Michael J. Morton

QSAR treatment of multiple toxicities: the mutagenicity and cytotoxicity of quinolines.

A series of 15 quinoline congeners were assayed for mutagenicity and cytotoxicity in the Ames test using strain TA100 bacteria. Statistical analysis of the data allowed simultaneous determination of the mutagenicity and cytotoxicity of each quinoline. These data were used to develop three quantitative structure-activity relationships (QSAR). In all three QSAR, the strength of the relationship between hydrophobicity (as measured by log P) and biological activity was similar as h was near 1 in all three cases. For the mutagenicity of these quinolines, both hydrophobic and steric interactions appear to be important. In contrast, the cytotoxicity is mainly affected by increasing hydrophobicity and by the addition of electron withdrawing substituents to the quinoline ring. Comparison to other QSAR from our laboratory and others lends support to these findings. Both simultaneous consideration of different biological activities and the comparison of newly developed QSAR with previous data for the purpose of lateral validation should be encouraged in future QSAR studies.

Toxicological Evaluation of Honey as an Ingredient Added to Cigarette Tobacco

A tiered testing strategy has been developed to evaluate the potential for new ingredients, tobacco processes, and technological developments to increase or reduce the biological activity that results from burning tobacco. In the manufacture of cigarettes, honey is used as a casing ingredient to impart both aroma and taste. The primary objective of this document is to summarize and interpret chemical and toxicological studies that have been conducted to evaluate the potential impact of honey on the biological activity of either mainstream cigarette smoke or cigarette smoke condensate. As part of ongoing stewardship efforts, cigarettes produced with honey (5% wet weight) as an alternative to invert sugar in tobacco casing material were subjected to extensive evaluation. Principal components of this evaluation were a determination of selected mainstream smoke constituent yields, Ames assay, sister chromatid exchange assay in Chinese hamster ovary cells, a 30-wk dermal tumor promotion evaluation of cigarette smoke condensate in SENCAR mice, and a 13-wk inhalation study of cigarette smoke in Sprague-Dawley rats. Comparative analytical evaluations demonstrated that the substitution of honey for invert sugar as a casing material in cigarettes had no significant impact on mainstream smoke chemistry. In addition, in vitro and in vivo studies demonstrated that cigarettes containing tobacco cased with honey had comparable biological activity to cigarettes containing invert sugar. Collectively, these data demonstrate that the use of honey as an alternative casing material in the manufacture of cigarettes does not alter the potential toxicity of cigarette smoke condensate (CSC) or cigarette smoke; therefore the use of honey as an ingredient added to cigarette tobacco is acceptable from a toxicological perspective.

Discriminatory power of standard toxicity assays used to evaluate ingredients added to cigarettes

A tiered approach for testing ingredients in a cigarette matrix was developed and includes chemical-analytical testing and a standard battery of biological toxicity assays. These assays were adapted for comparative evaluation of mainstream smoke from experimental cigarettes with or without ingredients at various inclusion levels. This adaptation to test cigarette mainstream smoke may impact assay response. Since it is difficult to a priori determine discriminatory power, it was evaluated using a large experimental dataset from a multi-year program of cigarette ingredient testing performed at two separate laboratories. A statistical method, minimum detectable difference (MDD), was used as a measure of assay discriminatory power. MDD of cigarette smoke constituents ranged from 6% to 29% of the average. Salmonella mutagenicity and cytotoxicity test MDDs ranged from 20% to 81% and 18% to 49%, respectively. Body weight gain in 90-day nose-only inhalation studies yielded an MDD of 30-40%. Histopathological findings with severity scores between 0.5 and 1.5 had the lowest MDDs of 23% and higher. In general, discriminatory power decreased with increasing biological complexity and toxicological relevance of the assay. Beyond statistical analysis, however, a weight-of-the-evidence analysis by experienced researchers is required for toxicological assessment of a cigarette ingredient.

