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Public Health Consequences of E-Cigarettes (2018)

Chapter: 5 Toxicology of E-Cigarette Constituents

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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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Suggested Citation:"5 Toxicology of E-Cigarette Constituents." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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5 Toxicology of E-Cigarette Constituents In general, electronic cigarettes often contain ingredients such as propylene glycol (PG) and glycerol, mixed with concentrated flavors and, optionally, a variable percentage of nicotine. Quantitative and qualitative studies have identified a wide variety of chemical components in the cartridges, refill solutions and aerosols of e-cigarettes. Herrington and colleagues (2015) have detected approximately 60 to 70 compounds (unidentified and identified) being observed in each liquid tested, only varying by several constituents throughout the liquid. Kucharska and colleagues (2016) have identified 113 chemicals in 50 brands of liquids. Even more compounds are observed in the aerosol over their respective solution because some chemicals are generated during the vaporization process. An aerosol generated from a single product tested by Herrington and Myers (2015) showed 18 additional compounds observed over the solution. Substances identified in e-cigarette liquids and aerosols include nicotine, solvent carriers (propylene glycol and glycerol), tobacco-specific nitrosamines (TSNAs), aldehydes, metals, volatile organic compounds (VOCs), phenolic compounds, polycyclic aromatic hydrocarbons (PAHs), flavorings, tobacco alkaloids, and drugs. Most reviewed studies have evaluated nicotine and impurities in the liquids such as TSNAs and nicotine-related impurities, while other studies focus on identifying potentially harmful chemicals in the aerosol, such as carbonyl compounds, VOCs, TSNAs, metals, and silicates. Various chemical substances and ultrafine particles known to be toxic, carcinogenic, and/or to cause respiratory and cardiac disease have been identified in e-cigarette aerosols, cartridges, refill liquids, and environmental emissions. Some of the identified TSNAs, aldehydes, metals, VOCs, phenolic compounds, PAHs, and tobacco alkaloids are harmful or potentially harmful constituents, and their general health risks are described below. HUMECTANTS (DELIVERY SOLVENTS) E-cigarettes use humectants as solvent carriers in e-liquids to produce aerosols that simulate combustible tobacco cigarette smoke. In addition to these humectants, water is a common ingredient of liquids. PG and glycerol (commonly referred to as a “vegetable glycerin” or VG in liquid formulations) are the most common vaporizing solvents used in e-cigarettes. Hutzler and colleagues (2014) analyzed 28 liquids of 7 manufacturers purchased in Germany and detected both propylene glycol and glycerol in all samples. Both PG and glycerol are also 5-1 PREPUBLICATION COPY: UNCORRECTED PROOFS

5-2 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES commonly used as humectant ingredients in manufactured cigarettes to control and maintain the moisture content of the cut tobacco filler (Uryupin et al., 2013). Users of e-cigarettes often report that PG produces better “throat hit” and carries flavor better than VG while VG is much smoother than PG. PG is physically much thinner than VG (Cheng, 2014; Etter, 2016; Li et al., 2016). Outside of usage in e-cigarette liquids, dermal exposure to PG and glycerol is more common than exposure via inhalation, as most consumer products containing PG and glycerol are liquids or creams. Thus, there are few animal or human studies providing evidence of the possible toxicity of inhaled PG or glycerol. Studies identifying PG and glycerol in e-liquids are described below, and toxicological evidence is described in the following sections. Hahn and colleagues (2014) used NMR methodology for analysis of 54 commercially available liquids for use in e-cigarettes. The study looked at several types of humectants, including dihydroxy (diols, glycols) and polihydroxy alcohols. PG and glycerol were detected in all samples at concentrations ranging from 0.4 to 98 g/100 g (average 57 g/100 g) and from 0.3 to 95 g/100 g (average 37 g/100 g), respectively. Generally, lower levels of another solvent, ethylene glycol (average 10 g/100 g), were detected. 1,3-Propanediol was detected only in seven samples in the concentration range of 3.3-10 g/100 g. 1,3-Butanediol and diethylene glycol were negative in all samples. The presence of the major compounds glycerol and propylene glycol corresponded to the labeling in the majority of cases, except three products contained no labeling information at all. Glycerol was not labeled on five products despite being present. Propylene glycol was not labeled in two products despite being present. In one case, “vegetal glycol” was labeled without specifying the exact chemical compound. Hutzler and colleagues (2014) analyzed 28 liquids of 7 manufacturers purchased in Germany and, like Hahn and colleagues, detected both propylene glycol and glycerol in all samples. Geiss and colleagues (2016) extrapolated lung concentration of propylene glycol and glycerol emitted from e-cigarettes using a smoking machine by measuring the average amounts condensed on the filter pad. The estimated lung concentrations were 160 and 220 mg/m3 for propylene glycol and glycerol, respectively. The most common symptom reported by e-cigarette users is a dry mouth and throat, which is considered to originate from the water-absorbing property of propylene glycol and glycerol. However, the health consequences of long-term exposure to propylene glycol and glycerol from e-cigarettes have not been investigated. Both compounds might pyrolyze, leading to the formation of carbonyl compounds (aldehydes), which contributes to potential health risk in e-cigarette users (for discussion about carbonyl compounds, see the subsequent section in this chapter). Propylene Glycol Propylene glycol (also known as 1,2-dihydroxypropane, 1,2-propanediol, methyl glycol, and trimethyl glycol) is a clear, colorless, slightly syrupy liquid at room temperature. It is practically odorless and tasteless. It is used by the chemical, food, and pharmaceutical industries as a humectant to absorb extra water and maintain moisture in certain medicines, cosmetics, or food products. It is also used as a solvent for food colors and flavors, and in the paint and plastics industries. PG has been widely used for decades as a solvent for many intravenous drugs, and in some oral preparations such as cough syrups. PG was listed as “Generally Recognized as Safe” (GRAS) by the Food and Drug Administration (FDA) in 1973 (HHS, 2015). Substances listed as GRAS are deemed as generally safe under conditions of intended use as a food additive. Thus, GRAS substances are safe for ingestion, but not necessarily for other routes of administration PREPUBLICATION COPY: UNCORRECTED PROOFS

TOXICOLOGY OF E-CIGARETTE CONSTITUENTS 5-3 like inhalation. PG may exist in air in the aerosol form, but must be heated or briskly shaken to produce a mist. PG is also used to create artificial smoke or fog used in firefighting training and in theatrical productions. Human Studies and Case Reports on PG Toxicity Some people have reported having an allergic reaction to PG. Some people have reported upper respiratory irritation after inhaling aerosolized PG for one minute (Wieslander et al., 2001), but the longer term health effects in humans are not well defined. Though some preclinical studies showed inhalation of PG and VG can be safe up to 28 days (Werley et al., 2011) or 18 months (Robertson et al., 1947), breathing aerosolized PG can also affect the risk of asthma development (Choi et al., 2010). For example, one woman exhibited signs of exogenous lipoid pneumonia (e.g., fever, productive cough, and labored breathing) after using e-cigarettes for half a year (McCauley et al., 2012). The e-cigarette’s oil-based humectants likely caused her pneumonia, as her symptoms improved when she quit the device (McCauley et al., 2012). PG is frequently used as a vehicle for intravenous delivery of antiseizure medications in pediatric populations, typically at concentrations of 40 to 80 percent v:v with saline (Lim et al., 2014). Thus, there have been numerous human studies on the toxicity of relatively large doses of both oral and intravenously administered PG. Lim and colleagues (2014) conducted a systematic literature review of case reports and other clinical studies on the toxicity of PG in pediatric populations. They identified numerous case reports and several small studies that identified a “toxidrome” for PG toxicity that can result following repeated, relatively high-dose intravenous administration of PG. The adverse effects include: hyperosmolarity, lactic acidosis, hemolysis, central nervous system (CNS) toxicity, and cardiac arrhythmia. In one particularly striking case study, an 11-year-old was given 2-4 ml per day of PG containing vitamin D, daily for 13 months. Estimated daily dose for this subject was 114 mg (2 ml dose) to 228 mg (4 ml dose) of PG per kg bw (Arulanantham and Genel, 1978; LaKind et al., 1999). After 13 months of repeated exposures, the child began to have seizures and lapsed into unconsciousness. Once the PG/Vitamin D preparation was stopped, the child recovered (LaKind et al., 1999). In another example, a 15-month-old infant receiving large doses of a vitamin C suspension in PG orally had episodes of unresponsiveness, diaphoresis, tachycardia, tachypnea, and hypoglycemia (Martin and Finberg, 1970). Based on analyses of case reports, Lim and colleagues (2014) attempted to arrive at a “safe” dose of PG for repeated administration of antiseizure drugs that are routinely compounded in 40 percent PG (see Table 5-1). They suggested maximum cumulative dose of 69 g/day in a pediatric population. Although such clinical studies on relatively high doses of orally and intravenously administered PG in pediatric populations is clinically relevant for those populations, it is perhaps of modest relevance to potential health consequence of inhalation of PG vapors from repeated vaping. However, diagnostic procedures, such as characterization of anion gap (or osmolal gap, defined as the discrepancy between the measured and calculated osmolalities) (Lim et al., 2014), and evaluation for the presence of lactic acidosis, could be of potential value in suspected cases of high dose PG toxicity from extensive vaping. PREPUBLICATION COPY: UNCORRECTED PROOFS

5-4 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES TABLE 5-1 Dose Limits of Commonly Used Drugs to Avoid Propylene Glycol Intoxication Based on a Maximum Amount of PG Equal to 69 g/day Maximum Daily Dose Drug Amount of PG (mg/ml) Adult Pediatrics Lorazepam 2 mg/ml 828 166 mg/day (7 mg/hr) 2.4 mg/kg/day (.01 mg/kg/hr) Phenobarbital 130 mg/ml 702 12.8 g/day (533 mg/hr) 183 mg/kg/day (7.6 mg/kg/hr) Pentobarbital 50 mg/ml 414.4 8.3 g/day (346 mg/hr) 119 mg/kg/day (4.9/kg/hr) Diazepam 5 mg/ml 414.4 832 mg/day (34.7 mg/hr) 12 mg/kg/day (0.5 mg/kg/hr) Phenytoin 50 mg/ml 414.4 8.3 g/day (346 mg/hr) 119 mg/kg/day (4.9 mg/kg/hr) TMP/SMX 16:80 mg/ml 414.4 2.7:13.3 g/day 39 mg/kg/day TMP component (1.6 mg/kg/hr) Etomidate 2 mg/ml 362.6 381 mg/day (16 mg/hr) 5.4 mg/kg/day (0.2 mg/kg/hr) NOTE: PG = propylene glycol; SMX = sulfamethoxazole; TMP = trimethoprim. SOURCE: Adapted from Lim et al., 2014. Pharmacokinetics of PG PG is well absorbed orally, and can also be absorbed through skin or mucus membranes from topical preparations. Following absorption, the kidneys eliminate 45 percent of the PG, and the liver metabolizes the remainder to lactic acid, pyruvic acid, or acetone. Thus, patients with impaired liver and/or kidney function are generally thought to be at increased risk for developing PG toxicity following high dose oral or intravenous administration. Speth and colleagues (1987) conducted a relatively detailed pharmacokinetic analysis of PG following intravenous administration of PG at different dose rates, administered over 4 hours. The elimination half-life of PG was dose dependent; at doses of either 3 or 4.5 g/m2 (over 4 hours) the terminal half-life was approximately 1.8 hour. However, at a dose rate of 7.5 g/m2 over 4 hours the half-life increased to approximately 3.1 hour, suggesting saturable elimination at dose rates above about 5 g/m2 (see Table 5-2). Yu and colleagues (1985) also reported elimination half-lives of PG following multiple large oral doses (20.7 g three times per day, or 41.4 g two times per day) of PG, with terminal half-lives of 3.8 ± 0.8 hours, with relatively large interpatient variability in plasma concentration. Blood concentrations of PG associated with hyperosmolality and anion gap have been reported, ranging from 177 to 1,520 g/ml (Fligner et al., 1985; Kelner and Bailey, 1985). However, Yu and colleagues (1985) did not observe any evidence of toxicity (hyperosmolality or lactic acidosis) in subjects with plasma concentrations as high as 425 g/ml. No studies have evaluated blood concentrations of PG in subjects using e-cigarettes or other vaping devices with PG as the humectant. Evidence of Health Effects from Occupational Exposures to PG There is relatively limited evidence of toxicity from occupational exposures to PG. However, glycols are used in theatrical fogs, so actors and performers in the entertainment industry may have routine exposures to relatively high concentrations of PG, as it is often a major component of these fogs. Varughese and colleagues (2005) studied 101 employees in 19 different locations PREPUBLICATION COPY: UNCORRECTED PROOFS

TOXICOLOGY OF E-CIGARETTE CONSTITUENTS 5-5 who were routinely exposed to such fogs. They measured the levels of exposure, lung function, and acute and chronic symptoms. The mean concentration of exposure for employees exposed only to PG-based fog on the testing day was 0.49 mg/m3 (maximum 3.22 mg/m3). They reported that theatrical fog exposures were significantly associated with chronic work-related wheezing and chest tightness. Although these acute effects appeared to be specific to PG-based fogs, most of the workers were also exposed to mineral oil. Thus, the authors were unable to distinguish the role of glycol or mineral oil fogs in the development of chronic effects and work-related symptoms from increasing chronic exposure. Another study addressed the same general issue regarding the safety of PG used in theatrical fog (Moline et al., 2000). Based on their analysis of symptoms reported by 218 theatrical actors, detailed integrated PG dose and peak exposure estimates were available. They found statistically significant associations between peak PG exposure and reported symptoms of mucus membrane irritation. They also found other respiratory symptoms, including throat and nasal symptoms associated with peak exposure but not integrated dose. The measured peak concentrations during “fogging” at on-stage locations ranged from less than 1 to 16 mg/m3. Estimates of actors’ “per performance” exposures to PG ranged from 0.1 to ~8 g/show (Moline et al., 2000). Wieslander and colleagues (2001) conducted a study to examine the effects of PG mist in aviation emergency training. Twenty-seven non-asthmatic volunteers were exposed in an aircraft simulator to a mist of PG at 309 mg/m3 (176-851 mg/m3 range) for 1 minute. Subjects were then evaluated for a range of pulmonary function tests and symptoms assessment. Although measures of pulmonary function (FEV1, vital capacity) were not significantly affected, symptoms reported included eye and throat irritation in some of the subjects. Four subjects also reported development of an irritating cough. The reported symptom of eye irritation was supported by measurement of tear film stability, which was decreased following PG exposure. The U.S. Occupational Safety and Health Administration established an interim 8-hour threshold limit value (TLV) of 10 mg/m3 for all organic mists (applicable to propylene glycol and glycerin) with no specific exposure limits or identified toxicity (OSHA, 2006). The Health Council of the Netherlands recommended exposure limit for PG of 50 mg/m3 over 8 hours. Although they noted a concern about short-term respiratory effects, the proposed limit was not based on observed adverse effects from workplace exposures (Health Council of the Netherlands, 2007). Thus, although occupational exposure limits have been proposed for PG, it is important to note that neither of these proposed exposure limits are based on evidence of adverse effects, but rather are “precautionary” in nature. Nevertheless, studies in some workplace populations relate symptoms of eye and throat irritation to acute, and possibly chronic, exposures to PG mist in the low mg/m3 concentrations. Relevance of Occupational Exposures and Clinical Case Reports of Pharmaceutical Exposures of PG to Exposures from E-Cigarettes Although the clinical case reports of PG exposures demonstrate that high-dose oral and intravenous exposure to PG can induce toxicity, the relevance of those studies to potential health effects of PG from e-cigarettes/vaping depends on the dose and pharmacokinetics of PG following inhalation exposure through e-cigarettes. Burstyn (2014) estimated the potential levels of exposure to PG from e-cigarettes, “assuming extreme consumption of the liquid per day via vaping (5 to 25 ml/day and 50-95 percent propylene glycol in the liquid)” and concluded that PREPUBLICATION COPY: UNCORRECTED PROOFS

