Elsevier

Toxicology in Vitro

Volume 28, Issue 2, March 2014, Pages 198-208
Toxicology in Vitro

Identification of toxicants in cinnamon-flavored electronic cigarette refill fluids

https://doi.org/10.1016/j.tiv.2013.10.006Get rights and content

Highlights

  • Most cinnamon-flavored refill fluids were cytotoxic to embryonic and adult cells.

  • Human embryonic stem cells were more sensitive than adult pulmonary fibroblasts.

  • Four chemicals were identified in cinnamon-flavored refill fluids.

  • Dipropylene glycol and vanillin had low cytotoxicity.

  • Cinnamaldehyde and 2-methoxycinnamaldehyde were cytotoxic in the MTT assay.

Abstract

In a prior study on electronic cigarette (EC) refill fluids, Cinnamon Ceylon was the most cytotoxic of 36 products tested. The purpose of the current study was to determine if high cytotoxicity is a general feature of cinnamon-flavored EC refill fluids and to identify the toxicant(s) in Cinnamon Ceylon. Eight cinnamon-flavored refill fluids, which were screened using the MTT assay, varied in their cytotoxicity with most being cytotoxic. Human embryonic stem cells were generally more sensitive than human adult pulmonary fibroblasts. Most products were highly volatile and produced vapors that impaired survival of cells in adjacent wells. Cinnamaldehyde (CAD), 2-methoxycinnamaldehyde (2MOCA), dipropylene glycol, and vanillin were identified in the cinnamon-flavored refill fluids using gas chromatography–mass spectrometry and high-pressure liquid chromatography (HPLC). When authentic standards of each chemical were tested using the MTT assay, only CAD and 2MOCA were highly cytotoxic. The amount of each chemical in the refill fluids was quantified using HPLC, and cytotoxicity correlated with the amount of CAD/product. Duplicate bottles of the same product were similar, but varied in their concentrations of 2MOCA. These data show that the cinnamon flavorings in refill fluids are linked to cytotoxicity, which could adversely affect EC users.

Graphical abstract

Flow diagram showing the steps used to identify the toxicants in cinnamon-flavored refill fluids.

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Introduction

Electronic cigarettes (EC), which deliver nicotine to users without burning tobacco, are rapidly gaining popularity worldwide (Ayers et al., 2011, Etter et al., 2011, McQueen et al., 2011). The original EC consisted of a cartridge with nicotine-containing fluid and an atomizer which aerosolized the cartridge fluid when heated by a battery (Trtchounian et al., 2010). In many newer models, the cartridge and atomizer are combined into a single unit, termed a “cartomizer” (Williams and Talbot, 2011). Cartridge/cartomizer fluid contains nicotine, flavorings, and a humectant, such as propylene glycol (Bahl et al., 2012, Laugesen, 2008). Nicotine concentrations usually range from 0 to 24 mg/ml. Used cartomizers can be replaced or refilled with fresh fluid, referred to as refill fluid (Bahl et al., 2012). Although the basic design of EC is similar across brands, significant variation in performance exists between and within brands (Trtchounian et al., 2010, Williams and Talbot, 2011). EC and their associated products are sold in shops, malls, and online where age verification is not always needed, making these products relatively accessible.

Several recent online surveys and interviews found that EC may help users limit or stop smoking conventional cigarettes (Etter, 2010, Etter and Bullen, 2011, Goniewicz et al., 2013, McQueen et al., 2011). Nevertheless, some users are concerned about the toxicity of EC (Etter, 2010, Etter and Bullen, 2011), while others acknowledge that EC are addictive and may not be completely safe, but consider them less harmful than conventional cigarettes (Goniewicz et al., 2013).

EC aerosol contains relatively few chemicals (Goniewicz et al., 2012, Laugesen, 2008, Westenberger, 2009), suggesting they are safer to use than conventional cigarettes. However, significant amounts of tin were present in the fluid of one brand of EC, and the corresponding aerosol contained metals, including metal nanoparticles (Williams et al., 2013). In a clinical case report, a woman was diagnosed with exogenous lipoid pneumonia seven months after she started using EC (McCauley et al., 2012), and her condition improved when she stopped EC use. Lipoid pneumonia was thought to be caused by inhaling aerosolized EC oil-based humectants, which lead to dyspnea, productive cough, and subjective fevers. A second recent study examined the effect of EC use on respiratory mechanics and the fraction of exhaled nitric oxide in healthy smokers. Individuals ad-lib puffed for 5 min, during which time EC use caused an increase in impedance, peripheral airway flow resistance, and oxidative stress (Vardavas et al., 2012). In a recent infodemiological study, numerous symptoms attributed to EC were self-reported in Internet forums by EC users (Hua et al., 2013). These studies show that the safety of EC cannot be assumed and that EC may cause their own set of health problems, which are not necessarily found with conventional cigarette use.