Insights from analysis for harmful and potentially harmful constituents (HPHCs) in tobacco products

A total of 20 commercial cigarette and 16 commercial smokeless tobacco products were assayed for 96 compounds listed as harmful and potentially harmful constituents (HPHCs) by the US Food and Drug Administration. For each product, a single lot was used for all testing. Both International Organization for Standardization and Health Canada smoking regimens were used for cigarette testing. For those HPHCs detected, measured levels were consistent with levels reported in the literature, however substantial assay variability (measured as average relative standard deviation) was found for most results. Using an abbreviated list of HPHCs, statistically significant differences for most of these HPHCs occurred when results were obtained 4-6months apart (i.e., temporal variability). The assay variability and temporal variability demonstrate the need for standardized analytical methods with defined repeatability and reproducibility for each HPHC using certified reference standards. Temporal variability also means that simple conventional comparisons, such as two-sample t-tests, are inappropriate for comparing products tested at different points in time from the same laboratory or from different laboratories. Until capable laboratories use standardized assays with established repeatability, reproducibility, and certified reference standards, the resulting HPHC data will be unreliable for product comparisons or other decision making in regulatory science.

Insights from a multi-year program designed to test the impact of ingredients on mainstream cigarette smoke toxicity.

Context: Cigarette tobacco ingredients may alter the distribution of chemical constituents present in smoke. When considering the toxicological relevance of potential ingredient-related effects on chemical and biological measurements assessing cigarette smoke toxicity, it is critical to understand the intrinsic variability of tobacco and cigarette smoke that is influenced by the environmental conditions during growing, agricultural practices during preparation, cigarette manufacturing tolerances, and stability of the assay methods. Objective: To understand possible effects of ingredients on cigarette smoke toxicity, various chemical and biological endpoints were measured in smoke from experimental cigarettes (added ingredient) to the intrinsic variability of control cigarettes (no added ingredient). Materials and methods: Data were collected during a multi-year program testing a variety of cigarette ingredients from several chemical classes. Chemical analysis of mainstream cigarette smoke,and biological procedures (Salmonella mutagenicity, cytotoxicity, and smoke inhalation) were performed using validated and controlled laboratory methods. The within-study and temporal variation of control cigarettes manufactured in parallel with experimental cigarettes was calculated and used to measure intrinsic variability. Results: The overwhelming majority of data generated from experimental cigarettes fell within the experiment variability represented by the pooled standard error of the entire multi-year dataset for the control cigarettes. Conclusion: The results of this evaluation add to a growing body of the literature regarding a weight of evidence assessment of cigarette ingredient toxicity. When assessed against the variability of assay methodology, natural agricultural change, and manufacturing control, the ingredients studied here demonstrated little relevant influence on the mainstream cigarette smoke toxicity endpoints measured.

Toxicological evaluation of dry ice expanded tobacco

A tiered testing strategy has been developed to evaluate the potential of tobacco processes, ingredients, or technological developments to change the biological activity resulting from burning tobacco. The strategy is based on comparative chemical and biological testing. Dry ice expanded tobacco (DIET) is an example of a common tobacco expansion process currently used in the manufacture of cigarettes to increase tobacco filling capacity. As part of the toxicological evaluation of DIET, test cigarettes containing DIET were compared with control cigarettes containing tobacco expanded with a traditional expansion agent (Freon-11, also known as trichlorofluoromethane). Testing included mainstream cigarette smoke chemistry studies, genotoxicity studies (Ames and sister chromatid exchange, SCE), a 13-week inhalation study in Sprague-Dawley rats, and a 30-week dermal tumor promotion study in SENCAR mice. Cigarettes containing DIET or Freon-11 expanded tobacco were similar in biological activity.

Toxicological evaluation of propane expanded tobacco

A tiered testing strategy has been developed to evaluate the potential for tobacco processes, ingredients, and other technological developments to increase or decrease the biological activity resulting from burning tobacco. The strategy is based on comparative chemical and biological testing. Propane expanded tobacco is an example of a processed tobacco used in the modern manufacture of cigarettes. Test cigarettes containing propane expanded tobacco were compared to control cigarettes containing tobacco expanded with a traditional expansion agent (Freon-11). The toxicological evaluation included chemistry studies using mainstream cigarette smoke (determination of selected constituent yields), in vitro studies using cigarette smoke condensate (Ames study in Salmonella typhimurium and sister chromatid exchange study in Chinese hamster ovary cells) and in vivo studies (13-week inhalation study of mainstream cigarette smoke in Sprague-Dawley rats and 30-week dermal tumor promotion study of cigarette smoke condensate in SENCAR mice). Although statistically significant differences in several smoke constituents were observed, most constituents from cigarettes containing 100% propane expanded tobacco were within market survey ranges. Furthermore, biological tests indicated that the cigarettes containing propane or Freon-11 expanded tobacco were not significantly different.

