A comparison of in vitro toxicities of cigarette smoke condensate from Eclipse cigarettes and four commercially available ultra low-"tar" cigarettes.

Eclipse is a cigarette that primarily heats rather than burns tobacco. R.J. Reynolds Tobacco Company (RJRT) has previously reported the results of in vitro toxicity studies comparing Eclipse with University of Kentucky 1R5F and 1R4F reference cigarettes. To characterize the differences between Eclipse and very low yielding/ultra low-"tar" (vULT) tobacco-burning cigarettes, RJRT conducted a comparative evaluation of the genotoxicity and cytotoxicity of mainstream cigarette smoke condensate (CSC) from Eclipse and three vULT tobacco-burning cigarettes (Now 83 Box, Merit Ultima and Carlton Soft Pack) as well as the leading ultra low-"tar" (ULT) brandstyle (Marlboro Ultra Lights) under four smoking regimens: (1) FTC-35 ml puff volume every 60 s for a 2 s duration (35/60/2); (2) 50/30/2, 0% vents blocked; (3) Massachusetts-45/30/2, 50% vents blocked; (4) Canadian-55/30/2, 100% vents blocked. Ames testing indicated that Eclipse CSC was less (P<0.05) mutagenic than CSC from the four cigarettes under all smoking regimens when compared on a revertants per mg Total Particulate Matter (TPM) basis. When mutagenicity was calculated on a revertants per cigarette basis the mutagenicity of Eclipse CSC was lower (P<0.05) than the mutagenicity of Merit Ultima, Carlton Soft Pack, and Marlboro Ultra Lights, regardless of the puffing regimen. On a per cigarette basis, the calculated mutagenicity of Eclipse was higher (P<0.05) than Now 83 Box cigarettes in the FTC and 50/30/2 regimens but lower (P<0.05) in the Massachusetts and Canadian regimens. Eclipse CSC was less (P<0.05) cytotoxic as measured in the neutral red assay (based on EC(50) values-microg TPM/ml media) than the CSC from the four test cigarettes regardless of the regimen used. Collectively, these data demonstrate that the toxicity of CSC from Eclipse is significantly reduced relative to the activity of CSC from the tested vULT cigarettes and the Marlboro Ultra Lights.

Subchronic inhalation by rats of mainstream smoke from a cigarette that primarily heats tobacco compared to a cigarette that burns tobacco.

A subchronic, nose-only inhalation study comparing the potential biological activity of mainstream smoke from a cigarette that primarily heats tobacco (Eclipse) to mainstream smoke from a 1R4F reference cigarette was conducted using Sprague-Dawley rats of each gender. Smoke exposures were for 1 h/day, 5 days/wk for 13 wk, at concentrations of 0, 0.16, 0.32, or 0.64 mg wet total particulate matter (WTPM)/L air. Smoke was generated at the Federal Trade Commission standard of a 2-s puff of 35 ml, taken once per minute. Clinical signs, body and organ weights, clinical chemistry, hematology, carboxyhemoglobin, serum nicotine, plethysmography, gross pathology, and histopathology were determined. Plethysmography indicated that respiratory rate was decreased at all concentrations of 1R4F smoke, but only at the high concentration of Eclipse smoke. Tidal volume was depressed and minute volume was lower for all smoke-exposed rats. Rats exposed to Eclipse smoke inhaled more smoke at the low and mid-concentration exposures than rats exposed to equivalent concentrations 1R4F smoke. Carboxyhemoglobin and serum nicotine were directly related to the exposure concentrations of carbon monoxide (CO) and nicotine in an exposure-dependent manner. Body weights were slightly lower in smoke-exposed rats, while no treatment-related effects were seen in clinical signs, clinical chemistry, hematology, or gross changes at necropsy. The only treatment-related effect seen in organ weights was an increase in heart weight in females in the Eclipse high-concentration exposure group, attributed to higher CO in the Eclipse exposure atmosphere. Higher CO resulted from the lower dilution of Eclipse smoke required to maintain WTPM concentrations equal to those of the 1R4F smoke, and not from a higher CO yield from Eclipse cigarettes. Nasal epithelial hyperplasia and ventral laryngeal squamous metaplasia were noted after exposure to either the 1R4F or Eclipse smoke. The degree of change was less in Eclipse smoke-exposed rats. Lung macrophages were increased to a similar extent in the Eclipse and 1R4F smoke-exposed groups. Brown/gold pigmented macrophages were detected in the lungs of rats exposed to 1R4F smoke, but not those exposed to Eclipse smoke. Subsets of rats from each group were maintained for an additional 13 wk without smoke exposures. Most of the changes noted at the end of the smoke exposures had disappeared, while those that remained were regressing toward normal. Evaluation of these findings indicated the overall biological activity of Eclipse smoke was less than 1R4F smoke at comparable exposure concentrations.

