Michael S. Werley

Characterization of potential impurities and degradation products in electronic cigarette formulations and aerosols

E-cigarettes are gaining popularity in the U.S. as well as in other global markets. Currently, limited published analytical data characterizing e-cigarette formulations (e-liquids) and aerosols exist. While FDA has not published a harmful and potentially harmful constituent (HPHC) list for e-cigarettes, the HPHC list for currently regulated tobacco products may be useful to analytically characterize e-cigarette aerosols. For example, most e-cigarette formulations contain propylene glycol and glycerin, which may produce aldehydes when heated. In addition, nicotine-related chemicals have been previously reported as potential e-cigarette formulation impurities. This study determined e-liquid formulation impurities and potentially harmful chemicals in aerosols of select commercial MarkTen(®) e-cigarettes manufactured by NuMark LLC. The potential hazard of the identified formulation impurities and aerosol chemicals was also estimated. E-cigarettes were machine puffed (4-s duration, 55-mL volume, 30-s intervals) to battery exhaustion to maximize aerosol collection. Aerosols analyzed for carbonyls were collected in 20-puff increments to account for analyte instability. Tobacco specific nitrosamines were measured at levels observed in pharmaceutical grade nicotine. Nicotine-related impurities in the e-cigarette formulations were below the identification and qualification thresholds proposed in ICH Guideline Q3B(R2). Levels of potentially harmful chemicals detected in the aerosols were determined to be below published occupational exposure limits.

The effect of menthol containing cigarettes on adult smokers' exposure to nicotine and carbon monoxide.

UNLABELLED There is limited information comparing biomarkers of exposure (BOE) to cigarette smoke in menthol (MS) and non-menthol cigarette smokers (NMS). OBJECTIVE To compare BOE to nicotine and carbon monoxide in MS and NMS. METHODS Cross-sectional, observational, ambulatory, multi-centre study in 3341 adult cigarette smokers. Nicotine equivalents (NE) in 24h urine, NE/cigarette, COHb and serum cotinine were measured. Statistical analyses included analysis of variance and Wilcoxon test. RESULTS Analyses of variance revealed no statistically significant effects of mentholated cigarettes on NE/24h, COHb, serum cotinine and NE/cigarette. On average MS smoked 15.0 and NMS 16.8 cigarettes/day. The unadjusted mean differences were as follows: MS had lower NE/24h (5.4%) and COHb (3.2%), higher serum cotinine (3.0%) and NE/cigarette (5.7%) than NMS. African-Americans MS smoked 40% fewer cigarettes, showed lower NE/24h (24%) and COHb (10%) and higher NE/cig (29%) and serum cotinine (8%) levels than their White counterparts. CONCLUSIONS Smoking mentholated cigarettes does not increase daily exposure to smoke constituents as measured by NE and COHb. These findings are consistent with the majority of epidemiological studies indicating no difference in smoking related risks between MS and NMS.

A comprehensive evaluation of the toxicology of cigarette ingredients: aliphatic and aromatic carboxylic acids.

Context: Aromatic and aliphatic carboxylic acids are present in tobacco and tobacco smoke. Objective: A battery of tests was used to compare the toxicity of mainstream smoke from experimental cigarettes containing eight aromatic and aliphatic carboxylic acids and the salt of one acid that were added individually at three different levels (lowest and highest target inclusions were 100 and 90,000 ppm, respectively). Materials and methods: Mainstream smoke from cigarettes containing each of the test ingredients was evaluated using analytical chemistry and assays to measure in vitro cytotoxicity (neutral red uptake) and Salmonella (five strains) mutagenicity. For four of the compounds (citric, lactic, benzoic acids, and sodium benzoate), 90-day rodent inhalation studies were also performed. Results: Although sporadic statistically significant differences in some experimental cigarette smoke constituents occurred, none resulted in significant changes in mutagenicity or cytotoxicity responses, nor in responses measured in the inhalation studies, except for lactic acid (LA). Inclusion of LA resulted in dose-dependent increase in water and caused a dose-dependent decrease in cytotoxicity. Incorporation of LA into cigarettes resulted in several dose-related reductions in histopathology, which were largely restricted to the nasal passages. Incorporation of LA also ameliorated some of the typical decrease in body weight gain seen in cigarette smoke-exposed rats. Conclusions: Inclusion of these ingredients at exaggerated use levels resulted in sporadic dose-related and treatment effects for some smoke constituents, but no toxicological response was noted in the in vitro and in vivo tests performed.

