SOT 2014 ecigs lorillard

POTENTIAL TOXICITY OF ELECTRONIC CIGARETTE LIQUIDS AND AEROSOLS AS MEASURED BY
FOUR IN VITRO ASSAYS
Leverette, R.D., Misra, M., Cooper, B.T. and Bennett, M.B.
Lorillard Tobacco Company, A.W. Spears Research Center, Greensboro, NC 27405, USA
ABSTRACT #1015
ABSTRACT
RESULTS
A.
blu™ M M -High
100
M arlboro SNUS
Copenhagen Snuff
0
Lozenge
Whole Smoke (WS) and Whole Aerosol (WA) exposures performed on a
dilution air flows ranging from 0.1 – 8.0 L / min.
VC10,
Ames Modules, with smoke
Smokeless Tobacco aqueous extracts prepared from commercially available products (Marlboro SNUS, Copenhagen Snuff, Nicorette ®
Lozenge Original 4 mg Nicotine). Samples suspended in Phosphate Buffered Saline @ 80 mg / mL (Dulbecco’s PBS, Gibco #14040,
+MgCl2 +CaCl2), incubated @ 37°C for 21-24 hrs @ 150 rpm shaking, centrifuged @ 12K g for 10 min to remove particulates, filter
sterilized, aliquoted and stored @ -80°C prior to analysis1.
3R4F
400
150
50
M arlboro SNUS
Nicorette  Lozenge
Control E-cig
25,000
0
20,000
Copenhagen Snuff
blu M M High
0
g / mL
25,000
M arlboro SNUS
blu M M -
20,000
blu M M High
blu CT High
15,000
blu M M -
blu CT-
100
10,000
blu CT High
M arlboro Gold
0
20
40
60
80
100
120
140
5,000
% Control (DMSO)
blu CT-
100
Table 1: NRU EC50 (µg / mL) values for
1R5F
M arlboro Gold
200
Nicorette  Lozenge
Control E-cig
3R4F
1R5F
300
Copenhagen Snuff
Micronucleus: Cell Viability
induction is not due to cytotoxicity at higher doses. All activities for E-cig liquids and smokeless tobacco extracts significantly different when compared to WTPM from
traditional tobacco burning cigarettes (p < 0.001).
MN frequency (%MN) determined using Cellomics® ArrayScan® VTI (Micronucleus Bioapplication, V.4). MN activity reported as % control
and compared using GraphPad Prism v. 5.04 (two tailed; for comparisons, statistical significance @ p < 0.05).
SMOKE & E-CIG AEROSOL DELIVERIES
WS & WA: VITROCELL® Laser Photometers positioned between dilution system and exposure module with 35 mm plates containing 4 mL
DMSO (dH2O for WA). Dilution air flow rates 0.1 – 8.0 L / min. For WS, Ex / Em: 355nm / 485nm measurements of smoke-exposed DMSO
extrapolated to WTPM standard curve2. WA (E-cig) dose correlated to glycerol captured in aerosol-exposed dH2O quantified with Free
Glycerol Reagent (Sigma # FG0100). Plot of laser photometer values (Volt*sec, Area Under Curve) versus quantified smoke particulate
(WS) or glycerol (WA) used to calculate delivered doses.

blu™ M M High
E.
400
Micronucleus: Cell Viability
200
M arlboro Gold
blu CT-
200
blu CT High
blu M M -
100
blu M M High
% Control (DMSO)
1R5F
3R4F
1R5F
150
M arlboro Gold
blu CT-
100
blu CT High
blu M M -
50
blu M M High
g / mL
g / mL
25,000
20,000
15,000
0
10,000
Control E-cig
0
20
40
60
80
100
120
140
5,000
25,000
20,000
Control E-cig


g / mL
3R4F
300
3,500
0
g / mL
Micronucleus (MN)
0
blu™ M M -Ø
100
3,500
3,000
2,500
2,000
1,000
0
1,500
blu™ M M High
blu™ CT High
3,000
blu™ M M -Ø
50