5-6 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES “levels of propylene glycol in inhaled air can reach 1-6 mg/m3”. With an assumption of complete absorption via inhalation, Burstyn concluded that “estimated levels of exposure to propylene glycol and glycerin are close enough to TLV to warrant concern.” However, putting these values in perspective with the clinical data from intravenous administration of PG in adults may be useful. Speth and colleagues (1987) reported that doses from 5-21 grams/day (see Table 5-2), which are comparable to the 5 to 25 ml/day calculated by Burstyn (2014), were not associated with any evidence of any adverse effects. In the Speth and colleagues (1987) study, peak plasma concentrations of PG ranged from 48 g/ml (5.1 g/day; ~88 mg/kg/d) to 425 g/ml (21 g/day; ~488 mg/kg/d). In one clinical report in a 60-year-old male showing toxicity and for whom blood concentrations were measured, serum levels of PG greater than 180 g/ml were reported to be associated with toxicity (Arbour and Esparis, 2000). Other investigators found clinical evidence of toxicity at serum PG concentrations that exceeded 250 g/ml (Hansen et al., 2015), although it is important to note that these are following intravenous administration. Nevertheless, absorption of PG via inhalation theoretically could be very rapid and largely complete, so the comparison of blood levels between patients administered PG intravenously over 4 hours to individuals with extensive vaping may not be unreasonable. In 1974, the World Health Organization recommended a maximum dose of 25 mg/kg/day of PG when ingested as a food additive. Thus, for a typical young adult with a body weight of 60 kg, this would be equivalent to 1.5 g/day, which is considerably less than the 5-25 ml/day “worst case” exposure to PG from vaping estimated by Burstyn (2014). There are no studies of clinical measures of potential PG toxicity (e.g., anion gap, lactic acidosis) among heavy users of e-cigarettes, or which have measured blood/serum levels of PG following use of vaping devices containing PG-based liquids. TABLE 5-2 Plasma Pharmacokinetics of Propylene Glycol Given as a 4-H Intravenous Infusion Maximum Plasma t1/2 Patient Dose MTQ Dose PG Dose PG Concentration (h) Cl (ml/min/1.73 AUC ( g * Initials (mg/m2) (g/m2) (g/day) ( g/ml) Vd (L) m2) h/ml/1.73 m2) Kr 120 3 5.1 60 1.8 36 305 261 Kr 120 3 5.1 58 1.4 58 390 318 Li 120 3 5.1 48 1.5 41 321 279 Kr 180 4.5 7.7 102 2.1 40 196 762 Li 180 4.5 7.7 131 1.8 52 339 390 Ri 180 4.5 7.2 116 1.9 41 269 518 Jw 300 7.5 12.6 218 3.1 58 206 1,080 Be 300 7.5 13.5 168 3.3 62 221 938 Rb 600 15 21.0 425 3.3 40 144 3,719 Mean 2.3 ± 0.7 NOTE: AUC = area under the plasma concentration–time curve, relative to 1.73 m2 body surface; CI = clearance, relative to 1.73 m2 body surface; MTQ = mitoquidone; PG = propylene glycol; t1/2 = half-life; Vd = apparent volume of distribution. SOURCE: Adapted from Speth et al., 1987. PREPUBLICATION COPY: UNCORRECTED PROOFS

TOXICOLOGY OF E-CIGARETTE CONSTITUENTS 5-7 Allergic Reactions to PG It has been known for years that some individuals can develop allergic reactions to PG following repeated dermal applications (Aberer et al., 1993; Catanzaro and Smith, 1991; Funk and Maibach, 1994; Lessmann et al., 2005; Warshaw et al., 2009). Although most dermal reactions to PG are the result of irritation, true immunologic reactions have been confirmed through patch testing. For example, in a patch test of 1,226 patients who received an application of 5 percent PG in Vaseline, or 10, 30, or 50 percent in water, 208 (17 percent) of the subjects had evidence of irritant and/or allergic dermatitis. Of those showing some dermal reaction, 195 were from irritation, but 13 exhibited an allergic reaction (Aberer et al., 1993). However, a more recent analysis of allergic dermatitis found an incidence of only 2.1 percent in a large sample size (5,083 subject in 2007-2008), and this was significantly decreased from previous years (3.8 percent of 4,095 subjects in 1996-1998) (Fransway et al., 2013). Whether PG could induce allergic reactions via inhalation from e-cigarettes has not been studied. In Vivo Animal Toxicology Studies of PG Because of its widespread use as a food additive and other industrial uses, PG was subjected to standard in vivo toxicological assays many years ago, and these studies, coupled with the relative lack of human evidence of toxicity of PG from its use as a food additive, form the basis for FDA’s listing of PG as GRAS. A study of male and female Sprague-Dawley rats found that larynx, trachea, and lung tissues were not affected by nose-only exposure to different levels of PG for 90 days (Suber et al., 1989). Additional studies of aerosolized PG found no effects on rat or monkey gross pathology, respiratory tract function, histology, or hematology and clinical chemistry (Robertson et al., 1947). LaKind and colleagues (1999) provide a comprehensive review of the animal toxicology data for PG prior to that date. Acute Toxicity PG is considered “practically non-toxic” orally, with acute lethal dose (LD50) values of 20 gm/kg or greater (see Table 5-3). Signs and symptoms of acute toxicity included increased respiratory rate, loss of equilibrium, CNS depression, analgesia coma, and death in 18 to 36 hours. Of more relevance are animal studies using inhalation exposures to PG. Konradova and colleagues (1978) evaluated the effects on airway epithelia of exposure of rabbits to a 10 percent aerosol of PG for 20 and 120 minutes. The 20-minute exposure had no visible effect on ciliated cells in the tracheal epithelium, but did produce alterations in goblet cells. Longer exposure, for 120 minutes, altered goblet cells and induced some visible alterations in ciliated epithelial cells. Another study examined the results of 15-minute inhalation exposure of dogs to either 10 or 20 percent aerosol of PG on hemodynamic effects and hemolysis (effects seen following large oral doses of PG). No effects on either endpoint were reported (MacCannell, 1969; Renne et al., 1992). Repeated Dose Exposures to PG to Evaluate Potential Reproductive Effects in Animals Three standard reproductive assays of PG have been performed, all in male and female mice, using repeated doses and multi-generation assessment for reproductive outcomes (Kavlock et al., 1987; Morrissey et al., 1989; OECD, 2001). None of these studies reported any statistically significant effects of PG exposure on measures of reproductive outcome in different strains of mice given 10,100 mg/kg/day for 14 weeks. PREPUBLICATION COPY: UNCORRECTED PROOFS

5-8 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES TABLE 5-3 Acute Lethal Dose (LD50) of Propylene Glycol in Rats, Mice, Guinea Pigs, and Rabbits Species Propylene Glycol LD50 (g/kg) Reference Rats 21.7 (Laug et al., 1939; Smyth et al., 26.4 1941; Weatherby and Haag, 1938) 33.5 Mice 24.8 (Laug et al., 1939; Bornmann, 31.9 1954) Guinea Pigs 18.35 (Smyth et al., 1941; Laug et al., 19.6 1939) Rabbits 19.3 (Weatherby and Haag, 1938) (based on data by Braun and Cartland, 1936) SOURCE: Adapted from LaKind et al., 1999. Repeated Dose Exposures to PG to Assess Developmental/Teratogenic Effects in Animals Several animal studies using standard teratogenicity protocols have been completed for PG. An FDA-sponsored study in pregnant CD-1 mice, Wistar rats, golden hamsters, and Dutch- belted rabbits found no evidence of teratogenicity at the highest doses tested (1,600, 1,600, 1,550, and 1,250 mg/kg/day for 10 days, respectively) (FDRL, 1973). Long-Term (Chronic) Bioassays on PG for Assessment of Organ System Function Because of the well-documented nephrotoxic effects of ethylene glycol, early studies on the toxicity of propylene glycol focused on potential effects of chronic PG exposure on kidney functions. Van Winkle and Newman (1941) administered PG in drinking water to female (5 percent PG, twice daily) and male dogs (600 ml of 10 percent PG, once daily) for up to 9 months. Animals were evaluated for liver and kidney function and by histopathology at the end of the experiment. No effects on liver or kidney were observed in any of the animals. A 2-year chronic bioassay in albino rats given PG in the diet at doses approximately equivalent to 1,225 or 2,450 mg/kg/day found no evidence of any organ system toxicity (LaKind et al., 1999; Morris et al., 1942). A 2-year feeding study in dogs given up to 2,000 mg/kg/day also found no significant effects on renal weight. However, a dose of 5,000 mg/kg/day was associated with increased urinary output and decreased water consumption, suggestive of adverse effects on kidney function (LaKind et al., 1999). Other chronic studies of PG in mice and dogs reported in Patty’s Industrial Hygiene and Toxicology (Clayton and Clayton, 1995) found no significant effects of PGs on any organ system. Inhalation Exposure Levels of PG from E-Cigarette Use Of importance to the question of the potential health effects of PG in the context of e- cigarette use are the actual concentrations and doses inhaled during a puff. Kienhuis and colleagues (2015) evaluated exposures to both PG and VG from a “shisha-pen” device. The authors define a shisha-pen as “an electronic cigarette (e-cigarette) variant that is advertised to mimic the taste of a water pipe, or shisha…. The shisha-pen operates in the same manner as an e- cigarette, it can be disposable or rechargeable and refillable, and it is available with and without nicotine” (Kienhuis et al., 2015, p. 1). They estimated that the PG exposure from one 50 to 70 mL puff would be from 430 to 603 mg/m3, and noted that “These exposure concentrations were PREPUBLICATION COPY: UNCORRECTED PROOFS

TOXICOLOGY OF E-CIGARETTE CONSTITUENTS 5-9 higher than the points of departure for airway irritation based on a human study (propylene glycol, mean concentration of 309 mg/m3) and a rat study (glycerol, no-observed adverse effect level of 165 mg/m3)” (Kienhuis et al., 2015, p. 1). As discussed above, Wieslander and colleagues (2001) exposed healthy human subjects in an aircraft simulator to a mist of PG at 309 mg/m3 (176-851 mg/m3 range) for 1 minute. This is similar to the range of PG concentrations in puffs of e-liquid from a shisha-pen device. No effects on lung function were noted by Wieslander and colleagues, although some subjects did complain of eye and throat irritation. This is consistent with web-based literature from vaping groups.1 Summary of Toxicological Effects of PG PG has long been considered “practically non-toxic,” consistent with the FDA’s inclusion of PG on the GRAS list. Animal studies, including chronic studies at very high levels, have consistently failed to identify any target organ, or other evidence of toxicity at doses less than several grams per kg/day. Although most of these studies were done decades ago and would not generally meet today’s “Good Laboratory Practices” standards, the large doses used, coupled with the consistent lack of any evidence of organ system effect or reproductive or developmental toxicity, provides strong support for the general lack of toxic effects of PG in humans from dietary or occupational exposures. However, there is limited, but consistent evidence from case reports that very high doses of PG administered orally or intravenously to humans can produce toxic effects that appear to be related to osmolar changes in the blood and lactic acid formation secondary to the metabolism of PG. Finding: Substantial toxicological data indicate that oral exposure to propylene glycol is not likely to be associated with adverse health effects. However, the data from inhalation exposure to propylene glycol are limited. In some individuals, exposure to propylene glycol aerosols in concentrations found in e-cigarettes has been shown to cause irritation to the eyes and throat. Glycerol Glycerol (also known as glycerin) is an oily, hygroscopic liquid with a warm, sweet taste. Although glycerol can be derived from naturally occurring fats and oils (“vegetable glycerin,” VG), synthetic glycerol is produced from petrochemical products in a multistep process. Glycerol is used in food products, nutritional supplements, pharmaceutical products, personal care products, and oral care products. As discussed above, most liquids used in e-cigarettes and other vaping devices contain a mixture of PG and glycerol. Typically, the mixtures are somewhere in the range of 30-50 percent glycerol, with the balance as PG. Among the vaping community, there is a perception, which is supported by acute toxicology studies, that PG is more irritating to upper respiratory airways than glycerol.2 FDA considers glycerol GRAS (HHS, 2017). 1 See, for example, http://www.whitecloudelectroniccigarettes.com/blog/vaping-throat-irritation (accessed January 5, 2018); http://ecigarettereviewed.com/allergies-conditions-and-e-liquid (accessed January 5, 2018); and http://www.ecigarette-politics.com/pg-sensitivity.html (accessed January 5, 2018). 2 See, for example, https://www.misthub.com/blogs/vape-tutorials/76788613-tutorial-propylene-glycol-pg-vs- vegetable-glycerin-vg-e-juice (accessed January 5, 2018); https://vapingdaily.com/best-vape-juices-and-e- liquids/pg-vs-vg (accessed January 5, 2018). PREPUBLICATION COPY: UNCORRECTED PROOFS

