Charles L. Gaworski

13-Week inhalation toxicity study of menthol cigarette smoke

Menthol is a common pharmaceutical, food and tobacco flavouring ingredient used for its minty characteristics and cooling effects. A 13-wk comparative nose-only smoke inhalation toxicity study was conducted using an American-style, cellulose acetate-filtered, non-menthol reference cigarette and a similarly blended test cigarette containing 5000 ppm synthetic l-menthol tobacco. Male and female Fischer 344 rats were exposed for 1 hr/day, 5 days/wk for 13 wk at target mainstream smoke particulate concentrations of 200, 600 or 1200 mg/m3, while control rats were exposed to filtered air. Internal dose biomarkers (blood carboxyhaemoglobin, serum nicotine and serum continine) indicated equivalent exposures were obtained for the two cigarettes. Effects typically noted in rats exposed to high levels of mainstream tobacco smoke were similar for both cigarette types and included reduced body weights (males slightly more affected than females), increased heart-to-body weight ratios and lung weights, and histopathological changes in the respiratory tract. Rats exposed to reference cigarette smoke displayed a dose-related increase in nasal discharge that was not observed in menthol smoke-exposed rats. All smoke-related effects diminished significantly during a 6-wk non-exposure recovery period. The results of this 13-wk smoke inhalation study indicated that the addition of 5000 ppm menthol to tobacco had no substantial effect on the character or extent of the biological responses normally associated with inhalation of mainstream cigarette smoke in rats.

TOXICOLOGIC EVALUATION OF HUMECTANTS ADDED TO CIGARETTE TOBACCO: 13-WEEK SMOKE INHALATION STUDY OF GLYCERIN AND PROPYLENE GLYCOL IN FISCHER 344 RATS

Glycerin (CAS no. 56-81-5) and propylene glycol (CAS no. 57-55-6) are commonly used as humectant ingredients in manufactured cigarettes to control and maintain the moisture content of the cut tobacco filler. The potential of these added humectants to affect the toxicity of cigarette smoke was investigated in a subchronic nose-only smoke inhalation study in rats. Filtered test cigarettes were prepared from an American-style tobacco blend containing either glycerin added at 5.1% w/w tobacco, propylene glycol at 2.2% w/w tobacco, or combinations of these humectants totaling 2.3%, 3.9%, and 7.2% w/w tobacco. Other groups of animals were exposed similarly to the smoke of reference cigarettes without added humectants, or to filtered air (sham control). Smoke exposures were conducted for 1 h/ day, 5 days/wk for 13 wk, at target smoke particulate concentrations of 350 mg/m 3. All smoke-exposed groups had equivalent increases in blood carboxyhemoglobin, serum nicotine, and serum cotinine relative to the air controls. Smoke-associated reductions in body weights and occasional increases in heart and lung weights were generally similar among the various exposure conditions at necropsy. Increases in serum alkaline phosphatase and decreases in serum glucose and cholesterol were observed among smoke-exposed females relative to air controls. However, no significant differences in these parameters were evident between the humectant-containing and reference cigarette smoke exposure groups. Assessments of respiration conducted after 3, 6, 9, and 12 wk of smoke exposure indicated an initial smoke-related but not humectant-related decrease in respiratory rate, tidal volume, and minute volume during the first 20 min of each smoke exposure. Respiratory-tract histopathology was consistent across sexes and smoke groups, comprising (1) diffuse and focal alveolar pigmented macrophages and chronic interstitial inflammation in the lung, (2) laryngeal epithelial hyperplasia, squamous metaplasia, and scab formation, and (3) epithelial hyperplasia in the anterior nose. Smoke-related histopathology resolved substantially during a 6-wk postexposure recovery period. Addition of the tested humectants to cigarettes, singly or in combination, had no meaningful effect on the site, occurrence, or severity of respiratory-tract changes or on the measured indices of pulmonary function. It was concluded that the addition of glycerin and propylene glycol to cigarettes does not significantly affect the biological activity of inhaled cigarette smoke in this rat model.

An evaluation of the toxicity of 95 ingredients added individually to experimental cigarettes: approach and methods

Context: Ingredients have been used in modern cigarette manufacturing to facilitate tobacco processing, provide flavor, and preserve tobacco. Concern has been raised regarding the use of ingredients in cigarette manufacturing due to the possible generation of toxic chemicals resulting from their combustion when added to tobacco. Objective: Investigate the impact of individual ingredients on cigarette smoke toxicity. Materials and methods: A total of 95 ingredients were tested individually through addition at different concentrations to the tobacco of experimental cigarettes. Mainstream cigarette smoke chemistry analysis, bacterial mutagenicity testing, and cytotoxicity testing were conducted. Additionally, 31 of the ingredients were tested in 90-day nose-only rat inhalation studies using mainstream cigarette smoke. Studies were designed following conventional toxicity testing methods employed for food additives and other consumer products. Results: The studies reported here demonstrate that high levels of some ingredients can change the quantity of some smoke constituents, altering the smoke chemistry profile. From the panel of biological endpoints monitored, these added ingredients produced minimal changes in the overall toxicity profile of mainstream cigarette smoke. In some cases, the addition of high levels of an ingredient caused a small reduction in toxicity findings, probably due to displacement of burning tobacco. Conclusion: The battery of testing results presented here is a useful addition to the available scientific information for cigarette ingredients and extends the dataset which can be used for evaluating their appropriate use.