blu™ CT-Ø
200
2,500
blu™ CT High
M arlboro Gold
2,000
blu™ CT-Ø
1R5F
300
1,500
M arlboro Gold
100
3R4F
1,000
1R5F
400
500
150
IL-8 Release
0
3R4F
IL-8 Release (%Control)
200
M
ar
lb
1R
or 5F
o
G
ol
d
C
on
bl
t
u  rol
bl
u  CT
-
C
T
H
bl
ig
u
h
bl  M
u
M
-
M
M
H
ig
h
3R
4F
0
C.
NRU
500
Cell Viability (% Control)
1
15,000
Cells exposed (absence of S9) to increasing doses of samples (E-cig liquids, smokeless extracts, pad-collected smoke and aerosols) and
incubated for 20 hrs @ 37°C, 5% CO2 followed by treatment with cytochalasin B (cytokinesis blocking agent) for 27.5 hrs @ 37°C, 5% CO2
(Cellomics® Micronucleus Kit K11-0001-1; Thermo Scientific). Cell viability determined by cytokinesis-block proliferation index (CBPI).
TA98
TA100
10,000
MICRONUCLEUS (MN)
Chinese hamster ovary (CHO-K1, ATCC# CCL-61) cells seeded (~2500 cells / well) and incubated (complete media: F-12K media + 10%
FBS) in 96-well plates overnight (37°C, 5% CO2) prior to exposures.
B.
2000
1750
1500
1250
1000
750
500
250
2
D.