5-10 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES Human Toxicology Studies The toxicology of glycerol was reviewed by the Organisation for Economic Cooperation and Development in 2002 (OECD, 2002). A study of 10 male and 4 female volunteers who were administered glycerin in orange juice with each meal at a dose of 1.3 to 2.2 g/kg/d for 50 days reported no evidence of toxicity or adverse effects on blood or urine production. Based on the highest administered dose, they estimated a no-observed-adverse-effect-level (NOAEL) for glycerol greater than or equal to 2.2 g/kg/d (CIR, 2014; OECD, 2002; Tourtellotte et al., 1972). When used as a drug, reported adverse effects following the oral administration of glycerin at unspecified doses included mild headache, dizziness, nausea, vomiting, thirst, and diarrhea. Headache is likely a result from dehydration (CIR, 2014). Venable and colleagues (CIR, 2015; Venable et al., 1980) evaluated 64 male employees involved in the manufacture of synthetic glycerin for potential effects on reproductive function. They found no differences in sperm counts and percentage of normal forms compared with a similar sized control group (n = 63) that had no known occupational exposures to glycerin. Absorption, distribution, metabolism, and excretion Glycerol is a natural product and endogenous component in the body, largely as triglycerides with fatty acids, but free glycerin is also naturally present in human plasma. Typical serum levels of glycerol in adult humans range from 0.05 to 0.1 mmol/L (Nelson et al., 2011). Exogenous glycerin is rapidly absorbed from the stomach and intestine, with distribution occurring throughout the extracellular space (CIR, 2015). The primary pathway of biotransformation is via glycerol kinase-mediated phosphorylation to -glycerophosphate in the liver (80 to 90 percent) and kidneys (10 to 20 percent). -Glycerophosphate is then transformed to form glucose (gluconeogenesis) and glycogen through intermediary metabolism pathways (Lin, 1977). Most of the dose of orally administered glycerol is metabolized in about 2.5 hours, with 7 to 14 percent of eliminated glycerol unchanged in urine. In the liver, exogenously administered glycerol can undergo lipogenesis (combining with free fatty acids to form triglycerides), and these fats can be distributed to adipose tissues. The turnover rate for glycerol is proportional to plasma concentration of glycerol (Bortz et al., 1972). Glycerol has been used clinically because of its ability to increase the osmotic pressure in plasma. Orally administered glycerol can reduce the volume of intraocular fluids in order to decrease intraocular pressure (IOP). The extent of IOP reduction depends on both the etiology and magnitude of the increased pressure and the glycerol dose. Glycerol’s osmotic effect has also been used to decrease in cerebrospinal fluid pressure (Tourtellotte et al., 1972). In Vivo Animal Toxicological Studies Acute toxicity As summarized in the 2015 Cosmetic Ingredients Review for Glycerol (CIR, 2015), oral LD50 values of glycerin ranged from 2.53 to 58.4 g/kg in rats. The highest dose used in one study was 24 g/kg, and no deaths were reported. Oral LD50 values reported for glycerin were 4.1 to greater than 38 g/kg in mice, 27 g/kg in rabbits, and 77.5 g/kg in guinea pigs (CIR, 2015). The dermal LD50 value of glycerin in rats was reported to be greater than 21.9 g/kg, and in rabbits, greater than18.7 g/kg. The approximate value for the time to death for 50 percent of the rats (LT50) for was 423 minutes for exposure to glycerin aerosols at 11.0 mg/L (CIR, 2015). Reported intraperitoneal LD50 values of glycerin were 4.42-10.1 g/kg in rats and 8.6-9.5 g/kg in mice. LD50 values of glycerin via subcutaneous administration were 100 mg/kg in rats and 91- 100 mg/kg in mice (CIR, 2015). PREPUBLICATION COPY: UNCORRECTED PROOFS

TOXICOLOGY OF E-CIGARETTE CONSTITUENTS 5-11 Repeated dose toxicity studies Because glycerol has been used extensively as a vehicle for drug delivery in many drug toxicology studies, Gad and colleagues (2006) surveyed four laboratories on their use of glycerin and other vehicles for in vivo experiments. They found the highest NOAEL was 500 mg/kg for guinea pigs and 15 g/kg for rats for 1 month of oral administration. A study in mice also reported a NOAEL for glycerin of 500 mg/kg for 90 days. Numerous repeated dose studies, ranging from a few days to 2 years, have been conducted. Glycerol was administered in the diet of rats for 2 years at 5 percent and 10 percent of the diet. There were no pathological or toxicological effects noted, although food consumption was increased in males (CIR, 2015). Undiluted glycerol caused a variety of irritant-related effects, including petechial hemorrhage and erosions in the small intestine that were dose dependent. In several short-term feeding experiments, 20 percent glycerol administered in the diet for 4 weeks had no adverse effects, although an increase in kidney weights and increased liver enzymes were observed in more than half of the animals. Renne and colleagues (1992) established a NOAEL for glycerol of between 115 and 2,300 mg/kg when administered in drinking water to rats for 44 days. In another short-term drinking water study, calcification in kidney tubules between the cortex and medulla was observed in 3 of 5 rats administered 3,335 mg/kg/d glycerin in drinking water for 6 months (CIR, 2014). A 3-day oral dosing study of glycerol in mixed-breed dogs established a NOAEL of 950 mg/kg/d. At the highest dose of 3,800 mg/kg/d, the stomach mucosa was severely hyperemic with petechial hemorrhages (Latven and Molitor, 1939). Another longer term feeding study in dogs using 35 percent glycerol in the diet found weight loss after 36 weeks. The weight loss continued after reduction of glycerol by 50 to 80 percent for the remainder of a 50-week study (CIR, 2015). Guinea pigs given 6,300 mg/kg/d of glycerol orally for 30-40 days showed no observable pathological changes (CIR, 2015; Ostwald, 1962). Inhalation–non-human One study exposed rats for 6 hours per day, 5 days per week for 2 weeks to concentrations of 0, 1,000, 1,930 and 3,910 mg/m3 of aerosolized VG (Renne et al., 1992). The authors reported minimal squamous metaplasia of the epiglottis in 2/25, 1/19, 4/20 and 10/21 rats at 0, 33, 167 and 662 mg/L, respectively; one male in the high-dose group showed mild squamous metaplasia. The authors did not observe macroscopic or systemic effects, or changes in organ weights (Renne et al., 1992). They determined a lowest observed adverse effect level for local irritant effects of the upper respiratory tract of 1,000 mg/m3. In another study by the same researchers, 11 rats exposed to the highest concentration of respirable VG for 13 weeks (6 hours per day, 5 days per week) similarly exhibited mild squamous metaplasia but did not display macroscopic changes or differences in organ weights (Renne et al., 1992). Male rats in the study showed reduced triglyceride levels, but there was no dose–response relationship (Renne et al., 1992). Based on this study, the inhalation NOAEL was 0.167 mg/L (Renne et al., 1992). Reproductive and developmental toxicity A two-generation reproductive study of 10 rats administered glycerol (0, 20 percent; ~2,000 mg/kg/d in drinking water) for 8 weeks before mating until weaning of pups (CIR, 2015). The researchers observed no adverse effects on the reproductive efficiency of the parents (F0 generation), or the growth, fertility, or reproductive performance of the untreated F1 generation offspring. In the F0 generation, all 10 females PREPUBLICATION COPY: UNCORRECTED PROOFS

5-12 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES became pregnant with similar litter size as the controls (9.0 versus 8.1). In the F1 generation, 9 of 10 females became pregnant. Additionally, there were no significant differences in the onset of estrus cycles, weight gain, and microscopic observations of the endocrine organs between the F1 and the F2 generations and the controls. Tissues from both the F1 and F2 generations showed no histological changes. Another study administered glycerin (13.1, 60.8, 282, and 1,310 mg/kg/d) by gavage to Wistar rats (n = 25-28) on days 6 through 15 of gestation (CIR, 2015). No adverse effects were observed in the dams (NTIS, 1974). The number of pregnancies, implantations, resorptions, litter sizes, weights, and sex ratio, and the incidences of external, visceral, and skeletal abnormalities were similar among treatment groups compared with controls. The NOAEL for maternal toxicity and teratogenicity was 1,310 mg/kg/d. A similar study administered glycerol (12.8, 59.4, 276, and 1,280 mg/kg/d) by gavage to CD-1 mice (n = 25) on days 6 through 15 of gestation. As with the study of Wistar rats, the researchers found no adverse effects in the dams (CIR, 2015), and the number of pregnancies, implantations, resorptions, litter sizes, weights, and sex ratio and incidences of external, visceral, and skeletal abnormalities were similar among treated mice compared with controls. The NOAEL for maternal toxicity and teratogenicity was 1,280 mg/kg/d. A study of Dutch-belted rabbits (n = 25) administered glycerol (11.8, 54.8, 254.5, and 1,180 mg/kg/d) by gavage on days 6 through 18 of gestation, and also reported no adverse effects in the dams (CIR, 2015). Again, the number of pregnancies, implantations, resorptions, litter sizes, weights, sex ratio, and external, visceral, and skeletal abnormalities were similar among treated rabbits compared with controls. The NOAEL for maternal toxicity and teratogenicity was 1,180 mg/kg/d. Male Fertility One study found that glycerol injected into the testes of rats (50-200 L and 862 mg/kg body weight) and monkeys (119 mg/kg body weight) suppressed spermatogenesis (CIR, 2015; Wiebe and Barr, 1984a,b; Wiebe et al., 1989). Genotoxicity and carcinogenicity Numerous studies have examined the mutagenic potential of glycerol in the Ames Salmonella assay, in dose ranges from 0.2 to 50 mg/plate, and using a variety of strains of S. typhimurium, with and without metabolic activation, and all reported negative results (CIR, 2015; Clark et al., 1979; Doolittle et al., 1988; Haworth et al., 1983; Ishidate et al., 1984; Stolzenberg and Hine, 1979; Yamaguchi, 1982). Carmines and Gaworski (2005) measured the mutagenicity of mainstream tobacco smoke condensate in the presence and absence of various concentrations of glycerol (5, 10, and 15 percent) and found no difference in mutagenicity in the presence or absence of glycerol. Glycerol also tested negative in the hypoxanthine-guanine phosphoribosyl transferase mutagenicity assay, sister chromatid exchange assay in CHO cells, and unscheduled DNA synthesis in rat hepatocytes, at concentrations up to 1 mg/ml (CIR, 2015). Another study looking at interlaboratory comparisons of the DNA damage assay in rat hepatocytes evaluated glycerol as one of three “negative” vehicles for administration of other carcinogens, and confirmed the lack of any effect on DNA damage in rat hepatocytes (CIR, 2015). An in vivo bone marrow chromosome aberration assay tested negative following intraperitoneal injection administration of 1,000 mg/kg glycerol (CIR, 2015). PREPUBLICATION COPY: UNCORRECTED PROOFS

TOXICOLOGY OF E-CIGARETTE CONSTITUENTS 5-13 A chronic bioassay in rats, with glycerin administered at concentrations up to 20 percent for 1 year or up to 10 g/kg for 2 years, failed to increase tumor incidence (CIR, 2015). Thus, there is substantial evidence indicating that glycerol itself is not mutagenic. However, when combusted, glycerin can form thermal decomposition products (see the section on carbonyl compounds for discussion of thermal decomposition products). Ethylene Glycol In addition to PG and glycerol, studies have also identified ethylene glycol as a solvent used in e-liquids. Ethylene glycol is odorless, clear, slightly viscous liquid that is commonly used as antifreeze in cooling and heating systems, in hydraulic brake fluids, and as an industrial solvent. Hahn and colleagues (2014) identified ethylene glycol in samples even though it was not listed on any labels. Hutzler and colleagues (2014) found that ethylene glycol replaced PG and glycerol as the dominant compound in five products. In a liquid from one particular manufacturer, the ethylene glycol content was as high as 76 percent. Four out of five products from this particular manufacturer revealed more than 70 percent ethylene glycol, whereas only 2 percent were detectable in the fifth. Seven products from three manufacturers contained 1 to 6 percent ethylene glycol, and in one additional sample again more than 30 percent was detected. Conversely, altogether 15 samples produced by 3 other manufacturers tested negative. Most e- cigarette liquids do not contain ethylene glycol and, where present, it is at levels that are not likely to contribute significantly to adverse health effects. Nonetheless, ethylene glycol is a respiratory irritant and is associated with markedly enhanced toxicological hazards when compared with conventionally used glycerol and propylene glycol (Gomes et al., 2002). FLAVORINGS There are more than 7,000 unique e-liquid flavors available to e-cigarette users (Zhu et al., 2014), and yet, little is known about them as there are few studies looking directly at exposure to flavorings. Furthermore, flavoring components are often not included in e-cigarette products’ ingredient lists. For example, one study of 54 e-liquids found many products labeled with “natural or artificial flavors,” and just four samples listed specific flavoring substances (Hahn et al., 2014). While the Flavor and Extracts Manufacturers Association considers many flavors to be GRAS in food products, at their levels of intended use these chemicals could still be harmful when they are aerosolized and inhaled, as such ingredients are not safety tested for exposure routes other than ingestion (Barrington-Trimis et al., 2016; FEMA, 2015). For instance, saccharides, which are used to make sweet e-liquid flavors that can appeal to children (Farley et al., 2014; King et al., 2014; Villanti et al., 2013), degrade and produce furans and aldehydes when heated (Soussy et al., 2016). Aldehydes may cause irritation to the respiratory tract (Tierney et al., 2016). One study of 28 e-liquids identified more than 140 volatile flavoring components at concentrations varying from 1 to 5 percent (10 to 50 mg/ml), and detected the formation of aldehydes (Hutzler et al., 2014). Another study that tested multiple flavors in two brands of single-use cigarettes found a similar concentration of flavor chemicals and identified aldehydes such as vanillin and ethyl vanillin (Tierney et al., 2016). Hahn and colleagues (2014) analyzed 54 e-liquids and distinguished ethyl vanillin in 13 samples and thujone in 2 samples. PREPUBLICATION COPY: UNCORRECTED PROOFS

