sample="quota" bates="2029239018" isource="pm" decade="1990" class="ui" date="19950000" In vivo and in vitro Studies of Mainstream Smoke from a New Cigarette Prototype Paul Ayres, Mark Higuchi, David Bombick, David Doolittle, C. Lee, B. Brown, A. Mosberg, and Gary T. Burger R.J. Reynolds Tobacco Company, Winston-Salem, NC, USA. John Sagartz. Winston-Salem, NC, USA Roger Renne, Battelle Northwest Laboratories, Richland, Washington, USA. Background A new cigarette has been developed that primarily heats tobacco. Only a small amount of tobacco in the heat source is burned (less than 25mg). Biological activity of the new cigarette was compared to the University of Kentucky 1R4F reference cigarette. Experimental Systems In vitro Analyses - tobacco smoke condensate » Ames bacterial mutagenicty » Sister chromatid exchange assay ( Chromosome re-arrangement) » Cytotoxicity In vivo Sprague-Dawley Rat - Subchronic nose-only inhalation study conducted at R. J. Reynolds Tobacco Company Syrian Golden Hamster - Subchronic nose-only inhalation study conducted at Battelle Northwest Laboratories Experimental Design Comparative Approach: Biological changes observed in smoke exposed laboratory animals were compared to sham exposed animals to determine smoke related effects. Smoke related effects were then compared between the TEST and REFERENCE cigarette groups. Experimental Design A new prototype cigarette that primarily heats tobacco - TEST cigarette Kentucky 1R4F cigarette - REFERENCE cigarette Equivalent wet total particulate matter (WTPM) concentration exposures were conducted Biological changes with the TEST cigarette were compared to the REFERENCE cigarette Cigarette Yields - FTC TEST 1R4F - REFERENCE 6.7 mg WTPM 10.8 mg WTPM 4.5 mg Tar 9.2 mg Tar 0.2 mg Nicotine 0.8 mg Nicotine 11.9 mg CO 9.8 mg CO Results: In vitro studies Ames Bacterial Mutagenicity - TA98, TA100, TA1537, TA1538 +/- S9 activation; 0-250 ug WTPM/plate TEST cigarette - negative 1R4F cigarette - positive +S9 as low as 50 ug WTPM/plate Sister Chromatid Exchange (CHO cells) +/- S9; 0-250 ug WTPM/mL TEST cigarette - negative 1R4F - positive +/- S9 as low as 25 ug WTPM/mL Cytotoxicity Neutral Red Assay (CHO cells) ; 0-15- ug WTPM/mL TEST cigarette - negative 1R4F cigarette - positive as low as 25 ug WTPM/mL Subchronic Inhalation Studies Sprague-Dawley rats » 32 juvenile male and female rats per group » 22 rats of both sexes killed at 90 days » 10 rats of both sexes remained on study for a 13 week reversibility period without exposure Syrian Golden hamsters » 32 juvenile male and female hamsters per group >> 22 hamsters of both sexes killed at 90 days » 10 hamsters of both sexes remained on study for a 13 week reversibility period without exposure Exposure Conditions Cigarette smoked under FTC conditions Smoke from cigarettes was diluted and controlled to target concentrations of WTPM Concentrations of WTPM used were: » 0.00 mg WTPM/L - SHAM exposure » 0.16 mg WTPM/L - LOW exposure » 0.32 mg WTPM/L - MID exposure » 0.64 mg WTPM/L - HIGH exposure Rats and hamsters were exposed nose-only 1 hour per day, 5 days per week, during a 13 week period. Exposure Characterization - Summary Data Rat Subchronic Inhalation Study The same type of equipment was used for the hamster study. Similar results were obtained. Methods - Biological Endpoints Measures of Exposure » Inhaled Mass of WTPM » Carboxyhemoglobin » Serum nicotine In-life observations » Clinical observations » Body weights Pathology » Gross changes, organ weights, organ weight ratios » Histopathology Measure of Exposure Inhaled Mass of WTPM Minute volume - the product of respiratory rate and tidal volume Normalized inhaled mass » minute volume x exposure concentration x duration of exposure; normalized by body weight Measure of Exposure Inhaled Mass of WTPM in Rats Measures of Exposure Rat and Hamster Inhaled mass of WTPM was higher in TEST exposed rats compared to REFERENCE Inhaled mass of WTPM was similar in TEST and REFERENCE exposed hamsters Carboxyhemoglobin - related to exposure in a concentration dependant manner for both TEST and REFERENCE exposed rats and hamsters Serum Nicotine - related to exposure in a concentration dependant manner for both TEST and REFERENCE