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.

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

The rapid development associated with Next Generation Tobacco Products (NGTP) has necessitated the development of high throughput methodologies to test their genotoxic potential in vitro when compared to conventional cigarette smoke (CS). An assessment of two Vitrocell® Mammalian 6/48 exposure modules in three independent experiments was made by comparing results from multiple dosimetric techniques applied to aerosol generated from 3R4F Kentucky Reference cigarettes, commercially available electronically heated tobacco product (eHTP) and Electronic Nicotine Delivery System (ENDS) using the Vitrocell® VC10®. Real-time aerosol particle concentration was assessed by means of light scattering photometers and expressed as area under the curve (∑AUC). Nicotine concentrations were determined analytically by LC/MS. Humectant amount and distribution was assessed for eHTP and ENDS by the quantification of free glycerol in a phosphate buffered saline (PBS) trap, whereas total particulate matter (TPM) was assessed in the 3R4F cigarettes by the fluorescence of the particulate at 485 nm in anhydrous dimethyl sulfoxide (DMSO) trap within the exposure. Dose was adjusted by means of the addition of ambient air to dilute the whole smoke/aerosol in L/min and sampled into the system at a rate of 5 mL/min. Dilution of CS ranged from 8.0 to 0.5 L/min and for the eHTP and ENDS ranged from 4 to 0 L/min (undiluted). Dosimetric analysis of the system showed good concordance within replicates (p-values ranged from p = 0.3762 to p = 0.8926) and showed that the Vitrocell® Mammalian 6/48 is a viable means for genotoxic assessment of aerosol generated from both conventional cigarettes and NGTP. Results demonstrate the need to tailor dosimetry approaches to different aerosols due to variations in the physio-chemical composition, with a multi-dosimetry approach recommended.

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.

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

The Vitrocell® VC10® smoke exposure system offers multiple platforms for air liquid interface (ALI) and air agar interface (AAI) exposure that mimic in vivo conditions for assessing toxicological impact of whole smoke using in vitro assays. The aim of this study was to investigate and compare multiple dosimetry techniques that may be employed during combustible cigarette whole smoke exposure using the Vitrocell® VC10® smoking robot. The following techniques were assessed: (1) quartz crystal microbalances (QCMs), (2) aerosol photometers (using area under curve, AUC), and (3) fluorescence of anhydrous dimethyl sulfoxide (DMSO)-captured smoke constituents. Results showed that each of the dosimetry techniques was able to distinguish different levels of whole smoke airflow in a concentration-related manner. When compared to each other, the three techniques showed a high level of consistency and all were considered efficient tools in quantifying dose during an exposure, although higher variation was observed at the higher airflows tested. Overall, the dosimetry tools investigated here provide effective measures of the whole smoke concentrations tested during the exposure.

The mutagenic assessment of mainstream cigarette smoke using the Ames assay: a multi-strain approach.

Salmonella typhimurium strains TA1535, TA1537, TA97, TA102 and TA104 were assessed for their suitability and use in conjunction with a Vitrocell(®) VC 10 Smoking Robot and 3R4F reference mainstream cigarette smoke. Little information exists on TA97, TA104, TA1535, TA1537 and TA102 using an aerosol 35mm spread-plate format. In this study, TA1535 and TA1537 were considered sub-optimal for use with a scaled-down format, due to low spontaneous revertant numbers (0-5 revertants/plate). In the context of a regulatory environment, TA97 is deemed an acceptable alternative for TA1537 and was therefore selected for whole smoke exposure in this study. However, there is no acceptable alternative for TA1535, therefore this strain was included for whole smoke exposure. TA1535, TA97, TA102 and TA104 were assessed for mutagenic responses following exposure to cigarette smoke at varying concentrations (using diluting airflow rates of 1.0, 4.0, 8.0 and 12.0L/min), and exposure times of 24 and 64min. A positive mutagenic response to cigarette smoke was observed in strain TA104 at both the 24 and 64min time points, in the presence of S-9, at the highest smoke concentration tested (1.0L/min diluting airflow). The three remaining strains were found to be unresponsive to cigarette smoke at all concentrations tested, in the presence and absence of metabolic activation. Cigarette smoke particulate deposition was quantified in situ of exposure using quartz crystal microbalance technology, enabling data to be presented against an associated gravimetric mass (µg/cm(2)). Finally, data obtained in this study were combined with previously published Ames data for TA98, TA100, YG1024, YG1042 and Escherichia coli (WP2 uvrA pKM101), generated using the same 35mm methodology. The combined data-set was used to propose an aerosol testing strategy, based on strain compatibility with the whole smoke aerosol, whilst maintaining the essence of the regulatory guidelines for the standard Ames assay.