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Electronic nicotine delivery systems (ENDSs) are designed as a non-combustible alternative to cigarettes, aiming to deliver nicotine without the harmful byproducts of tobacco combustion. As the category evolves and new ENDS products emerge, it is important to continually assess the levels of toxicologically relevant chemicals in the aerosols and characterize any related toxicology. Herein, we present a proposed framework for characterizing novel ENDS products (i.e., devices and formulations) and determining the reduced risk potential utilizing analytical chemistry and in vitro toxicological studies with a qualitative risk assessment. To demonstrate this proposed framework, long-term stability studies (12 months) analyzing relevant toxicant emissions from six formulations of a next-generation product, JUUL2, were conducted and compared to reference combustible cigarette (CC) smoke under both non-intense and intense puffing regimes. In addition, in vitro cytotoxicity, mutagenicity, and genotoxicity assays were conducted on aerosol and smoke condensates. In all samples, relevant toxicants under both non-intense and intense puffing regimes were substantially lower than those observed in reference CC smoke. Furthermore, neither cytotoxicity, mutagenicity, nor genotoxicity was observed in aerosol condensates generated under both intense and non-intense puffing regimes, in contrast to results observed for reference cigarettes. Following the proposed framework, the results demonstrate that the ENDS products studied in this work generate significantly lower levels of toxicants relative to reference cigarettes and were not cytotoxic, mutagenic, or genotoxic under these in vitro assay conditions. [ABSTRACT FROM AUTHOR]

A unique adult human mouth/throat physical model has been developed to study e-cigarette aerosol dynamics during inhalation. The 3D printed physical model was created from the CT scan of a 28 yr. old healthy male. Internal walls of the physical model were lined with a thin layer of cotton gauze that can be saturated with water to replicate the high relative humidity conditions in a human Oral/Pharyngeal cavity. Aerosol hygroscopic growth and deposition inside the physical model from a cartomizer style e-cigarette using a prototype e-liquid formulation was determined by measuring cumulative aerosol mass from five puffs (gravimetric) and for individual constituents (propylene glycol, glycerol, and nicotine) from a single puff (GC/MS analysis). Measurements were taken at constant temperature of 37 °C under both wet and dry inner wall conditions for a for a 3-sec. 55 mL puff. The condition of holding aerosol inside the physical model without airflow for a duration of 3-sec. was also included. For the same puffing conditions of 3-sec puff of 55 mL and a 3-sec. puff hold time, dry wall conditions resulted in a mean aerosol mass loss of 20.9 ± 3.8%, while the total aerosol mass was increased by 150.9 ± 19% under wet wall condition when compared to total aerosol mass entering the physical model entrance. The dramatic increase is due to water vapor uptake by the aerosol particles when flowing through the wetted physical model. Aerosol evolution of individual chemical constituent analysis appeared to vary as a function of volatility. [ABSTRACT FROM AUTHOR]

A total of 20 commercial cigarette and 16 commercial smokeless tobacco products were assayed for 96 compounds listed as harmful and potentially harmful constituents (HPHCs) by the US Food and Drug Administration. For each product, a single lot was used for all testing. Both International Organization for Standardization and Health Canada smoking regimens were used for cigarette testing. For those HPHCs detected, measured levels were consistent with levels reported in the literature, however substantial assay variability (measured as average relative standard deviation) was found for most results. Using an abbreviated list of HPHCs, statistically significant differences for most of these HPHCs occurred when results were obtained 4–6 months apart (i.e., temporal variability). The assay variability and temporal variability demonstrate the need for standardized analytical methods with defined repeatability and reproducibility for each HPHC using certified reference standards. Temporal variability also means that simple conventional comparisons, such as two-sample t -tests, are inappropriate for comparing products tested at different points in time from the same laboratory or from different laboratories. Until capable laboratories use standardized assays with established repeatability, reproducibility, and certified reference standards, the resulting HPHC data will be unreliable for product comparisons or other decision making in regulatory science. [ABSTRACT FROM AUTHOR]

