the effect of charcoal filters on gas vapour phase in ... · (pct wo 03/100417) cm7 c-ab *** 0 10...

2012 CORESTA CONGRESS Sapporo, Japan

SS03

The effect of charcoal filters on gas vapour

phase in vitro toxicity tested in different

air/liquid interface exposure systems

Roman Wieczorek

Imperial Tobacco

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Objective and experimental approach

To compare different fresh smoke exposure systems.

For this purpose, contribution of Gas Vapour Phase

(GVP) to fresh Whole Smoke (WS) toxicity in vitro

was determined for cigarettes with different filters.

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Methods - in vitro

Cell line Hep-G2 (ATCC HB-8065)

Human hepatoma cells were incubated in mineral medium

supplemented with Insulin-Transferrin-Selenium and 1%

serum substitute (Ultroser-G; BioSepra S.A.).

Cytotoxicity assay

NRU - after smoke exposure the cells were incubated in

mineral medium with 0.1% serum substitute for 65 hours.

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Methods - exposure of cells

Fresh Smoke Exposure Systems Bt020 smoke aerosol exposure system; Burghart GmbH; Wedel, Germany

Exposure vessels / chamber

• in round bottom wells of 96 Multiwell Plate (96MWP)

• in inserts (24MWP)

• BAT exposure chamber with transwells

(PCT WO 03/100417)

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Methods - smoke generation and dilution

Each puff of smoke is diluted, if necessary, directly in syringe by partly rejecting and refilling with charcoal filtered ambient air.

The volume of smoke transported to the exposure chamber is constant and independent of dilution factor.

Smoke dilution and transport after puff till to contact with cells takes less than 6 seconds.

Excess smoke actively removed by inter-puff flushing cycles with 80 ml charcoal filtered air.

Ambient air

(charcoal filtered)

Dilution of remaining smoke with

fresh air until next puff (80mL)

2- Dilution of smoke

1- Puff of smoke

3- Discarding of diluted smoke

4- Transport of diluted smoke

Smoking and dilution device

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discarding of smoke by

inter-puff flushing

cells in transwells

Methods - used exposure systems

24 wells Smoke Distribution Device

cells in inserts

Ambient air

(charcoal filtered)

first puff only all puffs

Discarding of smoke

by vacuum and

inter-puff flushing

96 wells Smoke Distribution Device

cells in round bottom wells

Smoking and dilution device

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cig.sp. EC50

filter ventilation

Contribution of GVP to WS - Cellulose Acetate filter

Toxicity of Fresh Whole Smoke

Test in 96 MWP (2/2/35)

The contribution level of GVP to

WS toxicity in cigarettes with

cellulose acetate filter stayed

roughly the same at about 33%,

independent of puff volume and

filter ventilation

25

50

75

100

125

0% 30% 45% 60% 74%

WS GVP

%Tox

2010 CORESTA, Edinburgh, Scotland (SSPT32) The effect of puff

volume on in vitro toxicity of mainstream cigarette smoke

higher EC50 lower toxicity

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Contribution of GVP Screening of cigarette toxicity using 96 MWP with 4µl buffer per well

Code Filter Dry Particulate Matter

DPM [mg/cig]

Charcoal [mg/filter]

CA acetate 13.9 0.0

LC acetate, carbon, cellulose 15.7 19.0

HCC acetate, carbon / cavity 14.6 85.0

HCD acetate, carbon / dalmatian 14.3 82.5

The contribution of GVP to WS toxicity correlates with the amount of charcoal in filter

CA LC HCC HCD

0

10

20

30

40

CA

LC

HCC

HCD

EC50 [µg/DPM]

WS

GVP

100 1000

Co

ntr

. o

f G

VP

to

WS

to

xic

ity [

%]

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Contribution of GVP (contd.)

Cigarette toxicity using 96 MWP with 2µl buffer per well

HCLF decreased the contribution GVP to WS toxicity by roughly 15-20%

Tests with 2 µl and 4 µl buffer per well do not change significantly the contribution level of GVP.

Cellulose Acetate

Filter

High Charcoal Loaded

Filter

CM7 CA HCD C-AB C-VB

0

10

20

30

40

%GVP/WS

CM7 Coresta Monitor 7

CA C-Acetate filter

HCD Carbon / dalmatian

C-AB Carbon / American Blend

C-VB Carbon / Virginia Blend

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Influence of fluid layer thickness on GVP effect

in 96MWP exposure system

CM7 C-AB

1µl 2µl 4µl 10µl 20µl

0

10

20

30

40

control

***

***

1µl 2µl 4µl 10µl 0

5

10

15

***

HEPES buffered PBS in wells protects cells against drying during smoke exposure

procedure.