Ames (S9+)
0
A.
% Control (DMSO)
EC50 (NRU) and IL-8 (pg / mL; % control) calculated and compared using GraphPad Prism v. 5.04 (two tailed; for comparisons, statistical
significance @ p < 0.05).
blu™ CT-Ø
100
blu™ CT-High
blu™ M M -Ø
50
blu™ M M -High
aerosols. Data points in each plot
represent the mean values ± SD from a
minimum
of
two
(2)
independent
experiments. All E-cig whole aerosol
0
0
100 200 300 400 500 600 700 800
g / mL
activities significantly different when
compared
to
whole
smoke
from
whole
300
smoke / aerosol
exposures
under development.
3R4F
1R5F
200
M arlboro Gold
blu™ CT-Ø
blu™ CT-High
100
blu™ M M -Ø
blu™ M M -High
0
100 200 300 400 500 600 700 800
TABLE 3: Whole Smoke & Whole E-cig
Aerosols
NRU
Sample
SE
EC50† (µg / mL)
171.2
6.8
3R4F
157.6
17.9
1R5F
164.5
28.1
Marlboro Gold
Table 3: NRU EC50 (µg / mL) values for
tobacco
smoke
burning
only
cigarette
(mean
±
whole
SE).
†No
cytotoxicity detected for E-cig whole
aerosols.
SUMMARY
MN (D, E). Data points in each plot represent the mean values ± SD from a minimum of two (2) independent experiments. MN cell viability (E) shown to verify lack of MN
NEUTRAL RED UPTAKE (NRU)4,5 & IL-86
A549 (human lung epithelial carcinoma cell line, ATCC# CCL-185) cells seeded (~15K cells / well) and incubated (complete media: F-12K +
10% FBS) in 96-well plates overnight (37°C, 5% CO2) prior to exposures.
WS & WA Exposures: A549 cells seeded in 35 mm culture dishes @ 400K cells / dish in 2 mL complete media and incubated overnight
(37°C, 5% CO2). Just prior to exposures, complete media removed and cells washed 1X in DPBS. Added 1 mL incomplete media (-FBS)
and exposed to either 5 tobacco burning cigarettes (8 puffs / cigarette) or 200 puffs from E-cig. After exposure added 1 mL complete
media and incubated for 20 – 24 hrs @ 37°C, 5% CO2 followed by NRU4,5 and IL-86 analyses.
done for whole smoke and whole
M arlboro-Gold
g / mL
Figure 1: In vitro activity of E-cig liquids, smokeless tobacco and lozenge aqueous extracts, and pad-collected smoke particulate (WTPM) in Ames (A), NRU (B), IL-8 (C), and
Activity reported as revertants per mg was calculated from the linear portion of the dose response curve and compared using GraphPad
Prism v. 5.04 (slope analysis, two tailed; for comparisons, statistical significance @ p < 0.05).
Cells exposed to increasing doses of samples (E-cig liquids, smokeless extracts, pad-collected smoke and aerosols) and incubated for 20
– 24 hrs @ 37°C, 5% CO2 followed by NRU4,5 and IL-86 analyses.
1R5F
preparations.
0
20
40
60
80
100
120
140
5,000
Revertant colonies counted after 48 hrs of incubation @ 37°C.
Ames (A), NRU (B) and IL-8 (C). MN not
detected for E-cig, smokeless and NRT
Revertants / mg
WS & WA Exposures: TA98 or TA100 @ ~2 – 4 X
bacteria / mL in 200 µL S9-Mix (5% v/v) were spread on fresh 0.4% minimal glucose
agar plates (35 mm) supplemented with 0.05 mM Histidine / Biotin, exposed to WS or WA from 3 tobacco burning cigarettes or 200 puffs
from E-cig.
109
3R4F
IL-8 Release
0
WTPM only (mean ± SE). †No cytotoxicity
g / mL
PAD-COLLECTED SMOKE & E-CIG AEROSOLS
Preincubation assays: 100 µL of Salmonella strains TA98 or TA100, 500 µL S9-Mix (5% v/v), 25 µL sample, 20 min preincubation @ 37°C,
250 rpm shaking followed with the addition of 0.05 mM Histidine / Biotin top agar (2.5 mL) and plated onto minimal glucose agar plates.
C.
IL-8 Release (% Control)
E.
Micronucleus (MN)
AMES(2,3):
S9-Mix: 33mM KCl, 8mM MgCl2, 5mM Glucose-6-phosphate, 4mM NADP, sodium phosphate buffer (0.1M, pH 7.4), S9 fraction @ 5% v/v
(Aroclor 1254-induced male Sprague-Dawley rat liver in 0.15M KCl; Moltox; Boone, NC).
150
traditional tobacco burning cigarettes
TABLE 1: E-cig Liquids &
Smokeless Extracts
NRU
Sample
EC50†
SE
(µg / mL)
195.7
8.0
3R4F
235.3
9.2
1R5F
201.5
11.2
Marlboro Gold
All cigarettes & E-cigs smoked on a VITROCELL® VC10 smoking robot under Canadian Intense (CI) puff profile: 55 mL puff volume, 2 sec
draw, 30 sec puff interval, 100% blocked air dilution.
VITROCELL®
smoke and whole E-cig aerosols in
200
M
g / mL
g / mL
M
D.
Electronic Cigarettes (E-cig):
E-cigs, Classic Tobacco (CT) and Magnificent Menthol (MM). Nicotine @ 0 mg / mL (Ø; rechargeable)
or 24 mg / mL (High; disposable). Batteries charged immediately prior to vaping (rechargeable only). Control E-cig: disposable E-cig
containing a glycerol / water mixture, no flavors or nicotine.
VITROCELL®
Lozenge
Figure 3: In vitro activity of whole
NRU
(p < 0.001). Micronucleus methods for
blu™
Wet Total Particulate Matter (WTPM) and E-cig aerosols collected on Cambridge filter pads, extracted in dimethylsulfoxide (DMSO) to a
final concentration of 40 mg / mL, stored at -80°C prior to analysis.
Nicorette

