Paul H. Ayres

Histological sectioning of the rodent larynx for inhalation toxicity testing

In rodents, the larynx is a major site of histopathologic alteration following inhalation exposure to particulates, vapors, and aerosols. Specifically, the epithelial lining of a narrowly delineated region on the ventral floor of the larynges of rats and mice appears to be especially vulnerable to inhaled materials, and is recognized as a preferred site for histopathological evaluation in inhalation studies. This site is located at the base of the epiglottis, cranial to the ventral laryngeal diverticulum (ventral pouch). The presence of underlying seromucinous glands is critical for histologic identification of this site. We report a histologic sectioning technique, using the ventral laryngeal diverticulum as the anatomical landmark, to obtain tissue sections from this area of predilection in rats and in mice.

Comparative subchronic inhalation study of smoke from the 1R4F and 2R4F reference cigarettes

A subchronic, nose-only inhalation study compared the effects of mainstream smoke from a 1R4F research cigarette to that of a 2R4F research cigarette. Male and female rats were exposed for 1 h/day, 5 days/wk, for 13 wk to mainstream smoke at 0, 0.06, 0.20, or 0.80 mg wet total particulate matter per liter of air. Clinical signs, body and organ weights, clinical chemistry, hematology, carboxyhemoglobin, serum nicotine, pulmonary plethysmography, gross pathology, and histopathology were determined. When histological changes resulting from exposure to smoke from the two types of cigarettes were compared, no biologically significant differences were observed. At the end of the exposure period, subsets of rats from each group were maintained without smoke exposures for an additional 13 wk (recovery period). At the end of the recovery period, there were no statistically significant differences in histopathological findings observed between the 1R4F and the 2R4F cigarettes. The complete toxicological assessment in this comparative inhalation study of 1R4F and 2R4F cigarettes suggests no overall biologically significant differences between the rats exposed to the two cigarettes.

SUBCHRONIC INHALATION STUDY IN RATS USING AGED AND DILUTED SIDESTREAM SMOKE FROM A REFERENCE CIGARETTE

AbstractMale Sprague-Dawley rats were exposed 6 hr/day, 5 days/week for up to 13 weeks 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/m3. Subgroups of animals were killed after 1 and 4 weeks of exposure. 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 target particulate concentrations were achieved; at the exaggerated exposure they resulted in CO concentrations in excess of 50 ppm. Particle size distributions showed that the aerosols were completely respirable: the mass median diameter values were less than 1 μm. The only pathological response observed was slight to mild epithelial hyperplasia in the rostral nasal cavity, in the exaggerated exposure group only. No effects we...

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.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 smokeexposed 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.

Morphological observations on the epithelium of the developing urinary bladder of the mouse and rat.

Bladders from fetal and neonatal BALB/cStCrlfC3H/Nctr mice and Sprague-Dawley rats were studied to establish the sequence of events in their morphological development by using scanning electron, transmission electron and light microscopy. On fetal day 18 or 19 the epithelium from the mouse and the rat displayed 2 or 3 distinct cell layers. With transmission electron microscopy a star-like contraction of the cell surface of the mouse bladder occurred which was not seen in the developing rat bladder. In both the mouse and the rat, some of the superficial cells sloughed between fetal day 18 or 19 and the day of birth. On the day of birth, the epithelium was composed of only 2 layers. The nuclei of both the superficial and basal layers contained prominent euchromatin, and mitotic figures were often present in the basal layer. By the 5th postnatal day, some of the superficial cells contained autophagic vacuoles, and the epithelium was still 2 cell layers thick. One week after birth the epithelium consisted of 2 to 3 cell layers. Three weeks after birth the epithelium was 3 cell layers thick and appeared as the adult pattern with both transmission and scanning electron microscopy. The study demonstrated that the fetal and neonatal mouse and rat urinary bladders undergo a series of rapid developmental changes and suggest that the fetal and neonatal urinary bladder may be a target organ susceptible to toxic insult.

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.

Relevant exposure to environmental tobacco smoke surrogate does not produce or modify secretory otitis media in the rat.

Parental smoking is a possible risk factor in the development of secretory otitis media (SOM) in children. This experiment was designed to determine, using rats as an experimental model, whether exposures to environmental tobacco smoke (ETS) produce SOM and whether ETS exposure affects the rate of clearance of an experimentally induced effusion. Male Sprague-Dawley rats were exposed to 3 different concentrations of aged and diluted sidestream smoke, a surrogate for ETS, from 1R4F research cigarettes for 6 hr per day for 5 days. Experimental SOM was induced bilaterally in subgroups of animals from each group, by cold air exposure to the external auditory canals. Ears of rats were examined during the in-life portion of the study. Histopathologic examination of the middle ear was conducted at the termination of the 5-day period. The production of SOM was not induced by ETS exposure, nor were there differences noted between the groups in the rates of clearance of the experimentally induced SOM. Short-term exposure to ETS did not affect the acquisition or clearance of SOM in the rat.

Comparative Inhalation Study in Rats Using Cigarettes Containing Tobacco Expanded with Chlorofluorocarbon-11 (CFC-11) or Hydrochlorofluorocarbon-123 (HCFC-123)

AbstractThis study was performed to evaluate the use of an alternate process (using hydrochlorofluorocarbon-123, HCFC-123) for expanding tobacco instead of the standard expansion process (using chlorofluorocarbon-11, CFC-11). Rats were exposed to smoke 1 hr/day, 5 days/week, for 13 weeks. The effects of smoke exposure, primarily on respiratory physiology and on histopathology of the respiratory tract, were as previously reported in the literature. No novel changes were noted. An independent review was made of the above conclusions, formed by the study pathologist. The conclusions made by the reviewing pathologist agreed with those made by the study pathologist. The data obtained in this inhalation study show that there are no differences between the responses noted in animals exposed to smoke from cigarettes containing tobacco expanded with HCFC-123 or with CFC-11.

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.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.

Quantative 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.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.