5-14 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES Other flavoring chemicals have been measured in e-liquids as well. For example, pulegone and eucalyptol were identified in menthol-flavored e-cigarettes (Lisko et al., 2015). Similar to combustible tobacco cigarettes, concentrations of menthol in this study varied from 3,700 to 12,000 g/g. Additionally, 40 percent of non-menthol products tested in the study had low levels of menthol (Lisko et al., 2015). Menthol properties include cooling and local anesthesia, as well as effects on drug absorption and metabolism, bronchodilation and respiration changes, and electrophysiology (Ahijevych and Garrett, 2004). Although little is known about the role of menthol in e-cigarettes, the effects of menthol in increasing the reinforcing effects of nicotine on tobacco smoking behavior were evidenced in both qualitative and quantitative empirical studies (Ahijevych and Garrett, 2010). For the menthol smokers, a greater exposure to nicotine and the particulate matter (tar) of the smoked cigarette was observed and can result in increased nicotine dependence and a greater chance of tobacco-attributable disease (Garten and Falkner, 2004). Exposure to Flavorings Broadly speaking, flavored tobacco use is associated with younger age; consumers perceive flavored tobacco products more favorably. Flavoring in tobacco products is considered an attractive characteristic and is associated with temporary experimentation and/or initiation of tobacco product use (Feirman et al., 2016; Kowitt et al., 2017). Flavors are extremely common among e-cigarette users, and are often named as a primary reason for e-cigarette use. For example, about 75 percent of regular e-cigarette users report using some non-tobacco flavor (Wang et al., 2015; Yingst et al., 2017). Despite the increasing popularity of e-cigarettes, little is known about users’ preferences, selection, and switching among various flavors. Farsalinos and colleagues (2013) conducted an online survey of more than 4,000 e-cigarette users and found that flavors, especially flavor variety, were an important factor in the maintenance of e-cigarette use by current and former smokers. Specifically, nearly half of the study subjects reported that limiting the range of available e-cigarette flavors would increase cravings for combustible tobacco cigarettes and would decrease their likelihood of reducing or quitting smoking. The results also indicated that smokers tended to start with tobacco-flavored products, and then would switch to multiple flavors as they transitioned from dual use to complete (or nearly complete) substitution of e-cigarettes from their usual cigarettes. Berg (2016) recruited 1,567 adults, ages 18 to 34 years, through Facebook ads targeting tobacco users and non-users. Fruity e-cigarette flavors were the most preferred among both smokers and non-smokers. Flavors appear to hold value to users. In a willingness-to-pay (WTP) study, removing flavors resulted in an 18 percent drop in WTP among exclusive e-cigarette users, compared with a 1 percent drop for dual users (Nonnemaker et al., 2016). In a discrete choice experiment context, flavor (cherry in particular) significantly increased intentions to purchase (Czoli et al., 2016). In the laboratory, participants worked harder for flavored puffs, meaning that flavors appear to enhance the reward/reinforcement value of nicotine (Audrain-McGovern et al., 2016). In a concept mapping study among vapers, five statement clusters around flavor use were identified: increased satisfaction and enjoyment, variety and customization, better feel and taste than cigarettes, food craving suppression, and social impacts (Soule et al., 2016). At the same time, data from novice users indicated that non-menthol flavorings were not associated with decreased cigarette consumption over 6 weeks of use (Litt et al., 2016). In e-liquids, flavor combinations are common and their classification is not straightforward. This has been a limitation in determining preferred flavors among e-cigarette PREPUBLICATION COPY: UNCORRECTED PROOFS

TOXICOLOGY OF E-CIGARETTE CONSTITUENTS 5-15 users, as common measures have not been used, resulting in widely divergent estimates across studies. A classification system with transparent decision rules that can be applied across product classes may yield more consistent findings to inform regulatory science (Yingst et al., 2017). The role of menthol in e-cigarette users has not been studied. However, for combustible tobacco cigarettes, African-American smokers report substantially greater preference for menthol cigarettes relative to smokers of European ancestry. This had led some to speculate that menthol may contribute to the greater incidence and severity of certain smoking-related diseases among African Americans. Certain studies also suggest that menthol may influence the rates of smoking initiation and cessation (TPSAC, 2011). Although studies have dealt with flavoring chemicals in e-cigarette products, there is little information on how these chemicals affect health during long-term exposures by inhalation. Studies have shown that users switch among flavors frequently. Additionally, the choice of flavor may change over the course of a smoker’s substitution of combustible tobacco with e- cigarettes, such that tobacco flavors are more popular when users start using e-cigarettes (Farsalinos et al., 2013). Flavoring compounds might also include substances of sensitizing, toxic, or irritating potency. Although few studies have examined the effects of flavoring substances administered by inhalation, there are some chemicals which, although approved for ingestion, have known adverse health effects when inhaled. Table 5-4 presents an overview of common flavorings and their inhalation toxicity. Examples of such chemicals include diacetyl, acetylpropionyl, acetoin, cinnamaldehyde, and benzaldehyde and are reviewed in details below. Diacetyl, acetylpropionyl (also known as 2,3-pentanedione), and acetoin are chemicals used by food manufactures to add creamy flavors like butter, caramel, butterscotch, piña colada, and strawberry to food products. Acetylpropionyl is structurally similar to diacetyl and therefore can be used as a flavoring substitute. However, these ingredients have been associated with adverse respiratory health outcomes. For example, investigations in microwave popcorn manufacturing plants found increased incidences of chronic cough and bronchitis, asthma, and bronchiolitis obliterans, a severe lung condition that can result in permanent pulmonary scarring and obstruction (Kreiss et al., 2002; NIOSH, 2016). Workers in these facilities inhaled diacetyl and acetoin when butter flavoring containing these chemicals was heated and became aerosolized (Kreiss et al., 2002; NIOSH, 2016). Workers with bronchiolitis obliterans have also been found in flavoring production companies (NIOSH, 2016). These flavoring ingredients have also been measured in e-cigarette liquids. For instance, a study of flavored e-cigarettes available in the United States identified at least one of these three chemicals in more than 90 percent of the tested e-cigarettes (Allen et al., 2016). Of the 51 samples, 46 flavors had acetoin (concentration ranging up to 529 g per e-cigarette), 39 contained diacetyl (up to 239 g per e-cigarette), and 23 flavors included acetylpropionyl (up to 64 g per e-cigarette) (Allen et al., 2016). Another study of 159 sweet-flavored liquids from 36 American and European manufacturers found diacetyl and/or acetylpropionyl in nearly three- quarters of sampled liquids and their aerosols (Farsalinos et al., 2015c). These samples indicated a median daily exposure of 56 g of diacetyl per day (IQR: 26-278 g/day); the median daily exposure to acetylpropionyl was 91 g per day (IQR: 20-432 g/day) (Farsalinos et al., 2015c). PREPUBLICATION COPY: UNCORRECTED PROOFS

5 5-16 PU UBLIC HEALTH CONSEQUEN H NCES OF E-CIG GARETTES T TABLE 5-4 Overview of Commo Flavorings and Their Inhalation Toxicity on d n Chemical CAS Flavor Respirattory Group Flavoring Che emical Numbe er Type Flavo Descriptor or Irritan nt Inhalation Toxicity n Naature Identical A Alcohols Geraniol 106-24-1 Floral Sweet, floral, fr ruity, rose, waxy,, citrus Menthol 2216-51- Mentholic Peppermint, coo oling, mentholic, , 5 minty Thymol 89-83-8 8 Herbal Herbal, thyme, phenolic, medici inal camphor Eugenol 97-53-0 0 Spicy Sweet, spicy, cl love, woody A Acids Butyric acid 107-92-6 Cheesy Sharp, dairy-lik cheesy, buttery ke, ry, Mouse LC > 500 C with a fruity nu uance mg/m3 Valeric acid 109-52-4 Cheesy Acidic and shar cheesy, sour m rp, milky, Mouse LC50 > 4,100 C tobacco, with fr ruity nuances mg/m3/2 h hr 2-Methylbutyric acid c 116-53-0 Acidic Acidic, fruity, d dirty, cheesy with a h fermented nuan nce E Esters Ethyl butyrate 105-54-4 Fruity Fruity, juicy fru pineapple, cog uit, gnac 2-Methylbutyra ate 105-37-3 Fruity Sweet, ethereal, rummy, grape, winey Methyl cinnama ate 103-26-4 Balsamic Sweet, balsam, strawberry, cherr rry, cinnamon Methyl salicyla ate 119-36-8 Minty Wintergreen, m mint L Lactones Gamma-nonala actone 104-61-0 Coconut Coconut, cream waxy, sweet, my, buttery, oily Delta-decalacto one 705-86-2 Coconut Coconut, cream fatty, buttery, my, milky, and nutty with a slightly fruity y nuance PREPUB BLICATION CO OPY: UNCORR RECTED PRO OOFS

T TOXICOLOGY OF E-CIGARET CONSTITU TTE UENTS 5-17 Chemical CAS Flavor Respirattory Group Flavoring Che emical Numbeer Type Flavo Descriptor or Irritan nt Inhalation Toxicity n A Aldehydes Geranial 141-27-5 Citrus Citrus, lemon Benzaldehyde 100-52-7 Fruity Almond, fruity, powdery, nutty, and , Mouse LC > 500 C benzaldehyde-like mg/m3 Rat LC > 5 mg/m3 500 Cinnamaldehyd de 104-55-2 Spicy Sweet, spice, ci innamon red, hots s, warm Vanilin 121-33-5 Vanilla Sweet, vanilla, creamy, chocolat te Mouse LC > 41,700 C g/kg/2 hr r Rat LC > 4 41,700 g/kg/4 hr r K Ketones Diacetyl 431-03-8 Buttery Sweet, creamy, buttery, pungent t, with a pungent caramellic nuanc ce yl Acetyl propiony 600-14-6 Buttery Buttery, nutty, t toasted, caramell lic, diacetyl and aceetoin notes Raspberry keton ne 5471-51- Fruity Sweet, berry jam raspberry, ripe m, e, 2 floral Heterocycles H O Oxygen Furfural 98-01-1 1 Bready Brown, sweet, wwoody, bready, n nutty, Human TCLO 310 C c containing caramellic with a burnt astringen h nt g/m3 nuance Rat LC50 1 ppm/6 175 hr 5-Methylfurfura al 620-02-0 Caramellic Sweet, caramellic, bready, brow wn, coffee-like Maltol 118-71-8 Caramellic Sweet, caramel, cotton candy, ja am, fruity, baked brread N Nitrogen 2-Acetypyrazin ne 22047- Popcorn Musty, roasted, corn chip, popco , orn, 25-2 nutty, potato-lik ke PREPUB BLICATION CO OPY: UNCORR RECTED PRO OOFS

5 5-18 PU UBLIC HEALTH CONSEQUEN H NCES OF E-CIG GARETTES Chemical CAS Flavor Respirattory Group Flavoring Che emical Numbeer Type Flavo Descriptor or Irritan nt Inhalation Toxicity n c containing 2,3,5- 14667- Nutty Nutty, musty, p powdery cocoa, p potato, Trimethylpyraz zine 55-1 musty 2-Acethylpyrrole 1072-83- Musty Musty, nutty-lik with a coumar ke rin 9 nuance S Sulfur 2-isopropyl-4- 15679- Fruity Musty alliaceou earthy sulfury us, y, c containing methylthiazole 13-7 slight fruity, co offee, meaty 2-isobuthylthiaz zole 18640- Green Green, vegetabl tomato-like w le, with 74-9 raw musty nuannces Sulfu Compounds fur M Mercaptans Furfuryl mercap ptan 98-02-2 Coffee Roasted coffee, sulfurous, with a , burnt match not te Thiomenthone 38462-22 2- Sulfurous Fruity, berry, an tropical with a nd 5 raspberry, mint nuance ty thiol p-Menthene-8-t 71159-90 0- Citrus Grapefruit, fres tropical, juicy,, sh, 5 mango S Sulfides Dimethyl sulfid de 75-18-3 Sulfurous Sulfurous, cream tomato, scall my, lop, Rat LC50 4 40,250 (DMS) berry fruity, veg getative nuances ppm Mouse LC50 3,1620 C g/m3 Tropathiane 67715-80 0- Tropical Green, tropical, galbanum, pinea , apple 4 Fla avor Synthetic Ethyl vanillin 121-32-4 Vanilla Sweet, creamy, vanilla, caramel Ethyl maltol 4940-11- Caramel Sweet, caramel, jam, strawberry y, 8 cotton candy -3- Ethyl 3-methyl- 77-83-8 8 Fruity Sweet, fruity, st trawberry, floral,, phenylglycidate e honey, fatty PREPUB BLICATION CO OPY: UNCORR RECTED PRO OOFS

TOXICOLOGY OF E-CIGARETTE CONSTITUENTS 5-19 Several studies looked at the cinnamaldehyde-containing e-liquids and e-cigarette aerosols. Cinnamaldehyde is the major chemical in cinnamon-flavored e-cigarette products but has been also found in tobacco-, sweet- (including caramel), and fruit-flavored e-liquids (Behar et al., 2016). Behar and colleagues (2016) evaluated the distribution, concentration, and toxicity of cinnamaldehyde in 39 e-liquids and aerosols generated from e-cigarettes. The study used the gas chromatography and mass spectrometry method and found that 20 of the 39 refill fluids contained cinnamaldehyde at concentrations that were cytotoxic to human embryonic and lung cells in the cell viability assay. The study also revealed that aerosol generated from a single product (“Cinnamon Ceylon”) from a cartomizer-style e-cigarette was cytotoxic. The same product has been shown to be more cytotoxic when aerosol was generated with battery output voltage settings of 5 V than of 3 V, potentially due to additional chemicals released at higher voltage settings, including 2,3-butandione (diacetyl) as confirmed in the study. Cinnamaldehyde depolymerized microtubules in human pulmonary fibroblasts. At concentrations that produced no effect in the cytotoxicity assay, cinnamaldehyde decreased cell growth, attachment, and spreading; altered cell morphology and motility; increased DNA strand breaks; and increased cell death. In general, studies described above have shown that, even at low concentrations, cinnamaldehyde in e-cigarette products is cytotoxic, genotoxic, and adversely affects cell processes and survival. These studies also indicate that cinnamaldehyde in e-cigarettes may impair homeostasis in the respiratory system. Benzaldehyde, which imparts a fruity taste, is an aromatic aldehyde commonly used in food and cosmetics. Studies suggest that oral and dermal exposure to benzaldehyde produces little to no toxicity; however, occupational exposure has been linked to irritation of the eyes and mucus membranes of the respiratory passages (MAK Commission, 2002). One study measured benzaldehyde in aerosol generated from an e-cigarette refilled with 145 flavored nicotine- containing solutions purchased from international online retailers (Kosmider et al., 2016). The solutions were classified into groups according to labelled flavor characteristics: berry/tropical fruit (n = 40), tobacco (n = 37), alcohol-related/drink (n = 15), chocolate/sweet flavor (n = 11), coffee/tea (n = 11), mint/menthol (n = 10), cherry (n = 10), and other, non-identifiable flavor varieties (e.g, “Indian Summer,” and “Cosmopolitan”) (n = 11). Benzaldehyde was present in 75 percent of 145 e-cigarette refill fluids, with the highest concentrations in cherry flavors. The benzaldehyde doses inhaled using 30 puffs from flavored e-cigarettes were often higher than doses inhaled from a combustible tobacco cigarette. The estimated median daily inhaled dose of benzaldehyde from cherry-flavored e-cigarettes was 70.3 g, a level of exposure greater than one thousand times lower than the permissible exposure limit (PEL) of benzaldehyde as defined by the U.S. Workplace Environmental Exposure Level Guides. CARBONYL COMPOUNDS It is important to evaluate the health effects of e-cigarettes when liquid is heated and aerosolized; under such conditions, chemical reactions may result in the formation of new compounds. For example, although refill liquids can contain carbonyl compounds such as reactive aldehydes, heating can enhance the concentrations of these compounds in the aerosol. Several studies have shown that e-cigarettes emit toxic carbonyl compounds, generated from thermal decomposition of e-liquid ingredients. Carbonyl compounds such as formaldehyde, acetaldehyde, acrolein, and glyoxal, which have been found in e-cigarette aerosols, are potentially hazardous and may induce various health effects on its users. Formaldehyde is PREPUBLICATION COPY: UNCORRECTED PROOFS

