For MOEs below 10,000, cancer risk needs to be considered. We calculated MOEs for β-myrcene, hydrocoumarin, estragole, and pulegone based on an available BMD that caused a 10% increase in tumor incidence in animal models and NOAELs and a user consumption of 3.4 or 5 mL of fluid/day for a body weight of 60 kg 15,24,28-30 . The MOEs for β-myrcene and hydrocoumarin were >10,000 in all samples , indicating a low cancer risk. In contrast, some products had pulegone and estragole concentrations that were well below 10,000, meaning there is a cancer risk associated with these products . Q Menthol and Two Apple had extremely low MOEs. In the current study, concentrations of the flavor chemicals were averaged and plotted as a function of their frequency . The dominant flavor chemicals separated into three groups. Ethyl maltol and triacetin were most frequently, followed by vanillin, corylone, menthol, ethyl vanillin, benzyl alcohol, and ethyl lactate, while carvone, furaneol, and isobutyl alcohol were infrequently used. The individual dominant flavor chemicals were compared across our current and two previous studies . Twenty-seven dominant flavor chemicals were identified in the present study bringing the total number across our three studies on refill fluids to 37 .12,14 Of these,planting blueberries in pots five flavor chemicals were used in at least one product at > 1 mg/mL in all three studies .
Ten dominant chemicals -3-Hexen-1-ol, corylone, ethyl acetate, ethyl butanoate, ethyl vanillin, furaneol, and isoamyl acetate were in two of the three studies, and 13 were found only in the current study. Other chemicals present in only one study of our prior studies at > 1 mg/mL included acetoin, allyl hexanoate, linalool, strawberry glycidate_A and _B, 14 and benzaldehyde, cinnamaldehyde, ethyl cinnamate, and panisaldehyde.12This study is the first to identify and quantify the flavor chemicals in refill fluids manufactured under one brand and purchased worldwide. In general, the flavor chemicals and their concentrations were similar in duplicate bottles of refill fluids from each country. One bottle of “Apple” from Kansas, USA was an exception in that it had twice the total concentration of flavor chemicals than “Apple” bottles purchased at other locations , These differences may be due to instability or reactivity of the flavor chemicals in these products, mislabeling, human error in compounding, or the use of different batches of ingredients during production at plants in Italy and China. While some of the “Ritchy” refill fluids that we previously purchased in Nigeria were counterfeits, 17 all the products in the current study were manufactured by Ritchy LTD. Generally, the flavor chemicals and their concentrations were similar irrespective of the country of purchase. One of our objectives was to determine which flavor chemicals are used frequently in refill fluids and to establish their concentration ranges by amalgamating data from our prior and current studies. We categorize flavor chemicals as “dominant” when they are 1 mg/ml or higher.
Dominant chemicals are likely added intentionally to create the desired flavor profile. Chemicals at low concentrations may be added intentionally or may be co-constituents of the dominant flavors. For example, pulegone, a potential carcinogen, 31 is often found at low concentrations in menthol-flavored products, but it is not likely added intentionally during manufacture. One hundred thirty-seven flavor chemicals were quantified in our prior12,14 and current studies . These refill fluids represent a convenience sample, 12 the most popular flavors in southern California vape shops, 14 and products manufactured by one company and sold worldwide . Of the 137 flavor chemicals identified in the three studies, 37 were present at concentrations > 1 mg/ml and were distributed among the studies . This number of flavor chemicals reinforces our earlier conclusions that a relatively small number of flavor chemicals are used in the manufacture of a broad range of EC refill fluid products. In contrast to our prior studies, triacetin was the most frequently used flavor chemical in the current LIQUA study, where it exceeded 44 mg/mL in one product. In all studies, esters were the most used chemical class with terpenes, ketones, alcohols, and aldehydes also identified. The five dominant flavor chemicals in our three studies have also appeared in products analyzed in other labs, supporting the conclusion they are commonly used. Most products have at least one flavor chemical that is > 1 mg/ml. Tobacco-flavored products are sometimes an exception, having few flavor chemicals at low concentrations. The LIQUA “Ry4 Tobacco” product was unusual in having four dominant flavor chemicals. Products that are a single flavor, such as menthol, peach, or cinnamon, often use one dominant flavor chemical to create the desired profile . An exception would be LIQUA “Two Apple” which had four dominant flavor chemicals. Products with names that obscure the flavor profile, such as Dewberry Cream14 or Cheesecake , often use multiple dominant flavor chemicals to create a more complex profile.