Quantitative analysis of potential transfer of continuous glass filament from eclipse prototype 9-014 cigarettes.

This study was designed to determine if the Eclipse prototype 9-014 cigarettes, which use a special form of continuous glass filament (CGF) as an insulator around the carbon heat source, yield CGFs via mainstream smoke. A previously developed method (Higuchi et al., 2000) that employed electrostatic precipitation-with a greater than 99% collection efficiency of mass-was used to capture CGFs transferred to mainstream smoke. The cigarettes were smoked using an exaggerated puffing condition more than twice the Federal Trade Commission (FTC) standard. Prior to smoking, cigarettes were subjected to handling procedures that simulated commercial shipping conditions. Using a modified standard addition method, and utilizing a mixture of water and glycerol as a mock condensate, CGFs were intentionally added to a series of (mock condensate) samples to develop knowledge of CGF recovery efficiency. The linear regression model of the recovered CGFs demonstrated a recovery efficiency of 86%. This efficiency rate was applied to the number of CGFs recovered from samples of smoke condensate and associated background samples. The number of CGFs in smoke condensate collected from the Eclipse 9-014 prototype was approximately 0.32 +/- 0.17 CGFs per cigarette (+/- standard deviation), including the background counts of CGFs, and 0.16 CGFs per cigarette when corrected for background contributions. The number of CGFs found in the smoke condensates for this prototype was statistically (p =.00031) distinguishable from zero and background in these experiments. The low number of CGFs seen in the transfer data from this prototype studied, the unique physical characteristics of the filaments (e.g., controlled physical dimensions), and the absence of biological activity of similar glass filaments/fibers indicate that biologically significant exposure to the Eclipse smoker does not occur.

Analysis of potential transfer of continuous glass filament from Eclipse cigarettes.

This study was designed to determine if a prototype of the Eclipse cigarettes, which uses a special form of continuous glass filament (CGF) as an insulator around the carbon heat source, yielded CGFs via mainstream smoke. A method was developed that used electrostatic precipitation with a greater than 99% collection efficiency of mass to capture CGFs transferred to mainstream smoke. The cigarettes were smoked using an exaggerated puffing condition that was more than twice the Federal Trade Commission (FTC) standard. The cigarettes were subjected to handling procedures that simulated commercial shipping conditions prior to smoking. CGFs were intentionally added to a series of smoke condensate samples to determine CGF recovery efficiency. The recovery efficiency was determined for a series of four internal standards added to smoke condensate. The recovery efficiency was 86% for the Eclipse 5-014 prototype. The number of CGFs in smoke condensate collected from the Eclipse 5-014 prototype was approximately 0.06 +/- 0.02 CGFs per cigarette (+/- standard deviation), including the background counts of CGFs and 0.03 CGFs per cigarette, when corrected for background contributions. The number of CGFs found in smoke condensates for this prototype was not statistically distinguishable from zero or background in these experiments, which were capable of detecting transfer rates of greater than 0.2 CGFs per cigarette.

Comparative study of DNA adduct formation in mice following inhalation of smoke from cigarettes that burn or primarily heat tobacco.

