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Context: In addition to tobacco and cigarette ingredients, there are many non-tobacco components and processes used to manufacture commercial cigarettes. Proposed cigarette components and manufacturing process changes were evaluated using a tiered toxicological testing program., Objective: The toxicological testing and evaluation of proposed changes to selected non-tobacco cigarette components and manufacturing processes are described in this special report., Materials and Methods: Selected non-tobacco cigarette components and manufacturing processes were evaluated using experimental and control cigarettes. These experimental cigarettes were evaluated using mainstream smoke chemistry, bacterial mutagenicity and cytotoxicity assays. In some evaluations, 90-day nose-only mainstream smoke inhalation studies using rats were performed., Results: Some proposed design changes were not implemented, or limitations on their use were established. Most study results, however, were similar to those previously reported in the scientific literature for design changes in cigarette construction., Conclusion: The studies reported in the series of publication demonstrate that our testing approach is feasible for evaluation of cigarette component and manufacturing process changes.

Context: Adhesives are used in several different manufacturing operations in the production of cigarettes. The use of new, "high-speed-manufacture" adhesives (e.g. vinyl acetate based) could affect the smoke chemistry and toxicology of cigarettes, compared with older "low-speed-manufacture" adhesives (e.g. starch based)., Objective: This study was conducted to determine whether the inclusion of different levels of three adhesives (ethylene vinyl acetate, polyvinyl acetate and starch) in experimental cigarettes results in different smoke chemistry and toxicological responses in in vitro and in vivo assays., Materials and Methods: A battery of tests (analytical chemistry, in vitro and in vivo assays) was used to compare the chemistry and toxicology of smoke from experimental cigarettes made with different combinations of the three adhesives. Varying levels of the different side-seam adhesives, as well as the transfer of adhesives from packaging materials, were tested., Results: There were differences in some mainstream cigarette smoke constituents as a function of the level of adhesive added to experimental cigarettes and between the tested adhesives. None of these differences translated into statistically significant differences in the in vitro or in vivo assays., Conclusion: The use of newer "high-speed-manufacture" vinyl acetate-based adhesives in cigarettes does not produce toxicological profiles that prevent the adhesives from replacing the older "low-speed-manufacture" adhesives (such as starch).

Context: Historical work indicates that cigarette circumference may affect the toxicological profile of experimental cigarettes., Objective: Studies were conducted to examine the effect of different cigarette circumferences on (1) selected mainstream smoke constituents including concentrations of tobacco specific nitrosamines (TSNA) in smoke and (2) mutagenicity and cytotoxicity of cigarette smoke condensate., Materials and Methods: Analytical chemistry, Salmonella mutagenicity and cytotoxicity assays were used to evaluate the composition and biological activity of mainstream smoke from experimental, non-filtered cigarettes manufactured with four different circumferences (17.0-27.1 mm)., Results: Most smoke constituents, including TSNA, decreased with decreasing cigarette circumference; however, amounts of hydrogen cyanide increased in a non-circumference dependent manner. Mutagenicity and cytotoxicity also decreased slightly with decreasing cigarette circumference., Conclusion: Cigarette circumference may have a minor role in the toxicological profile of experimental cigarettes, with a so-far-unidentified mechanism.

Context: Cigarettes often have a small identifying mark (monogram) printed either on the cigarette paper toward the filter end of the cigarette or on the tipping paper., Objective: A battery of tests was used to compare the toxicology of mainstream smoke from experimental cigarettes manufactured with different monogram inks. Cigarettes with different concentrations of different pigments were compared with cigarettes without ink, and with a control ink., Materials and Methods: Smoke from each of the experimental cigarettes was evaluated using analytical chemistry and in vitro bacterial mutagenicity (Salmonella, five strains, ± S9) and cytotoxicity (neutral red uptake) assays., Results: No differences were observed between experimental cigarettes printed with three different pigment loads of iron oxide-based Black pigment and non-printed cigarettes. In general, no dose response was observed. However, increases in certain smoke constituents were found to correlate with Pigment Yellow 14 (also known as benzidine yellow) and Pigment Blue 15 (copper phthalocyanine). Increases in bacterial mutagenicity were observed for high-level print of Pigment Yellow 14 in TA98 and TA1537 and the high-level print of Pigment Blue 15 in TA98. In vitro cytotoxicity of mainstream smoke was unaffected by the presence of monogram ink on cigarettes., Conclusion: Statistically significant dose-responsive constituent changes and an increase in mutagenicity were observed with inclusion of Pigment Yellow 14 and Pigment Blue 15. Other pigments showed minimal toxicological activity.

