There are several whole smoke exposure systems used to assess the biological and toxicological impact of tobacco smoke in vitro. One such system is the Vitrocell® VC 10 Smoking Robot and exposure module. Using quartz crystal microbalances (QCMs) installed into the module, we were able to assess tobacco smoke particle deposition in real-time. We compared regional deposition across the module positions and doses delivered by six VC 10s in four independent laboratories: two in the UK, one in Germany and one in China. Gauge R&r analysis was applied to the total data package from the six VC 10s. As a percentage of the total, reproducibility (between all six VC 10s) and repeatability (error within an individual VC 10) accounted for 0.3% and 7.4% respectively. Thus Gauge R&r was 7.7%, less than 10% overall and considered statistically fit for purpose. The dose-responses obtained from the six machines across the four different locations demonstrated excellent agreement. There were little to no positional differences across the module at all airflows as determined by ANOVA (except for one machine and at three airflows only). These results support the on-going characterisation of the VC 10 exposure system and suitability for tobacco smoke exposure in vitro.

BackgroundThe development of whole smoke exposure systems have been driven by the fact that traditional smoke exposure techniques are based on the particulate phase of tobacco smoke and not the complete smoke aerosol. To overcome these challenges in this study, we used a Vitrocell® VC 10 whole smoke exposure system. For characterisation purposes, we determined smoke deposition in relationship to airflow (L/min), regional smoke deposition within the linear exposure module, vapour phase dilution using a known smoke marker (carbon monoxide) and finally assessed biological responses using two independent biological systems, the Ames and Neutral Red uptake (NRU) assay.ResultsSmoke dilution correlates with particulate deposition (R2 = 0.97) and CO concentration (R2 = 0.98). Regional deposition analysis within the linear exposure chamber showed no statistical difference in deposited mass across the chamber at any airflows tested. Biological analysis showed consistent responses and positive correlations with deposited mass for both the Ames (R2 = 0.76) and NRU (R2 = 0.84) assays.ConclusionsWe conclude that in our study, under the experimental conditions tested, the VC 10 can produce stable tobacco smoke dilutions, as demonstrated by particulate deposition, measured vapour phase smoke marker delivery and biological responses from two independent in vitro test systems.

To date there are no widely accepted methods for the toxicological testing of complex gaseous mixtures and aerosols, such as cigarette smoke, although some modifications to the standard regulatory methods have been developed and used. Historically, routine testing of cigarettes has primarily focused on the particulate fraction of cigarette smoke. However, this fraction may not accurately reflect the full toxicity and mutagenicity of the smoke aerosol as a whole, which contains semi-volatiles and short-lived products of combustion. In this study we have used a modified version of the bacterial reverse-mutation (Ames) assay for the testing of mainstream smoke generated from 3R4F reference cigarettes with a Vitrocell(®) VC 10 exposure system. This method has been evaluated in four strains of Salmonella typhimurium (TA98, TA100, YG1024 and YG1042) and one strain of Escherichia coli (WP2 uvrA pKM101) in the absence and presence of a metabolic activation system. Following exposure at four concentrations of diluted mainstream cigarette-smoke, concentration-related and reproducible increases in the number of revertants were observed in all four Salmonella strains. E. coli strain WP2 uvrA pKM101 was unresponsive at the four concentrations tested. To quantify the exposure dose and to enable biological response to be plotted as a function of deposited mass, quartz-crystal microbalances were included in situ in the smoke-exposure set-up. This methodology was further assessed by comparing the responses of strain YG1042 to mainstream cigarette-smoke on a second VC 10 Smoking Robot. In summary, the Ames assay can be successfully modified to assess the toxicological impact of mainstream cigarette-smoke.

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BackgroundTobacco smoke toxicity has traditionally been assessed using the particulate fraction under submerged culture conditions which omits the vapour phase elements from any subsequent analysis. Therefore, methodologies that assess the full interactions and complexities of tobacco smoke are required. Here we describe the adaption of a modified BALB/c 3T3 neutral red uptake (NRU) cytotoxicity test methodology, which is based on the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) protocol for in vitro acute toxicity testing. The methodology described takes into account the synergies of both the particulate and vapour phase of tobacco smoke. This is of particular importance as both phases have been independently shown to induce in vitro cellular cytotoxicity.FindingsThe findings from this study indicate that mainstream tobacco smoke and the gas vapour phase (GVP), generated using the Vitrocell® VC 10 smoke exposure system, have distinct and significantly different toxicity profiles. Within the system tested, mainstream tobacco smoke produced a dilution IC50 (dilution (L/min) at which 50% cytotoxicity is observed) of 6.02 L/min, whereas the GVP produced a dilution IC50 of 3.20 L/min. In addition, we also demonstrated significant dose-for-dose differences between mainstream cigarette smoke and the GVP fraction (P < 0.05). This demonstrates the importance of testing the entire tobacco smoke aerosol and not just the particulate fraction, as has been the historical preference.ConclusionsWe have adapted the NRU methodology based on the ICCVAM protocol to capture the full interactions and complexities of tobacco smoke. This methodology could also be used to assess the performance of traditional cigarettes, blend and filter technologies, tobacco smoke fractions and individual test aerosols.