Cell Viability (% Control)
blu™ M M -Ø
1R
or 5F
o
G
ol
d
C
on
bl
tr
u
o
bl  C l
u
T
C
TH
bl
ig
u
bl  M h
u
M
-
M
M
-H
ig
h
Nicorette

blu™ CT-High
200
ar
lb
Copenhagen Snuff
blu™ CT-Ø
B.
TA98
TA100
3R
4F
M arlboro SNUS
Revertants / mg
blu™ M M -High
M arlboro Gold
300
300
1,000
4,000
7,000
10,000
13,000
16,000
19,000
22,000
25,000
28,000
blu™ M M -Ø
50
1R5F
200
blu™ CT-High
6500
5500
4500
3500
2500
1500
500
15
10
5
0
3R4F
400
0
blu™ CT-Ø
Ames (S9+)
IL-8 Release
100
M arlboro Gold
100
0
100
200
300
400
500
1,000
4,000
7,000
10,000
13,000
16,000
19,000
22,000
25,000
28,000
Cell Viability (% Control)
1R5F
0
WHOLE SMOKE & WHOLE E-CIG AEROSOLS
C.
3R4F
150
3R
4F
ar
lb 1R
or 5
o F
G
o
C ld
bl o n
t
u
bl  rol
u C
T
C -
bl T H
u
bl  igh
u M
M
M M- 
a
M
C
op rlb
o H
N en ro igh
ic ha S
or
N
et gen US
te  S
Lo nuf
ze f
ng
e
Revertants / mg
TA98
TA100
NRU
IL-8 Release (% Control)
B.
2000
1750
1500
1250
1000
750
500
250
6
4
2
0
15,000
CIGARETTE SMOKE PREPARATIONS & EXPOSURES:
Tobacco burning cigarettes (3R4F, 1R5F, Marlboro Gold) conditioned (60% relative humidity, ~23°C) at least 18 hours prior to smoking.
Ames (S9+)
10,000
MATERIALS & METHODS
A.
0
20
40
60
80
100
120
140
5,000
parameters (55 mL puff volume, 2 second puff duration, 30 second puff interval, 100% blocked air dilution). At the
levels tested, exposures with neat E-cig liquids and pad-collected aerosols showed no-to-extremely low activity in
the Ames, NRU, MN and IL-8 assays when compared to responses from the traditional tobacco burning cigarettes.
Results from E-cig samples without nicotine were very similar in all assays, indicating that the presence of
nicotine, at the levels tested, did not significantly contribute to any cytotoxic and genotoxic effects observed at
high doses. Whole smoke and whole E-cig aerosols were tested in the Ames, NRU and IL-8 assays, again resulting
in no-to-extremely low activity when compared to traditional tobacco burning cigarettes. Overall, under the
experimental conditions used to evaluate traditional tobacco burning cigarettes, E-cigs did not produce any
meaningful toxicological effects as measured by four in vitro endpoints: Ames, NRU, IL-8, and Micronucleus.
E-CIG LIQUIDS & SMOKELESS EXTRACTS
% Control (DMSO)
The popularity of electronic cigarettes (E-cig) continues to increase worldwide, with several major tobacco
companies entering the E-cig market. A typical E-cig delivers a flavored propylene glycol or glycerol based
aerosol, with or without nicotine, via vaporization by a battery-powered heating element. Currently, data regarding
the potential toxicity of E-cigs is limited. To further our understanding, an in vitro battery of established assays
was used to examine the mutagenicity (Ames), cytotoxicity (Neutral Red Uptake; NRU), genotoxicity
(Micronucleus; MN) and inflammatory (IL-8 release) response of a set of glycerol-based commercial E-cigs,
specifically the neat E-cig liquids, pad-collected aerosols and freshly generated whole aerosols. Pad-collected
smoke condensates and whole smoke from traditional tobacco burning cigarettes (3R4F, 1R5F and one
commercial cigarette) as well as aqueous extracts from smokeless tobacco and nicotine replacement therapy
products were included for comparison. All E-cigs and traditional cigarettes were smoked under Canadian Intense
TABLE 2: Pad-Collected Smoke &
E-cig Aerosols
NRU
Sample
EC50†
SE
(µg / mL)
195.7
10.5
3R4F
237.4
14.1
1R5F
203.8
9.8
Marlboro Gold