Introduction: Few clinical studies involving cigarettes have provided a comprehensive picture of smoke exposure, test article characterization, and insights into sensory properties combined. The purpose of these pilot studies was to determine whether cigarettes with different levels of nicotine but similar tar levels would affect sensory experience or smoking behavior so as to significantly alter levels of selected biomarkers of exposure (BOE). Methods: In 2 confined, double-blind studies, 120 adult smokers switched from Marlboro Gold cigarettes at baseline to either 1 of 2 lower nicotine cigarettes or 1 of 2 higher nicotine cigarettes and then to the other cigarette after 5 days. Urinary excretion of exposure biomarkers (nicotine equivalents [NE], total and free 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol [NNAL], 1-hydroxypyrene, and 3-hydroxypropyl mercapturic acid) as well as carboxyhemoglobin and plasma cotinine were measured at baseline, Day 5, and Day 10. Daily cigarette consumption was monitored and sensory characteristics were rated for each cigarette. Results: With higher nicotine yield, urine NE, urine total NNAL, and plasma cotinine increased while nonnicotine BOE decreased without changes in cigarette consumption. In contrast, with lower nicotine yield, urine NE, urine total NNAL, and plasma cotinine dropped while nonnicotine BOE and cigarettes per day increased. Higher nicotine cigarettes were rated harsher and stronger than at baseline while lower nicotine cigarettes were less strong. All 4 test cigarettes were highly disliked. Conclusions: These studies demonstrate that abrupt increases or decreases in nicotine and the resulting sensory changes impact BOE through changes in intensity or frequency of smoking. [ABSTRACT FROM PUBLISHER]

Context: In addition to tobacco and cigarette ingredients, there are many non-tobacco components and processes used to manufacture commercial cigarettes. Proposed cigarette components and manufacturing process changes were evaluated using a tiered toxicological testing program. Objective: The toxicological testing and evaluation of proposed changes to selected non-tobacco cigarette components and manufacturing processes are described in this special report. Materials and methods: Selected non-tobacco cigarette components and manufacturing processes were evaluated using experimental and control cigarettes. These experimental cigarettes were evaluated using mainstream smoke chemistry, bacterial mutagenicity and cytotoxicity assays. In some evaluations, 90-day nose-only mainstream smoke inhalation studies using rats were performed. Results: Some proposed design changes were not implemented, or limitations on their use were established. Most study results, however, were similar to those previously reported in the scientific literature for design changes in cigarette construction. Conclusion: The studies reported in the series of publication demonstrate that our testing approach is feasible for evaluation of cigarette component and manufacturing process changes. [ABSTRACT FROM AUTHOR]

Context: Historical work indicates that cigarette circumference may affect the toxicological profile of experimental cigarettes. Objective: Studies were conducted to examine the effect of different cigarette circumferences on (1) selected mainstream smoke constituents including concentrations of tobacco specific nitrosamines (TSNA) in smoke and (2) mutagenicity and cytotoxicity of cigarette smoke condensate. Materials and methods: Analytical chemistry, Salmonella mutagenicity and cytotoxicity assays were used to evaluate the composition and biological activity of mainstream smoke from experimental, non-filtered cigarettes manufactured with four different circumferences (17.0-27.1 mm). Results: Most smoke constituents, including TSNA, decreased with decreasing cigarette circumference; however, amounts of hydrogen cyanide increased in a non-circumference dependent manner. Mutagenicity and cytotoxicity also decreased slightly with decreasing cigarette circumference. Conclusion: Cigarette circumference may have a minor role in the toxicological profile of experimental cigarettes, with a so-far-unidentified mechanism. [ABSTRACT FROM AUTHOR]

