Optimisation of in vitro standard

testing of E-Vapour and Heated

Tobacco Products

Roman Wieczorek, Edgar Trelles Sticken

2015 CORESTA Joint Study Groups Meeting

Jeju Island, South Korea, 4-8 October

- ST44 -

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Objective

2

How to optimise existing in vitro methods for testing of

– Smoked Tobacco Products

– Heated Tobacco Products (HTP)

– Electronic Vapour Products (EVP)

The use of accepted and adapted in vitro methods is still the

key to demonstrate the reduced in vitro toxicity of such products

Due to the low toxicity of vapour produced by EVPs the

sensitivity of in vitro test systems towards gas components is of

crucial importance and their effects must be recognised

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Experimental Methods

3

Organisms

Ames - Salmonella typhimurium OECD strains (TA98, TA100, TA102, TA1535 and TA1537)

NRU - Hep-G2 (human liver) for aerosol exposure, TPM and non tobacco materials (NTM)

IVM - V79 (hamster lung) for aerosol exposure

- TK6 (human lymphoblast) for condensates and NTMs

CM7 - CORESTA Monitor Test Piece 7 11-12 puffs

DBC - Dark Blended Cigarette 8-9 puffs

HTP1 - Heated Tobacco Product 1 10 puffs

HTP2 - Heated Tobacco Product 2 10 puffs

EVP - Electronic Vapour Product up to 300 puffs

Products (commercially available)

Smoking regimen 2 puffs per minute / 55 ml puff volumes

• Tobacco products: 2 second puff duration (bell shape)

• EVP: 3 second puff duration (square wave)

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Optimised Exposure in Ames Test

Experimental setup:

Bacteria suspension – per test 120 ml of overnight

cultures are centrifuged and re-suspended in 12 ml PBS to

obtain increased cell densities

Concentrated bacteria suspension (microsuspension assay)

enhance the sensitivity to mutagenic compounds and

concurrently reduces toxic effects of the test aerosol

The fresh aerosol is directed through bacteria suspension

in a glass tube placed in an impinger. After treatment the

bacteria are incubated in the presence and absence of

external metabolic activation system S9

4

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OECD - S. typhimurium strains with aerosol

The Salmonella microsuspension test with TA100 in ITG-Biolab is a common

procedure for testing of aerosol mutagenicity.

This method is validated and accredited according to ISO17025.

Reproducibility in 9 independent replicates was determined between 16 - 26%

CM7 was used for Whole Smoke and GVP

Vapour generated by EVP from formaldehyde spiked liquid (1%) was used as positive control for Whole Vapour and its GVP

(The formaldehyde concentration in the vapour positive control is significantly higher than detected sometimes in EVPs)

5

TA98 TA100* TA102* TA1535 TA1537

Whole Smoke/Vapour + + + - -

Gas Vapour Phase weak + - - -

OECD recommended S. typhimurium strains were tested for their

response to fresh generated smoke / formaldehyde vapour.

* Formaldehyde as mutagenic aldehyde in Ames test was described in: D. Dillon et al. The effectiveness of Salmonella strains TA100,

TA102 and TA104 for detecting mutagenicity of some aldehydes and peroxides; Mutagenesis vol13 no. 1 pp. 19-26, 1998

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Fresh aerosol mutagenicity with TA100

6

HTP2 was less mutagenic than whole smoke of CM7 but similar to the DBC.

HTP1 showed much less mutagenic effects.

However, the vapour of EVP did not show any mutagenic response.

The mutagenic response with aerosol from HTP1 was detected with S. typhimurium

TA100 and TA102 as well.

Positive control for vapour testing

Assays with vapour containing formaldehyde confirm the high sensitivity of

the Salmonella microsuspension Ames test to whole vapour and its gas

phase as well.

The gaseous formaldehyde represents about 60% of mutagenicity of whole

vapour.

CM7

DBC

HTP1

HTP2

EVP

1 0 0 2 0 0

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

9 0 0 1 0 0 0 1 1 0 0 1 2 0 0

p u ff n u m b e r

re

ve

rta

nts

/p

late

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Demands for optimised cytotoxicological

testing using NRU assay

Due to the modified composition of particulate matter

especially from EVPs, the standard test procedure for

condensate testing is of only limited suitability

Direct exposure of cells to the freshly generated

smoke/aerosol at air liquid interface (ALI) seems to be the

most practicable alternative approach

The sensitivity of in vitro test systems towards gas phase

components is of crucial importance

Cell exposure method in 96 multiwell plate has to be adapted

for a long term treatment (up to 120 minutes)

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View from above

Side view on well with collagen and HepG2 cells

NRU – prolonged ALI exposure in 96 well plate

Organotypic matrix in form of Collagen-I is a proven material for

maintenance of cells under optimal conditions and enables

prolonged exposure to aerosol under ALI conditions.

8

Collagen-I layer (0.1%)

Round bottom of 96 well

Neutral red stained HepG2 cells

on top of Collagen-I surface

HepG2 cells on a Collagen-I matrix are exposed

to aerosol under ALI conditions in a newly

constructed smoke exposure machine (SEIVS).

Multiwell plate with

round bottom wells

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Smoke Exposure In Vitro System (SEIVS)(assembled by Burghart TABAKTECHNIK)

24 well Aerosol Distribution Device

96 well Aerosol Distribution Device

Smoking pumps 1-5

Mixing

pump

Exposure

chamber 1

Exposure

chamber 2

Diluting

pump 1

Diluting

pump 2

2 exposure chambers with separate dilution systems allow

parallel exposure to the same aerosol e.g.