The genotoxic potential of mainstream smoke from a test cigarette (TOB-HT) that primarily heats tobacco and a representative tobacco-burning cigarette (Kentucky reference 1R4F) was compared in male B6C3/F1 mice after nose-only inhalation exposure. Mice were exposed 1 hr per day, 5 days/ week for a 4 week period to mainstream smoke at concentrations of 0, 0.16, 0.32, and 0.64 mg total particulate matter/liter of air. Micronuclei formation in bone marrow and peripheral blood polychromatic erythrocytes (PCE) of animals exposed to either the TOB-HT or 1R4F cigarette was not significantly different compared with control animals exposed nose-only to filtered and humidified air (sham controls). DNA adduct measurement by the 32P-post-labeling method indicated an exposure-dependent increase in lung adducts of animals exposed to 1R4F cigarette smoke at all three concentrations with the mid and high exposure groups exhibiting statistically significant increases (P < 0.05) in adduct formation compared to sham-exposed animals. The concentration of DNA adducts in the lungs of animals exposed to the TOB-HT cigarette was not significantly increased (P < 0.05) at any concentration compared to sham-exposed controls. A statistically significant (P < 0.05) concentration-dependent formation of DNA adducts was also observed in the heart tissues of animals exposed to smoke from the 1R4F cigarette at all three concentrations, but no significant increase in adduct formation was observed in heart DNA of the animals exposed to the TOB-HT cigarette (P < 0.05). Under the conditions of this experiment, the mainstream smoke from the TOB-HT cigarette was demonstrated to be less genotoxic in mice than mainstream smoke from the 1R4F cigarette, which is representative of cigarettes in the current U.S. market.

Design, construction, and evaluation of an inhalation system for exposing experimental animals to environmental tobacco smoke.

An inhalation system was designed to expose experimental animals to aged and diluted sidestream smoke (ADSS), used as a surrogate for environmental tobacco smoke (ETS). The construction of the smoke generator and of the smoke dilution systems is described. Target ADSS concentrations in a 90-day inhalation study were 0.1, 1, and 10 mg/m3 of respirable suspended particulates (RSP). Data is presented on the physical and chemical composition of the smoke presented to animals at or near these target RSP concentrations. The design of the inhalation laboratory was shown to result in highly reproducible respirable aerosols that were effective surrogates of ETS.

Long-term systemic hemodynamic effects of cotinine in rats.

Cigarette smoking has been epidemiologically correlated with lower BP in humans, despite the acute BP effects of nicotine. Cotinine, a primary metabolite of nicotine, has been suggested as a mediator of the BP-lowering effect of smoking. To determine the cardiovascular effects of cotinine itself, arterial BP was monitored continuously in chronically instrumented Sprague-Dawley rats before, during, and after 14 days of intraarterial (i.a.) infusion of cotinine or placebo. Cardiovascular data were collected and analyzed by microcomputer for diurnal changes and for light- and dark-cycle averages. Heart rate (HR) was higher in both precotinine and placebo groups during the dark cycle than during the light cycle. HR was significantly lower (p < 0.025) during cotinine infusion in the cotinine group as compared with placebo. Five days after cotinine infusion, however, HR was not different from that of placebo controls. Arterial BP was not different between cotinine- and placebo-treated rats at any time period. The difference between HR but not arterial BP suggested that baroreceptor activity might differ after cotinine administration. Baroreceptor activity, assessed by analysis of HR changes evoked by phenylephrine, did not differ before and during cotinine administration, however. We conclude that cotinine did not decrease BP in rats under the present experimental conditions, but that cotinine was probably responsible for the observed bradycardia.

Fourteen-day inhalation study in rats, using aged and diluted sidestream smoke from a reference cigarette. I. Inhalation toxicology and histopathology.