Interestingly, LIQUA “Peach” and “Q Pina Colada” have very similar flavor chemicals with triacetin being the dominant flavor chemical in both. Presumably, some of the flavor chemicals with lower concentrations contribute to the taste and enable the users to distinguish between the two flavors. In general, the total concentration and the total number of flavor chemicals in LIQUA “Q” and “HP” products were lower than in the regular LIQUA products. The concentrations of flavor chemicals in some LIQUA products were higher than those typically used or permitted in other consumer goods, such as fragrances and food. Triacetin, ethyl maltol, and corylone were used at concentrations averaging 6 mg/mL, 4 mg/mL, and 2 mg/mL, respectively . While triacetin should not exceed 2% in cosmetics for external use, 38 its concentration in LIQUA “Mint” was 4.4% . Ethyl maltol concentrations in edible products and cosmetics should not exceed 0.015%. However, LIQUA concentrations were 0.015% or higher in 60% of the products containing ethyl maltol, with one product containing 2.6%. These concentrations exceed the MTT NOAEL for ethyl maltol. Ethyl maltol has been linked to free radical formation, 41 which could increase the cytotoxicity of these products. Likewise, the maximum average concentration of corylone in chewing gum for example, is 0.015 mg/mL, 22 while in some LIQUA refill fluids, concentrations ranged between 0.03 to 10.2 mg/mL. Flavor chemicals that were not dominant may also have significant health effects, including the potential to cause cancer with chronic use. Hydrocoumarin , a derivative of coumarin which is prohibited in human food increased kidney and liver neoplasms in male rats and female mice, respectively.β-myrcene is a naturally occurring acyclic monoterpene which increased kidney and liver neoplasms in male rats and mice, resulting in its prohibition in food. Because the MOEs for hydrocoumarin and β-myrcene in LIQUA products were >10,000, they do not appear to present a cancer risk to EC users. In contrast, the MOEs for both pulegone and estragole were far below 10,000 in some LIQUA products, consistent with cancer risk. The “Q” version of refill fluids, which are Ritchy’s higher quality products, had the lowest MOEs, indicating that more expensive products are not necessarily safer. Pulegone levels in other EC products have likewise produced MOEs below the safe threshold. Pulegone, a naturally occurring oxygenated monoterpene, is a major constituent of pennyroyal plant oil extracts and several other mint plants and has been classified as a type 2B carcinogen by the International Agency for Research on Cancer. Estragole a naturally occurring chemical found in spices, plants, and essential oils,blackberries in containers is a rodent hepatocarcinogen at high doses. While the Joint FAO/WHO Expert Committee on Food Additives concluded further research is needed to assess the risk of estragole to humans, the European Medicines Agency recommended keeping exposures to the lowest levels possible.Other flavor chemicals that are not carcinogens may cause health effects, even at low concentrations. Diacetyl and cinnamaldehyde were less frequently found in LIQUA products than in our other studies and ranged in concentration between 0.005 – 0.057 mg/mL and 0.003 – 0.112 mg/mL, respectively. While probably not added intentionally, diacetyl causes bronchiolitis obliterans in humans, and cinnamaldehyde is highly cytotoxic in vitro, having IC50s within the LIQUA range when tested in the MTT assay with human embryonic stem cells and human pulmonary fibroblasts .Cinnamaldehyde also inhibits ciliary beating in bronchial epithelial cells and impairs innate immune function.Triacetin, the most frequently used flavor chemical in the LIQUA products, ranged in concentration from 0.005 to 44.333 mg/mL, a concentration significantly higher than triacetin in our other EC studies.
Triacetin is a clear, colorless, oily GRAS human food and cosmetic additive that produces eye and skin irritation in humans but is non-toxic in animals when administered orally or dermally. While triacetin has relatively low cytotoxicity in vitro, 14 upon heating, it produces acetic acid, which catalyzes the formation of acrolein, formaldehyde hemiacetals, and acetaldehyde from propylene glycol and glycerol. We are currently determining if triacetin increases the concentrations of reaction products in LIQUA aerosols. While our cytotoxicity data is based on refill fluids, other factors may affect results when heated aerosol are used. For example, additional chemicals that can be toxic, such as 2, 3 butanedione, acetaldehyde, formaldehyde, and acrolein, may form upon heating and could alter cellular responses. In addition, 100% of the flavor chemicals may not transfer to aerosol so that users are exposed to lower concentration than those in the fluids. These factors notwithstanding, in one study that compared refill fluids and aerosols, the cytotoxicity of the fluids accurately predicted that of the aerosols in 74% of the samples when one EC devise was tested. The cytotoxicity of refill fluids generally correlates with the concentration of cytotoxic flavor chemicals, and this was observed in the current study for “Two Apples” and “Ry4 Tobacco” in the both the MTT and cell growth assays. These products contained high concentrations of ethyl maltol, benzyl alcohol, ethyl vanillin, and corylone, which were themselves directly correlated with cytotoxicity in the MTT assay. In contrast, “Peach,” with high levels of triacetin , was not cytotoxic in the MTT or proliferation assays, even though a concentration of triacetin lower than 20 mg/ml was cytotoxic when tested individually as an authentic standard. This observation may be explained by the fact that three of the “Peach” chemicals that were cytotoxic individually produced no effect when tested in a mixture . A similar neutralizing effect was observed when carvone and triacetin were combined . Both mixtures in Figure 6 contained high concentrations of triacetin, which may decrease cytotoxicity in mixtures or the presence of solvents. Previously, a similar unexpected decrease in cytotoxicity was observed when benzyl alcohol, which was cytotoxic by itself, was used in a refill fluid. This type of antagonism usually occurs when the chemicals in a mixture interact with each other to inhibit uptake or interaction with a target. Antagonism appears to be rare in EC refill fluid mixtures; however, it should be studied further as “Peach” aerosols may be cytotoxic due to reaction products formed during heating. The MTT assay measures mitochondrial reductase activity and is widely used to evaluate mitochondrial function and cell health.The inhibition of cell growth by “Two Apples” and “RY4 Tobacco” may have occurred due to the reduction in ATP levels by poorly functioning mitochondria. Although not measured in this study, disruption of mitochondrial function can lead to increases in reactive oxygen species, inflammation, altered expression of genes in the electron transport chain, abnormal Ca2+ elevation, and glutathione depletion. These changes underlie diseases of the respiratory system including chronic obstructive pulmonary disease, asthma, and lung cancer.The use of flavor chemicals and their concentrations may differ for refill fluids made by other companies. In addition, it is possible that LIQUA products had additional flavor chemicals that were not on our target list. In summary, flavor chemicals in LIQUA products were generally similar in all countries of purchase. The flavor chemicals on our target list varied in total flavor chemical concentration and the number of flavor chemicals per product in 103 of the refill fluids we analyzed. No target and non-target flavor compound was detected in two tobacco flavored refill fluids . Twenty seven flavor chemicals were dominant , and triacetin was the most frequently used, often at high concentrations. Thirty-seven chemicals not identified in our prior work were present in LIQUA products.