- Whole smoke and GVP

- Different dilution levels per each plate

- Using 24 and 96 multiwell plates

- Suitable for different in vitro assays at a time

Smoke flow through the SEIVS

Puff dependent exposure by covering of wells

Each pump can be used for separate dilution of aerosol

SEIVS - Top view on the exposure chambers and pump units

Cambridge filter

(optional)

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Results from parallel exposure WS/WV & GVP

10

HepG2 cells were incubated for 67 hours after exposure

Total cytotoxicity and the contribution

of GVP to Whole aerosol cytotoxicity

Reciprocal EC50 (log) of whole smoke/vapour from selected tobacco products

0,001 0,01 0,1 1 10

DBC

CM7

HTP1

EVP

1/EC50 (log puff number)

Higher 1/EC50 higher Toxicity

Cytotoxicity of WS and GVP

from HTP1

Curve progressions of whole smoke and their GVP

generated from the same smoking runs

30% GVP/WV

19% GVP/WS

9% GVP/WS

16% GVP/WS

0 2 0 4 0 6 0

0

2 0

4 0

6 0

8 0

1 0 0

W S

G V P

p u ff n u m b e r

% c

yto

tox

icit

y

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Fresh aerosol cytotoxicity

11

• The whole smoke of CM7 and dark blend cigarette is more cytotoxic in

comparison to the tested HTP1.

1 hour of exposure

HepG2 cells puffed with ambient air for about 2.5 hours showed similar toxicity to EVP.

EC50

- 0.71

- 0.41

- 9.75

- 128.9

CM7

DBC

HTP1

EVP

0 5 1 0 1 5 2 0

0

2 0

4 0

6 0

8 0

1 0 0

5 0 1 0 0 1 5 0 2 0 0

p u ff n u m b e r

% c

yto

tox

icit

y

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Genotoxicological examination

with V79 cells

Exposure of V79 cells in the IVM assays for

genotoxicity testing is performed using the

SEIVS machine

The cells in IVM assays exposed to the

freshly generated smoke/aerosol at air

liquid interface (ALI) in inserts are placed in

a 24 multiwell plate

After exposure the cells are incubated over

3.5 hours in the presence of S9 following

post-incubation over 22 hours

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Effect of whole aerosol and GVP

generated by the same run with V79 cells

13

5 1 0 1 5 2 0

0 .0

0 .2

0 .4

0 .6

p u ff n u m b e r

inc

rea

se

of

MN

W S

G V P

0 5 1 0 1 5 2 0

0 .0

0 .2

0 .4

p u ff n u m b e r

inc

rea

se

of

MN

(%

)

The evaluation method is validated and

accredited according to ISO17025.

Reproducibility in 9 independent replicates for smoke

of CM7 was determined at 25.5%

Whole Smoke and GVP of HTP1 Whole vapour and GVP of formaldehyde spiked liquid (1%)

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Fresh aerosol genotoxicity with V79 cells

14

• The whole smoke of CM7 is more genotoxic in comparison to the tested HTP1.

• EVP did not show any mutagenic response.

* EC-MN is the number of puffs which

results in 3-fold increase against

background level of micronuclei

CM7

HTP1

EVP

EC-MN*

- 0.20

- 9.65

- not determinable

3-fold increase of MN

against background level**

0 5 1 0 1 5 2 0

0 .0

0 .2

0 .4

0 .6

5 0 1 0 0 1 5 0

p u ff n u m b e r

inc

rea

se

of

MN

(%

)

**Expert Opin. Drug Metab. Toxicol. (2011) 7(12):1513-1520

Ewald Roemer et al. Toxicol Mech Methods. 2015 May 19:1-14

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In Vitro Micronucleus assay with TK6

p53-competent human B lymphoblastoid cell line TK6 is introduced as a

“better predictive” alternative to V79 hamster cells in the IVM assay *

Condensate of test piece CM7 induces dose dependent increases of

micronuclei with and without S9

15

Phase

contrastDAPI

Higher toxicity than 50% (RICC) lead to a

decrease of cells with MN and an increase of

cells with apoptotic morphology

TPM from CM7 test piece shows positive

effects. However, for comparative studies

between different TPMs the dose levels will

need fine adjustment to meet pre-apoptotic

toxicity levels

Further information are presented at STPOST 14

Apoptotic

TK6 cell

* J. Whitwell et al. / Mutation Research 789-790 (2015) 7–27

Tox induced decrease in the

number of MN cells. High

abundance of apoptotic cells.

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Conclusions

In order to be able to determine the in vitro toxicity of tobacco smoke and vapour

products the NRU assay, Ames test and IVM assay were optimised:

Positive vapour control for fresh aerosol in vitro testing is developed and

implemented

Ames test procedure is optimised for smoke/vapour and their gas phase components

in particular

In the NRU assay the HepG2 cells are seeded on a collagen-I matrix for prolonged

exposure to aerosol under ALI conditions

Additionally p53-competent human B lymphoblastoid cell line TK6 is introduced for

TPM testing as an alternative to V79 hamster cells in the IVM assay

In NRU and IVM assay a new constructed smoke exposure machine (SEIVS) allows

parallel testing of whole aerosols and their gas phase

16

www.imperialtobaccoscience.com/

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