Context: Adhesives are used in several different manufacturing operations in the production of cigarettes. The use of new, 'high-speed-manufacture' adhesives (e.g. vinyl acetate based) could affect the smoke chemistry and toxicology of cigarettes, compared with older 'low-speed-manufacture' adhesives (e.g. starch based). Objective: This study was conducted to determine whether the inclusion of different levels of three adhesives (ethylene vinyl acetate, polyvinyl acetate and starch) in experimental cigarettes results in different smoke chemistry and toxicological responses in in vitro and in vivo assays. Materials and methods: A battery of tests (analytical chemistry, in vitro and in vivo assays) was used to compare the chemistry and toxicology of smoke from experimental cigarettes made with different combinations of the three adhesives. Varying levels of the different side-seam adhesives, as well as the transfer of adhesives from packaging materials, were tested. Results: There were differences in some mainstream cigarette smoke constituents as a function of the level of adhesive added to experimental cigarettes and between the tested adhesives. None of these differences translated into statistically significant differences in the in vitro or in vivo assays. Conclusion: The use of newer 'high-speed-manufacture' vinyl acetate-based adhesives in cigarettes does not produce toxicological profiles that prevent the adhesives from replacing the older 'low-speed-manufacture' adhesives (such as starch). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Context: Cigarettes often have a small identifying mark (monogram) printed either on the cigarette paper toward the filter end of the cigarette or on the tipping paper. Objective: A battery of tests was used to compare the toxicology of mainstream smoke from experimental cigarettes manufactured with different monogram inks. Cigarettes with different concentrations of different pigments were compared with cigarettes without ink, and with a control ink. Materials and methods: Smoke from each of the experimental cigarettes was evaluated using analytical chemistry and in vitro bacterial mutagenicity ( Salmonella, five strains, ±S9) and cytotoxicity (neutral red uptake) assays. Results: No differences were observed between experimental cigarettes printed with three different pigment loads of iron oxide-based Black pigment and non-printed cigarettes. In general, no dose response was observed. However, increases in certain smoke constituents were found to correlate with Pigment Yellow 14 (also known as benzidine yellow) and Pigment Blue 15 (copper phthalocyanine). Increases in bacterial mutagenicity were observed for high-level print of Pigment Yellow 14 in TA98 and TA1537 and the high-level print of Pigment Blue 15 in TA98. In vitro cytotoxicity of mainstream smoke was unaffected by the presence of monogram ink on cigarettes. Conclusion: Statistically significant dose-responsive constituent changes and an increase in mutagenicity were observed with inclusion of Pigment Yellow 14 and Pigment Blue 15. Other pigments showed minimal toxicological activity. [ABSTRACT FROM AUTHOR]

Context: Recent technological advances allow ventilation holes in (or adjacent to) cigarette filters to be produced using lasers instead of using the mechanical procedures of earlier techniques. Objective: Analytical chemistry can be used to compare the composition of mainstream smoke from experimental cigarettes having filters with mechanically produced ventilation holes to that of cigarettes with ventilation holes that were produced using laser technology. Materials and methods: Established procedures were used to analyze the smoke composition of 38 constituents of mainstream smoke generated using standard conditions. Results: There were no differences between the smoke composition of cigarettes with filter ventilation holes that were produced mechanically or through use of laser technology. Conclusion: The two methods for producing ventilation holes in cigarette filters are equivalent in terms of resulting mainstream smoke chemistry, at two quite different filter ventilation percentages. [ABSTRACT FROM AUTHOR]

Context: Manufacture of cigarettes results in tobacco by-products, some of which can be processed and added back to cigarettes. Such additions (known as reconstituted tobacco or reconstituted leaf) have been shown to reduce tar yields. A new process (termed 'Deli' cast sheet) is a potential refinement of the reconstitution process. Objective: Compare toxicity of smoke from experimental cigarettes made with reconstituted leaf with that from cigarettes made with Deli cast sheet. Materials and methods: Analytical chemistry, Salmonella mutagenicity and cytotoxicity assays were used to evaluate the composition biological activity of mainstream smoke from experimental cigarettes made with Deli cast sheet or with reconstituted leaf. The effect of different amounts of guar and propylene glycol in Deli cast sheet was also evaluated. Results: Small increases in the amount of nitrogen oxides were found as a result of inclusion of the Deli cast sheet when compared with reconstituted leaf; no differences in cytotoxicity or mutagenicity were found. Conclusion: The Deli process neither significantly modified chemical composition of smoke nor affected its biological activity, as measured by the mutagenicity and cytotoxicity assays used here. [ABSTRACT FROM AUTHOR]

