The growth in the number of smokers currently using next generation products (NGPs) has
significantly increased, predominantly e-cigarettes but also novel tobacco heating products
(THPs) that heat rather than burn tobacco. Using a series of in vitro test methods, we have
assessed the toxicological and biological effects of two commercially available NGPs, an e-
cigarette (Vype ePen) and a THP (glo), designed to reduce toxicant exposures. Responses
were compared to a commercially a 3R4F reference cigarette (Figure 1).
Generation of test matrices
Three different test matrices were used for in vitro assessments: total particulate matter
(TPM)/aerosol collected matter (ACM), whole aerosol (WA), and aqueous aerosol extract
(AqE). These were produced according to the puffing regimes detailed in Table I.
Approximately 150 mg of TPM or ACM were collected on 44 mm Cambridge filter pads
(Whatman, UK). DMSO (Sigma-Aldrich, UK) was used to elute the TPM or ACM from the
pads to a stock concentration of 24 mg/mL. The extracts were stored in single-use
volumes at -80oC until required.
AqE was produced by bubbling aerosol from each product through 20 mL of non-
supplemented DMEM/F12 medium (Gibco, USA) in a glass impinger.
For WA exposures, a Vitrocell VC10 Smoking Robot (Vitrocell Systems, Germany) was
used for the Ames assay, as previously described.1 A Borgwaldt RM20S exposure system
was used for the cytotoxicity assay, as detailed previously.
Ames bacterial reverse mutation assay
TPM exposures were conducted to the principles of OECD 471, using S. typhimurium
strains TA98 and TA100, + metabolic activation (S9). For product WA exposures, the
Ames assay was employed with S. typhimurium tester strains TA98 and TA100 using a
modified methodology as previously described.
Mouse lymphoma assay (MLA)
TPM was assessed following OECD 490, + S9 with short 3 h exposures and longer 24 h –
S9 exposures.
Neutral red uptake (NRU) cytotoxicity assay
TPM cytotoxicity was assessed using BALB/c 3T3 mouse fibroblasts3.. WA cytotoxicity was
assessed using human bronchial epithelial cells (H292) exposed at the air-liquid interface
(ALI) for 1 h.
Bhas cell transformation assay
The potential of TPM from the products to induce tumour development was evaluated
using the Bhas 42 cell transformation assay, promoter protocol.
Luciferase-based reporter gene assay to assess oxidative stress
Antioxidant response element (ARE) transcriptional activation in stably transfected H292
cells were assessed after 6 and 24 h treatment.
Multiparametric analysis using high-content screening (HCS) approaches
The Cellomics Arrayscan VTi platform was used to assess 10 endpoints in normal human
bronchial epithelial cells (NHBEs) after 4 or 24 h exposures, as previously described.
Figure 1. Products used in this study, (a) a THP (THP1.0;
glo), (b) an e-cigarette (Vype ePen) and (c) a reference
cigarette 3R4F
Glo demonstrated significantly
reduced toxicological response
compared to 3R4F in
genotoxicity, cytotoxicity and
cell transformation assays4
Table 2. Glo (THP1.0) was
negative across all assays,
under each of the conditions
tested. In contrast, 3R4F was
positive for each endpoint.
Table 3. High content screening
Endpoint Exposure time (h)
3R4F
THP1.0
ATP 4 60* -
24 120 -
Cell count 4 - -
24 - -
Glutathione content 4 120 -
24 - -
Mitochondrial mass 4 - -
24 - - Mitochondrial membrane potential
4 120 - 24 - -
Nuclear size 4 - -
24 - -
ROS formation 4 - -
24 - -
DNA structure 4 - -
24 - - DNA damage (p-H2AX)
4 - - 24 60* -
Stress kinase (p-c-Jun)
4 - - 24 - -
Values are the minimum required TPM concentration
(µg/mL) to elicit a ≥ 1.5-fold increase in assay signal from
the 0.5% DMSO vehicle control or a 30% decrease in signal
for the ATP, cell count, glutathione content, mitochondrial
mass or mitochondrial membrane potential assay
endpoints. Stars indicate a TPM-concentration dependant
response6.
Figure 5. Neutral red uptake determined cell
cytotoxicity of H292 cells after 1 hour exposure
to a range of dilutions of the two test articles
generated on the Borgwaldt RM20S smoking
machine. Cell cytotoxicty is expressed as a
function of (a) aerosol dilution, and (b) nicotine
levels measured in the media following
exposure.
