Toxicological Assessment of Flavor Ingredients in E-Vapor Products.

Many flavor ingredients are often used in potentially reduced-risk tobacco products (such as e-vapor products). Although most are "generally recognized as safe (GRAS)" when used in food, there is limited information available on their long-term health effects when delivered by inhalation. While obtaining route-of-exposure-specific toxicological data on flavor ingredients is critical to product evaluation, the large number of individual flavor ingredients available and their potential combinations render classical toxicological assessment approaches impractical, as they may require years of preclinical investigations and thousands of laboratory animals. Therefore, we propose a pragmatic approach in which flavor ingredients are initially assigned to groups of structurally related compounds (Flavor Groups), from which flavor group representatives (FGR) are then selected and tested individually and as a mixture in vitro and in vivo. The premise is that structurally related compounds would have comparable metabolic and biological activity and that the data generated using FGRs could support the toxicological assessment of other structurally related flavor ingredients of their respective Flavor Groups. This approach is explained in a step-wise manner and exemplified by a case study, along with its strengths, limitations as well as recommendations for further confirmatory testing. Once completed, this FGR approach could significantly reduce the time and resources required for filling the data gap in understanding the health risks of many flavor ingredients while also minimizing the need for laboratory animals.

Influence of Nitrite on Formation of Tobacco-Specific Nitrosamines in Electronic Cigarette Liquids and Aerosols.

Tobacco-specific nitrosamine (TSNA) formation occurred during aerosol generation from select commercial cig-a-like e-cigarette products. To understand the drivers behind the potential formation of TSNAs in electronic cigarette (e-cigarette) aerosols and e-liquids, model e-liquid systems were generated in the lab to demonstrate that nitrite can react with nicotine and minor alkaloids to form TSNAs in e-liquids. In the presence of nitrite and nicotine, TSNA levels in e-liquids increased over time and the process was accelerated by elevated temperature. Additionally, TSNAs formed during aerosol generation when nitrite was present in the corresponding e-liquids. The commercial e-cigarette products that showed higher levels and formation of TSNAs were observed to contain nitrite and minor alkaloid impurities in the corresponding e-liquids. This study provides valuable information about drivers for TSNA formation in e-liquids and e-cigarette aerosols that may be applied to the evaluation and quality assurance of e-cigarette products.

Formation of Diacetyl and Other α-Dicarbonyl Compounds during the Generation of E-Vapor Product Aerosols.

Exposure to diacetyl (DA) has been linked to the respiratory condition bronchiolitis obliterans. Previous research has demonstrated that DA and other α-dicarbonyl compounds can be detected in both the e-liquids and aerosols of e-vapor products (EVPs). While some EVP manufacturers may add these compounds as flavor ingredients, the primary objective of this work was to determine the potential for the formation of α-dicarbonyl compounds during the generation of aerosols from EVPs where no DA or other α-dicarbonyl compounds are added to the e-liquid. A novel ultraperformance liquid chromatography-mass spectrometry-based analytical method for the determination of DA, acetyl propionyl, glyoxal, and methylglyoxal was developed and validated. Next, eight commercially available cig-a-like-type EVPs were evaluated for α-dicarbonyl formation. Increased levels of α-dicarbonyls were observed in the aerosols of all evaluated EVPs compared to their respective e-liquids. Mechanistic studies were conducted using a model microwave reaction system to identify key reaction precursors for DA generated from propylene glycol (PG) and carbon-13-labeled glycerin (GLY). These studies, along with the corresponding retrosynthetic analysis, resulted in the proposed formation pathway where hydroxyacetone is generated from PG and/or GLY. Hydroxyacetone then participates in an aldol condensation with formaldehyde where formaldehyde can also be generated from PG and/or GLY; the resultant product then dehydrates to form DA. This proposed pathway was further investigated through in situ synthetic organic experiments within the model microwave reaction system. This work establishes that DA is formed in the aerosol generation process of the EVPs tested though at levels below toxicological concern.

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

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.

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.

A comprehensive evaluation of the toxicology of monogram inks added to experimental cigarettes.

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.

A comprehensive evaluation of the toxicology of cigarette ingredients: essential oils and resins.

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.

A comprehensive evaluation of the toxicology of cigarette ingredients: cocoa-derived ingredients.

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.

A comprehensive evaluation of the toxicology of cigarette ingredients: aromatic and aliphatic alcohol compounds.

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.

Systemic immunosuppression following a single pharyngeal aspiration of 1,2:5,6-dibenzanthracene in female B6C3F1 mice.