Context: Literature suggests that the width of tobacco strips in cigarettes may affect the smoke chemistry and toxicology of such products. Objective: A comprehensive analysis of smoke from experimental cigarettes can be used to determine whether different cut widths of tobacco result in different toxicological activity. Materials and methods: A battery of tests was used to compare the chemistry and in vitro and in vivo toxicology of smoke from experimental cigarettes made with tobacco cut to different widths. Results: Different cut widths of tobacco did not elicit consistent and significant differences in cigarette smoke chemistry, responses in in vitro mutagenicity or cytotoxicity assays or most endpoints in 90-d rat inhalation studies. Of note, however, were atypical in-life observations and slightly depressed body weights observed in two rat inhalation studies. Conclusion: Most of our data indicate that different cut widths of tobacco used in cigarettes are unlikely to change the toxicity of mainstream cigarette smoke; however, without additional investigation, the atypical in-life observations and depression in body weights cast doubt on the toxicological acceptability of cutting the tobacco into wider shreds. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Context: Three heterocyclic nitrogen compounds, 2,3-diethylpyrazine (DEP), 2,3,5,6-tetramethylpyrazine (TMP), and 2-acetyl pyridine (AP), are naturally present in tobacco and are also added to tobacco as flavor ingredients. Objective: A battery of tests was used to compare the toxicity of mainstream smoke from experimental cigarettes containing the three heterocyclic nitrogen compounds added individually at three different levels. The lowest target inclusion level of the ingredient was 10 ppm, and the highest was 10,000 ppm. Material and methods: Smoke from experimental and control cigarettes was evaluated in analytical smoke chemistry, in vitro cytotoxicity, and mutagenicity assays. Results: The cigarettes with added DEP produced some minor (approximately 10%) changes in smoke chemistry when compared with the cigarettes containing no DEP. Smoke chemistry was effectively unchanged by the addition of either AP or TMP. Cytotoxicity, assessed by the neutral red uptake assay using both gas-vapor and particulate phases of smoke, was unaffected by the addition of any of the test ingredients. Mutagenicity, assessed in five strains of Salmonella treated with mainstream cigarette smoke condensate, also was unaffected by any of the test ingredients. Conclusions: Despite the exaggerated ingredient levels relative to commercial-use levels, there was a lack of a toxicological response for the three heterocyclic nitrogen compounds in the test systems used. [ABSTRACT FROM AUTHOR]

Context: Aromatic carbonyls are typically used in the processing or flavoring of tobacco used in the manufacture of cigarettes. Objective: A battery of tests was used to compare the toxicity of mainstream smoke from experimental cigarettes containing different added levels of aromatic carbonyl compounds. Materials and methods: Ten aromatic carbonyl compounds, nine of which have been reported in tobacco or in tobacco smoke, were added individually to experimental cigarettes at three different levels. The tenth compound, not found naturally in tobacco, was 2-phenoxyethyl isobutyrate. The lowest target inclusion level was 100 ppm and the highest was 10,000 ppm. Smoke from each of the 10 experimental cigarette types was evaluated using analytical chemistry, in vitro cytotoxicity, and mutagenicity testing. For one of the compounds, ethyl vanillin, a 90-day smoke inhalation study using rats was also performed. Results: Smoke chemistry was effectively unchanged by the addition of any of the compounds. Cytotoxicity, assessed by the neutral red uptake assay and using both gas-vapor and particulate phases of smoke, was unaffected by the addition of any of the test compounds. Mutagenicity, assessed by five strains of Salmonella typhimurium treated with smoke condensate, also was unaffected by any of the test compounds. In the rat inhalation study, there were effectively no differences between cigarettes without added ethyl vanillin and cigarettes containing ~8000 ppm of ethyl vanillin. Conclusion: Even at the exaggerated inclusion levels in cigarette tobacco used in these tests, no adverse toxicological responses occurred for any of aromatic carbonyl compounds tested. [ABSTRACT FROM AUTHOR]

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 (ββ-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. [ABSTRACT FROM AUTHOR]

