Electronic cigarette aerosol induces significantly less cytotoxicity than tobacco smoke

Figures & data

Figure 1. Borgwaldt RM20S smoking machine. (i) Cigarette smoke generator. (ii) Original four-syringe system. (iii) Four-syringe extension. (iv) Air-flow controller. (v) Cell culture media maintained at 37 °C. (vi) British American Tobacco’s exposure chamber housed at 37 °C, attached to the smoke diluter and culture media (modified from Adamson et al., 2011).

Figure 2. British American Tobacco’s standard exposure chamber used for in vitro exposures to aerosol at the air–liquid interface (a; Adamson et al., 2011 and b; Thorne & Adamson, 2013). Modifications to accommodate the three quartz crystal microbalance units (lid removed) (c; Adamson et al., 2013).

Figure 3. Vype ePen e-cigarettes are attached to the adapted Borgwaldt RM20S by bypassing the cigarette smoke generator.

Table 1. Specification of products and parameters.

	Product
Characteristics	3R4F	eStick	ePen
Aerosol	Reference cigarette smoke	E-cigarette aerosol	E-cigarette aerosol
Manufacturer	University of Kentucky (USA)	Vype (Nicoventures, UK)	Vype (Nicoventures, UK)
Length (mm)	84	84	153
Diameter (mm)	8	8	20 (10 at mouth piece)
Nicotine content (e-liquid)	N/A	36 mg/mL^a	18 mg/mL (1.8%)^a
Nicotine content (emissions)	0.7/2.0 mg/cig^b	0.079 mg/puff^c	0.055 mg/puff^c
TPM (3R4F)/ACM (e-cigs)	9.4/44.1 mg/cig^b	2.82 mg/puff^c	4.28 mg/puff^c
Puff number	8/10^b	120-150	>200
Voltage options? (v)	N/A	No	Yes (3.6 or 4.0)
Voltage used in study (v)	N/A	3.7 nominal	4
Cartridge used^a	N/A	Blended Tobacco^a	Blended Tobacco^a
Rechargeable?	N/A	Yes	Yes
Pre-coil activation	N/A	None (puff activated)	1 Sec (button activated)

TPM: total particulate matter; ACM: aerosol collected mass.

a As stated on the pack.

b Dependent on smoking regimen used (ISO vs. HCI); ISO data from University of Kentucky (2016) and HCI data from Eldridge et al. (2015).

c Based on 80/3/30 regimen.

Table 2. Specification of Regimens and experimental parameters.

	Study phase
Characteristics	Adapted Borgwaldt RM20S deposition assessment				Cytotoxicity assessment
Test product	Vype eStick		Vype ePen		3R4F	Vype ePen
Regimen	mISO	mHCI^a	mISO	mHCI	HCI	mHCI
Puff volume (mL)	35	55	35	55	55	55
Puff duration (s)	3	3	2^b	2^b	2	2^b
Puff interval (s)	60	30	60	30	30	30
Puff profile	Rectangular	Rectangular	Rectangular	Rectangular	Bell	Rectangular
Filter ventilation blocking	N/A	N/A	N/A	N/A	100%	N/A
Deposition exposure duration (min)/puff#	15/15	15/30	15/15	15/30	N/A	N/A
Cytotoxicity deposition exposure duration (min)/puff#	N/A	N/A	N/A	N/A	15/30	15/30
Cytotoxicity exposure duration (min)/puff#	N/A	N/A	N/A	N/A	60/120	60/120
Dilution range (smoke/aerosol:air, vol:vol)	1:40 - 1:5	1:40 - 1:5	1:40 - 1:5	1:40 - 1:5	1:5000 - 1:30	1:100 - 1:2

HCI: health Canada intense; mHCI: modified Health Canada intense; mISO: modified International Organization for Standardization; N/A: not applicable.

a CORESTA recommended method no. 81 (CORESTA, 2015).

b Button activated 1 s before puff commencement.

Figure 4. Deposited mass concentration of Vype eStick and Vype ePen e-cigarette aerosol generated over 15 minutes under two different regimens and various dilutions between 1:40 and 1:5 aerosol:air vol:vol, as quantified by quartz crystal microbalances within the in vitro exposure chamber. Data are represented as means and 95% confidence intervals of the fit, represented by the shaded region. Experiments were represented in the graphs by the individual points (n = 4 – 6 exposures per dilution). Abbreviations – HCI: Health Canada intense; ISO: International Organization for Standardization.

Table 3. Deposited eStick and ePen aerosol mass generated from a 15 minute exposure using modified ISO and HCI puffing regimens.

Aerosol dilutionaerosol:air (vol:vol)	Deposited mass (μg/cm^2)
	eStick		ePen
	mISO	mHCI	mISO	mHCI
1:40	0.23	0.63	0.72	1.28
1:20	0.54	0.99	1.83	3.02
1:10	1.48	2.06	4.18	7.24
1:5	3.17	4.99	11.47	14.32

mHCI: modified Health Canada intense; mISO: modified International Organization for Standardization.

