Patrick Vanscheeuwijck, PhD

Director Pre-Clinical Toxicology

Non‐Clinical Toxicology of Nicotine and Aerosol from a Heated Tobacco Product

Page: 2

Offering adult smokers satisfying products that reduce risk

• Smoking is addictive and causes a number of serious diseases

• Worldwide it is estimated that more than one billion people will continue to smoke in the foreseeable future*

• Successful harm reduction requires that current adult smokers be offered a range of Reduced Risk Products (RRPs) so that consumer acceptance can be best fulfilled

• Our Heated Tobacco Product (HTP) is a RRP.*The Tobacco Atlas 3rd Edition. American Cancer Society, 2009Figure adapted from Clive Bates presentation to E-Cigarette Summit (19 Nov 2013)

HARM REDUCTIONPRODUCT

ACCEPTANCE AND USAGE

REDUCED-RISK PRODUCT =X

PMI’s Goal for Harm Reduction

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Reduced-Risk Products (“RRPs”) is the term the company uses to refer to products with the potentialto reduce individual risk and population harm in comparison to smoking cigarettes. PMI’s RRPs are invarious stages of development and commercialization, and we are conducting extensive and rigorousscientific studies to determine whether we can support claims for such products of reduced exposureto harmful and potentially harmful constituents in smoke, and ultimately claims of reduced diseaserisk, when compared to smoking cigarettes.

Before making any such claims, we will rigorously evaluate the full set of data from the relevantscientific studies to determine whether they substantiate reduced exposure or risk. Any such claimsmay also be subject to government review and authorization as is the case in the US today.

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Outline & Scope

Short description of design and methods used in a series of in vivo inhalation studies assessing theeffects of nicotine aerosol, conventional cigarette smoke and aerosols from potentially Risk-Reduced Product (pRRP) [or candidate Modified Risk Tobacco Product] – Heated Tobacco Product

Description of effects of nicotine (in combination with pyruvic acid) using pre-clinical and systemstoxicology endpoints

Toxicity of cigarette smoke exposure and the effect of a HTP – interpretation of findings usingnicotine-exposure knowledge

Application of a mouse disease model for cardiovascular and respiratory disease – effects ofcigarette smoke exposure, switching to HTP aerosol exposure and cessation

HTP: Heated Tobacco Product

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Methods – in vivo studies

Regulatory rat inhalation studies (OECD protocols; 28-day, 90-day*, OECD GLP)• Aerosol generation: using appropriate standards

– Smoking machines: conventional cigarettes, reduced risk products

– Collison nebulizers: nicotine/salt solution, nicotine containing liquids

• Aerosol characterization: demonstrate that aerosol is representative and follows guidelines– Determination of aerosol markers (e.g. TPM, CO, carbonyls), breathing zone

– Particle size distribution

• Bio-monitoring: demonstrate that exposure is representative– Respiratory physiology

– Urinary particulate and gas phase biomarkers of exposure (e.g. nicotine metabolites, mercapturic acid metabolites from acrolein, benzene, acrylonitrile, NNK) blood carboxyhemoglobin)

*OECD Guidelines for the Testing of Chemicals, Section 4.Test No. 413: Subchronic Inhalation Toxicity: 90-day Study, OECD 2009Test No. 412: Subacute Inhalation Toxicity: 28-Day Study, OECD 2009

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Methods ‐ design

OECD TG 412/413 Endpoints –Standard Toxicology1. In-life observations2. Hematology3. Clinical chemistry4. Organ weights5. Inflammatory cells in BALF6. Histopathology

OECD ‘Plus’-Systems Toxicology1. Transcriptomics2. Genomics3. Lipidomics4. Proteomics

Subset of dataEg Histopathology or Inflammatory Data

Computational analysisComputational Network BiologyBiological Impact Factor Analysis

Systemic toxicity

• Test atmospheres presented in low/medium/high concentration: up to MTD: 50 µg Nicotine/l* for nicotine-containing aerosols – 23 µg Nicotine/l for cigarette smoke; concentration-response