Recent in vitro studies of cytotoxicity suggest that EC products differ in their potential to adversely affect health. In our prior in vitro screen, EC refill fluids varied widely in their cytotoxicity when tested with human embryonic stem cells (hESC), mouse neural stem cells (mNSC), and human pulmonary fibroblasts (hPF) (Bahl et al., 2012). The stem cells were generally more sensitive to refill fluids than differentiated adult lung cells. The same study also showed that the flavoring chemicals and their concentrations varied among refill fluids of the same flavor both within and between manufacturers. In addition, the cytotoxicity of EC refill fluids correlated with the number and concentration of chemicals used for flavoring.

In our prior refill fluid screen, Cinnamon Ceylon was the most cytotoxic of 36 products that were tested (Bahl et al., 2012). The purpose of the current study was to determine if cinnamon-flavored EC refill fluids are generally cytotoxic and to identify the toxicant(s) in Cinnamon Ceylon. Eight additional cinnamon-flavored refill fluids were screened for cytotoxicity. The chemicals in Cinnamon Ceylon were determined using GC–MS, and authentic standards of the identified chemicals were tested to establish the potency of each. The amount of each chemical in the cinnamon-flavored refill products was quantified with HPLC, and correlations were made between the concentrations of the chemicals and the cytotoxicity of each product tested.

Two cell types were used to evaluate cytotoxicity. hESC, which resemble post-implantation epiblast cells (Nichols and Smith, 2009), were chosen as a model for an early stage of prenatal development and could therefore be useful in identifying products that may be embryotoxic. hPF were used to model effects that could occur in lungs following inhalation of EC refill fluid vapors. It is well established that conventional cigarette products can effect lung fibroblasts and lead to disease development (Hallgren et al., 2010, Selman and Pardo, 2002, Kitamura et al., 2011, Togo et al., 2008). These cell types were also used in our prior study (Bahl et al., 2012) and therefore allow comparison to prior our work and to planned future work involving aerosols.

Section snippets

Sources of refill fluids and chemicals

Ten cinnamon-flavored EC refill products (inventory numbers = #22, #42, #53, #54, #58, #60, #61, #62, #65, #69) were purchased from online vendors. Refill fluid #53 and #69, Sinful Cinnamon, are duplicate purchases from Tasty Puff (Albuquerque, NM). Refill fluid #60, Cinnamon, and #61, Cinnabun, were both purchased from e-cigexpress (Orlando, FL). Refill fluids #22, Cinnamon Ceylon FlavourArt, #42 Cinnamon, and #54, Cinnamon FlavourArt, were purchased from Freedom Smoke USA (Tucson, AZ), #58,

Cinnamon Ceylon produced a vapor effect

EC refill fluids contain volatile organic chemicals that can transfer to adjacent wells and effect cell viability (Behar et al., 2012a), thus causing an erroneous leftward shift in dose response curves. In the initial MTT screen with Cinnamon Ceylon, a vapor effect was observed (not shown). To quantify how much Cinnamon Ceylon was transferred to adjacent wells, wells containing 1% or 0.3% Cinnamon Ceylon solution and wells containing only water in a cross pattern in a 96-well plate were read at

Discussion

The rapid growth in worldwide sales of EC and their associated products make it important to understand their effects on human health (Etter et al., 2011, Hua et al., 2013, Williams et al., 2013). This study evaluated the volatility and cytotoxicity of 10 cinnamon-flavored EC refill fluids, compared their cytotoxicity using prenatal (hESC) and adult (hPF) models, and identified chemicals in these fluids that are causing cytotoxicity. Nicotine concentration did not correlate with cytotoxicity,

Conflict of Interest

The authors have no conflict of interest to declare.

Acknowledgments

This work was supported by the Tobacco-Related Disease Research Program of California (TRDRP XT-0118), and the California Institute of Regenerative Medicine (NE-A0005A-1E). Rachel Behar and Barbara Davis were supported by Cornelius Hopper Fellowships from the Tobacco-Related Disease Research Program of California and Dean’s Fellowships. Dr. Sabrina Lin was supported by a Tobacco-Related Disease Research Program of California postdoctoral Fellowship. We thank Michael Dang and Jisoo Kim for

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