Context: Cocoa-derived ingredients are used in cigarette tobacco. Objective: A battery of tests was used to compare toxicity of mainstream smoke from experimental cigarettes containing different added levels of cocoa-derived ingredients. Materials and Methods: Five cocoa-derived ingredients chocolate (CH), cocoa (COC), cocoa-grand prix black (CGPB), cocoa nibs tincture (CNT) and cocoa shells extract (CSE) were added individually to experimental cigarettes at three different levels. Smoke from each of the experimental cigarette types was evaluated using analytical chemistry; in vitro cytotoxicity and mutagenicity testing were performed for four of the five compounds. For CH, COC and CNT, 90-day smoke inhalation studies were performed with 6-week recovery periods. Results: No consistent changes were found in the analytical chemistry results. Results of the cytotoxicity and mutagenicity were unaffected by any of the ingredients. Two of the three inhalation studies showed very few differences between the groups. The inhalation study with COC showed several increases in mean histopathology severity scores in groups exposed to different levels of COC, compared with the controls. These apparent effects of COC on histopathology lesion severity scores were only present in a single sex and none were dose-related, which is not consistent with a true increase in biological activity. Also there were effectively no differences in the patterns of recovery for any of the compounds. Conclusions: Even at high inclusion levels there was a lack of toxicological response in these COC derived ingredients. [ABSTRACT FROM AUTHOR]

Context: A total of 32 essential oils and resins were added individually to experimental cigarettes. Objective: A battery of tests was used to compare the toxicity of mainstream smoke from these experimental cigarettes. The lowest target inclusion level was 100 ppm and the highest was 100,000 ppm. Materials and methods: Smoke from each of the experimental cigarette was evaluated using analytical chemistry and in vitro bacterial ( Salmonella, five strains) mutagenicity and cytotoxicity (neutral red uptake) assays. For seven of the ingredients (carob bean, carob bean extract, carrageenan, chamomile flower Hungarian oil, guar gum, peppermint oil, and spearmint oil), 90-day smoke inhalation studies with rats were also performed. Results: In general, inclusion levels resulted in minimal changes in smoke chemistry; the exceptions were PO and SO, where reductions to 40--60% of control values were noted, possibly indicating a tobacco displacement effect. Cytotoxicity and mutagenicity were unaffected by any of the test ingredients, except for a dose-related reduction in cytotoxicity for SO. There were very few statistically significant differences within any of the seven inhalation studies; when present, the differences were sporadic and inconsistent between sexes. The addition of SO appeared to depress body weight gain and increase the atrophy of olfactory epithelia, but only in males. Conclusion: The essential oils and resins tested here as ingredients in experimental cigarettes show minimal toxicological sequelae, even at high inclusion levels. The highest inclusion level for SO showed some equivocal responses. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Context: Two ammonia compounds, diammonium phosphate and ammonium hydroxide, are typically used in the processing or flavoring of tobacco used in the manufacture of cigarettes. Objective: A battery of tests was used to compare the toxicity of mainstream smoke from experimental cigarettes containing different added levels of diammonium phosphate (target maximal inclusion level, 50,000 ppm) or both ammonium hydroxide (target maximal inclusion level 11,160 ppm) and diammonium phosphate. Materials and methods: The tests included analytical chemistry, with over 40 constituents in mainstream cigarette smoke; in vitro bacterial ( Salmonella) mutagenicity and cytotoxicity (neutral red uptake) assays, and 90-day smoke inhalation studies using rats. Diammonium phosphate acted as a burn retardant, and consequently, the highest planned inclusion level could not be used. Ammonium hydroxide could not be added to cigarettes at meaningfully different levels. Results: Apart from a substantial reduction in smoke concentrations of formaldehyde seen in the smoke chemistry analysis and animal exposure characterization, there were very few endpoints in any of the analyses that showed significant differences as a result of the addition of either of the two ammonia compounds. These differences, when present, occurred only sporadically, with no evidence of any dose-response relationships. Conclusion: The results of these experiments show that the ammonia compounds, diammonium phosphate and ammonium hydroxide, when added to cigarette tobacco, even at high inclusion levels, have minimal toxicological sequelae. [ABSTRACT FROM AUTHOR]

