Volume 24 (2011): Issue 4 (January 2011)

Currently, several in vitro tests are widely used to measure toxicological properties of mainstream smoke (Neutral Red Uptake Assay, Micronucleus assay, Ames Test). These tests are necessary to assess cytotoxicity, genotoxicity, and mutagenicity, but are time consuming. This is essentially due to the preparation and the handling of cells. It is difficult to use these in vitro tests as screening method for product testing and development. For a better assessment of the cytotoxicity of the vapor phase, a rapid cell-free method has been developed. This paper describes a capillary electrophoresis cell-free method, based on the depletion of an anti-oxidant L-gamma-glutamyl-L-cysteinylglycine (GSH), applied to an aliquot of vapor phase phosphate buffered saline (PBS)-trapped cigarette smoke (as recommended for in vitro testing). The correlation between this method and the survival/viability test (Neutral Red cytotoxicity) is excellent (coefficient of correlation (r = 0.99)).

Changes in exposure to cigarette smoke and smoking behavior were assessed in adult smokers participating in a multi-center, cross-sectional study who spontaneously switched to > 3 mg lower or higher machine measured ‘tar’ yield. Of 2,542 consenting smokers only 23 down-switchers (DWNSW) and 68 up-switchers (UPSW) met study eligibility criteria. Biomarkers of exposure (BOE) to selected smoke constituents were measured. Large variability was observed in the BOEs (e.g. CV% for nicotine equivalents (nicotine and five of its metabolites, NE) per day ranged from 59% to 78%). On average, DWNSW smoked two more cigarettes/day (+ 9%) that had ~ 5.9 mg lower ‘tar’ yield. Mean NE/day were 12.0 ± 6.2 mg/day compared to 13.9 ± 8.2 mg/day after down switching. Slightly lower levels of NE/cigarette (-8%), total NNAL/day and per cigarette were observed (-18% and -23%) in the DWNSW's. UPSW smoked two fewer cigarettes/day (-13%) with higher ‘tar’ yield (~ 8.4 mg higher ‘tar’). NE/day was 12.5 ± 9.7 vs. 12.8 ± 9.0 mg/day. Total NNAL values per day and per cigarette were lower (-24% and -17%). Due to the large variability and insufficient power to detect significant differences in exposure based on post-hoc power calculations, no definitive conclusions can be drawn from this study. These results suggest that it might not be feasible to conduct a definitive assessment of changes in exposure among spontaneous switchers.

Joint experimental work carried out in 2006 by the CORESTA Special Analytes Task Force compared yield data on a wide range of smoke constituents obtained from reference cigarettes according to the existing methods used by participants. This work identified that the methodologies that were used to determine yields of selected volatiles in mainstream smoke under the ISO smoking regime would benefit from further joint experiments. This report describes the output from the 2008 Joint Experiment on selected volatiles in smoke (1,3-butadiene, benzene, toluene, acrylonitrile, and isoprene). Its objectives were to investigate the main weaknesses and influencing factors in methodologies used by the participating laboratories and their effects on yield variability before deciding on one to take forward to a CORESTA recommended method. The Task Force considered this step was necessary before progressing to a full collaborative study using a recommended method. An experimental protocol was devised to investigate several factors such as the use of different calibration standards and the efficiencies of different trapping systems. The effects of other general factors identified from supplied methodology information as differing across laboratories were also analysed. A statistical assessment was made of their possible influence on smoke yields and yield reproducibility across different laboratories and is discussed in this report. Between-laboratory variability has been reduced since the last study indicating that some laboratories have improved their methodology although extremely high values for the among-laboratory variability were still found for acrylonitrile (> 100%) and 1,3-butadiene (~ 80%) when related to the mean yields. The means to reduce the variability in acrylonitrile and 1,3-butadiene yields are not apparent from the data and interpretations made in this study. However, when the different laboratories use the same methodology during the development of a recommended method at the next development stage then it is hoped that this high level of variability for acrylonitrile and 1,3-butadiene will be reduced to similar levels to those found for benzene, toluene, and isoprene. As in previous work, it was recognised that although a more intense smoking regime may be introduced into the regulatory arena in the future, it was decided that the current ISO smoking regime should be used for this joint experiment. A wider range of product styles will be investigated when the Task Force works towards a recommended method to take account of differing blends and designs and the potentially greater product variability of commercial products. This will provide robust estimates of within-laboratory repeatability and among-laboratory reproducibility and is intended to be reported in a later paper.

The influence of cigarette design on the content of phenols in mainstream tobacco smoke was studied. The most abundant phenols - catechol, hydroquinone, phenol, o-, m-, and p-cresol, and resorcinol - were determined by HPLC with fluorescence detection. Hydroquinone and catechol made the most significant contribution to the total content of phenols with maximum values of 135.0 µg/cig and 95.7 µg/cig, respectively. The highest total content of phenols (330.9 µg/cig) was measured in the smoke of a Virginia tobacco cigarette. The total content of phenols (µg/cig) in cigarette mainstream smoke decreased linearly with increased filter ventilation, R² = 0.9536. The results obtained indicate that filtration and ventilation can strongly influence the mainstream tobacco smoke content of phenol and its less polar derivatives, o-, m-, and p-cresol, which were reduced by up to 85%. Hydroquinone and catechol are less affected and only cigarettes with the special “recessed charcoal filter system” and cigarettes with filter ventilation over 50% showed significant reductions. On a per mg ‘tar’ basis the largest contributor to phenols in cigarette mainstream smoke was the selection of the tobacco type. The use of any standard commercial filter on an unfiltered cigarette can substantially reduce the yield of phenols in cigarette mainstream smoke. The use of special filters (e.g., the “recessed charcoal filter system”) or high levels of cigarette ventilation does not reduce the amount of phenols in tobacco smoke considerably when normalized on a per mg ‘tar’ basis.

Based on a two-year field trial in the region of Campania (Southern Italy) the effects of water volume and nitrogen fertilization on the yield and quality of Burley tobacco (Nicotianatabacum L.) were investigated with reference to the following traits: cured leaf yield, price index, yield value, leaf area, specific leaf weight, burning capacity, color parameters, total alkaloid, nitrate and chloride leaf content. The experimental design was a factorial comparison among three water volumes (40, 80 and 120% evapotranspiration (ET)), four nitrogen fertilization levels (0, 80, 160 and 240 kg ha^-1) and two genotypes (cv TN86 and the hybrid R7-11). The yield of cured leaves rose with the increase in water and nitrogen availability, albeit at a decreasing rate. With the increase in water volume, the price index, burning capacity, specific leaf weight, total alkaloid and nitrate content decreased, while leaf area and chloride content increased. Up to a rate of 160 kg ha^-1, nitrogen fertilization increased the price index, yield value, burning capacity, leaf area, specific leaf weight, total alkaloid and nitrates, and reduced leaf chloride content especially at 40% ET water volume. Both, nitrogen fertilization and water volume had little influence on leaf color. The year had considerable effects on yield, leaf area and color parameters, with higher values in the rainier season. In the two years, genotype TN86 showed higher stability for yield and yield value, lower alkaloid and higher nitrate content in the leaf than the R7-11 hybrid.