Figure 4. Activation of the H292-ARE-Luc2P RGA following exposure
to 3R4F and THP1.0. Data shown are mean fold changes in response
normalized to the vehicle control (0.83% DMSO). Activation following
(a) 6h exposure to 3R4F and THP1.0 (b) 24h exposure to 3R4F and
THP1.0 (c) 6h exposure to THP1.0 (d) 24hr exposure to THP1.0.6
• Across all the in vitro techniques employed a clear positive response was observed
with 3R4F cigarette smoke particulate matter, aqueous extract and whole aerosol
• NGP particulate matter, aqueous extract and whole aerosol showed reduced or no
activity in the in vitro assays at doses equivalent or higher than 3R4F reference
cigarette
• The data generated add to growing evidence that suggests THPs may provide a less
risky alternative to traditional cigarettes, however further studies investigating the longer
terms effects on consumers is required to substantiate disease relevant risk reduction
References 1. Thorne, D., Crooks, I., Hollings, M., Seymour, A., Meredith, C., Gaҫa, M. 2016. The mutagenic assessment
of electronic-cigarettes and tobacco smoke using the Ames assay in strains TA98 and TA100. Mutat. Res.
812:29-36
2. Azzopardi, D., Patel K., Jaunky, T., Santopietro, S., Camacho, O.M., McAughey, J., Gaҫa, M. 2016.
Electronic cigarette aerosol induces significantly less cytotoxicity than tobacco smoke. Tox. Mech. Methods
26, 477–491.
3. Taylor, M., Carr, T., Oke, O., Jaunky, J., Breheny, D., Lowe, F., Gaça, M. 2016. E-cigarette aerosols induce
lower oxidative stress in vitro when compared to tobacco smoke. Toxicol. Mech. Methods 26, 465–476.
4. Assessment of tobacco heating product THP1.0 Part 7: Comparative in vitro toxicological evaluation.
Thorne, D et al., 2018. Reg Tox Pharm. 93, 84-91.
5. Assessment of tobacco heating product THP1.0 Part 5: In vitro dosimetric and cytotoxic assessment.
Jaunky, T et al., 2018 Reg Tox Pharm. 93, 52-61
6. Assessment of tobacco heating product THP1.0 Part 6: A comparative in vitro study using contemporary
screening approaches. Taylor, M et al., 2018. Reg Tox Pharm 93, 62-70
A e r o s o l d ilu t io n (a e ro s o l:a ir , 1 :x )
Ce
ll v
iab
ilit
y (
% S
HA
M c
on
tro
l)
0
2 5
5 0
7 5
1 0 0
110100100010000
3 R 4 F
T H P 1 .0
a b
Table 2. Regulatory toxicity testing: in vitro cytotoxicity, mutagenicity
and tumour promotion.
Treatment
condition
NRU1
TPM
AMES2 TPM AMES3 WA MLA4 TPM Bhas5
TPM
-S9 -S9 +S9 +S9 3h –
S9
3h+
S9
24h-
S9
-S9
3R4F + +
(TA98,
TA100)
+
(TA98,
TA100,
TA1537)
+ (TA98,
TA100)
+ + + +
THP1.0 - - - - - - - -
TPM= total particulate matter
WA= whole aerosol
1= tested up to 240 μg/mL over 24 h
2= tested up to 2400 μg/mL over 72 h plate incorporation and preincubation
3= tested to equivalent doses using QCM technology
4= tested up to 240 μg/mL +/-S9 over 3 treatment conditions
5= tested up to 120 μg/mL for 10 days
WA cytotoxicity assessment demonstrated that 3R4F produced a concentration-related
decrease in cell viability, resulting in complete cytotoxicity at the top concentrations tested.
Glo (THP1.0) induced significantly less cytotoxicity at comparable and higher levels of
nicotine delivered to the cells5 (Figure 5).
Correspondence: [email protected]
d
0 5 0 1 0 0 1 5 0 2 0 0
0 .0
2 .5
5 .0
7 .5
1 0 .0
1 2 .5
( g /m L )
Fo
ld c
ha
ng
e
3 R 4F
T H P 1 .0
0 5 0 1 0 0 1 5 0 2 0 0
0
1 0
2 0
3 0
4 0
( g /m L )
Fo
ld c
ha
ng
e
3 R 4F
T H P 1 .0
0 5 0 1 0 0 1 5 0 2 0 0
0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
3 .0
3 .5
4 .0
( g /m L )
Fo
ld c
ha
ng
e
T H P 1 .0
0 5 0 1 0 0 1 5 0 2 0 0
0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
3 .0
3 .5
4 .0
( g /m L )
Fo
ld c
ha
ng
e
T H P 1 .0
a
c
b
[L o g ] N ic o t in e in m e d ia (n g / m l)
Ce
ll v
iab
ilit
y (
% S
HA
M c
on
tro
l)
0 2 4 6
0
2 5
5 0
7 5
1 0 0
3 R 4 F
T H P 1 .0
David Smart, Damien Breheny, Stella Bozhilova, Tomasz Jaunky, Mark Taylor, Simone Santopietro, Anya Terry,
David Thorne, James Murphy, Marianna Gaca, Analucia Saraiva.