Context: To comply with state requirements, cigarette manufacturers have added low-permeability bands to the cigarette paper. These bands can extinguish the cigarette when it is no longer being puffed by a smoker., Objective: This study was conducted to evaluate the toxicology resulting from the addition of different types of bands to experimental cigarettes., Materials and Methods: A battery of assays that are typically used in toxicology studies with cigarette smoke, namely smoke chemistry, in vitro mutagenicity and cytotoxicity, and inhalation studies with rats, were used to evaluate different band characteristics added to cigarette paper., Results: Although differences in the amount of band material was associated with an increase in some metals measured in mainstream tobacco smoke, it was not dose responsive to any band design parameter (base paper permeability, band width, band spacing, band chalk amount, or citrate). Occasional, minor differences were produced by the different types of bands; overall, there was no increased toxicity., Conclusion: Although there were increases and decreases in some mainstream smoke constituents, the in vitro and in vivo testing performed demonstrated that low-permeability bands on cigarettes do not modify the toxicity of smoke inhaled by smokers.

Context: Manufacture of cigarettes results in tobacco by-products, some of which can be processed and added back to cigarettes. Such additions (known as reconstituted tobacco or reconstituted leaf) have been shown to reduce tar yields. A new process (termed "Deli" cast sheet) is a potential refinement of the reconstitution process., Objective: Compare toxicity of smoke from experimental cigarettes made with reconstituted leaf with that from cigarettes made with Deli cast sheet., Materials and Methods: Analytical chemistry, Salmonella mutagenicity and cytotoxicity assays were used to evaluate the composition biological activity of mainstream smoke from experimental cigarettes made with Deli cast sheet or with reconstituted leaf. The effect of different amounts of guar and propylene glycol in Deli cast sheet was also evaluated., Results: Small increases in the amount of nitrogen oxides were found as a result of inclusion of the Deli cast sheet when compared with reconstituted leaf; no differences in cytotoxicity or mutagenicity were found., Conclusion: The Deli process neither significantly modified chemical composition of smoke nor affected its biological activity, as measured by the mutagenicity and cytotoxicity assays used here.

Context: Literature suggests that the width of tobacco strips in cigarettes may affect the smoke chemistry and toxicology of such products., Objective: A comprehensive analysis of smoke from experimental cigarettes can be used to determine whether different cut widths of tobacco result in different toxicological activity., Materials and Methods: A battery of tests was used to compare the chemistry and in vitro and in vivo toxicology of smoke from experimental cigarettes made with tobacco cut to different widths., Results: Different cut widths of tobacco did not elicit consistent and significant differences in cigarette smoke chemistry, responses in in vitro mutagenicity or cytotoxicity assays or most endpoints in 90-d rat inhalation studies. Of note, however, were atypical in-life observations and slightly depressed body weights observed in two rat inhalation studies., Conclusion: Most of our data indicate that different cut widths of tobacco used in cigarettes are unlikely to change the toxicity of mainstream cigarette smoke; however, without additional investigation, the atypical in-life observations and depression in body weights cast doubt on the toxicological acceptability of cutting the tobacco into wider shreds.