• Endpoints:

*50 µg/l nic corresponds to 13.6 mg/kg, daily dose; way above acute toxicity levels- or the nicotine amount from approx. 130 cig/day for a 60 kg human

Executed according to OECD GLPs

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MethodsExposure – nebulized nicotine –nicotine‐salt solutions

Fres

h

Aero

sol

E xha

led

A ir

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Effects of nicotine – 28‐day inhalation study (OECD TG 412)Exposure and Bio‐monitoringUrinary nicotine metabolites demonstrate appropriate exposure to ‘respirable’ aerosol

Test atmosphere concentrations Particle size determination Urinary nicotine metabolites

Means ± SEM; ***p ≤ 0.001

Means ± SD

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Effects of nicotine – 28‐day inhalation study (OECD TG 412)Systemic effects

Means ±SEM; *p≤0.05, r**p ≤ 0.01, ***p ≤ 0.001 relative to control

Nicotine concentration-dependent changes in:– Body weight development (approx. 5% body weight gain reduction in ‘high’ concentration group)

– Organ weights (relative to body weight): Liver, higher than controls; Spleen, lower

– Clinical chemistry: Alkaline phosphatase and alanine aminotransferase: higher than controls; cholesterol and glucose: lower than controls – all in normal ranges

– Hematology: neutrophils, higher than controls; lymphocytes, lower – all in normal ranges

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Effects of nicotine – 28‐day inhalation study (OECD TG 412)Histopathology of non‐respiratory tract organsLiver effects: nicotine concentration-dependent cytoplasmic vacuolation – vacuoles contain PAS-positive material, likely to consist of glycogen granules

Means ±SEM; *p≤0.05, r**p ≤ 0.01, ***p ≤ 0.001 relative to control

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Effects of nicotine – 28‐day inhalation study (OECD TG 412)Summary & Conclusion

• Exposure to nicotine aerosols, alone or in combination with pyruvic acid, results in mild changes in toxicity in a 28-day inhalation toxicology study with respect to:– Systemic effects: increased liver weight, blood neutrophil counts, activity of liver enzymes and decreased

blood levels of cholesterol and glucose

– Histopathological effects: increased vacuolation of and glycogen content in hepatocytes

Changes in liver gene expression and lipidomics provide mechanistic explanation for the changes observed, i.e. alterations in xenobioitic and lipid metabolism

Toxicity of aerosols of nicotine and pyruvic acid (separate and combined) in Sprague–Dawley rats in a 28-day OECD 412 inhalation study and assessment of systems toxicology.

Blaine Phillips, Marco Esposito, Jan Verbeeck, Stephanie Boue, Anita Iskandar, Gregory Vuillaume, Patrice Leroy, Subash Krishnan, Ulrike Kogel, Aneli Utan, Walter K. Schlage, Monali Bera, Emilija Veljkovic, Julia Hoeng, Manuel C. Peitsch, and Patrick Vanscheeuwijck.

Inhalation toxicology 2015 Aug;27(9):405-31, PMID 26295358

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Methods – Conventional cigarette smoke and aerosol from a HTP 90‐day inhalation study (OECD TG 413)

BV1"

WA

ST

E

Approximately 3.9m

App

roxi

mat

ely

1.2m

Control room

Clean corridor

Exposure room Fres

h

Aero

sol

E xha

led

A ir

Potentially Reduced-Risk product: Aerosol from Heatsticks and Tobacco Heating System, HTP, commercialized as iQOS(also designated as P1 or THS 2.2)

Conventional cigarettes:Smoke from University of Kentucky Standard Reference Cigarette 3R4F

Assessment of smoke/aerosol – Health Canada Intense smoke protocol

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Smoke chemistry – determination of 58 constituents in conventional cigarette smoke and HTP (THS2.2) aerosol

• Smoke chemistry data shows a strong reduction of HPHC* in THS2.2 relative to 3R4F (data per mg nicotine, 100 % is data from 3R4F)