5-20 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES classified as a human carcinogen (Group 1) by the International Agency for Research on Cancer (IARC), and acetaldehyde is classified as possible carcinogenic to humans (Group 2B) (Bekki et al., 2014). Glycidol is a probable carcinogen and acrolein causes irritation of the nasal cavity and damages the lining of the lungs (ATSDR, 2007; NTP, 2007). How formaldehyde-releasing agents (hemiacetals) behave in the respiratory tract is currently unknown. Glyoxal and methylglyoxal show mutagenicity. The amount of carbonyl compounds in e-cigarettes varied significantly not only among different brands but also among different samples of the same products. Although, in most cases, detected levels of carbonyl compounds were lower than those in combustible tobacco cigarette smoke, very high levels of formaldehyde were also reported in e-cigarette aerosols (a comparison of toxicants from combustible tobacco cigarette smoke and e-cigarette aerosols is discussed in Chapter 18) (Canistro et al., 2017; Gillman et al., 2016). Uchiyama and colleagues (2010, 2013) measured carbonyl compounds in e-cigarette aerosols using high-performance liquid chromatography (HPLC) (Bekki et al., 2014; Ohta et al., 2011; Uchiyama et al., 2010, 2013). The authors tested 13 brands of Japanese e-cigarettes and detected several derivative peaks of carbonyl compounds, including formaldehyde, acetaldehyde, acetone, acrolein, propanal, crotonaldehyde, butanal, glyoxal, and methylglyoxal (Bekki et al., 2014; Ohta et al., 2011; Uchiyama et al., 2013). Four out of the 13 e-cigarette brands did not generate any carbonyl compounds. The other nine e-cigarette brands generated various carbonyl compounds. The maximum concentrations of formaldehyde, acetaldehyde, acrolein, propanal, glyoxal, and methylglyoxal were 140, 120, 40, 46, 23, and 21 g/10 puffs, respectively. Goniewicz and colleagues (2014) measured 15 carbonyl compounds in aerosol generated from 12 e-cigarette brands. Only 4 carbonyl compounds (formaldehyde, acetaldehyde, acrolein and o-methylbenzaldehyde) were found in aerosols and these compounds were identified in nearly all examined e-cigarettes. The content of formaldehyde ranged from 2.0 mg to 56.1 mg, acetaldehyde from 1.1 mg to 13.6 mg, and acrolein from 0.7 mg to 41.9 mg per e-cigarette (150 puffs). Kosmider and colleagues (2014) tested 13 samples of aerosol generated from Polish e- cigarettes, and detected formaldehyde and acetaldehyde in 8 of them. The amounts of formaldehyde and acetaldehyde in e-cigarette aerosols at a lower voltage were on average 13- and 807-fold lower than those in combustible tobacco cigarette smoke, respectively. E-cigarette aerosols generated from PG-based e-liquids were found to have the highest levels of carbonyls. Furthermore, different e-cigarettes showed large variations in carbonyl levels. Hutzler and colleagues (2014) measured formaldehyde in e-cigarette aerosol and estimated that exposure to formaldehyde can even be comparable with combustible tobacco cigarettes. They measured 20 to 50 g of formaldehyde per 10 puffs in the final fractions, which roughly corresponds to the expected exposure from smoking one combustible tobacco cigarette. Flora and colleagues (2016) tested the aerosols of four MarkTen® e-cigarettes (rechargeable with disposable cartridges) for potential degradation products. They found formaldehyde levels that varied from 0.09 to 0.33 g/puff. The same research team found that formaldehyde residues in both the gas (approximately 30 percent) and liquid (approximately 70 percent) phases of an aerosol (Flora et al., 2017). Blair and colleagues (2015) measured acrolein in aerosol from e-cigarettes and tobacco smoke and found that five puffs of an e-cigarette emitted 0.290 ± 0.018 g of acrolein while nine puffs on a combustible tobacco cigarette emitted 2.61 ± 0.16 g of this toxicant. There was a PREPUBLICATION COPY: UNCORRECTED PROOFS

TOXICOLOGY OF E-CIGARETTE CONSTITUENTS 5-21 substantial range in the relative standard deviations reported for all mean value measurements, suggesting inconsistencies across products in the release of these chemicals. Papousek and colleagues (2014) measured acrylamide and acrolein in tobacco smoke and three e-cigarette aerosol samples. The e-cigarette aerosol samples contained no detectable levels of acrylamide. Acrolein levels in combustible tobacco cigarette smoke varied from 4.48 to 8.27 g per cigarette while levels detected in an equivalent sample of e-cigarette aerosol varied from 0.17 to 3.70 g. Sleiman and colleagues (2016) detected up to 31 compounds, including formaldehyde, acetaldehyde, glycidol, acrolein, acetol, and diacetyl, in e-cigarette aerosols from different devices. Emission rates were significantly higher for a single-coil versus a double-coil device, ranging from tens to thousands of nanograms of toxicants per milligram of e-liquid aerosol. Tayyrah and Long (2014) tested 55 harmful and potentially harmful constituents in e- cigarette aerosol (blu and SKYCIG brands) and quantified three carbonyls (acrolein, acetaldehyde, and propionaldehyde) at levels 86 to 544 times lower than combustible tobacco cigarette smoke. Table 5-5 summarizes experimental studies to determine carbonyl compounds in electronic cigarette aerosols, their set-ups (i.e., methods to trap and analyze carbonyls, e-liquids used), and results. Because carbonyl compounds were primarily detected in aerosol and only traces have been reported in e-liquids, it has been suggested that these compounds are generated when e- liquid ingredients are heated. Figure 5-1 illustrates the pathways and byproducts formed during thermal dehydration of propylene glycol and glycerin as postulated by Sleiman and colleagues (2016). Hutzler and colleagues (2014) incubated e-cigarette liquids at various temperatures and found levels of acetaldehyde and formaldehyde from 10-fold to 20-fold higher at the temperature of 150°C compared with ambient temperatures for samples containing PG. They did not observe this effect at 100°C. Several studies looked at the potential mechanisms for generating carbonyl compounds in e-cigarettes. In addition to temperature and effects from potential overheating, air flow and catalytic properties of metal heating coils may influence the occurrence of decomposition products. As described in the section on humectants, propylene glycol, and glycerol can be a source of carbonyl compounds. It has been shown that the oxidation and fragmentation of PG and glycerol contained in e-liquids when they come in contact with the heating coil generates carbonyl compounds (Bekki et al., 2014; Geiss et al., 2016; Goniewicz et al., 2014; Ohta et al., 2011; Uchiyama et al., 2013). Lower liquid levels within the cartridges or tanks also seems to be associated with the occurrence of carbonyls, because low liquid levels may increase air flow and could therefore promote overheating of the wire if no safety features are incorporated to maintain a constant and lower temperature. Results reported by Geiss and colleagues (2016) confirmed that the PG oxidation is involved primarily in the formation of acetaldehyde, while the oxidation of glycerol typically generates acrolein. Oxidation of both PG and glycerol can generate formaldehyde, although a predominance of glycerol can be observed. Glycerol forms acrolein and acetaldehyde as oxidation-byproducts only at higher coil temperatures. PREPUBLICATION COPY: UNCORRECTED PROOFS

TOXICOLOGY OF E-CIGARETTE CONSTITUENTS 5-22 TABLE 5-5 Overview Table Comparing Experimental Set-Ups and Results of the Current Study with Comparable Studies Conducted in the Past Methodology for Carbonyl Study Trapping/Analysis Type of E-Cigarette(s) Liquid(s) Used Determined Carbonyl Emissions Geiss et al. (2016) Machine smoking (50 ml, 3.0 Third-generation e-cigarette Glycerol (50%), propylene Formaldehyde: 24-2,559 ng s, 30 s, 10 puffs), direct with variable voltage/wattage glycol (40%), water, puff 1 trapping on DNPH-sorbent, (5 W, 10 W, 15 W, 20 W, 25 fragrance, nicotine Acetaldehyde: 13-350 ng puff 1 HPLC W tested). Heating element Acrolein: 2.5 ng puff 1 (at 20 W) with 1.6 resistance, 2,200 mAh battery Tayyarah and Long Machine smoking (55 ml, 30 Two disposable and three (1) Glycerol/propylene Expressed as total carbonyls: (2014) s, 99 puffs), smoke/aerosol rechargeable e-cigarettes; glycol (20:70%), water, < 900 ng puff 1 collected in two DNPH- no detailed information of e- nicotine, fragrance; Acetaldehyde: 320 ng puff 1 containing impingers, HPLC cigarette properties available (2) Glycerol (80%), water, Acrolein: 150 ng puff 1 nicotine, fragrances Propionaldehyde: 110 ng puff 1 Kosmider et al. (2014) Machine smoking (70 ml, 1.8 Second-generation e-cigarette (1) primarily glycerol; (2) Formaldehyde: 3.2-3.9 ng puff 1 s, 17 s, 30 puffs), direct with variable voltage (3.2 glycerol and propylene Acetaldehyde: 1.3-7.1 ng puff 1 trapping on DNPH-sorbent V/4.3 W, 4 V/6.7 W, and 4.8 glycol; (3) primarily Acetone: 3.9-19.7 ng puff 1 tubes, HPLC V/9.6 W tested); heating propylene glycol Acrolein: < DL element with 2.4 resistance, Propionaldehyde: < DL 900 mAh battery Bekki et al. (2014) Machine smoking (55 ml, 2 s, 13 Japanese e-cigarette No detailed information Formaldehyde: 660-3,400 ng 30 s, 10 puffs), direct brands; no detailed available puff 1 trapping on cartridges information of e-cigarette Acetaldehyde: 20-2,600 ng (hydroquinone and DNPH), properties available puff 1 HPLC Acrolein: 110-2,000 ng puff 1 (at 20 W) Propionaldehyde: 40-1,500 ng puff 1 Goniewicz et al. (2014) Machine smoking (70 ml, 1.8 11 popular Polish brands; no No detailed information Formaldehyde: 21-374 ng puff 1 s, 10 s, 15 puffs), sorbent detailed information of e- available Acetaldehyde: 13-91 ng puff 1 trapping, HPLC cigarette properties available Acrolein: 4.6-201 ng puff 1 (at 20 W) Hutzler et al. (2014) Machine smoking (55 ml) 3 s, First-generation e-cigarette; Prefilled cartridges; no Formaldehyde: ~300 ng puff 1 30 s, puffing until no vapors no detailed information of e- detailed information Acetaldehyde: ~500 ng puff 1 observable), collected in two cigarette properties available available Acrolein: 500-2,500 ng puff 1 DNPH-containing impingers, (only when overheating) HPLC Propionaldehyde: 100-1,100 ng puff 1 (only when overheating) NOTE: DNPH = 2,4-Dinitrophenylhydrazine; HPLC = high-performance liquid chromatography. SOURCE: Adapted from Geiss et al., 2016. PREPUBLICATION COPY: UNCORRECTED PROOFS

5-23 PUBLI HEALTH CONSEQU IC H UENCES OF E-CIGARET F TTES FIGURE 5-1 Postulate pathways and byproduc formed du ed a cts uring thermal dehydration o propylene of glycol and glycerin. d SOURCE Sleiman et al., 2016. E: a Gillman and colleagues (2 G c 2016) demon nstrated that glycerol ca undergo th t an hermal decompo osition to for reactive aldehydes, in rm a ncluding formmaldehyde, acetaldehyd and acrole de, ein. The exten of formati is depen nt ion ndent upon bo the powe (watts) of the coil and the design of oth er f d the devic itself. Esti ce imated exposures to tota aldehydes from daily c al consumption of 3 grams of e- n liquid rannged from le than 0.1 to 41 mg/day Formaldeh ess t y. hyde was th predomina aldehyde he ant e present, with the high estimate exposure to be 22 mg w hest ed g/day. The au uthors report a 750-fold ted differenc in total ald ce dehyde produ uction betwe different devices, us een t sing the same e-liquid. F For the devic that gener ce rated the high levels of aldehydes, the estimat daily dos exceed th hest o , ted ses he OSHA Occupational Health PEL for formald O L dehyde by 10 0-fold. Canistro and colleagues (2017) also found that he C ( f eating of VG produces te G emperature- dependen amounts formaldehyd acetaldehy and acr nt f de, yde, rolein (see T Table 5-6). WWhen rats we ere exposed via inhalatio to e-cigarette vapors (11 cycles pe day, 5 day per week for 4 weeks), a on ( er ys statistically significan 4-fold inc nt crease in the formation o 8-hydroxy of y-deoxyguan nosine was foound in the lun along with other evi ngs, w idence of oxidative stres in these an ss nimals. Thus it is likely that s, glycerol in e-liquids, under some circumstances that are b e both device- and power- (watt) - - dependen can under thermal decompositi to genera reactive a nt, rgo ion ate aldehydes caapable of contribut ting to oxida ative tissue in njury, includ ding potentia DNA da ally amage. However, for oth her devices, the levels of aldehyde were very low relative to both typica indoor air and the leve t f w w, o al els found in combustible tobacco cig e garette smok It should be noted tha the conditions that res ke. at sulted in very high levels of aldehydes were extrem and not ty h f w me ypically attaiined during nnormal consu umer use. Nevertheless, the potential exists for e-c e cigarette deviices to form very high le m evels of aldehyde under extr es reme conditio ons. PR REPUBLICA ATION COP UNCO PY: ORRECTED PROOFS D

