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 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.

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.

Assessment of novel tobacco heating product THP1.0. Part 7: Comparative in vitro toxicological evaluation.

In vitro studies have been widely used to support the toxicological evaluation of chemicals and complex mixtures including cigarette smoke. In this study, the total particulate matter and whole aerosol from a Kentucky reference 3R4F cigarette and two commercially available tobacco heating products (THPs) were assessed using in vitro mutagenicity, cytotoxicity and tumour-promoting activity assays. The Ames assay assessed mutagenicity using Salmonella typhimurium tester strains TA98, TA100, TA1535, TA1537 and TA102 ± metabolic activation (S9). The mouse lymphoma assay was used with short 3 h and longer 24 h exposures. The Bhas 42 cell transformation assay was incorporated as an in vitro alternative for detecting tumour promoters, and the neutral red uptake cell viability assay provided an acute measure of cytotoxicity. To complement the approach, the Ames assay was also employed with S. typhimurium tester strains TA98, TA100, TA1535, TA97 and TA102 using a scaled down methodology for the assessment of aerosols. All the in vitro techniques employed produced a clear positive response with cigarette smoke and in contrast, a negative response to THPs at doses equivalent to or higher than a cigarette smoke test matrix. The data show little difference between the THPs assessed suggesting parity between products.

Assessment of novel tobacco heating product THP1.0. Part 6: A comparative in vitro study using contemporary screening approaches.

Cigarette smoking is a major risk factor for many adverse health conditions. Novel tobacco heating products (THPs) heat tobacco, reducing exposure to many of the harmful combustion toxicants in conventional cigarette emissions. In vitro studies have been employed to support the toxicological evaluation of chemicals and complex mixtures, including cigarette smoke. The use of automated robotics platforms for in vitro toxicological screening complements traditional testing approaches. Multiparametric toxicity and oxidative stress endpoints were used to assess in vitro biological responses elicited after exposure to total particulate matter (TPM) from two commercially available THPs, and the reference tobacco product 3R4F, in human bronchial epithelial cells. A luciferase-based reporter gene assay was used to assess antioxidant response element (ARE) transcriptional activation in stably transfected H292 cells after 6 and 24 h exposures. High-content screening was used to assess 10 endpoints normal human bronchial epithelial cells after 4 or 24 h exposures. 3R4F TPM stimulated significant increases in ARE activation (p < 0.005) and moderate activity in HCS cell-based assays compared to THP at comparable doses. THPs showed little or no activity in all assays. HCS techniques can extend safety assessments providing information quickly in the early stages of product innovation and development.

Assessment of tobacco heating product THP1.0. Part 5: In vitro dosimetric and cytotoxic assessment.

Tobacco heating products (THPs) represent a subset of the next-generation nicotine and tobacco product category, in which tobacco is typically heated at temperatures of 250-350 °C, thereby avoiding many of the harmful combustion-related toxicant emissions of conventional cigarettes. In this study, we have assessed aerosol generation and cytotoxicity from two commercially available THPs, THP1.0 and THS, relative to tobacco smoke from 3R4F reference cigarettes, using an adapted Borgwaldt RM20S Smoking Machine. Quantification of nicotine in the exposed cell-culture media showed greater delivery of nicotine from both THPs than from the cigarette. Using Neutral Red Uptake assay, THPs demonstrated reduced in vitro cytotoxicity in H292 human bronchial epithelial cells as compared with 3R4F cigarette exposure at the air-liquid interface (p < 0.0001). Both THPs demonstrated a statistically similar reduction in biological response, with >87% viability relative to 3R4F at a common aerosol dilution (1:40, aerosol:air). A similar response was observed when plotted against nicotine; a statistical difference between 3R4F and THPs (p < 0.0001) and no difference between the THPs (p = 0.0186). This pre-clinical in vitro biological testing forms part of a larger package of data to help assess the safety and risk reduction potential of next-generation tobacco products relative to cigarettes, using a weight of evidence approach.

Assessment of enamel discoloration in vitro following exposure to cigarette smoke and emissions from novel vapor and tobacco heating products.