E-cigs VS. Tobacco WTPM: At doses up to 100 X higher than typical cigarette smoke exposures, E-cig liquids and pad-collected
aerosols had no-to-extremely low in vitro activity (Ames, NRU, IL-8 & MN) when compared to WTPM from tobacco burning cigarettes
(WTPM activity was up to ~ 6000 X higher than E-cigs).
E-cigs VS. Smokeless & NRT: E-cig liquids demonstrated similar no-to-extremely low in vitro activity as aqueous extracts from a
commercial nicotine lozenge and commercial smokeless tobacco products (SNUS & Snuff).
E-cigs VS. Tobacco Whole Smoke: Direct exposure of freshly generated E-cig aerosols did not produce any significant levels of in
vitro activity (Ames, NRU & IL-8) when compared to fresh whole smoke from tobacco burning cigarettes (cigarette whole smoke
activity up to ~ 1300 X higher than E-cigs). Micronucleus whole smoke / aerosol exposure methods currently under development.
Effect of Nicotine: In vitro activities (Ames, NRU, IL-8 & MN) measured for E-cig exposures, with and without nicotine, were similar for
all sample types, indicating that the presence of nicotine, at the levels tested, did not significantly contribute to any toxicological
effects.
Effect of Flavors: In vitro activities (Ames, NRU & MN) for the commercial E-cigs were indistinguishable from control (glycerol /
water); indicating these flavors (CT & MM), at the levels tested, had no detectable impact on the cytotoxicity and genotoxicity
endpoints utilized in this study.
Liquid VS. Aerosol: In vitro results for E-cigs, in this study, were similar for the different exposure methods (liquids, pad-collected &
freshly generated whole aerosols); demonstrating no detectable impact on the in vitro toxicological responses when the liquids were
aerosolized.
Overall: Under the experimental conditions used to evaluate traditional tobacco burning cigarettes, E-cigs did not produce any
meaningful toxic effects as measured by four in vitro endpoints: Ames (bacterial mutagenicity), NRU (cytotoxicity), IL-8
(inflammation) and Micronucleus (genotoxicity). These results demonstrate the potential for E-cigs to significantly reduce the
toxicological impact when compared to traditional tobacco burning cigarettes.
REFERENCES
Table 2: NRU EC50 (µg / mL) values for
1.
Rickert, W.S. et al (2007) A comparative study of the mutagenicity of various types of tobacco products. Regulatory Toxicology and Pharmacology, 48, 320-330.
†No
2.
Aufderheide, M & Gressmann, H. (2007) A modified Ames assay reveals the mutagenicity of native cigarette mainstream smoke and its gas vapour phase.
Experimental and Toxicologic Pathology, 58, 383-392.
3.
Maron, D. M. & Ames, B. N. (1983) Revised methods for the Salmonella mutagenicity test. Mutation Research, 113, 173-215.
4.
Borenfreund, E. & Puerner, J.A. (1985) Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicology Letters, 24, 119-124.
5.
Bombick, D.W. & Doolittle D.J. (1995) The role of chemical structure and cell type in the cytotoxicity of low molecular weight aldehydes and pyridines. In Vitro
Toxicology 8, 349-356.
6.
Human IL-8 ELISA Kit. For the quantitative determination of human IL-8 concentrations in serum, plasma, cell culture supernatant and other biological fluids.
Catalog #EL10008. Doc. #S7.5(02)IL-8. Abazyme, LLC.
WTPM
only
(mean
±
SE).
cytotoxicity detected for E-cig padcollected aerosols.
Figure 2: In vitro activity of pad-collected smoke particulate (WTPM) and E-cig aerosols in Ames (A), NRU (B), IL-8 (C), and MN (D, E). Data points in each plot represent
the mean values ± SD from a minimum of two (2) independent experiments. MN cell viability (E) shown to verify lack of MN induction is not due to cytotoxicity at the
higher doses. All activities for E-cig pad-collected aerosols significantly different when compared to WTPM from traditional tobacco burning cigarettes (p < 0.001).
53rd Annual Meeting of the Society of Toxicology; Phoenix, AZ. March 23 – 27, 2014
© 2014 Lorillard

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