5-24 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES TABLE 5-6 Volatile Compounds Detected in E-Cigarette Aerosol Chamber 1 Chamber 5 Standard Standard Mean Deviation Mean Deviation Statistical Significance 1,2-Propanediamine 0.83 0.08 1.09 0.11 ns Acrolein 0.02 0.00 0.03 0.02 ns Indole 0.19 0.24 0.18 0.02 ns Acetole* 0.07 0.03 0.07 0.00 ns 3-Hexen-1-ol* 0.05 0.00 0.06 0.02 ns Diacetyl* 0.03 0.01 0.08 0.01 ns Propylene Glycol (PG) 87.71 1.03 88.66 0.19 ns 1-Methoxy-2-propyl acetate 0.07 0.04 0.05 0.01 ns Methyl propionate* 0.20 0.01 0.21 0.06 ns Propanoic acid, 1-methylpropyl ester 0.09 0.00 0.09 0.02 ns Nicotine 6.36 0.62 6.54 0.18 ns Glycerin (VG) 4.36 1.68 2.98 0.05 ns PG/VG 21.80 8.63 29.80 0.43 ns NOTES: Volatile compounds (VOCs) detected in the first and last treatment chambers during exposure to e-cig vapor. Values are expressed as a percentage (%) of total peak area of VOCs; factorial analysis of variance (ANOVA) was performed to study the effect of exposure cycling on the formation of VOCs. Statistically different means were investigated (Turkey’s test, p < 0.05); * = flavor compounds. SOURCE: Canistro et al., 2017. Some e-cigarette devices allow users to change the power of the device or output voltage of the battery to increase aerosol production and nicotine delivery. The battery output voltage, and consequently the heat generated on the coil, has been reported to affect the quantity of carbonyls formed. Kosmider and colleagues (2014) showed that increasing the voltage from 3.2 V to 4.8 V resulted in an increase from 4 to more than 200 times in the levels of formaldehyde, acetaldehyde, and acetone. The levels of formaldehyde in aerosol generated from high-voltage devices were nearly identical to those in combustible tobacco cigarette smoke (1.6-52 g per cigarette) (see Figure 5-2). Increasing levels of carbonyl compounds were observed for a voltage over 3 V (Bekki et al., 2014; Ohta et al., 2011). Thus, commercial e-cigarettes with 4-5 V batteries may generate carbonyl compounds. The battery output voltage significantly affects the concentration of carbonyl compounds in the e-cigarette aerosol, and high-voltage e-cigarettes may expose users to high levels of carbonyl compounds. Formaldehyde also reacts with PG and glycerol during aerosolization to produce hemiacetals. Jensen and colleagues (2015) analyzed commercial e-liquid aerosolized with the use of a tank system e-cigarette featuring a variable voltage battery. They detected no formation of any formaldehyde-releasing agents at 3.3 V. However, at 5.0 V, they detected a mean (± SE) of 380 ± 90 g per sample (10 puffs) of formaldehyde as hemiacetals. Similarly, Sleiman and colleagues (2016) found that when they increased the voltage applied to a single-coil device from 3.3 to 4.8 V, the mass of e-liquid consumed doubled from 3.7 to 7.5 mg puff 1 and the total aldehyde emission rates tripled from 53 to 165 g/puff, with acrolein rates growing by a factor of 10. PREPUBLICATION COPY: UNCORRECTED PROOFS

TOXICO OLOGY OF E-CIGARETT CONSTIT E TE TUENTS 5-25 FIGURE 5-2 Effects of nicotine solvent and batt o tery output vo oltage on leve of carbony compounds els yl s released from electroni cigarettes ( g/15 puffs; n = 3; puff du f ic uration 1.8 s, puff volume 70ml, puff intervals 17 s). 1 SOURCE Kosmider et al., 2014. E: e Flora and colleagues (201 evaluated the effect o e-cigarett heating co temperatu 17) d of te oil ure on formaaldehyde form mation. Usin an infrare camera to measure the maximum heat coil ng ed o e temperatu and Four ure rier-transfor infrared spectrometer to measure gas-phase f rm s r formaldehyd de, the autho found tha in some of the comme ors at, o ercial e-ciga arettes tested the levels o formaldeh d, of hyde were grea than tho detected in combustib tobacco cigarettes, a as high a 14.1 g/pu ater ose ble and as uff. The study found that e-cigarettes produce low amounts o formaldeh y t s w of hyde at temp peratures beloow 350°C, but as the temmperature inc creases, the levels of for l rmaldehyde a rise stee also eply. The au uthors conclude that the hi levels of formaldehy observed in some e-c ed igh f yde d cigarettes tes in the st sted tudy were like due to he ely eating coil te emperatures above 350°C C. Geiss and col G lleagues (2016) also repo orted correla ation betwee the amoun of carbon en nts nyl compoun emitted by e-cigarett with the temperature of the heatin coil. The authors used nds b tes t ng infrared thermograph to determ the temp t hy mine perature of th heating-coil and had a experienc he an ced e-cigarett user cond a subject sensoria quality evaluation of t aerosol g te duct tive al the generated at each temperatu The stud found a steep increas in the gene ure. dy s se erated carbo onyls when a applying a battery-o output of at le 15 W co east orresponding to 200-250 on the h g 0°C heating coil. A 20 W, the e- At e PR REPUBLICA ATION COP UNCO PY: ORRECTED PROOFS D

5-26 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES cigarette user provided a negative sensorial quality evaluation, suggesting that an e-cigarette user would be unlikely to apply such wattage in real world use. Wang and colleagues (2017) investigated how PG and glycerol influence carbonyl compounds formation under precisely controlled temperatures in the absence of nicotine and flavor additives. At reactor temperatures equal to or greater than 215°C for both PG and glycerol, the authors detected significant amounts of formaldehyde and acetaldehyde. Only e-liquids containing glycerol at temperatures exceeded 270°C produced acrolein. At 318°C, 2.03 ± 0.80 g of formaldehyde, 2.35 ± 0.87 g of acetaldehyde, and a trace amount of acetone were generated per milligram of PG; at the same temperature, 21.1 ± 3.80 g of formaldehyde, 2.40 ± 0.99 g of acetaldehyde, and 0.80 ± 0.50 g of acrolein were detected per milligram of glycerol. Other factors causing elevated carbonyl levels should also be considered. It is expected that both the heating element and wicking material will deteriorate with use, which could lead to more thermal degradation (Guthery, 2016). Sleiman and colleagues (2016) found that, after an e- cigarette device was used several times, carbonyl emissions increased by more than 60 percent, and attributed this effect to the buildup of polymerization byproducts that degraded upon heating. Flavoring compounds may also play a role. Using three popular brands of e-cigarettes filled with both flavored and unflavored e-liquids, Khlystov and Samburova (2016) measured several toxic aldehydes and showed that the formation of aldehydes during e-cigarette use comes primarily from thermal decomposition of flavoring compounds. They also found that the production of aldehydes was also found to be exponentially dependent on concentration of flavoring compounds. Sucrose, a sweetener and flavor enhancer detected in e-liquids in concentrations from 0.76 to 72.93 g/g, has been also suggested as potential ingredient that may thermally degrade to produce carbonyl compounds (Kubica et al., 2014). Several studies have examined the potential exposure to carbonyl compounds from e- cigarettes. Using American Conference of Governmental Industrial Hygienists (ACGIH) standards, Khylstov and Samburova (2016) assessed e-cigarette users’ exposure risk to carbonyls from e-cigarettes. ACGIH defines the threshold limit value-ceiling (TLV-C) as the concentration that should not be exceeded during any part of the working exposure; the TLV-C for formaldehyde is 0.3 mg m 3, and, for acrolein, is 0.23 mg m 3. To compare exposure to these aldehydes from one puff, the authors divided the amount per puff by 500 mL, the average tidal volume of a healthy adult, and found that all flavored products of a single brand exceeded the ACGIH formaldehyde ceiling level by factors of 190-270 and the acrolein ceiling level by factors of 11-24, depending on the flavor used. Three of five liquids of the second brand tested exceeded the formaldehyde ceiling level by 2.0-13-fold, depending on the liquid flavor. No acrolein was detected in the second brand tested. All flavored products of the third brand tested exceeded the formaldehyde ceiling level by 2.9-66-fold and four products of the same brand exceeded the acrolein ceiling by 1.5-6.0-fold. The authors concluded that one puff of any of the tested flavored e-cigarette liquids exposes the smoker to dangerous levels of these two aldehydes. Jensen and colleagues (2015) extrapolated the formaldehyde dose from levels of formaldehyde-releasing agents (hemiacetals) detected in aerosol generated at high voltage. The high battery output voltage setting (5V) used by Jensen and colleagues resulted in excessive breakdown of PG to formaldehyde. The estimated daily dose of formaldehyde-releasing agent for an e-cigarette user vaping at a rate of 3 ml per day would be as high as 14.4 ± 3.3 mg. This dose is much higher than the estimated daily dose of formaldehyde from combustible tobacco cigarettes, which is approximately 3 mg per pack of 20 combustible tobacco cigarettes (150 g PREPUBLICATION COPY: UNCORRECTED PROOFS

TOXICOLOGY OF E-CIGARETTE CONSTITUENTS 5-27 per cigarette). Under the assumption that the risk per unit associated with inhaling formaldehyde- releasing agents is the same as the risk associated with inhaling gaseous formaldehyde, the authors estimated that long-term e-cigarette use is associated with an incremental lifetime cancer risk from inhaling formaldehyde of 4.2 × 10 3. This risk is from 5 to 15 times higher than the risk associated with inhaling formaldehyde during long-term combustible tobacco smoking. Wang and colleagues (2017) estimated that the daily exposure to formaldehyde and acetaldehyde for an e-cigarette user vaping at 215°C could reach 105 ± 117 g and 36 ± 42 g, respectively. This estimated daily formaldehyde exposure is above the “No Significant Risk Level” of 40 g/day from the California Office of Environmental Health Hazard Assessment (OEHHA, 2013). Same authors estimated that if the e-cigarette heating temperature exceeds 270°C, the formaldehyde generated from 10 50-ml puffs could reach levels similar to those from combustible tobacco smoking (comparisons of exposure to potentially toxic substances from e- cigarettes with combustible tobacco cigarette smoking are described in more detail in Chapter 18 on harm reduction). In summary, when e-liquids are heated and aerosolized, they can produce chemical reactions that could form carbonyl compounds such as reactive aldehydes, which are considered to have toxic effects on human health. At temperatures within the range of most e-cigarette products (150°-350°C), formaldehyde, acetaldehyde, and acrolein levels have been detected at levels that have raised concerns for chronic health endpoints (Jensen et al., 2015). MINOR TOBACCO ALKALOIDS Although the main alkaloid found in tobacco-derived products, including e-liquids, is nicotine, several minor tobacco alkaloids have been identified. The process by which nicotine in e-liquids is extracted from tobacco may produce some impurities including minor alkaloids: nornicotine, anatabine, anabasine, cotinine, nicotine-N-oxides, myosmine, beta-nicotyrine, and beta-nornicotyrine. These minor alkaloids may arise from biosynthetic processes in the living plant or by bacterial action or oxidation during tobacco processing (Gorrod and Jacob, 1999). Etter and colleagues (2013) analyzed samples of e-liquids from 20 bottles of 10 different brands using ultra-high performance liquid chromatography, and found that the level of minor tobacco alkaloids products constituted 1-2 percent of the nicotine content in most samples. The most common substances found were cis-N-oxide, trans-N-oxide, myosmine, anatabine, and anabasine. The authors hypothesized that oxidative degradation of nicotine during the manufacturing of the ingredient or of the final liquids, interactions with packaging material, inadequate handling and storage, or an unstable formulation could have resulted in the high amounts of nicotine-related impurities measured. Testing nicotine-containing e-liquids, Lisko and colleagues (2015) found minor tobacco alkaloids in all samples, and observed that their relative concentrations varied widely among manufacturers. eSmoke brand e-liquids had the highest concentrations of the minor tobacco alkaloids (6.3-48.2 g/g nornicotine, 8.7-62.7 g/g myosmine, 21.2-152 g/g anabasine, 63.1- 485 g/g anatabine, and 2.4-20.7 g/g isonicoteine). Other products tested contained considerably lower concentrations of minor tobacco alkaloids. These variations could be due to use of more pure nicotine extract of minimization of nicotine oxidation. These minor tobacco alkaloid concentrations in e-liquids are much lower when compared with combustible tobacco cigarettes, which have minor tobacco alkaloid concentrations in the range of 659-986 g/g for nornicotine, 8.6-17.3 g/g for myosmine, 127-185 g/g for anabasine, 927-1390 g/g for PREPUBLICATION COPY: UNCORRECTED PROOFS