*HPHC: Harmful or Potentially Harmful Constituents

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• Test atmosphere characterization – gas phase and particulate matter markers indicate that:‒ Target nicotine concentrations met‒ HPHCs in test atmospheres reflect aerosol chemistry data

Sham

3R4F

_L_8

3R4F

_M_1

5

3R4F

_H_2

3

THS2

.2_L

_15

THS2

.2_M

_23

THS2

.2_H

_500

15

30

45

60

mea

n co

ncen

tratio

n of

nico

tine

( g/

L +/

- SD

)

0 8 15 23 500

100

200

300

400Sham3R4FTHS2.2

test atmosphere concentration of nicotine (g/L)

mea

n co

ncen

tratio

n of

CO

(ppm

+/-

SD)

8 15 23 500.0

0.5

1.0

1.5

2.03R4FTHS2.2

test atmosphere concentration of nicotine (g/L)m

ean

conc

entra

tion

ofac

rolei

n (

g/L

+/- S

D)

Nicotine concentrations in test atmospheres

Acrolein concentrations in test atmospheres

CO concentrations in test atmospheres

Cigarette smoke and HTP aerosol– 90‐day inhalation study (OECD TG 413)Exposure

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Cigarette smoke and HTP aerosol– 90‐day inhalation study (OECD TG 413) ‐Biomarkers of exposure

• Bio-monitoring parameters indicate that:– Urinary biomarkers of nicotine correlate with nicotine levels in test atmosphere– Urinary biomarkers of HPHC correlate with chemical composition of aerosols – The HTP causes much less upper respiratory tract irritation resulting in higher uptake of aerosol

0 8 15 23 500

100

200

300

400

500Sham3R4FTHS2.2

test atmosphere concentration of nicotine (g/L)

conc

entra

tion

of S

PMA

(ng/

ml,

mea

n +/

- SD

)0 8 15 23 50

0

5000

10000

15000

20000Sham3R4FTHS2.2

test atmosphere concentration of nicotine (g/L)

conc

entra

tion

of H

PMA

(ng/

ml,

mea

n +/

- SD

)

Urinary benzene metabolite

Urinary acrolein metabolite

Data for male rats only, after 4 weeks of exposure. The data for female rats and different collection time points are very similar.

0 8 15 23 500

2000

4000

6000

8000Sham3R4FTHS2.2

test atmosphere concentration of nicotine (g/L)

tota

l nic

otin

e m

etab

olite

s(n

mol

, mea

n +/

- SD

)

Urinary nicotine metabolites

0 8 15 23 5050

100

150

200

250Sham3R4FTHS2.2

test atmosphere concentration of nicotine (g/L)

resp

irato

ry m

inute

volum

e( m

l/ m

in, m

ean

+/- S

EM)

Respiratory Minute Volume

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Cigarette smoke and HTP aerosol– 90‐day inhalation study (OECD TG 413) ‐Systemic toxicity

• No remarkable toxic effects in THS 2.2

• Body weight effect in males only

• Nicotine concentration-related increases in– Neutrophil count in blood

– Relative weight of liver

– Liver enzyme activity (normal range)

• Mild liver effects

Effects similar to those of nicotine exposure

-20 0 20 40 60 80 100 120 140200

300

400

500

600Sham3R4F_L_83R4F_M_153R4F_H_23THS2.2_L_15THS2.2_M_23THS2.2_H_50

90d exposure<-----------------------------><-------------->

90d exposure+ 42d recovery

Study Days

mea

n bo

dy w

eight

(g)

Body weight development, male rats

Alkaline phosphatase activity, female rats

* p < 0.05 as compared with SHAM at a given time point, # p < 0.05 as compared with 3R4F at target nicotine 15 g/l at a given time point, § p < 0.05 as compared with 3R4F at target nicotine 23 g/l at a given time point, and + p < 0.05 as compared with the same group at the 90-day exposure time point

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Cigarette smoke and HTP aerosol– 90‐day inhalation study (OECD TG 413) ‐Lung inflammation

• Very low numbers of inflammatory cells are recruited in the lungs (BALF) of THS2.2 aerosol-exposed rats