Context: Recent technological advances allow ventilation holes in (or adjacent to) cigarette filters to be produced using lasers instead of using the mechanical procedures of earlier techniques., Objective: Analytical chemistry can be used to compare the composition of mainstream smoke from experimental cigarettes having filters with mechanically produced ventilation holes to that of cigarettes with ventilation holes that were produced using laser technology., Materials and Methods: Established procedures were used to analyze the smoke composition of 38 constituents of mainstream smoke generated using standard conditions., Results: There were no differences between the smoke composition of cigarettes with filter ventilation holes that were produced mechanically or through use of laser technology., Conclusion: The two methods for producing ventilation holes in cigarette filters are equivalent in terms of resulting mainstream smoke chemistry, at two quite different filter ventilation percentages.

There is considerable evidence that inhaled toxicants such as cigarette smoke can cause both irreversible changes to the genetic material (DNA mutations) and putatively reversible changes to the epigenetic landscape (changes in the DNA methylation and chromatin modification state). The diseases that are believed to involve genetic and epigenetic perturbations include lung cancer, chronic obstructive pulmonary disease (COPD), and cardiovascular disease (CVD), all of which are strongly linked epidemiologically to cigarette smoking. In this review, we highlight the significance of genomics and epigenomics in these major smoking-related diseases. We also summarize the in vitro and in vivo findings on the specific perturbations that smoke and its constituent compounds can inflict upon the genome, particularly on the pulmonary system. Finally, we review state-of-the-art genomics and new techniques such as high-throughput sequencing and genome-wide chromatin assays, rapidly evolving techniques which have allowed epigenetic changes to be characterized at the genome level. These techniques have the potential to significantly improve our understanding of the specific mechanisms by which exposure to environmental chemicals causes disease. Such mechanistic knowledge provides a variety of opportunities for enhanced product safety assessment and the discovery of novel therapeutic interventions.

Toxicological comparisons were made of three commercial cigarettes, namely Marlboro full flavor, Marlboro Lights, and Marlboro Ultra Lights, with the 1R4F reference cigarette. The main comparison was a 90-d inhalation study with mainstream smoke at 150 mg total particulate matter per cubic meter, in Sprague-Dawley rats using 6 h/d and 7 d/w exposures. The principal endpoint was histopathology of the respiratory tract, along with examinations of free lung cell counts after broncho-alveolar lavage. Additional studies on mainstream smoke included Salmonella mutagenicity, cytotoxicity of particulate and gas/vapor phases, and analytical chemistry. The exposures produced effectively the same responses in each of the four groups, and the histopathology results in the commercial cigarette groups were also effectively the same. The 1R4F was also tested at 75 and 200 mg/m(3), and most of the histopathology results obtained here showed dose-response relationships. The free lung cell responses were similar in the 1R4F/commercial cigarette comparison, and there were dose-related changes in the 1R4F groups, most notably for neutrophils. Most of the changes produced in the 90-d of exposure were resolved in a 42-d post-inhalation period. Responses in the in vitro and analytical assays for the four cigarettes were in general similar, when data were expressed either per mg TPM or per mg tar yield. There were judged to be no toxicologically meaningful differences between the profiles evaluated at similar smoke concentrations for the three commercial cigarettes and for the 1R4F using these assays.

A review of the published work with charcoal-filtered cigarettes indicates that there are reductions in the concentrations for many gas-vapor phase constituents found in mainstream smoke. However, charcoal filters provided no apparent capacity for reduction of smoke particulate phase components. The reductions in gas-vapor phase smoke chemistry analytes generally correspond with findings of reduced toxicological activity, principally related to a reduction in the cytotoxic action of the volatile smoke constituents. Results of a short-term clinical study show small reductions in the biomarkers of the gas-vapor phase smoke constituents in subjects smoking charcoal-filtered cigarettes, compared to subjects smoking non-charcoal filtered cigarettes. The very limited epidemiology data (a single study) fail to demonstrate a conclusive beneficial effect of charcoal-filtered cigarette products compared to non-charcoal filtered cigarette products. Review of the scientific literature is hindered due to the lack of documentation regarding the activity of the charcoal used in the filter, and the inconsistency in product designs used between the various different disciplines (chemistry, pre-clinical, clinical and epidemiology) that have conducted studies with charcoal filtered cigarettes. There do not appear to be any published studies using a combination of data from the different disciplines based on a consistently designed charcoal cigarette filter. Although the literature presently available would suggest that smoke filtration provided by current charcoal filter techniques alone may not be substantial enough to reduce smoking-related disease, the data are limited. Therefore, for the reduction of smoking-induced disease, it is difficult to come to a definitive conclusion regarding the potential health benefits of using charcoal as a smoke filtration technology.

