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: david_smart@bat.com

d

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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