5-28 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES anatabine and 23.4-45.5 g/g for isonicoteine (comparisons between e-cigarettes and combustible tobacco cigarettes are described in more detail in Chapter 18 on harm reduction). Flora and colleagues (2016) tested the liquids and aerosols of four MarkTen® e-cigarettes (rechargeable with disposable cartridges) for potential impurities and degradation products. They found that liquids contained 11-19 g/g of nicotine-N-oxides, undetectable levels to 9.4 g/g of cotinine, 14-31 g/g of nornicotine, and 7.4-13.0 g/g of myosmine. Regueiro and colleagues (2016) tested 12 e-cigarette liquids purchased from different vendors in the European Union. Among the nicotine-related compounds studied, the authors detected only anatabine, cotinine, myosmine, and nornicotine in any of the samples, and at concentrations in the microgram per milliliter level. Nicotine dehydrogenation also results in another alkaloid: nicotyrine. Considerable quantities of this nicotine analogue have been measured in an analysis of various e-cigarette aerosols (Martinez et al., 2014). Nicotyrine has been shown to hinder nicotine metabolism in mice (Stålhandske and Slanina, 1982). Therefore, its presence in e-cigarette aerosols could diminish smoking cravings by aiding nicotine absorption in the lungs, restraining metabolism, and consequently maintaining nicotine levels (Martinez et al., 2014). Nicotine purity varies by grade and manufacturer. The American E-Liquid Manufacturing Standards Association requires members to use U.S. Pharmacopeia (USP)- certified nicotine in e-liquids, although the group does not have regulatory authority (AEMSA, 2014). According to USP standards, nicotine solutions cannot exceed 0.5 percent (5 mg/g) of a single impurity, nor 1 percent (10 mg/g) of total impurities (U.S. Pharmacopeia, n.d.). Nicotine- related impurities are less toxic than nicotine, but the health effects of these minor tobacco alkaloids to e-cigarette users, especially at high levels, is unknown. TOBACCO-SPECIFIC NITROSAMINES TSNAs are potent carcinogenic chemicals (Hecht, 1998; Hecht and Hoffmann, 1988), which are derived from tobacco leaves and formed during the curing process via nitrosation of amines. Low levels of TSNAs have been reported in e-cigarette liquids and aerosol, typically at levels similar to those found in pharmaceutical nicotine products. This is probably attributed to the use of pharmaceutical grade nicotine that most manufacturers claim to use. This grade of nicotine is highly purified to remove the majority of impurities, including TSNAs. Using liquid chromatography-tandem mass spectrometry, Kim and Shin (2013) detected TSNAs in 105 refill liquid brands from 11 e-cigarette companies purchased in the Korean market. They measured TSNAs in concentration ranges of 0.34-60.08 g/L (64.8 percent detection frequency) for N'-nitrosonornicotine (NNN), 0.22-9.84 g/L (88.6 percent detection frequency) for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 0.11-11.11 g/L (54.3 percent detection frequency) for N'-nitrosoanabasine (NAB), and 0.09-62.19 g/L (75.2 percent detection frequency) for N'-nitrosoanatabine. Farsalinos and colleagues (2015b) evaluated the presence of selected tobacco-derived chemicals in liquids produced by extracting flavor from cured tobacco leaves and found that total nitrosamine concentrations varied from 2.5 to 38.5 ng/ml. In another study, Farsalinos and colleagues (2015a) also compared the levels of TSNAs in three commercial e-liquids and the aerosol from three 100-puff sets from each liquid trapped in filter pads. In two of the liquids, NAB was found at trace levels (1.2 and 2.3 ng/g); the third contained 1.5 ng/g NAB and 7.7 ng/g NNN (Farsalinos et al., 2015a). The authors found no TSNAs in the aerosol from the 100-puff sets. Finally, Goniewicz and colleagues (2014) analyzed PREPUBLICATION COPY: UNCORRECTED PROOFS

TOXICOLOGY OF E-CIGARETTE CONSTITUENTS 5-29 aerosol generated from 12 brands of e-cigarette and identified two nitrosamines (NNN and NNK) in all but three products. The NNN yelds ranged from 0.8 ng to 4.3 ng and NNK yields from 1.1 ng to 28.3 ng per 150 puffs. FREE RADICALS AND REACTIVE OXYGEN SPECIES (ROS) Reactive oxygen species (ROS), including free radicals, can stem from normal biologic processes as well as from external sources, such as tobacco smoke. ROS cause oxidative stress, which damages cellular proliferation, metabolism, and health and can be involved in the development of several cardiovascular (e.g., atherosclerosis), respiratory (e.g., chronic obstructive pulmonary disease, asthma), and neurodegenerative disorders (e.g., Parkinson’s disease, multiple sclerosis) as well as diabetes, rheumatoid arthritis, and some types of cancers (e.g., lung, colorectal) (Domej et al., 2014; HHS, 2010; Kehrer and Klotz, 2015; Kirkham and Rahman, 2006; Messner and Bernhard, 2014; Phaniendra et al., 2015; Prescott and Bottle, 2017; Pryor, 1997). E-cigarette users may be exposed to both highly reactive and more stable ROS during use. Activating the e-cigarette’s heating element and aerosolizing the e-liquid produce ROS; these species are drawn into the lungs directly from the device (Lerner et al., 2015b). This process is affected by the age of the heating element (Lerner et al., 2015a). Oxidants can also derive from a device’s lithium ion battery, similar to that used in combustible tobacco cigarette filters and e-cigarette cartomizers (Lerner et al., 2015a). Goel and colleagues (2015) identified free radicals from all e-cigarettes and e-liquids tested (at 3.3V, 2.5 × 1013 to 10.3 × 1013 radicals per puff), as well from glycerol and propylene glycol and during dry puff scenarios. Sussan and colleagues (2015) found 7 × 1011 free radicals per puff. In their mouse model, these free radicals caused oxidative stress and airway inflammation and disrupted anti-bacterial and anti-viral responses. Lerner and colleagues (2015b) similarly detected free radicals in a popular e-cigarette brand. In examining unaerosolized e-liquids, the authors found tobacco flavors were weaker oxidizers than sweet or fruity flavors (Lerner et al., 2015b). OTHER TOXICANTS Volatile Organic Compounds (VOCs) and Phenols Lim and Kim (2017) tested flavored e- liquids (283), nicotine liquids (21), and disposable cartridges (12) and detected 14 VOCs, including alcohols. Specifically, they detected VOCs in the following concentration ranges: benzene (0.008-2.28 mg/L), toluene (0.006-0.687 mg/L), ethylbenzene (0.01-1.21 mg/L), m-xylene (0.002-1.13 mg/L), p-xylene (0.007-2.8 mg/L), o- xylene (0.004-2.27 mg/L), styrene (0.011-0.339 mg/L), ethyl acetate (0.3-669.9 mg/L), ethanol (16-38,742 mg/L), methanol (66-3375 mg/L), pyridine (0.077-99.7 mg/L), acetylpyrazine (0.077-147 mg/L), 2,3,5-trimethylpyrazine (0.008-96.8 mg/L), and octamethylcyclotetrasiloxane (0.1-57.2 mg/L). According to the authors, the use of petrogenic hydrocarbons as a solvent in the extraction of flavor compounds and nicotine from natural plants may have produced benzene (classified as a group 1 carcinogen by IARC), toluene, ethylbenzene, m-xylene, p-xylene, and o- xylene. The maximum detected concentrations of benzene, methanol, and ethanol in the samples were higher than their authorized maximum limits as residual solvents in pharmaceutical PREPUBLICATION COPY: UNCORRECTED PROOFS

5-30 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES products. Farsalinos and colleagues (2015b) evaluated the presence of selected tobacco-derived chemicals in liquids produced by extracting flavor from cured tobacco leaves and found nitrate (levels varied from undetectable to 317.9 g/ml) and small amounts of phenols (total average 1.5 g/ml), including catechol, m-cresol and o-cresol, and phenol. Goniewicz and colleagues (2014) measured 11 VOCs in aerosol generated from 12 brands of e-cigarettes. Among 11 VOCs analyzed, only two (toluene and m,p-xylene) were found in almost all examined e-cigarettes. The yields of toluene ranged from 0.2 mg to 6.3 mg per one e-cigarette (150 puffs). Although the m,p-xylene levels found in analyzed samples of e-cigarette aerosol ranged from 0.1 mg to 0.2 mg/150 puffs, it was also found on the same level in blank samples. Microorganisms and Residual Solvents Varlet and colleagues (2015) analyzed 42 models from 14 brands of refill liquids for e- cigarettes for the presence of micro-organisms, diethylene glycol, ethylene glycol, hydrocarbons, ethanol, and solvents. All of the products tested contained some potentially toxic compounds. The authors detected diethylene glycol, ethylene glycol, and ethanol at levels within limits permitted for food and pharmaceutical products. The authors also found terpenic compounds and residual solvents such as 1,3-butadiene, cyclohexane, and acetone in some products. In compliance with norms, none of the liquids contained yeast, mold, aerobic microbes, Staphylococcus aureus, or Pseudomonas aeruginosa. Furans The thermal degradation of sugars can produce toxic furans, such as 5- hydroxymethylfurfural and furfural. Furfural is known to cause irritation to the upper respiratory tract in humans (Arts et al., 2004), and both furanic compounds showed tumorigenic activity in mice (Irwin, 1990; Surh and Tannenbaum, 1994; Surhet al., 1994). Soussy and colleagues (2016) investigated the formation of furanic compounds in e-cigarette aerosols using e-liquids of varying sweetener concentrations and devices under different power settings and puff durations. The authors detected both 5-hydroxymethylfurfural and furfural in the aerosols of sweet-flavored e-liquids. Levels of furans in the e-cigarette emissions were significantly correlated with power of the device and sweetener concentration, but not puff duration. The formation of furanic compounds from a sugar alcohol was negligible. Phthalates A recent study found diethyl phthalate (DEP) and diethylhexyl phthalate (DEHP) in e- liquids, although the quantified levels in the study’s sample were below phthalate exposure limits (Oh and Shin, 2015). DEP can be a solvent or plasticizer and is found in variety of consumer products, including fragrances, cosmetics, and detergent bases. DEHP is a plasticizer often used in making polyvinylchloride products. These antiandrogenic, estrogen-like compounds have been shown to initiate early breast development; IARC classifies DEHP as “possibly carcinogenic to humans” (IARC, 2000, p. 529). Researchers hypothesize DEP and DEHP could have originated from the e-liquid packaging or during the e-liquid production process (Oh and Shin, 2015). PREPUBLICATION COPY: UNCORRECTED PROOFS

TOXICOLOGY OF E-CIGARETTE CONSTITUENTS 5-31 Caffeine E-liquid flavors like coffee, tea, chocolate, and energy drinks, which are associated with having caffeine, often contain caffeine at concentrations significantly lower than their dietary counterparts. Lisko and colleagues (2015) measured caffeine concentrations in 44 flavored e- liquids from cartridges, disposables, and refill solutions. The researchers chose flavors traditionally associated with caffeine, marketed as energy boosters, or labeled as containing caffeine by the manufacturer. They detected caffeine in 42 percent of coffee-flavored products, 66 percent of tea-flavored products, and 50 percent of chocolate-flavored e-liquids in concentrations ranging from 3.3 g/g to 703 g/g. They did not detect caffeine in energy drink- flavored e-liquids. Eleven of twelve products marketed as energy enhancers contained caffeine in concentrations that varied substantially, ranging from 31.7 g/g to 9,290 g/g. Although the estimated caffeine exposures from e-cigarettes are at levels significantly lower than those from drinking caffeinated beverages, very little is known about the effects of caffeine inhalation, and health risks cannot be estimated. Pharmaceutical Drugs In addition to the toxicants described above, although rare, e-cigarette users may also be exposed to pharmacologic components in their devices’ e-liquids. For example, one study found evidence of a weight loss medication (rimonabant) originally approved in Europe in an analysis of e-liquids (Hadwiger et al., 2010). This treatment has been associated with adverse neurologic events such as seizures and suicide, and is not approved by FDA (2007). Furthermore, this study also found e-liquid can contain an analogue (amino-tadalafil) to the active ingredient in Cialis, an erectile dysfunction drug (Hadwiger et al., 2010). The potential exposure to medicinal compounds in some e-liquids places users at risk of experiencing undetermined or harmful health effects. SYNTHESIS • Many chemicals, other than nicotine, have been identified in liquids and aerosols generated from e-cigarettes. • Compounds not listed on labels have been identified in e-liquids. • Several hazardous compounds have been found in liquids and in the heated aerosol produced by e-cigarettes, including formaldehyde, acetaldehyde, and acrolein, which are known cancer-producing toxicants. • Of greater concern are the added flavorings that are considered safe for use in food, but have not been widely tested for sensitizing, toxic, or irritating potency. • E-cigarettes are a source of extremely high-particle doses in the human respiratory system. Fine particles are emitted when humectants (mostly propylene glycol and glycerol) are aerosolized.3 3 As described in Chapter 3, the particle count in e-cigarette aerosols may not be substantially different than main stream combustible tobacco smoke. However, whereas e-cigarette aerosol particulate consists largely of aqueous droplets and vapors of humectants, particulate matter in combustible tobacco smoke are complex, largely organic constituents that include known or suspected carcinogens. Thus, it would be incorrect to assume that the long-term health risks of the two aerosols were similar just because particle count was similar. PREPUBLICATION COPY: UNCORRECTED PROOFS

5-32 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES Conclusion 5-1. There is conclusive evidence that in addition to nicotine, most e- cigarette products contain and emit numerous potentially toxic substances. Conclusion 5-2. There is conclusive evidence that, other than nicotine, the number, quantity, and characteristics of potentially toxic substances emitted from e-cigarettes is highly variable and depends on product characteristics (including device and e-liquid characteristics) and how the device is operated. Conclusion 5-3. There is substantial evidence that except for nicotine, under typical conditions of use, exposure to potentially toxic substances from e- cigarettes is significantly lower compared with combustible tobacco cigarettes. METALS As discussed above, research on the chemical constituents of e-cigarettes has generally focused on nicotine, the carcinogens formaldehyde and acetaldehyde, flavoring compounds, and particles. An increasing number of studies have also found toxic metals such as lead, nickel, and chromium in e-liquid emissions (Aherrera et al., 2017; Farsalinos et al., 2015b; Goniewicz et al., 2014; Hess et al., 2017; Lerner et al., 2015a; Mikheev et al., 2016; Williams et al., 2013, 2017). Metal exposure may originate from several parts of the device, including the metallic coil, a complex alloy that heats the e-liquid to produce the aerosol that is inhaled by the user (Aherrera et al., 2017; Hess et al., 2017; Olmedo et al., 2018; Williams et al., 2017). Other parts of the device, such as the joints and wires, could also contribute. For example, Kanthal, an alloy frequently used in e-cigarettes, contains aluminum, chromium, and iron. Other common alloys are Ni-200, which is made of nickel, and Nichrome, which includes chromium and nickel. Furthermore, metals such as tin have been found in the joints (Williams et al., 2017). E-liquids may also contain metals at varying concentrations. For instance, some e-liquid solutions contain arsenic (Beauval et al., 2016; Mikheev et al., 2016). A small number of studies have investigated the role of e-cigarette aerosols in metal exposure. Most of these studies have evaluated one or two devices to measure metals in e- cigarette emissions and assess which metals are in higher concentrations compared with other metals, as well as to compare metals found in e-cigarette emissions and tobacco smoke. For example, Saffari and colleagues (2014) used quartz filters to study emission rates of a European tank-style device and found evidence of several metals. The authors detected boron (mean emission rate, ng/h: 964), cadmium (0.480), chromium (28.1), lanthanum (3.21), lead (96.2), nickel (131), potassium (7765), silver (20.9), titanium (50.2), and zinc (1142), but did not identify aluminum, copper, iron, or tin. However, the particle-sampling method the authors used in this study could have failed to distinguish metals during the aerosol phase. A study by Goniewicz and colleagues (2014) assessed metal concentrations in aerosols from a pharmaceutical nicotine inhaler and 12 e-cigarettes. Metals, including cadmium (concentrations varied from undetectable to 0.22 g/150 puffs), lead (0.03 to 0.57 g/150 puffs), and nickel (0.11 to 0.29 g/150 puffs) were found in most of the samples tested. Mikheev and colleagues (2016) used quartz filters and inductively coupled plasma mass spectrometry to study metals in aerosols from a tank-style device and cigalike products. The authors measured antimony (0.05 to 0.50 ng/mg), arsenic (0.01 to 0.70 ng/mg), chromium (0.40 to 5.0 ng/mg), copper (0.05 to 5.0 ng/mg), nickel (0.05 to 5.0 ng/mg), tin (0.02 to 0.50 ng/mg), and zinc (1.50 to 50.0 ng/mg) in most samples, but did not measure lead. In another study, Williams and colleagues (2013) detected PREPUBLICATION COPY: UNCORRECTED PROOFS