Remarks: •Data for male rats; the data for female rats are very similar.•* p < 0.05 as compared with SHAM at a given time point, # p < 0.05 as compared with 3R4F at target nicotine 15 g/l at a given time point, § p < 0.05 as compared with 3R4F at target nicotine 23 g/l at a given time point, and + p < 0.05 as compared with the same group at the 90-day exposure time point

Means ± SEM

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Cigarette smoke and HTP aerosol– 90‐day inhalation study (OECD TG 413)Histopathology of respiratory tract organs

Rel

ativ

eS

ever

ity

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Cigarette smoke and HTP aerosol– 90‐day inhalation study (OECD TG 413)Histopathology of respiratory tract organs ‐ Nose

Page: 20

Cigarette smoke and HTP aerosol– 90‐day inhalation study (OECD TG 413)Histopathology of respiratory tract organs – Bronchi and lungs

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Methods ‐ design

OECD TG 412/413 Endpoints –Standard Toxicology1. In-life observations2. Hematology3. Clinical chemistry4. Organ weights5. Inflammatory cells in BALF6. Histopathology

OECD ‘Plus’-Systems Toxicology1. Transcriptomics2. Genomics3. Lipidomics4. Proteomics

Subset of dataEg Histopathology or Inflammatory Data

Computational analysisComputational Network BiologyBiological Impact Factor Analysis

Systemic toxicity

• Test atmospheres presented in low/medium/high concentration: up to MTD: 50 µg Nicotine/l* for nicotine-containing aerosols – 23 µg Nicotine/l for cigarette smoke; concentration-response

• Endpoints:

*50 µg/l nic corresponds to 13.6 mg/kg, daily dose; way above acute toxicity levels- or the nicotine amount from approx. 130 cig/day for a 60 kg human

Executed according to OECD GLPs

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System Toxicology ResearchIdentify and Represent Disease Mechanisms

22

Understanding of disease onset and progression

Chronic Cigarette Smoke

Exposure

Molecular changes

Disruption ofBiological

Mechanism

Cell / Tissue

Changes

Physiological changes

Disease (CVD, COPD, Lung cancer)

Biological Networks – Systems Biology/ToxicologyAnalytical Chemistry Medicine

Chronic exposure to cigarette smoke affects a number of biological networks associated with smoking-related diseases in a causal chain of Key Events known as Adverse Outcome

Pathways

Gh

β-catenin signaling

NFKB signaling

Cytokine signaling

Inflammation

Cigarette smoke

DNA damage

gtpof(Kras) gtpof(Hras)

taof(Myc)

taof(Mycn)

kaof(PI3K)

PDGF family

Fgf2

SMC differentiation regulation

β-Adrenergicsignaling

taof(Snai1)

Fhit

Response to oxidative

stress

catof(Sod1)

taof(Nrf2)

Hypoxia-induced angiogenesis

ROS

Apoptotic signaling

Oxidative phosphorylationLiterature-derived

knowledge

Data-derivedknowledge Identify & Build

Biological Networkmodels

Hoeng et al., Case Study: The Role of Mechanistic Network Models in Systems Toxicology. Drug DiscoveryToday, 2013, 19:183-192. (PMID: 23933191)

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Methods ‐ Systems Toxicology AssessmentUse Disease Mechanism Understanding for Product Assessment

23Hoeng J et al. A network-based approach to quantifying the impact of biologically active substances. Drug Discovery Today, 2012, 17:413-418 (PMID: 22155224)

Multi-OmicsData

Systems Response Profile

Inflammation

Cell Proliferation

Cell Stress

Apoptosis

DNA Damage

Necroptosis

Senescense

Autophagy

Previously BuiltBiological Network

models

Network PerturbationAmplitudes

Exposure

Sham

3R4F

pRRP

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MethodsFrom Gene Expression Profiles to Biological Impact Factor

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Effects of nicotine – 28‐day inhalation study (OECD TG 412)Application of systems toxicology on liver changes

System response profile of liver transcriptomics reveal up- and down-regulated gene expression

