QRA-based prioritization of

mainstream cigarette smoke toxicants

Xiang Li, Pingping Shang, Fuwei Xie, Maoxiang Zhu, Zhihua Yang, Cong Nie, Huimin Liu, Jianping Xie*

* Correspondence to Jianping Xie (ztridicp@126.com)

Zhengzhou Tobacco Research Institute of CNTC

CORESTA Congress 2018

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Outline

1. Background

2. Priority toxicants based on in vitro toxicity testing

3. QRA-based prioritization of mainstream cigarette

smoke toxicants

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1. Background

➢ In 2008, 7 representative toxicants were screened and

validated by CNTC to characterize the harmfulness of

mainstream cigarette smoke

➢ CO, HCN, NNK, NH3, B[a]P, phenol, and crotonaldehyde

➢ A hazard index based on these 7 representative toxicants was

developed later to regulate cigarette products of CNTC

➢ The relevant results were presented in 2008 CORESTA

Congress

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1. Background

➢ Recently, the “Tar” level was decreased year by year

➢ The delivery of smoke toxicants was also decreased

gradually

➢ To characterize exactly the harmfulness of cigarette smoke, 7

representative toxicants should be validated based on recent

brands

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2. Priority toxicants based on in vitro toxicity testing

➢ Determining 29 toxic chemicals selected from Health Canada

testing list in mainstream cigarette smoke

➢ 3 in vitro toxicity tests

➢ Establishing the relationship between the delivery of chemical

constituents and toxicological data

➢ Finding out the main toxic chemicals and developing the risk

index evaluation system of mainstream cigarette smoke

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Routine analytic

components (3)

Tar, Nicotine, Carbon monoxide

Inorganic compounds

(4)

HCN，NH3，NO，NOx

PAHs (3) Benzo[a]pyrene, Benzo[a]anthracene, Chrysene

TSNAs (4) NNN，NAT，NAB，NNK

Volatile Carbonyls (8) Formaldehyde, Acetaldehyde, Acetone, Acrolein, Propionaldehyde,

Crotonaldehyde, 2-Butanone, Butyraldehyde

Volatile phenols (7) Hydroquinone, Resorcinol, Catechol, Phenol, p-Cresol, m-Cresol,

o-cresol

Toxic Chemicals Testing List (29)

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Toxicity testing

Toxicity testing,

according to CORESTA recommended methods:

➢ Ames assay

➢ Micronucleus assay

➢ Cytotoxicity assay

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Cigarette samples

Types Number

Chinese Virginia type 0 4 17 95

Chinese blended type 2 7 5 6

International Virginia type 1 6 6 1

International blended type 2 7 3 1

Summation 5 24 31 103

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Screening and validation of representative toxicants

Data processing methods:

➢ Harmful constituents grouping: linear correlation analysis

➢ Selection of representative harmful constituents: genetic algorithm (GA)

➢ Establishment of Mathematical Models: multiple Linear regression

➢ Model test: internal , external

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Screening and validation of representative toxicants

Selection of representative harmful constituents:

For each toxicological assay

➢ The method of GA is used to model 100 times repeated calculation, and 7

variables are selected for each calculation.

➢ Select 50 results according to the principle of "small error".

➢ According to the "difference" principle, 10 out of 50 results are selected for

statistical analysis.

➢ Frequency of occurrence of statistical variables to identify the harmful

constituents that contribute most to the stability or predictive ability of the

quantitative model for each toxicological assay.

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Screening and validation of representative toxicants

Selection of representative harmful constituents:

Constituents who appear more than 7 times:

➢ CO

➢ HCN

➢ NNK

➢ NH3

➢ B[a]P, Chrysene

➢ Crotonaldehyde, Acetone, Acrolein, Propionaldehyde, 2-Butanone,

Butyraldehyde

➢ Phenol, Hydroquinone, Catechol, p-Cresol, m-Cresol, o-cresol

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Screening and validation of representative toxicants

Mathematical relationship model between the data from three toxicological

assays and 7 representative toxicants:

Y = -0.027*CB[a]P + 0.086*C1/2NH3 – 0.514*C-1

CO – 1.38*C-1NH3 + 9.72*10-8*C2

B[a]PC2Phenol+ 4.32*10-6*C2

B[a]PC2NH3 + 4.19*10-

8*C2PhenolC

2NNK + 3.23*C-1

COC-1B[a]P – 2.94*10-6*CNNKC2

Phenol -5.16*10-5*C1/2CrotonaldehydeC

2NH3 + 0.585*C1/2

B[a]PC-1NH3 –

0.011*C2COC-1

HCN + 2.45*10-6*C2HCNC-1

NNK + 0.057*C2B[a]PC-1

HCN – 3.85*10-4*C2B[a]PC-1

NNK – 0.053*C2NH3C

-1HCN – 2.23*10-

3*C2NH3C

-1B[a]P

Model for cytotoxicity assay

Y = 1.14*10-2*C2COC2

B[a]P + 3.94*10-5*C2HCNC2

NH3 - 1.44*10-3*C2NH3C

2NNK - 2.91*104*C-1

PhenolC-1

NNK - 6.31*CB[a]P C1/2HCN +

78.47*CHCNC-1CO + 55.93*CHCNC-1

Crotonaldehyde + 726.28*C1/2CrotonaldehydeC

-1NNK -0.05*C2

HCNC-1NNK + 36.32*C2

NNKC-1HCN

Model for Ames assay

Y = 3.00*10-2*CPhenolCNH3 + 2.18*10-5*C2COC2

B[a]P - 1.12*10-4*C2Crotonaldehyde*C2

B[a]P - 1.12*10-4*C2PhenolC

2NH3 - 332.49*C-

1CrotonaldehydeC

-1NNK - 2.53*103*C-1

HCNC-1B[a]P + 44.35*C-1

HCNC-1Phenol - 1.01*CCOC1/2

NH3 + 1.18*10-2*CNH3C2

CO + 1.55*10-

6*CNNKC2HCN - 15.66*CCrotonaldehydeC

-1CO + 80.36*CCrotonaldehydeC

-1HCN + 3.17*CHCNC-1

CO - 16.74*CPhenolC-1

CO - 7.92*CPhenolC-

1Crotonaldehyde+ 260.48*CPhenolC

-1HCN + 7.91*10-2*C2

CrotonaldehydeC-1

NNK - 8.28*10-3*C2NNKC-1

Crotonaldehyde

Model for micronucleus assay

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Screening and validation of representative toxicants