Figure 5. Estimated mass (a) and nicotine (b) deposition of Vype ePen e-cigarette aerosol and 3R4F reference cigarette smoke generated over 60 minutes under modified and standard HCI regimens and various dilutions between 1:100 – 1:2 aerosol:air, vol:vol and 1:2500 – 1:30 smoke:air, vol:vol, respectively, as quantified by quartz crystal microbalances within the in vitro exposure chamber. Experiments are represented by the individual points (n = 4–6). Abbreviation – HCI: Health Canada Intense.

Table 4. Estimated deposited 3R4F and ePen smoke/aerosol and nicotine mass generated using the standard and modified HCI regimens respectively.

Aerosol dilution(smoke/aerosol:air, vol: vol)	15 minute deposited mass (μg/cm^2)		Estimated 60 minute deposited mass (μg/cm^2)		^aEstimated 60 minute deposited nicotine(μg/cm^2)
	3R4F	ePen	3R4F	ePen	3R4F	ePen
1:5000			^b0.77		0.07
1:2500	0.26		1.03		0.09
1:630	0.34		1.37		0.12
1:500	0.41		1.65		0.14
1:330	0.67		2.66		0.23
1:240	0.78		3.14		0.27
1:140	1.00		4.02		0.35
1:100		0.53		2.13		0.04
1:80	1.60		6.40		0.56
1:50	2.26		9.03		0.79
1:40	3.11	1.28	12.44	5.10	1.09	0.09
1:30	4.51		18.05		1.58
1:20		3.02		12.07		0.21
1:10		7.24		28.95		0.49
1:5		14.32		57.29		0.98
1:2		45.46		181.83		3.10

^aA conversion factor of mass*0.087 (3R4F) and mass*0.017 (ePen) was applied to the 60 minute deposited mass values. ^bEstimation based on the relationship between deposited mass and aerosol concentration using a linear fit model (R²=.99).

Figure 6. Changes in NCI-H292 cell viability (% of the air control) after exposure to various dilutions (Smoke/aerosol:air, vol:vol) of Vype ePen e-cigarette aerosol (n = 6 exposures per dilution) and 3R4F cigarette smoke (n = 8 exposures per dilution). Data are expressed according to (a) aerosol dilution (ePen EC₅₀=1:5, 3R4F EC₅₀=1:153 smoke/aerosol:air, vol:vol), (c) estimated deposited mass (ePen EC₅₀=52.1, 3R4F EC₅₀=3.1 μg/cm²) and (e) estimated deposited nicotine (ePen EC₅₀=0.89, 3R4F EC₅₀=0.27 μg/cm²). Data are represented as means (circles) and standard deviations (bars). Regression fit of the linear regions of Vype ePen e-cigarette (n = 6) and 3R4F cigarette aerosols (n = 8) NCI-H292 cell viability curves expressed according to (b) aerosol dilution, (d) estimated deposited mass and (f) estimated deposited nicotine. Data are represented as means and 95% confidence intervals of the fit, represented by the shaded region. Experiments are represented by the individual points.

Table 5. 3R4F and ePen EC₅₀ values expressed as smoke/aerosol dilution respectively, estimated deposited mass and estimated deposited nicotine.

Product	Aerosol dilution (aerosol:air, vol:vol) EC50	Estimated deposited mass (μg/cm^2) EC50	Estimated deposited nicotine (μg/cm^2) EC50
3R4F	1:153	3.1	0.27
ePen	1:5	52.1	0.89

Adamson J, Azzopardi D, Errington G, et al. (2011). Assessment of an in vitro whole cigarette smoke exposure system: the Borgwaldt RM20S 8-syringe smoking machine. Chem Cent J 5:50–60.

 PubMed Web of Science ®Google Scholar

Thorne D, Adamson J. (2013). A review of in vitro cigarette smoke exposure systems. Exp Toxicol Pathol 65:1183–93.

 PubMed Web of Science ®Google Scholar

Adamson J, Thorne D, McAughey J, et al. (2013). Quantification of cigarette smoke particle deposition in vitro using a triplicate quartz crystal microbalance exposure chamber. Biomed Res Int 2013:685074.

 PubMed Web of Science ®Google Scholar

University of Kentucky: 3R4F reference cigarette. Available from: https://ctrp.uky.edu/resources/pdf/webdocs/3R4F%20Preliminary%20Analysis.pdf [last accessed 1 June 2016].

 Google Scholar

Eldridge A, Betson TR, Vinicius Gama M, McAdam K. (2015). Variation in tobacco and mainstream smoke toxicant yields from selected commercial cigarette products. Regul Toxicol Pharmacol 71:409–27.

 PubMed Web of Science ®Google Scholar

CORESTA. (2015). CORESTA recommended method No 81. Routine analytical machine for e-cigarette aerosol generation and collection – Definitions and Standard Conditions. CRM No 81. Available from: https://www.coresta.org/sites/default/files/technical_documents/main/CRM_81.pdf [last accessed 20 July 2016].

 Google Scholar