Up-regulated:

• Xenobiotic metabolism

• Oxidative phosphorylation

• Fatty acid/cholesterol metabolism

• Cell cycle

Down-regulated:

• Intracellular signaling

Lipidomics confirm downregulated glycerolipids and sterols

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Cigarette smoke and HTP aerosol– 90‐day inhalation studySystems biology evaluation – Systems Response Profile analysis of lung (90 days)

Page: 27

Cigarette smoke and HTP aerosol– 90‐day inhalation studySystems biology evaluation – Network Perturbation Analysis of lung (90‐days)

Page: 28

• Pre-clinical toxicology: Exposure of rats to aerosol from THS2.2 – even at a multiple of the aerosol concentration - results in a dramatically lower biological effects as compared to exposure to 3R4F in– Systemic toxicity; where effects were observed, they are related to nicotine exposure

– Lung inflammation

– Histopathology of respiratory tract organs

• Systems toxicology: Exposure of rats to aerosol from THS2.2 results in a dramatically lower biological network perturbations in the transcriptomes of the nasal epithelium and lung tissue

Cigarette smoke and HTP aerosol– 90‐day inhalation studySummary and conclusions

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ApoE-P1 Switching Study Design

[Time]

[Dis

ease

Ris

k]

Point ofintervention

Comparing switching to the HTP P1* with ongoing smoking and benchmarking against cessation

MethodsSwitching Study in an Animal Model of Disease – cardiovascular disease/emphysema

Mice:- ApoE-/- mice: animal model for aspects of CVD and COPDExposure:- 8 months with dissections at 1,2,3,6 and 8 months- Test atmosphere nicotine concentrations: approx. 30 µg/l- Whole body, 3 x 1 h/day

3R4FCombustible Cigarette: 3R4F

Fresh Air3R4FCESSATIONHTP3R4FSWITCHING

HTPHTPFresh AirControl: Sham/Air

Month 2 Month 8Start

Groups InhalationPoint of intervention

Non-clinical study

*P1: HTTP:THS2.2 or iQOS

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Switching Study in an Animal Model of DiseaseResult Summary: Disease Endpoint ‐ Lung function and Lung Volume

Lung volume (absolute, measured)

0 40.0

0.5

1.0

1.5Sham3R4F

ZRHCessation

Switch

1 2 3 6 8Month

Dis

plac

ed li

quid

wei

ght

(g)

3R4FCessationSwitchingHTPAir

Groups

Month 1 Month 2 Month 3

Month 6 Month 8

Means ± SEM

Page: 31

Switching Study in an Animal Model of DiseaseResult Summary: Tissue changes – Lung Emphysema

*: Statistically significant compared to sham*: Statistically significant compared to 3R4F:CONT at DT2

Histopathology Score 

Page: 32

Total cells absolute

0 1 2 3 4 5 6 7 8

0

10

20

30Sham3R4F

THS 2.2Cessation

Switch

Tota

l Cel

ls (x

105 )

+/- S

EM

Switching Study in an Animal Model of DiseaseResult Summary: Disease Mechanism ‐ Lung Inflammation

Study monthStudy month

Differential counts

Alveolar macrophages absolute

0 2 4 6 8

0

5

10

15Sham3R4F

THS 2.2Cessation

Switch

Alv

eola

r mac

roph

ages

(x 1

05 ) +/

- SEM

Neutrophils absolute

0 2 4 6 8

0

2

4

6

8Sham3R4F

THS 2.2Cessation

SwitchN

eutr

ophi

ls (x

105 )

+/- S

EM

Alveolar dendritic cells absolute

0 2 4 6 8

0.0

0.5

1.0

1.5

2.0Sham3R4F

THS 2.2Cessation

Switch

Alv

eola

r den

driti

c ce

lls (x

105 )

+/- S

EM

Lymphocytes absolute

0 2 4 6 8

0

1

2

3

4

5Sham3R4F

THS 2.2

Cessation

Switch

Lym

phoc

ytes

(x 1

05 ) +/

- SEM

3R4FCessationSwitchingHTPAir

Groups

Free lung cells in Broncho-alveolar lavage fluid (BALF)

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Switching Study in an Animal Model of DiseaseResult Summary: Disease Mechanism ‐ Lung Inflammation

3R4F P1 Cess. Switch.