This work was performed to verify whether or not the inhalation response to cigarette smoke in animal species for assessing carcinogenic potential in humans reflects the strong epidemiological evidence in human smokers. Significant increases in the numbers of malignant tumors of the respiratory tract were not seen in rats, mice, hamsters, dogs, or nonhuman primates exposed for long periods of time to very high concentrations of mainstream cigarette smoke. The results are clearly at variance with the epidemiological evidence in smokers, and it is difficult to reconcile this major difference between observational studies in humans and controlled laboratory studies.

The genotoxic effects of 90-day nose-only exposures to smoke from new cigarettes, which heat but do not burn tobacco (New), or from reference cigarettes, which burn tobacco, were evaluated in Sprague-Dawley rats by examining the cytogenetic endpoints of sister-chromatid exchanges (SCE), chromosome aberrations, and micronuclei in bone-marrow cells. The concentrations of wet total particulate matter (WTPM) and carbon monoxide in the smoke from both cigarette types were similar. The mainstream smoke from both New and reference cigarettes was adjusted to WTPM concentrations of approx. 200 and 400 micrograms/l for low and high smoke exposure. Rats were exposed to smoke 1 h per day, 5 days per week for 13 consecutive weeks. Inhalation of smoke by the exposed animals was confirmed by analysis of blood carboxyhemoglobin and plasma nicotine. Examination of bone-marrow cells following the final day of exposure showed that smoke from neither the New nor reference cigarette induced a positive response in the SCE, chromosome aberration, or micronucleus assays in rats.

Smoke condensates derived from cigarettes containing different amounts of the tobacco supplement Cytrel were painted onto the banks of mice 3 times a week for 104 weeks. Cigarette samples contained 0, 25, 50 or 100% Cytrel, blended with tobacco, and 3 dose levels were used for each sample. Statistical analyses were made of tumour incidence using a mathematical model based on the Weibull distribution. Good dose relationships were observed for each product; and for samples containing Cytrel there was a reduction in smoke condensate production and in tumourigenicity (per unit weight of smoke condensate) approximately proportional to the level of inclusion. Histopathological examinations of tumours indicated a reduction in malignancy of approximately 50% for all-Cytrel samples when compared with all-tobacco samples.

Eight groups of 30 male and 30 female rats were exposed 1 hr per day, 5 days per week for 13 weeks, to smoke from reference (tobacco burned) or test (tobacco only heated) cigarettes, at nicotine concentrations of 5, 15, or 30 micrograms/liter of air. Similar smoke concentrations of wet total particulate matter and carbon monoxide were produced in each of the test/reference comparisons. There was a pronounced depression of minute ventilation of animals in the reference groups, but not in the test animals. Blood carboxyhemoglobin concentrations were similar in animals exposed to smoke from test and reference cigarettes. Plasma concentrations of nicotine and cotinine in the test groups were higher than in the reference groups. There were no differences between the smoke-exposed groups in terms of body weight or feed consumption. At necropsy, an increase in heart weight was noted in both high exposure groups. There were notable differences in histopathology, with fewer and less-pronounced changes in the test groups than in the reference groups. Many of the histopathological responses induced in the reference groups were absent in the test groups. Overall, the study demonstrated a substantial reduction in the biological activity of smoke from the test cigarette when compared with the reference.