TOXICOLOGY OF E-CIGARETTE CONSTITUENTS 5-33 aluminum, iron, nickel, silver, and tin in particles greater than 1 m from one brand’s 22 cigalike cartomizers; nanoparticles (less than 100 nm) had chromium, nickel, and tin. The authors also used inductively coupled plasma optical emission spectrometry to identify lead (0.017 g/10 puffs). One of the key hypotheses is that metals in the coil leach during the heating process into the generated aerosol. For instance, Williams and colleagues (2013) describe the coil in their study of 22 cartomizers as a nickel-chromium filament soldered with tin to a thicker, silver- coated copper wire. The thick, copper-silver wire was also attached to the air tube and mouthpiece at tin solder joints (Williams et al., 2017).The same study group detected 35 of 36 selected elements in electronic hookahs and disposable e-cigarettes; in comparison, the authors found 15 of these elements in combustible tobacco cigarette smoke (Williams et al., 2017). Some metals, like copper, lead, nickel, and tin, were quantified at significantly higher concentrations in e-cigarette aerosols than combustible tobacco smoke, while levels of cadmium were lower. In an analysis of disposable e-cigarettes wires and joints using electron microscopy and energy- dispersive X-ray spectroscopy, nickel, chromium, copper, silver, zinc, iron, aluminum, tin, calcium, and lead were clearly detected in different parts of the device (see Figure 5-3). While many of the studies on e-cigarettes and metals have been done with first or second generation devices, a recent study has compared metal concentrations in e-liquid before being in contact with the device to metal concentrations in the aerosol generated after heating the coil of 56 modified e-cigarette devices from daily e-cigarette users (Olmedo et al., 2018). In the study, major increases in metal concentrations were found in aerosol samples compared with e-liquid samples for lead and zinc (increases greater than 2,000 percent) and chromium, nickel, and tin (increases greater than 600 percent). The finding of lead in e-cigarette aerosol samples, a metal not listed among the components of heating coils but that can be present in metal alloys or maybe in some other parts of the device, can be of concern. Aerosol mass concentrations for the detected metals (nickel, chromium, lead, manganese) spanned several orders of magnitude and exceeded current occupational or environmental standards for 50 percent of samples or more. In that study, 10 percent of the e-liquid samples had detectable arsenic concentrations and the levels remained similar in the aerosol (Olmedo et al., 2018). E-liquids may also acquire metals after they come in contact with e-cigarette coils. For example, one study found cadmium (mean concentration varied from 0.42 to 205 g/L), chromium (53.9 to 2,110 g/L), lead (4.89 to 1,970 g/L), manganese (28.7 to 6,910 g/L), and nickel (0.059 to 22.6 g/L) in e-liquids touching unused cartomizer coils from five different cigalike brands (Hess et al., 2017). By measuring five devices of each of five brands, this study illustrates the substantial variability within and across brands, especially for chromium, manganese, nickel, and lead (see Figure 5-4). Beauval and colleagues (2016) found generally low concentrations of metals in a study of e-liquids, with the exceptions of copper, nickel, and zinc (20, 16, and 200 /L, respectively). Furthermore, arsenic was measured in 57 percent of samples (mean concentration of 1.57 g/L). PREPUBLICATION COPY: UNCORRECTED PROOFS

5-34 PUBLI HEALTH CONSEQU IC H UENCES OF E-CIGARET TTES FIGURE 5-3 Scanning electron mic g croscopy and energy dispe d ersive X-ray s spectroscopy analysis of disposable EC/EH wire and joints. e es SOURCE Williams et al., 2017. E: t PR REPUBLICA ATION COP UNCO PY: ORRECTED PROOFS D

TOXICO OLOGY OF E-CIGARETT CONSTIT E TE TUENTS 5-35 FIGURE 5-4 Distribut tion of metal concentration within and across brand of disposab e-cigalike ns d ds ble devices. NOTE: Horizontal line within boxe indicate medians; boxes interquartile ranges; erro bars, values H es es s, or s within 1.5 times the int 5 terquartile ran solid circ nge; cles, outlying data points. g SOURCE Hess et al., 2017. E: To assess if metals are lea T m aking from the coil or ot t ther parts of the device, a direct f comparis of the me son etals in the e-liquid befo entering i contact w the coil w metals i e ore in with with in the aeros generated after heatin the coil would be nee ded. This research, inclu sol d ng w uding the comparis of aeroso concentrat son ol tions with metal standar in occupa m rds ational or ennvironmental l settings, is pending publication in the peer-re p n eviewed liter rature. So far, only one published study has compared m o d c metal concent trations in e- -cigarette emission to metal bi ns iomarker con ncentrations in an e-ciga s arette study. In that study conducted y, d among 64 daily e-cig 4 garette users (59 using se econd- and th third-generat tion devices and 5 using first-gene eration cigallikes), the lev of chrom vels mium and ni ickel were m measured in s several samp ples collected from the e-cigarette dev (dispenser, aerosol, and tank) u d vice used by the p participant, a as well as in several bio n omarkers col llected non-iinvasively: u urine, saliva, and exhaled breath , d condensa (EBC); data on e-ciga ate d arette use; an samples f nd from their e- -cigarette deevice (dispennser e-liquid, aerosol, and tank e-liqui (Aherrera et al., 2017 Median n d id) a 7). nickel and ch hromium lev vels were 0.73 and 0.39 g/g creatinin respective in urine , 2.25 and 1. 3 g ne, ely, .53 g/L in saliva, and 1. .25 and 0.29 g/L in EBC In adjusted models, tertiles 2 and 3 of aerosol nickel conc C. d centrations wwere associate with 16 pe ed ercent and 72 percent hig 2 gher urine niickel and 20 percent an 321 perce 02 nd ent higher saaliva nickel compared wi the lowes tertile. Ter c ith st rtile 3 of aer rosol chromi ium levels w were associate with 193 percent high saliva chr ed p her romium. An earlier time to first vape in the morn e e ning and more frequent co change were associate with high urine nick Tertile 2 of e-liquid e oil w ed her kel. d consump ption per wee and voltag were asso ek ge ociated with higher saliv nickel levels than terti 1. va ile Therefore, this study presents evi idence that participants’ internal dos of chromi p se ium and nickkel PR REPUBLICA ATION COP UNCO PY: ORRECTED PROOFS D

5-36 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES were positively associated with e-cigarette aerosol concentrations. Additional research is needed to evaluate the association between metal levels in e-cigarette emissions and metal biomarkers, as well as comparing metal biomarker levels in e-cigarette users and a comparable group of non- users. Exposure to metals through e-cigarettes is relevant as certain metals can cause serious health effects. For example, lead exposure is associated with neurotoxicity (Garza et al., 2006) and cardiovascular disease (Navas-Acien et al., 2007), and chromium (VI) and nickel have been associated with respiratory diseases such as lung cancer (IARC, 2012, 2017; Jaishankar et al., 2014). Nickel can also induce an allergic response in some individuals. Several cases of nickel- induced allergic dermatitis have been related to e-cigarette use (Maridet et al., 2015; Ormerod and Stone, 2017). Another concern is that metal absorption is markedly higher through inhalation, as compared with ingestion, and that while some of the metals found in e-cigarette aerosol are essential elements when ingested (zinc, manganese, Cu and chromium [III]), exposure to these metals through inhalation tends to be toxic (Goyer and Lavoie, 2001; Tchounwou et al., 2012). For chromium, no study has measured the valence state and it is currently unknown if the form of chromium in the aerosol is chromium (III) or chromium (VI). The implications could be major as chromium (VI) is an established carcinogen. Few studies have measured the toxic characteristics of metals in e-cigarette aerosols, although in principle metal toxicity would not necessarily change compared with metal exposure from other sources. In one of the few studies testing this metal e-cigarette toxicity, an in vitro study of copper nanoparticles from e-cigarette aerosols, it was found that copper nanoparticles increased mitochondrial oxidative stress and DNA fragmentation, supporting their critical toxic role (Lerner et al., 2016). Metals have also been involved as one the possible reason explaining cellular damage, generation of reactive oxygen species, and activation of global defense systems observed in vitro experiments (Bharadwaj et al., 2017; Lerner et al., 2015a). Limitations of the current research include the small number of studies, the small sample size, the evaluation mostly of first generation devices, the limited investigation on which characteristics of the device and patterns of use (e.g., wattage, temperature) could be major contributors of exposure, and the evaluation of devices that are selected by the investigators but which do not necessarily reflect what the consumers are using. Only one study has measured metal biomarkers in e-cigarette users, comparing chromium and nickel concentrations in e-liquid and e-cigarettes aerosols obtained from the participant’s devices (mostly tank-style and mod devices) to the corresponding metal biomarker (Aherrera et al., 2017). Synthesis An increasing but still limited number of studies have detected metals in e-liquid and aerosol samples generated by e-cigarette devices. Some of the key metals include chromium, nickel, lead, manganese, aluminum, tin, and iron. The coils and other parts of the device could be a source of metals, which could be leaking to the aerosol. Cadmium, which is a metal typically found in e-cigarettes, is found at a markedly lower level than in combustible tobacco cigarettes. However, the number of metals appears to be large, even larger than for combustible tobacco cigarettes. There is also substantial variability in metal levels, which can be substantially high in some instances. Overall the number of studies is small and the relevance for tank-style and mod devices is limited, as most studies have assessed first and second generation devices. One biomarker study evaluating e-cigarette devices actually used by the users supports that metals can be inhaled, contributing to metal internal dose, at least for chromium and nickel. While it is PREPUBLICATION COPY: UNCORRECTED PROOFS

TOXICOLOGY OF E-CIGARETTE CONSTITUENTS 5-37 well established that metals are highly toxic for multiple organs and systems through inhalation, no studies have evaluated the specific health effects of metals in e-cigarettes, except in the study of copper nanoparticles from e-cigarettes and mitochondrial oxidative stress and DNA fragmentation. Conclusion 5-4. There is substantial evidence that e-cigarette aerosol contains metals. The origin of the metals could be the metallic coil used to heat the e- liquid, other parts of the e-cigarette device, or e-liquids. Product characteristics and use-patterns may contribute to differences in the actual metals and metal concentrations measured in e-cigarette aerosol. Conclusion 5-5. There is limited evidence that the number of metals in e- cigarette aerosol could be greater than the number of metals in combustible tobacco cigarettes, except for cadmium, which is markedly lower in e-cigarettes compared with combustible tobacco cigarettes. REFERENCES Aberer, W., T. Fuchs, K. P. Peters, and P. J. Frosch. 1993. Propylene glycol: Cutaneous side effects and test methods. Literature and results of a multicenter study of the German contact allergy group (DKG). Dermatosen in Beruf und Unwelt 41:25-27. AEMSA (American E-Liquid Manufacturing Standards Association). 2014. E-liquid manufacturing standards. http://www.aemsa.org/wp-content/uploads/2014/02/AEMSA- Standards_Version-1-8.pdf (accessed October 9, 2017). Aherrera, A., P. Olmedo, M. Grau-Perez, S. Tanda, W. Goessler, S. Jarmul, R. Chen, J. E. Cohen, A. M. Rule, and A. Navas-Acien. 2017. The association of e-cigarette use with exposure to nickel and chromium: A preliminary study of non-invasive biomarkers. Environmental Research 159:313-320. Ahijevych, K., and B. E. Garrett. 2004. Menthol pharmacology and its potential impact on cigarette smoking behavior. Nicotine & Tobacco Research 6(Suppl 1):S17-S28. Ahijevych, K., and B. E. Garrett. 2010. The role of menthol in cigarettes as a reinforcer of smoking behavior. Nicotine & Tobacco Research 12(Suppl 2):S110-S116. Allen, J. G., S. S. Flanigan, M. LeBlanc, J. Vallarino, P. MacNaughton, J. H. Stewart, and D. C. Christiani. 2016. Flavoring chemicals in e-cigarettes: Diacetyl, 2,3-pentanedione, and acetoin in a sample of 51 products, including fruit-, candy-, and cocktail-flavored e- cigarettes. Environmental Health Perspectives 124(6):733-739. Arbour, R., and B. Esparis. 2000. Osmolar gap metabolic acidosis in a 60-year-old man treated for hypoxemic respiratory failure. Chest 118(2):545-546. Arts, J. H., H. Muijser, M. J. Appel, C. Frieke Kuper, J. G. Bessems, and R. A. Woutersen. 2004. Subacute (28-day) toxicity of furfural in Fischer 344 rats: A comparison of the oral and inhalation route. Food and Chemical Toxicology 42(9):1389-1399. Arulanantham, K., and M. Genel. 1978. Central nervous system toxicity associated with ingestion of propylene glycol. Journal of Pediatrics 93(3):515-516. ATSDR (Agency for Toxic Substances and Disease Registry). 2007. Acrolein. https://www.atsdr.cdc.gov/toxfaqs/tfacts124.pdf (accessed December 22, 2017). PREPUBLICATION COPY: UNCORRECTED PROOFS

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Millions of Americans use e-cigarettes. Despite their popularity, little is known about their health effects. Some suggest that e-cigarettes likely confer lower risk compared to combustible tobacco cigarettes, because they do not expose users to toxicants produced through combustion. Proponents of e-cigarette use also tout the potential benefits of e-cigarettes as devices that could help combustible tobacco cigarette smokers to quit and thereby reduce tobacco-related health risks. Others are concerned about the exposure to potentially toxic substances contained in e-cigarette emissions, especially in individuals who have never used tobacco products such as youth and young adults. Given their relatively recent introduction, there has been little time for a scientific body of evidence to develop on the health effects of e-cigarettes.

Public Health Consequences of E-Cigarettes reviews and critically assesses the state of the emerging evidence about e-cigarettes and health. This report makes recommendations for the improvement of this research and highlights gaps that are a priority for future research.

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