Toxicological assessment of a prototype e-cigaret device and three flavor formulations: a 90-day inhalation study in rats

Abstract A prototype electronic cigaret device and three formulations were evaluated in a 90-day rat inhalation study followed by a 42-day recovery period. Animals were randomly assigned to groups for exposure to low-, mid- and high-dose levels of aerosols composed of vehicle (glycerin and propylene glycol mixture); vehicle and 2.0% nicotine; or vehicle, 2.0% nicotine and flavor mixture. Daily targeted aerosol total particulate matter (TPM) doses of 3.2, 9.6 and 32.0 mg/kg/day were achieved by exposure to 1 mg/L aerosol for 16, 48 and 160 min, respectively. Pre-study evaluations included indirect ophthalmoscopy, virology and bacteriological screening. Body weights, clinical observations and food consumption were monitored weekly. Plasma nicotine and cotinine and carboxyhemoglobin levels were measured at days 28 and 90. After days 28, 56 and 90, lung function measurements were obtained. Biological endpoints after 90-day exposure and 42-day recovery period included clinical pathology, urinalysis, bronchoalveolar fluid (BALF) analysis, necropsy and histopathology. Treatment-related effects following 90 days of exposure included changes in body weight, food consumption and respiratory rate. Dose-related decreases in thymus and spleen weights, and increased BALF lactate dehydrogenase, total protein, alveolar macrophages, neutrophils and lung weights were observed. Histopathology evaluations revealed sporadic increases in nasal section 1–4 epithelial hyperplasia and vacuolization. Following the recovery period, effects in the nose and BALF were persistent while other effects were resolved. The no observed effect level based upon body weight decreases is considered to be the mid-dose level for each formulation, equivalent to a daily TPM exposure dose of approximately 9.6 mg/kg/day.

A comprehensive evaluation of the toxicology of cigarette ingredients: carbohydrates and natural products.

Context: Eleven carbohydrates and natural product ingredients were added individually to experimental cigarettes. Objective: A battery of tests was used to compare toxicity of mainstream smoke from these experimental cigarettes to matched control cigarettes without test ingredients. Materials and Methods: Smoke fractions from each cigarette type were evaluated using analytical chemistry; in vitro cytotoxicity (neutral red uptake) and in vitro bacterial (Salmonella) mutagenicity (five strains) testing. For 10 ingredients (β-cyclodextrin, cleargum, D-sorbitol, high fructose corn syrup, honey, invert sugar, maltodextrin, molasses, raisin juice concentrate, and sucrose), 90-day nose-only smoke inhalation studies using rats were also performed. Results: In general, addition of each ingredient in experimental cigarettes resulted in minimal changes in smoke chemistry; the exceptions were D-sorbitol and sucrose, where reductions in amount of 60% to 80% of control values for some smoke constituents were noted. Additionally, each ingredient resulted in small increases in smoke formaldehyde concentrations. Except for a reduction in cytotoxicity by inclusion of maltodextrin and an increase by inclusion of plum juice concentrate, the cytotoxicity and mutagenicity results were unaffected by addition of the other ingredients in experimental cigarettes. There were also very few statistically significant differences within any of the 10 inhalation studies, and when present, the differences were largely sporadic and inconsistent between sexes. Conclusion: The carbohydrates and natural products tested here as ingredients in experimental cigarettes as a class increased formaldehyde, but resulted in minimal toxicological responses, even at high inclusion levels compared with the levels used in commercial cigarette products.