Effect of filtration by activated charcoal on the toxicological activity of cigarette mainstream smoke from experimental cigarettes

Activated charcoal (AC) filtration reportedly decreases the yields of smoke vapor phase constituents including some identified as human carcinogens and respiratory irritants. Non-clinical studies including chemical smoke analysis, in vitro cytotoxicity and mutagenicity (bacterial and mammalian cells), and in vivo subchronic rat inhalation studies were carried out using machine smoking at ISO conditions with lit-end research cigarettes containing AC filters. The objective was to assess whether AC filter technology would alter the established toxicity profile of mainstream smoke by increasing or decreasing any known toxicological properties, or elicit new ones. The reduced yield of vapor phase irritants from AC filter cigarettes correlated with markedly decreased in vitro cytotoxicity and in vivo morphology of the nose and lower respiratory tract. Increased yields of particulate phase constituents (e.g. polycyclic aromatic hydrocarbons) in AC filtered smoke were noted in comparison to controls in some studies. The in vitro bacterial mutagenicity of AC filtered smoke particulate preparations was occasionally increased over control levels. Laryngeal epithelial thickness was increased in some rats inhaling AC filtered smoke in comparison to controls, an effect perhaps related to higher inspiratory flow. When tested under more intense Massachusetts Department of Public Health smoking conditions, AC filter associated reductions in vapor phase constituent yields were smaller than those seen with ISO conditions, but the effect on in vitro cytotoxicity remained.

An immunotoxicity assessment of food flavouring ingredients

A rapid screening protocol incorporating key elements of the US National Toxicology Programs immunotoxicity tier testing strategy was used to evaluate the effects of 35 commonly used food flavouring ingredients on humoral and cell-mediated immune responses. The test compounds were administered intragastrically on a daily basis for 5 days at three dose levels to female CD-1 or B6C3F1 mice, 6-8 wk old. A host resistance assay (Listeria monocytogenes bacterial challenge) was conducted to assess cell-mediated immunity. Humoral immunity was measured by the antibody plaque-forming cell (PFC) response to sheep erythrocytes. Body weights, lymphoid organ weights and spleen cellularity were also measured. Cyclophosphamide (80 mg/kg) served as an immunosuppressive positive control agent. The results indicated that the majority of the flavouring ingredients tested did not modulate the cell-mediated or humoral immune response. However, at very high dose levels, two of the materials tested, peppermint oil and citral dimethyl acetal, did increase mortality rate and reduce survival time in the host resistance assay. Neither of these materials significantly altered the PFC response. This rapid, economical screening battery for potential immunotoxicants proved to be a useful means of evaluating a large number of structurally diverse compounds and mixtures to prioritize them for more definitive testing.

Developmental toxicity evaluation of inhaled citral in Sprague-Dawley rats

Citral is a commonly used fragrance and flavour ingredient that has demonstrated a potential for teratogenicity in chick embryo screening studies. To investigate potential mammalian developmental toxicity, pregnant Sprague-Dawley rats were exposed to citral by inhalation for 6 hr/day on gestation days 6-15 at mean concentrations of 0, 10 or 34 ppm as vapour, or 68 ppm as an aerosol/vapour mixture. Dams were killed on gestation day 20 and the foetuses were removed and evaluated for gross, visceral and skeletal malformations. Exposure to 68 ppm was maternally toxic, with reduced body-weight gains, ocular opacity, breathing difficulty, nasal discharge and salivation noted in the dams. No maternal toxicity was seen at the lower vapour exposure levels. The number of corpora lutea, implantations, resorptions, foetal viability, litter size, and sex ratio were not adversely affected by citral at any exposure level tested, and no exposure-related malformations were observed. At a maternally toxic exposure level, a slight reduction in mean foetal body weight and a slight increase in the incidence of hypoplastic bones were noted. Results of this study indicate that citral does not produce developmental toxicity in the rat when administered by inhalation at concentrations up to a maternally toxic exposure level.

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.