Sprague-Dawley rats were exposed 6 hr per day for 14 consecutive days to aged and diluted sidestream smoke (ADSS), used as a surrogate for Environmental Tobacco Smoke (ETS), at concentrations of 0.1 (typical), 1 (extreme), or 10 (exaggerated) mg of particulates per cubic meter. Animals were exposed nose-only, inside whole-body chambers, to ADSS from the 1R4F reference cigarette. End-points included histopathology, CO-oximetry, plasma nicotine and cotinine, clinical pathology, and organ and body weights. The only pathological response observed was slight to mild epithelial hyperplasia and inflammation in the most rostral part of the nasal cavity, in the high-exposure group only. No effects were noted at medium or low exposures. The minimal changes noted were reversible, using a subgroup of animals kept without further treatment for an additional 14 days. Overall, the end-points used in the study demonstrated that there was no detectable biological activity of ADSS at typical or even 10-fold ETS concentrations and that the activity was only minimal at very exaggerated concentrations (particle concentrations 100 times higher than typical real-world concentrations).

A new method for obtaining electrocardiograms in unrestrained crocodilian reptiles.

A new procedure is described for acquiring measurements of electrocardiographic parameters in unrestrained crocodilians. These measurements are difficult to obtain in freely moving animals; hence, electrocardiographic activity under natural conditions has not been previously quantified. In this investigation, twelve American alligators were equipped with subcutaneous electrodes. The lead wires were sutured to each animal's skin and the extracutaneous wires coiled and held in place against the animals' dorsal surfaces with waterproof elastic bandages. The electrodes were connected to an ECG analyzer only at the time of measurement. The presence of the leads and harness did not appear to interfere with the movements of the animals either in the animal room or during testing. This method allows for more precise measurements of cardiac activity under conditions which closely resemble those of crocodilians in their natural state.

DNA adduct formation in mice following dermal application of smoke condensates from cigarettes that burn or heat tobacco.

A prototype cigarette that heats tobacco (test cigarette), developed by R.J. Reynolds Tobacco Company, has yielded consistently negative results in several in vivo and in vitro genetic toxicology tests. The objective of the present study was to evaluate the potential of cigarette smoke condensate (CSC) from the test cigarette to induce DNA adducts in mouse tissues and compare the results with those obtained with CSC from a reference tobacco-burning cigarette (1R4F). CD-1 mice were skin-painted with CSC from reference and test cigarettes three times a week for 4 weeks. The highest mass of CSC applied was 180 mg "tar" per week per animal for both reference and test cigarette. DNA adducts were analyzed in skin and lung tissues using the 32P-postlabeling method with the P1 nuclease modification. Distinct diagonal radioactive zones (DRZ) were observed in the DNA from both skin and lung tissues of animals dosed with reference CSC, whereas no corresponding DRZ were observed from the DNA of animals dosed with either test CSC or acetone (solvent control). The relative adduct labeling (RAL) values of skin and lung DNA from reference CSC-treated animals were significantly greater than those of the test CSC-treated animals. The RAL values of the test CSC-treated animals were no greater than those of solvent controls. The negative results in DNA adduct assays with test CSC are consistent with all previous results of in vivo and in vitro genetic toxicology testing on this cigarette and provide additional evidence that smoke condensate from the test cigarette is not genotoxic.

Histologic changes in the respiratory tract induced by inhalation of xenobiotics: physiologic adaptation or toxicity?

Toxicologists and pathologists are often faced with the dilemma of categorizing changes observed in the respiratory tract of laboratory animals as either "adaptive" or "toxic." However, it is often difficult to interpret the nature of a given change as either "adaptive" or "toxic." Certain lesions or changes in the respiratory tract are to be expected from the concentration of materials given or the experimental design of a study. Careful analysis suggests that some of these changes may be more properly described as adaptive rather than toxic within the context of a given study or situation. Tissue changes discussed in this paper include squamous metaplasia of laryngeal epithelium, goblet cell change in respiratory epithelium, macrophage accumulation within alveoli, and bronchiolization of alveolar epithelium. Examples provided show that some of these changes observed in inhalation studies are similar in severity but slightly increased in frequency over sham control animals. The introduction of exogenous material into the respiratory tract of laboratory animals in an experimental setting should be expected to result in certain changes. The challenge scientists must accept is to interpret these changes so that toxic events may be separated from adaptive changes. In order to meet this challenge, studies incorporating several species and novel technologies may have to be utilized.

Ninety-day inhalation study in rats, comparing smoke from cigarettes that heat tobacco with those that burn tobacco.

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.