Toxicological evaluation of aerosols of a tobacco extract formulation and nicotine formulation in acute and short-term inhalation studies

Abstract A formulation of tobacco extract containing 4% nicotine (TE) and similar nicotine formulation containing vehicle and 4% nicotine (NF) were evaluated using animal inhalation assays. Two 4-h inhalation exposures at 1 and 2 mg/L aerosol exposure concentrations, respectively, of the tobacco extract with 4% nicotine formulation showed that the LC50 was greater than 2 mg/L, the maximum concentration tested. All inhalation exposures were conducted using the capillary aerosol generator (CAG). Increasing aerosol TPM concentrations (0, 10, 50, 200, 1000 mg/m3 TE and 0, 50, 200, 500, 1000 mg/m3 NF) were generated via the CAG and used to expose groups of male and female rats for 4-h per day for 14 days. In life monitors for potential effects included clinical observations, weekly body weights and food consumption. Post mortem evaluations included gross tissue findings, hematology, clinical chemistry, serum plasma and nicotine levels, absolute and normalized organ and tissue weights, and histopathology of target organs. Treatment-related changes were observed in body weights, hematology, clinical chemistry, organ weights and histopathological findings for TE at the 200 and 1000 mg/m3 exposure levels, and in the 500 and 1000 mg/m3 exposure groups for NF. Under the conditions of these studies, the no-observed-adverse-effect level in the rat was approximately 50 mg/m3 for the TE aerosol-exposed groups, and approximately 200 mg/m3 in the NF aerosol-exposed groups.

A comprehensive evaluation of the toxicology of experimental cigarettes manufactured with banded papers

Abstract Context: To comply with state requirements, cigarette manufacturers have added low-permeability bands to the cigarette paper. These bands can extinguish the cigarette when it is no longer being puffed by a smoker. Objective: This study was conducted to evaluate the toxicology resulting from the addition of different types of bands to experimental cigarettes. Materials and methods: A battery of assays that are typically used in toxicology studies with cigarette smoke, namely smoke chemistry, in vitro mutagenicity and cytotoxicity, and inhalation studies with rats, were used to evaluate different band characteristics added to cigarette paper. Results: Although differences in the amount of band material was associated with an increase in some metals measured in mainstream tobacco smoke, it was not dose responsive to any band design parameter (base paper permeability, band width, band spacing, band chalk amount, or citrate). Occasional, minor differences were produced by the different types of bands; overall, there was no increased toxicity. Conclusion: Although there were increases and decreases in some mainstream smoke constituents, the in vitro and in vivo testing performed demonstrated that low-permeability bands on cigarettes do not modify the toxicity of smoke inhaled by smokers.

Evaluation of a novel inhalation exposure system to determine acute respiratory responses to tobacco and polymer pyrolysate mixtures in Swiss-Webster mice

Modern cigarette production processes are highly automated and yield millions of cigarettes per day. The forming cigarette and its components contact many different materials in the production process, some of which may leave minute residues. The potential for small inclusions of non-cigarette materials such as wood, plastic, cardboard and other materials exists from the bulk handling and processing of tobacco, in spite of vigilant workers and numerous online systems designed to keep the tobacco stream clean. Currently, there are no published methods that describe an approach to evaluate the potential toxicological impact of these non-tobacco residues and inclusions on the biological activity from exposure to the complex mixture of tobacco smoke. There are, however, many methods which describe toxicological evaluation approaches for pure materials, particularly synthetic polymers. We used the Deutsche Institute fur Normung (DIN) 53-436 tube furnace and nose-only exposure chamber in combination to conduct pilot studies in Swiss-Webster mice in order to develop a standardized methodology for the evaluation of sensory irritation and other potentially useful biological endpoints for predicting any potential hazards. Sensory and/or pulmonary irritation was assessed based on respiratory function parameters using the ASTM E981-84 method described by in mice, exposed to test atmospheres of 100% tobacco pyrolysate or tobacco/polymer pyrolysate mixtures. Other biological evaluations included respiratory function parameters, clinical signs, body weights, bronchoalveolar lavage fluid analysis, carboxyhemoglobin, blood cyanide concentrations and histopathology of the respiratory tract. These pilot studies have demonstrated that such an approach can detect biological changes resulting from exposure to unique tobacco/polymer pyrolysates. Small differences were detected in the sensory irritation responses (respiratory function), activation state of pulmonary macrophages, and histopathological findings in the nose of mice exposed to 100% tobacco or tobacco/polymer pyrolysates. Analytical measurements were also performed in order to characterize the test atmospheric changes that could occur from inclusion of the polymer into the tobacco. These included DIN-generated wet total particulate matter (TPM) DIN-Generated wet TPM (DWTPM), nicotine, cyanide, formaldehyde, acetaldehyde, acrolein, carbon monoxide, carbon dioxide, and nitrogen oxides. We attempted to correlate analyte differences in the test atmospheres with the resulting biological findings in the mice. The results demonstrated that this approach could detect minimal toxicological effects in mice exposed to test atmospheres of 100% tobacco or 70%/30% tobacco/polymer pyrolysates.