exposed rats and hamsters. Clinical Observations Body Weights Clinical Observations No significant smoke related clinical observations were observed in either rats or hamsters for either the TEST or REFERENCE cigarette. Body Weight Rat - No significant differences in body weights between comparable TEST and REFERENCE smoke exposed groups Hamster - In male hamsters, body weights of the mid and high exposure groups of the REFERENCE cigarette were depressed relative to the TEST cigarette. Results Pathology » Gross observations, organ weights, organ weight ratios » Histopathology Findings at Necropsy - Rats and Hamsters There were no significant findings at 90 days in either the TEST or REFERENCE groups of rats or hamsters in terms of: » Gross observations » Organ weights » Organ weight ratios Comparative Approach Statistical Analysis of Histological Findings Kolmogorov-Smirnov test - incidence and degree of change are compared between groups; p < 0.05 Smoke related changes compared on exposure basis for affected organs » Low TEST compared to low REFERENCE » Mid TEST compared to mid REFERENCE » High TEST compared to high REFERENCE Smoke Related Histological Changes Rat and Hamster Rat » Nasal 1 section - epithelial hyperplasia » Ventral larynx - squamous metaplasia » Lung - brown/gold pigmented macrophages Hamster » Nose, turbinates - hyperplasia of respiratory epithelium » Larynx, epiglottis - squamous metaplasia, hyperplasia, and hyperkeratosis » Larynx, ventral pouch - hyperplasia » Larynx, dorsal lateral walls - squamous metaplasia » Lung - pigmented macrophages Significant Findings - Rat Study TEST Responses Relative to REFERENCE Nasal 1, epithelial hyperplasia (turbinate) - low, mid, and high TEST of both sexes (except low female) reduced Ventral larynx, squamous metaplasia - low TEST reduced (males only) ; mid and high TEST not different Lungs, pigmented macrophages - low, mid, and high TEST of both sexes reduced Significant findings - Rat study TEST Responses Relative to REFERENCE Ventral larynx, squamous metaplasia - low TEST reduced (males only); mid and high TEST not different Significant Findings - Rat Study TEST Responses Relative to REFERENCE Lungs, pigmented macrophages - low, mid, and high TEST of both sexes reduced Significant Findings - Hamster Study TEST Response Relative to REFERENCE Nose, turbinates, hyperplasia respiratory epithelium - high and mid TEST of both sexes reduced; no response at low Larynx, epiglottis, squamous metaplasia and hyperplasia - high TEST of both sexes reduced; mid and low TEST not different Larynx, protuberance, squamous metaplasia, hyperplasia, and hyperkeratosis - low, mid, and high TEST of both sexes reduced Larynx, ventral pouch, hyperplasia - high TEST of both sexes reduced Larynx, dorsal lateral walls, squamous metaplasia - high TEST of both sexes reduced, mid TEST not different, no response at low Significant Findings - Hamster Study TEST Relative to REFERENCE Responses Larynx, epiglottis, squamous metaplasia and hyperplasia - high TEST of both sexes reduced; mid and low TEST not different Significant Findings -Hamster Study TEST Responses Relative to REFERENCE Larynx, protuberance, squamous metaplasia, hyper plasia, and hyper keratosis - low, mid, and high TEST of both sexes reduced Significant Findings - Hamster Study TEST Response Relative to REFERENCE Larynx, ventral pouch, hyperplasia - high TEST of both sexes reduced Larynx, dorsal lateral walls, squamous metaplasia - high TEST of both sexes reduced, mid TEST not different, no response at low Summary of Results In vitro studies of tobacco smoke condensate - TEST cigarette was negative; REFERENCE cigarette was positive in all assays. Rat - TEST cigarette responses were reduced in nasal tissues, larynx, and lung compared to REFERENCE, even though rats exposed to TEST smoke inhaled more smoke than REFERENCE exposed rats. Hamster - TEST cigarette responses were reduced in nasal tissues and four sites of the larynx compared to REFERENCE exposed hamsters. Conclusions Based on in vitro assays and subchronic inhalation studies in two species, the TEST cigarette that primarily heats tobacco was shown to have reduced biological activity compared to the REFERENCE cigarette.