Pulmonary Function in Nonsmokers Following Exposure to Sidestream Cigarette Smoke

Ten healthy male and 10 healthy female, “never-smoking” subjects (ages 21—50) participated in a 5-day environmental room study to determine if an acute exposure to a high level of fresh diluted sidestream smoke (FDSS) would alter pulmonary function. On Monday, Tuesday, Thursday and Friday, the twenty subjects sat in an environmental room for 7.33 hours and were exposed to fi ltered and humidified air. On Wednesday, the twenty subjects were exposed in an environmental room for 7.33 hours to an average respirable suspended particle (RSP) concentration of 179 micrograms per m3 of FDSS generated by machine smoking Kentucky 1R4F reference cigarettes. This level of FDSS is 3.3 times the 95th percentile concentration of workplace environmental tobacco smoke exposure levels previously measured in the US. FVC and FEV1 decreased approximately 1.6% (p < 0.05) in both males and females after exposure. Similarly, PEF decreased approximately 1.3% (p < 0.03) following exposure. The observed decrease in pulmonary function was consistent with a “stress” related norepinephrine-induce d alteration in blood flow leading to transient bronchoconstriction. Alternatively, a cholinergic refl ex due to activation of bronchopulmonar y C fibers may have also played a role in the transient bronchoconstriction. These small exposure-related decrements in pulmonary function were reversible.

Correlation of hematologic markers of inflammation and lung function: a comparison of asymptomatic smokers and nonsmokers

Increased inflammation of the peripheral airways has been implicated as a cause of pulmonary function impairment. However, little information is available on the correlation between subclinical decrements of pul monary function and inflammation in asymptomatic individuals. A relationship between markers of inflam mation and lung function may be useful in predicting the early onset of lung function impairment. The purpose of this study was to investigate the correlation of hematolo gic markers of inflammation and spirometry in asympto matic smokers and nonsmokers. The specific objectives of this study were twofold. The first objective was to quantify and compare the spirometric measures of lung function in smokers and nonsmokers having similar demographic and lifestyle characteristics. The second objective was to define the correlation between these spirometric measure ments and hematologic markers of inflammation (white blood cells, monocytes, basophils, PGE1, IgG, and IgE). Systemic blood samples and spirometric measurements were obtained from 61 age-matched (33 ± 9 years) healthy, asymptomatic smokers and nonsmokers, with similar self- reported lifestyles (i.e., food, alcohol, vitamin consump tion and exercise). Both male and female smokers self- reported a higher coffee consumption (P < 0.05) compared to nonsmokers. Male smokers self-reported a trend toward current blue-collar versus white-collar occupation when compared with the nonsmokers. Body weight (77.6± 16.6 kg) did not differ between the smokers and non smokers. The male nonsmokers were taller than the male smokers (P < 0.05). All subjects were asymptomatic and had clinically normal spirometry. Compared to male nonsmokers, the male smokers had lower FEF25-75% and FEF75-85% values (P<0.05). No additional spirometric measurements, including FEV1/FVC, FEV1 and FVC were significantly different. The female smokers did not differ from the female nonsmokers (P<0.05) in any of the spirometric endpoints measured. Thirteen statistically significant (P < 0.05) correlations involving inflammatory (white blood cells, monocytes, basophils, and PGE1) or immunologic endpoints (IgE) and spirometric measure ments were observed in female smokers, female non smokers and male nonsmokers. No statistically significant correlations involving immunologic or inflammatory endpoints were observed in the male smokers. A better mechanistic understanding of the observed relationship between elevated hematologic inflammatory endpoints and reduced lung function may provide valuable insight into the clinical significance of these correlations.

The Effect of Cigarette Circumference on Ignition Propensity

The 1984 Cigarette Safety Act created a Technical Study Group (TSG) to determine the feasibility of developing cigarettes with a minimum propensity to ignite upholstered furniture. The TSG found that combinations of certain cigarette characteristics were important in reducing the ignition pro pensity. Through a statistically designed study, those characteristics were ranked as follows: tobacco packing density, paper permeability, and cir cumference. The objective of the present study was to investigate the effect of cigarette cir cumference alone on ignition propensity. Cigarettes were tested by four methods: the NBS mockup method, a controlled-fabric mockup test, a flow- through mockup method, and a liquid crystal test. Results showed that while the NBS mockup test could not discriminate among the cigarettes, the other methods could, and results call into question the TSGs conclusion that smaller circumference cigarettes reduce the likelihood of ignition.