product test matrices. These cell lines gave varied responses to 3R4F TPM with the most robust response using V79 cells. The use of an extended exposure/recovery period was seen to increase assay sensitivity for CHO and V79 cell lines but was less clear for TK6 cells. Negative responses were observed for all products except 3R4F across all treatment conditions in V79 cells. The most potent response to cigarette smoke was following extended treatment with recovery, suggesting this may be a more appropriate treatment for the future assessment of tobacco and nicotine product test matrices.