Chemical, physical, and in vitro characterization of research cigarettes containing denicotinized tobacco

The use of very low nicotine tobacco cigarettes is currently being investigated as a possible harm reduction strategy. Here, we report the smoke chemistry, toxicity, and physical characteristics of very low nicotine cigarettes that were made using blended tobacco processed through a supercritical CO2 fluid extraction, which resulted in elimination of 96% of nicotine content (denicotinized (denic) tobacco). Three types of test cigarettes (TCs) were manufactured with tobacco filler containing 100% denic tobacco (TC100), 50% denic tobacco and 50% unextracted tobacco (TC50/50), and 100% unextracted tobacco (TC0). Mainstream smoke (MS) was generated for measurement of 46 analytes and cytotoxicity and mutagenicity determination. Analysis of physical characteristics of TCs demonstrated they were well made with <5% variability among cigarettes for most parameters measured. We observed significant changes in the levels of smoke constituents, including decreases in formaldehyde, nitrosamines, and phenol, and increases in aliphatic hydrocarbons, aliphatic nitrogen compounds, aromatic amines, halogen compounds, and metals. Use of denic tobacco resulted in changes in the chemical composition of MS, but these changes did not modify biological activity as measured in the mutagenicity and cytotoxicity assays.

Toxicological responses in SW mice exposed to inhaled pyrolysates of polymer/tobacco mixtures and blended tobacco

Modern cigarette manufacturing is highly automated and produces millions of cigarettes per day. The potential for small inclusions of non-cigarette materials such as wood, cardboard packaging, plastic, and other materials exists as a result of bulk handling and high-speed processing of tobacco. Many non-tobacco inclusions such as wood, paper, and cardboard would be expected to yield similar pyrolysis products as a burning cigarette. The aircraft industry has developed an extensive literature on the pyrolysis products of plastics, however, that have been reported to yield toxic by-products upon burning, by-products that have been lethal in animals and humans upon acute exposure under some exposure conditions. Some of these smoke constituents have also been reported in cigarette smoke. Five synthetic polymers, nylon 6, acrylonitrile–butadiene–styrene (ABS), nylon 12, nylon 6,6, and acrylonitrile–butadiene (AB), and the natural polymer wool were evaluated by adding them to tobacco at a 3, 10, and 30% inclusion level and then pyrolyzing the mixture. The validated smoke generation and exposure system have been described previously. We used the DIN 53-436 tube furnace and nose-only exposure chamber in combination to conduct exposures in Swiss-Webster mice. Potentially useful biological endpoints for predicting hazards in humans included sensory irritation and pulmonary irritation, respiratory function, clinical signs, body weights, bronchoalveolar lavage (BAL) fluid analysis, carboxyhemoglogin, blood cyanide concentrations, and histopathology of the respiratory tract. Chemical analysis of selected smoke constituents in the test atmosphere was also performed in order to compare the toxicological responses with exposure to the test atmospheres. Under the conditions of these studies, biological responses considered relevant and useful for prediction of effects in humans were found for sensory irritation, body weights, BAL fluid analysis, and histopathology of the nose. There was a marked sensory irritation response that recovered slowly for some polymers. Sustained body weight depression, lesions of the respiratory epithelium of the nose, and morphological changes in pulmonary alveolar macrophages (PAM) were observed after exposure to some polymer/tobacco pyrolysates. These responses were increased compared to exposure to tobacco pyrolysate alone. No moribundity or mortality occurred during the study. The data suggest that polymeric inclusions pose a minimal additional toxicologic hazard in humans.