Context: Various aromatic and aliphatic alcohol compounds are found 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 alcohol compounds that were added individually to experimental cigarettes at three different levels. The lowest target inclusion level was 100 ppm and the highest level was 24,400 ppm. Materials and methods: Mainstream smoke from each of the cigarette types was evaluated using analytical chemistry and assays to measure in vitro cytotoxicity (neutral red uptake) and Salmonella (five strains) mutagenicity. For three of the compounds (benzyl alcohol, propyl paraben, and rum flavor), 90-day smoke inhalation studies with 6-week recovery periods were also performed using rats. Results: Inclusion of eugenol produced several dose-related reductions in concentrations of selected smoke constituents. Cytotoxicity and mutagenicity were unaffected by any of the test ingredients, except for dose-related reductions in cytotoxicity of the gas vapor phase produced by the inclusion of eugenol. The three smoke inhalation studies showed a few sporadic differences between the groups and there were no differences in the patterns of recovery for any of the ingredients. Conclusions: Despite using exaggerated inclusion levels of the eight aliphatic and aromatic alcohol compounds in experimental cigarettes, there was minimal toxicological response, which is consistent with published reports of studies using mixtures of compounds added to tobacco. [ABSTRACT FROM AUTHOR]

Abstract: Propylene glycol (PG) is a humectant commonly used in cigarettes. Previous toxicological examinations of the effects on the addition of PG to tobacco used mixtures with several other flavoring agents. In the present work, toxicological comparisons were made of experimental cigarettes containing no added PG against otherwise similar cigarettes with three different amounts of PG added to the tobacco. The main toxicological comparison was a sub-chronic inhalation study with mainstream smoke in Sprague–Dawley rats (exposures of 150mg/m3 of total particulate matter, 6h exposure per day, for 90 consecutive days). The target PG concentrations in the tobacco of the four cigarette types were 0, 4, 7 and 10%. Additional studies with mainstream smoke were bacterial mutagenicity (5 Salmonella strains, both with and without metabolic activation, particulate phase only), cytotoxicity of both particulate and gas/vapor phases (using the neutral red uptake assay), and analytical chemistry (41 analytes). The graded inclusion of PG into experimental cigarettes resulted in increases in the smoke concentrations of propylene oxide, at very low concentrations. Broadly similar responses were seen across the four cigarette types, and the responses were similar to those previously described in the scientific literature. The addition of PG to experimental cigarettes reduced concentrations of some smoke components (e.g. nicotine), but had minimal effects on the biological responses. Most of the changes produced in the 90-days of exposure were resolved in a 42-day post-inhalation period. [Copyright &y& Elsevier]

Electronic nicotine delivery systems (ENDS) are generally recognized as less harmful alternatives for those who would otherwise continue to smoke cigarettes. The potential toxicity of aerosols generated from JUUL Device and Virginia Tobacco (VT3) or Menthol (ME3) JUULpods at 3.0% nicotine concentration was assessed in rats exposed at target aerosol concentrations of 1400 μg/L for up to 6 h/day on a 5 day/week basis for at least 90 days (general accordance with OECD 413). 3R4F reference cigarette smoke (250 μg/L) and Filtered Air were used as comparators. JUUL ENDS product aerosol exposures at >5x the 3R4F cigarette smoke level resulted in greater plasma nicotine and cotinine levels (up to 2x). Notable cigarette smoke related effects included pronounced body weight reductions in male rats, pulmonary inflammation evidenced by elevated lactate dehydrogenase, pro-inflammatory cytokines and neutrophils in bronchoalveolar lavage fluid, increased heart and lung weights, and minimal to marked respiratory tract histopathology. In contrast, ENDS aerosol exposed animals had minimal body weight changes, no measurable inflammatory changes and minimal to mild laryngeal squamous metaplasia. Despite the higher exposure levels, VT3 and ME3 did not result in significant toxicity or appreciable respiratory histopathology relative to 3R4F cigarette smoke following 90 days administration. [ABSTRACT FROM AUTHOR]