0 2 4 6 80

1

2

3

4

MMP activity

Sham

3R4FTHS 2.2Cessation

Switch

Month

MM

P ac

tivity

(m

U/m

l) +/

- SD

3R4FCessationSwitchingP1Air

Groups

Multiple analyte profiling in Broncho-alveolar Lavage Fluid

Page: 34

3R4F

Cessation

Switch

P1

Switching Study in an Animal Model of Disease Result Summary: Differential Gene Expression ‐ Lung 

Page: 35

Switching Study in an Animal Model of DiseaseResult Summary: Disease Mechanisms – Network Perturbations ‐ Lung

Page: 36

*

**

## #### ##

### # ##

Different from sham:* : p<0.05**: p<0.01

Different from 3R4F:# : p<0.05##: p<0.01

Sha

m

25.0 %

3R4F

38.5 %

HTT

P: P

1

27.5 %

Ces

satio

n

28.1 %

Sw

itch

29.9 %

Aortic Arches at month 8

Air 3R4F P1 Cess. Swit. Air 3R4F P1 Cess. Swit.

Switching Study in an Animal Model of Disease Result Summary: Disease Endpoint ‐ Aortic Plaque Growth

Image analysis of stained atherosclerotic plaques

Page: 37

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Means ± SEM

0

5

10

15

20

25

30

Means ± SEM

0

1

2

3

4

5

6

7

8

Means ± SEM

Plaq

ue V

olum

e (m

m3 )

Plaq

ue S

urfa

ce (m

m2 )

Mea

n O

cclu

sion

(%)

Air 3R4F P1 Cess. Swit. Air 3R4F P1 Cess. Swit. Air 3R4F P1 Cess. Swit.

Plaque Volume Aortic Arch Occlusion Plaque Surface

3D-Reconstructions

Switching Study in an Animal Model of Disease Result Summary: Disease Endpoint ‐ Aortic Plaque size at Month 83D Reconstruction from High Resolution micro-Computed Tomography

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Switching Study in an Animal Model of Disease Summary and Conclusions

• The ApoE-/- mouse model is suitable for studying smoke-related aspects of cardiovascular disease and COPD

• Continuous exposure to aerosol from a pRRP for up to 8 months does not increase cardiovascular disease, inflammation and emphysema

• Switching from cigarette smoke exposure after 2 months to fresh air exposure reverses the onset of disease as measured in apical, functional, and molecular endpoints

• Switching from cigarette smoke exposure to pRRP aerosol exposure reverses the onset of disease in a similar manner as cessation

Use of Apoe−/− Mice in an 8-Month Systems Toxicology Inhalation/Cessation Study to Investigate Cardiovascular and Respiratory Exposure Effects of a Candidate Modified Risk Tobacco Product, THS 2.2, Compared with Conventional Cigarettes Phillips B, Veljkovic E , Boué S , Schlage WK, Vuillaume G, Martin F, Titz B , Leroy P, Buettner A, Elamin A, Oviedo A, Cabanski M, Guedj E, Schneider T, Talikka M, Ivanov NV , Vanscheeuwijck P, Peitsch MC, Hoeng J.Toxicological Sciences (2016) 149, 411-32 (PMID 26609137)

Page: 39

Overall conclusion

• Exposure of rodents to cigarette smoke results in clear toxicological effects and development of known diseases

• Nicotine, when administered via inhalation exposure, causes limited toxicological effects after 28 or 90 days

• Aerosol from a pRRP, HTP elicits much lower toxicity (overall more than 80%) than cigarette smoke, even at a multiple of the nicotine exposure concentration. Most effects can be attribute to nicotine

• Aerosol from a pRRP, HTP does not lead to smoke-related disease and switching from smoke to pRRP aerosol exposure is almost identical to cessation.

Page: 40

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