Prechronic Inhalation Toxicity Studies of Isobutyl Nitrite

Isobutyl nitrite (IBN) is a volatile liquid that has become increasingly popular as an inhaled recreational drug. To investigate short-term toxic effects and establish exposure parameters for chronic inhalation studies, F344/N rats and B6C3F1 mice were exposed to IBN vapors on a 6 hr/day + t90, 5 days/week schedule. Twelve exposures were administered at concentrations of 0, 100, 200, 400, 600, and 800 ppm IBN. This exposure series resulted in mortality in rats exposed to greater than or equal to 600 ppm and mice exposed to 800 ppm. Animals exposed at the lower concentrations developed hyperplasia of the bronchiolar and nasal turbinate epithelium (rats and mice) and lymphocytic atrophy in the spleen and thymus (mice). Longer term, 13-week, subchronic exposures were conducted at concentrations of 0, 10, 25, 75, 150, and 300 ppm IBN. Exposure to 300 ppm IBN reduced the body weight gains in both sexes of rats and in female mice. IBN-related clinical pathology changes included reduced RBC counts accompanied by moderate increases in mean corpuscular volume and reticulocyte counts, increased WBC counts, and mildly increased methemoglobin concentration. Bone marrow hyperplasia was observed in all groups of IBN-exposed rats, while in mice only females at greater than or equal to 150 ppm IBN displayed this change. Excessive splenic pulp hematopoiesis was noted in mice at all IBN exposure levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Prechronic Inhalation Toxicity Studies of 2-Mercaptobenzimidazole (2-MBI)in F344/N Rats

2-Mercaptobenzimidazole (2-MBI), used in rubber processing, is a suspect carcinogen structurally related to ethylene thiourea. The inhalation toxicity of 2-MBI was evaluated in male and female F344/N rats exposed 6 hr/day, 5 days/week to respirable aerosols generated by spray atomization of aqueous suspensions of the 2-MBI powder and subsequent drying of the resulting aerosols. Twelve exposures at target concentrations of 0, 6.3, 12.5, 25.0, 50.0, or 100 mg/m3 of 2-MBI produced a dose-related reduction in body weight gains, thyroid follicular cell hyperplasia, adrenal cortex fatty change, and pituitary atrophy. Sub-chronic exposures were conducted at target concentrations of 0, 3.1, 6.2, 12.5, 25.0, and 50.0 mg/m3 of 2-MBI. Rats at greater than or equal to 25 mg/m3 displayed hunched posture, hypoactivity, and reduced body weight gain, with compound related mortality at the highest exposure level. Anemia; increased SGPT, SGOT, alkaline phosphatase, sorbitol dehydrogenase, BUN, and cholesterol; and reduced free fatty acid were seen in rats at greater than or equal to 25 mg/m3. Increased thyroid weight and thyroid follicular cell hyperplasia were noted in both sexes at greater than or equal to 6.2 mg/m3, with reduced triiodothyronine and thyroxine levels in both sexes at greater than or equal to 12.5 mg/m3. Thyroid follicular cell hyperplasia was also seen in rats at 3.1 mg/m3. Thymus weights were significantly reduced in both sexes at all exposure levels with liver weight increases at greater than or equal to 6.2 mg/m3. Exposure-related histopathologic changes included pituitary cytoplasmic vacuolization, adrenal cortex necrosis, lymphoid depletion, thymic atrophy, liver cell hypertrophy, renal mineralization and tubular atrophy, and hypocellularity of the bone marrow.

Immunomodulatory screening test of corn oil administered orally to female mice: effect of timing of dosing within 24 hours.

The effect of varying the dose-delivery time within a 24 h period (12:12 light-dark cycle) on the immunomodulatory properties of corn oil administered by gavage to 120 B6C3F1 female mice was investigated. Mice, housed in six separate boxes equipped with timers to regulate light onset and offset (staggered by 4 h increments), were treated for 5 consecutive days by intragastric (i.g.), administration of 5 mL/kg corn oil. Negative and positive control mice were given sham injections (needle inserted, but no injection). Sheep red blood cells (SRBCs) were injected intraperitoneally (i.p.) on the fifth day. Three days later, positive control mice were given cyclophosphamide intraperitoneally (80 mg/kg). Four days after SRBC injection, mice were weighed and killed, and spleens and thymuses were removed and weighed. Spleens were brought to single-cell suspensions and tested for an antibody response to the SRBC. Plaque-forming cells (PFCs), as measured per spleen, per 10(6) viable spleen cells or per 10(6) total spleen cells, exhibited significant circadian rhythms for mice given corn oil, but not for sham-gavage- and cyclophosphamide-treated mice. The peak response (acrophase, phi) occurred at 21 h, 22 h, and 23 h after lights on (HALO), respectively, with PFC values significantly different between the different time points. Corn oil and sham gavage affected the circadian pattern of antibody production; there was a high-amplitude (21-27%) rhythm observed when mice were treated with corn oil and no rhythm when mice received the sham-gavage treatment. In addition to testing mice near the end of the daily dark span and/or early light span to obtain a maximum immune response, this finding points to the importance of including as controls a group of animals that are not treated at all and a group given vehicle alone, rather than only sham-treated animals, for comparison with experimentally treated animals.