The buffer covers the cells on the bottom of well but the cells in edge region are directly

exposed to smoke.

The lower the buffer volume in wells the higher the air/liquid interface area.

Overlaid cells in round bottom wells are less sensitive to GVP than to WS.

Contr

. of G

VP

to W

S toxic

ity

[%]

Contr

. of G

VP

to W

S toxic

ity

[%]

buffer

per well

buffer

per well

buffer over cells

10 µl

buffer

per well

2 µl

buffer

per well

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Comparison of methods Contribution of GVP to WS toxicity in 96MWP with 2 µl buffer

25

75

50

0

1 10 100 1000

% cig. smoke

% C

yto

toxic

ity

CM7_WS CM7_GVP C-AB_WS C-AB_GVP

1175

2282

13.4

16.0

High Charcoal Loaded Filter

Acetate Filter

C-AB

CM7

Gas Vapour

Phase* [µg/cig]

TPM

[mg/cig] Filter Code

* Sum of 12 selected analytes

***

CM7 C-AB 0

10

20

30

40

Contr

. of G

VP

to W

S toxic

ity

[%]

Box plot

*** P value < 0.001

Dose response diagram of toxicity

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Comparison of methods Contribution of GVP to WS toxicity in inserts (24MWP)

25

75

50

0

1 10 100 1000

% cig. smoke

% C

yto

toxic

ity

CM7_WS CM7_GVP C-AB_WS C-AB_GVP CM7 C-AB

***

0

10

20

30

40

Contr

. of G

VP

to W

S toxic

ity

[%]

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Comparison of methods Contribution of GVP to WS toxicity in transwell system

25

75

50

0

1 10 100 1000

% cig. smoke

% C

yto

toxic

ity

CM7_WS CM7_GVP C-AB_WS C-AB_GVP

BAT exposure chamber

(PCT WO 03/100417)

CM7 C-AB

***

0

10

20

30

40

50

Contr

. of

GV

P t

o W

S toxic

ity [

%]

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Exposure

system CM7 [%]

C-AB [%]

96MWP-U wells 27 8.5

24MWP-Inserts 32 27

BAT-Transwell 32 17

Comparison of methods - summary

The contribution of GVP to WS toxicity:

constant for CM7 cellulose acetate filter at 30%

variable for charcoal filter cigarettes

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0 50 100 150 200

Differences in GVP of C-AB in comparison to CM7 testpiece

Substance CM7 [µg/cig]

C-AB [µg/cig]

Acrolein 81.9 31.1

HCN 127.4 46.9

Acetaldehyde 803 556

Acetonitrile 114.2 42.4

Benzene 53.4 16.8

Selected GVP substances of

CM7 in comparison to C-AB

Direct comparison of

GVP toxicity in NRU in vitro

assay (EC50) between

CM7 and C-AB

96MWP

Inserts

Transwells

CM7

CM7

CM7

C-AB

76%

70%

71%

C-AB

C-AB

EC50 [mg DPM]

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Comparison of sensitivity of the exposure systems Puff specific EC50 (CM7)

0 0,5 1

96MTP-U wells

24MTP-Ins

BAT-Transw.

0 1 2 3 4

96MTP-U wells

24MTP-Ins

BAT-Transw.

[puff] [puff]

Whole Smoke toxicity Gas Vapour Phase toxicity

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All three tested air/liquid exposure systems are able to show:

- dose response effects with WS and GVP.

- significantly higher WS and GVP toxicity of CM7 testpiece compared to charcoal filter

cigarette.

- similar decrease of GVP toxicity.

- significantly higher contribution level of GVP to WS toxicity of CM7 testpiece compared

to charcoal filter cigarette.

The contribution of GVP to WS toxicity amounts to 30% for cellulose acetate

filter. For charcoal filter cigarettes different effects were observed.

The level of medium in the round bottom wells had a direct influence on the

sensitivity of the cells. Up to a volume of 4 µl, the contribution of GVP to WS

toxicity seems to be constant.

Conclusions

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It was observed, that the level of medium below the growth membrane of

insert/transwell affects the liquid meniscus at the edge of the membrane. As a

consequence, the exposure conditions can change due to different covering of

the cells with fluid.

The exposure system with 96MWP in round bottom wells and growth membrane

delivers “meaningful” data.

Nevertheless, further tests for better comparability of results are needed.

Conclusions (contd.)

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Thanks to

ITG tox and analytical team

&

BAT for providing their smoke exposure

chamber

… and for your kind attention

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the effect of charcoal filters on gas vapour phase in ... · (pct wo 03/100417) cm7 c-ab *** 0 10...

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