British American Tobacco R&D Centre, Southampton, SO15 8TL, UK
THE ASSESSMENT OF A RANGE OF NEXT GENERATION TOBACCO
AND NICOTINE PRODUCTS USING PRE-CLINICAL
IN VITRO TOOLS
HCI HCIm CRM81
Puff volume 55 ml 55 ml 55 ml
Puff frequency 2 puffs/min 2 puffs/min 2 puffs/min
Puff duration 2 s 2 s 3 s
Vent blocking Yes No N/A
Table 1. Puffing regimes used, HCI
(3R4F), HCIm (THP) and CRM81
(e-cigarette)
Figure 2. Exposure to 3R4F reference
cigarette smoke caused mutations in a
dose dependent manner; Vype ePen
gave no response, even after 900 puffs.
Vype ePen vapour induced less
cytotoxicity than 3R4F reference
cigarette. 3R4F smoke induced
promotion activity; Vype ePen was
negative at all concentrations tested.1,2
Figure 3: Vype ePen induced no oxidative stress in human lung H292 epithelial cells
compared to 3R4F reference cigarette.3
a.
b.
c.
Toxilatin 2018
THE ASSESSMENT OF A RANGE OF NEXT GENERATION TOBACCO AND NICOTINE
PRODUCTS USING PRE-CLINICAL IN VITRO TOOLS.
David Smart, Damien Breheny, Stella Bozhilova, Tomasz Jaunky, Mark Taylor, Simone
Santopietro, Anya Terry, David Thorne, James Murphy, Marianna Gaca, Analucia Saraiva
British American Tobacco R&D Centre, Regents Park Road, Southampton, SO15 8TL, UK
Introduction: The growth in the number of smokers currently using next generation
products (NGPs) has significantly increased, predominantly e-cigarettes but also novel
tobacco heating products (THPs) that heat rather than burn tobacco. As NGPs do not burn
tobacco (or in the case of e-cigarettes, do not contain tobacco), the toxicant profile of their
aerosols is greatly reduced in comparison to cigarettes and therefore hold promise as
reduce risk products. The concept of the risk continuum can be used as a tool to rank the
reduced risk potential of a range of tobacco and nicotine products relative to cigarettes.
The US Food and Drug Administration (FDA), is currently the only national regulator that
has provided draft guidance with which to assess the harm reduction potential of NGPs as
Modified Risk Tobacco Products. In support of this guidance, we recently published an
integrated assessment framework which includes the use of pre-clinical, clinical and
population studies to assess the reduced risk potential of NGPs at the individual and
population level.
Objective and Methods: Using a series of in vitro test methods, we have assessed the
toxicological and biological effects of two commercially available NGPs, an e-cigarette
(Vype ePen) and a THP (gloTM), designed to reduce toxicant exposures. Responses were
compared to a commercially a 3R4F reference cigarette. Products were assessed using
particulate, whole aerosols and aqueous extracts across a battery of in vitro genetic
toxicological assays including the Ames and mouse lymphoma assay both assessing
mutagenicity, the Bhas cell transformation assay to assess tumour-promoting activity and
neutral red uptake measuring cytotoxicity. Human cellular based in vitro assays modelling
some of the key events for smoking related diseases such as chronic obstructive
pulmonary disease and atherosclerosis and included oxidative stress (antioxidant
response element (ARE) activation in lung epithelial reporter cells and endothelial cell
migration (wound healing). A high content screening (HCS) approach was used to assess
10 different toxicity endpoints such as DNA damage and intracellular glutathione content,
in primary human bronchial epithelial cells (HBEC) to support findings.
Results and Conclusions: The data from these assays indicated that the biological
response to NGP aerosol was significantly lower relative to the reference cigarette.
However a series of clinical and population studies measuring the longer terms effects of
these new products on consumers is required to substantiate disease relevant risk
reduction.
.
British American Tobacco
R&D Centre
Southampton SO15 8TL
United Kingdom
www.bat.com
www.bat-science.com