PURPOSE: To evaluate in vitro enamel sample discoloration following exposure to a scientific reference cigarette (3R4F) or emissions from next generation tobacco and nicotine products (NGPs) such as electronic cigarettes (EC) and tobacco heating products (THP). METHODS: Bovine enamel blocks (6.5 × 6.5 mm) were prepared and pre-incubated with human or artificial saliva, to form a pellicle layer before exposure to either particulate matter (PM) or whole aerosols. PM was prepared by capturing 3R4F cigarette smoke (CS), a commercial THP (THP1.0) or a novel vapor product (NVP)/next generation e-cigarette aerosols on Cambridge filter pads followed by elution with dimethyl sulfoxide (DMSO). Ten enamel samples were exposed to each PM for 14 days. For aerosol exposure, 12 enamel samples were exposed (200 puffs per day, for 5 consecutive days) to 3R4F CS or THP1.0 and NVP aerosols. Control samples were incubated with DMSO (PM study) or phosphate buffered saline (PBS, aerosol study). Individual enamel sample color readings (L*, a*, b*) were measured at baseline and on each exposure day. Mean ΔL*, Δa*, Δb* and ΔE values were calculated for each product or control. A one-way ANOVA was used to assess the differences between the products and controls. The Tukey procedure for pairwise comparisons was also used. RESULTS: At all timepoints, 3R4F PM and CS induced enamel discoloration that was statistically significant (< 0.0001) when compared to THP1.0 or NVP. After 14-day PM exposure, mean ΔE values were 29.4± 3.6, 10.5 ± 2.3, 10.7 ± 2.6 and 12.6 ± 2.0 for 3R4F, THP1.0, NVP and DMSO control respectively. After 5-day CS or aerosol exposure, mean ΔE values were 26.2 ± 3.2, 3.6 ± 1.9, 3.4 ± 1.3, 5.3 ± 0.8 for 3R4F CS, THP1.0, NVP or PBS control, respectively. Both exposure methods demonstrated that THP1.0 and NVP induced minimal staining, mean ΔL* , Δa* , Δb* and ΔE values were comparable to DMSO or PBS controls. CLINICAL SIGNIFICANCE: For the first time, diverse NGPs across the risk continuum were assessed in vitro for their impact on enamel staining. CS exposure significantly increased the level of bovine enamel sample discoloration, whereas THP1.0 or NVP exposure resulted in values comparable to the controls.

Cigarette smoke induced genotoxicity and respiratory tract pathology: evidence to support reduced exposure time and animal numbers in tobacco product testing.

Many laboratories are working to develop in vitro models that will replace in vivo tests, but occasionally there remains a regulatory expectation of some in vivo testing. Historically, cigarettes have been tested in vivo for 90 days. Recently, methods to reduce and refine animal use have been explored. This study investigated the potential of reducing animal cigarette smoke (CS) exposure to 3 or 6 weeks, and the feasibility of separate lung lobes for histopathology or the Comet assay. Rats were exposed to sham air or CS (1 or 2 h) for 3 or 6 weeks. Respiratory tissues were processed for histopathological evaluation, and Alveolar type II cells (AEC II) isolated for the Comet assay. Blood was collected for Pig-a and micronucleus quantification. Histopathological analyses demonstrated exposure effects, which were generally dependent on CS dose (1 or 2 h, 5 days/week). Comet analysis identified that DNA damage increased in AEC II following 3 or 6 weeks CS exposure, and the level at 6 weeks was higher than 3 weeks. Pig-a mutation or micronucleus levels were not increased. In conclusion, this study showed that 3 weeks of CS exposure was sufficient to observe respiratory tract pathology and DNA damage in isolated AEC II. Differences between the 3 and 6 week data imply that DNA damage in the lung is cumulative. Reducing exposure time, plus analyzing separate lung lobes for DNA damage or histopathology, supports a strategy to reduce and refine animal use in tobacco product testing and is aligned to the 3Rs (replacement, reduction and refinement).

A comparative assessment of cigarette smoke aerosols using an in vitro air-liquid interface cytotoxicity test.

This study describes the evaluation of a modified air-liquid interface BALB/c 3T3 cytotoxicity method for the assessment of smoke aerosols in vitro. The functionality and applicability of this modified protocol was assessed by comparing the cytotoxicity profiles from eight different cigarettes. Three reference cigarettes, 1R5F, 3R4F and CORESTA Monitor 7 were used to put the data into perspective and five bespoke experimental products were manufactured, ensuring a balanced and controlled study. Manufactured cigarettes were matched for key variables such as nicotine delivery, puff number, pressure drop, ventilation, carbon monoxide, nicotine free dry particulate matter and blend, but significantly modified for vapor phase delivery, via the addition of two different types and quantities of adsorptive carbon. Specifically manufacturing products ensures comparisons can be made in a consistent manner and allows the research to ask targeted questions, without confounding product variables. The results demonstrate vapor-phase associated cytotoxic effects and clear differences between the products tested and their cytotoxic profiles. This study has further characterized the in vitro vapor phase biological response relationship and confirmed that the biological response is directly proportional to the amount of available vapor phase toxicants in cigarette smoke, when using a Vitrocell® VC 10 exposure system. This study further supports and strengthens the use of aerosol based exposure options for the appropriate analysis of cigarette smoke induced responses in vitro and may be especially beneficial when comparing aerosols generated from alternative tobacco aerosol products.