1,2:5,6-Dibenzanthracene (DBA) is ubiquitous in our environment as a contaminant produced by incomplete combustion of organics from sources such as forest fires, cigarette smoke, and asphalt paving, and it is more immunosuppressive of the T-dependent antibody-forming cell (AFC) response than the well-studied polycyclic aromatic hydrocarbon, benzo(a)pyrene. The systemic immunosuppressive effects of DBA were investigated following a single pharyngeal aspiration (pa) in female B(6)C(3)F(1) mice. The immunotoxic effects of DBA were evaluated using numerous assays of varying complexity to evaluate innate (natural killer [NK] cell activity), cell-mediated (T-lymphocyte proliferation, mixed leukocyte response [MLR], cytotoxic T-lymphocyte [CTL] activity, delayed-type hypersensitivity [DTH]), and humoral immunity (B-lymphocyte proliferation, T-dependent antibody responses). A single pa of DBA at doses up to 30 mg/kg had no effect on NK cell activity, anti-CD3 antibody-mediated T-lymphocyte proliferation, the MLR, or B-lymphocyte proliferation. DBA at 30 mg/kg suppressed Concanavalin A (ConA)-stimulated T-lymphocyte proliferation and the CTL response. DBA exposure reduced cytokine production in spleen cell culture supernatants after in vitro stimulation with ConA or lipopolysaccharide (LPS). Immunosuppression was observed at lower doses in the holistic assays. The DTH response to Candida albicans was significantly decreased at 3.0 mg/ kg DBA, while the AFC response was intermittently suppressed at 1.0 mg/kg, with no effect observed at 0.3 mg/kg. These results demonstrate that a single pa of DBA produces systemic immunotoxicity, and of the assays utilized, the holistic assays (i.e., DTH, AFC) appear to be most sensitive to the immunosuppressive effects of DBA.

Modulation of murine innate and acquired immune responses following in vitro exposure to electrospun blends of collagen and polydioxanone.

In light of cell sourcing issues and the lack of a bioreactor comparable to the body, many in the field of tissue engineering have focused their efforts on designing biomaterials capable of in situ regeneration. The theory is that, by using the body as both the bioreactor and the source for cell infiltration, scaffolds composed of bioresorbable materials can be remodeled into native tissue. Thus, research into the effects of such materials on the host immune response is increasingly important. This study applies an immunotoxicological approach to evaluate the effects of electrospun blends of polydioxanone (PDO) and collagen type I on murine innate and acquired immune responses. Results indicated that these materials had few effects on innate immune responses, yet they produced significant immunomodulatory effects in multiple endpoints evaluating both branches of acquired immunity (i.e., cell-mediated and humoral immunity). Specifically, collagen content appeared to be responsible for suppression of cell-mediated immunity, while blends of PDO and collagen appeared to be more suppressive of antibody-forming cell responses than either PDO or collagen alone. These results demonstrate the importance of completing evaluations into the immunotoxicological effects of biomaterials, and they suggest that such testing should become a primary focus when evaluating a material's potential foruse in tissue engineering applications.

In vitro evaluations of innate and acquired immune responses to electrospun polydioxanone-elastin blends.

Immune response testing of biomaterials is an essential component of biocompatibility assessment, particularly when the materials of interest are used to design bioresorbable scaffolds with the potential to promote in situ regeneration. Current trends in immune response testing of biomaterials typically examine few elements of the immune system, and they often undertake a mechanistic approach without first determining if material exposure results in physiologically relevant modulation of both innate and acquired immunity. Here, we present a comprehensive in vitro evaluation of biomaterial-induced modulation of acquired (i.e. cell-mediated and humoral) and innate immune responses following exposure to electrospun blends of polydioxanone (PDO) and elastin (ELAS). Results indicated that in vitro exposure of murine spleen cells to PDO-ELAS blends produced statistically significant immunosuppression in multiple cell-mediated and humoral endpoints. Results suggested that ELAS is the primary cause of cell-mediated immunosuppression. In contrast, PDO and ELAS were equally suppressive of humoral immune responses, while blends of the two were more immunosuppressive than either pure polymer alone. Evaluations of innate immune responses demonstrated few significant effects, with statistically significant immunosuppression observed in natural killer cell activity but not in macrophage functional assays. This work presents an approach for assessing potential modulation of immune responses resulting from exposure to biomaterials, and such evaluations are essential to obtaining comprehensive assessments of biocompatibility.