An improved method for the isolation of rat alveolar type II lung cells: Use in the Comet assay to determine DNA damage induced by cigarette smoke.

Smoking is a cause of serious diseases, including lung cancer, emphysema, chronic bronchitis and heart disease. DNA damage is thought to be one of the mechanisms by which cigarette smoke (CS) initiates disease in the lung. Indeed, CS induced DNA damage can be measured in vitro and in vivo. The potential of the Comet assay to measure DNA damage in isolated rat lung alveolar type II epithelial cells (AEC II) was explored as a means to include a genotoxicity end-point in rodent sub-chronic inhalation studies. In this study, published AEC II isolation methods were improved to yield viable cells suitable for use in the Comet assay. The improved method reduced the level of basal DNA damage and DNA repair in isolated AEC II. CS induced DNA damage could also be quantified in isolated cells following a single or 5 days CS exposure. In conclusion, the Comet assay has the potential to determine CS or other aerosol induced DNA damage in AEC II isolated from rodents used in sub-chronic inhalation studies.

NucleaRDB: information system for nuclear receptors.

The NucleaRDB is a Molecular Class-Specific Information System that collects, combines, validates and disseminates large amounts of heterogeneous data on nuclear hormone receptors. It contains both experimental and computationally derived data. The data and knowledge present in the NucleaRDB can be accessed using a number of different interactive and programmatic methods and query systems. A nuclear hormone receptor-specific PDF reader interface is available that can integrate the contents of the NucleaRDB with full-text scientific articles. The NucleaRDB is freely available at http://www.receptors.org/nucleardb.

Corrigendum: Applying new approach methodologies to assess next-generation tobacco and nicotine products.

[This corrects the article DOI: 10.3389/ftox.2024.1376118.].

Applying new approach methodologies to assess next-generation tobacco and nicotine products.

In vitro toxicology research has accelerated with the use of in silico, computational approaches and human in vitro tissue systems, facilitating major improvements evaluating the safety and health risks of novel consumer products. Innovation in molecular and cellular biology has shifted testing paradigms, with less reliance on low-throughput animal data and greater use of medium- and high-throughput in vitro cellular screening approaches. These new approach methodologies (NAMs) are being implemented in other industry sectors for chemical testing, screening candidate drugs and prototype consumer products, driven by the need for reliable, human-relevant approaches. Routine toxicological methods are largely unchanged since development over 50 years ago, using high-doses and often employing in vivo testing. Several disadvantages are encountered conducting or extrapolating data from animal studies due to differences in metabolism or exposure. The last decade saw considerable advancement in the development of in vitro tools and capabilities, and the challenges of the next decade will be integrating these platforms into applied product testing and acceptance by regulatory bodies. Governmental and validation agencies have launched and applied frameworks and "roadmaps" to support agile validation and acceptance of NAMs. Next-generation tobacco and nicotine products (NGPs) have the potential to offer reduced risks to smokers compared to cigarettes. These include heated tobacco products (HTPs) that heat but do not burn tobacco; vapor products also termed electronic nicotine delivery systems (ENDS), that heat an e-liquid to produce an inhalable aerosol; oral smokeless tobacco products (e.g., Swedish-style snus) and tobacco-free oral nicotine pouches. With the increased availability of NGPs and the requirement of scientific studies to support regulatory approval, NAMs approaches can supplement the assessment of NGPs. This review explores how NAMs can be applied to assess NGPs, highlighting key considerations, including the use of appropriate in vitro model systems, deploying screening approaches for hazard identification, and the importance of test article characterization. The importance and opportunity for fit-for-purpose testing and method standardization are discussed, highlighting the value of industry and cross-industry collaborations. Supporting the development of methods that are accepted by regulatory bodies could lead to the implementation of NAMs for tobacco and nicotine NGP testing.

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.

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 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.

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.