Model internal test:

➢ Model self-prediction

➢ Leave One Out Cross Validation (LOOCV)

LOOCV for micronucleus assay

Model test Cytotoxicity Ames Micronucleus

R, Self-

prediction0.90 0.86 0.77

R, LOOCV 0.84 0.82 0.65

0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 0.05

0.01

0.015

0.02

0.025

0.03

0.035

0.04

0.045

0.05

细胞毒性的LOOCV模型

测量值

预测值

R = 0.83513

400 600 800 1000 1200 1400 1600 1800

400

600

800

1000

1200

1400

1600

1800

AMEs的LOOCV模型

测量值

预测值

R = 0.82478

1 2 3 4 5 6 7 8

1

2

3

4

5

6

7

8微核率的LOOCV模型

测量值

预测值

R = 0.65325

LOOCV for Ames assayLOOCV for cytotoxicity assay

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Screening and validation of representative toxicants

Model external test:

➢ 15 independent external samples were selected as the prediction

R= 0.80，RMSECV= 140R= 0.78，RMSECV= 0.0036

500 600 700 800 900 1000 1100 1200 1300 1400 1500

600

700

800

900

1000

1100

1200

1300

1400

AMEs的模型预测

测量值预测值

24

39

14

49

201

48

2862

72

74

3

581

18

R = 0.80344

y = x

Ames

0.01 0.015 0.02 0.025 0.03 0.035

0.012

0.014

0.016

0.018

0.02

0.022

0.024

0.026

0.028

0.03

0.032

细胞毒性的模型预测

测量值

预测值

31

7

2752

37

61

47

19

784620

80

67

70

39

R = 0.78362

y = x

Cytotoxicity

0 1 2 3 4 5 6 7

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

微核率的模型预测

测量值

预测值

7049

4750

63

31

73

40

65

33

37

7

15

41

66

R = 0.71682

y = x

Micronucleus

R= 0.72，RMSECV= 1.2

The internal and external models have good predictive abilitys.

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3. QRA-based prioritization of mainstream cigarette smoke toxicants

➢ Assessment principle:

✓ According to the risk assessment framework of US NRC

✓ Referring to the risk assessment procedures of EPA, EFSA and JECFA

➢ Assessment procedure:

✓ Hazard identification

✓ Dose-response assessment

✓ Exposure assessment

✓ Risk Characterization 危

害

识

别

剂 量

- 反

应

关

系

评

定

暴 露

评

定

风险度表征Risk

Characterization

Do

se-r

esp

on

se a

ssessm

en

t

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3. QRA-based prioritization of mainstream cigarette smoke toxicants

Hazard identification

Toxicants Toxic effect

CO Hypoxia, anoxia

Crotonaldehyde Mucosal irritation, neurological dysfunction

HCN Dyspnea, intracellular asphyxia

NH3 espiratory mucosal irritation, cardiac arrest

NNK Strong carcinogenesis

PhenolMucosal erosion, function damage

of central nervous system, liver and kidney

B[a]P Carcinogenesis, teratogenicity and mutagenesis

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3. QRA-based prioritization of mainstream cigarette smoke toxicants

Dose-response assessment

➢ Data source:

✓ Authoritative database toxicity information (US EPA、Cal EPA、IARC、

JECFA)

✓ Peer-reviewed literatures

➢ Key parameters :

✓ Non-cancer risk: RfD/RfC

✓ Cnacer risk: IUR、CPFs

✓ MOE: POD（NOAEL、LOAEL、BMCL/BMDL）

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3. QRA-based prioritization of mainstream cigarette smoke toxicants

Dose-response assessment

Toxicants Parameter Value Unit Species Toxicity endpointData

source

CO REL 2.30E+04 g/m3 Human Mild palpitation Cal EPA

HCN REL 3.40E+02 g/m3 MachinCentral nervous system

depressionCal EPA

Phenol REL 2.00E+02 g/m3 RatNeuropathy,

hepatopathyCal EPA

NH3 RfC 1.00E+02 g/m3 HumanRespiratory system

injuryUS EPA

Crotonaldehyde RfD 1.08E-02mg/

（kg×day）Rat Death US EPA

Safe Dose (for HQ calculation)

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3. QRA-based prioritization of mainstream cigarette smoke toxicants

Dose-response assessment

Toxicants Parameter Value Unit Species Toxicity endpointData

source

B[a]P IUR 1.10E-03 （g/m3）-1 hamsteRespiratory system

tumorCal EPA

NNK CPF 1.81E+01 （mg/kg-d）-1 Rat Lung cancerNaufal et al.

2009

Cancer Potency Factor (for ILCR calculation)

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3. QRA-based prioritization of mainstream cigarette smoke toxicants

Dose-response assessment

Toxicants Parameter Value Unit Species Toxicity endpoint Data source

CO LOAEL 2.30E+04 g/m3 Human Mild palpitation Cal EPA

HCN NOAEL 3.40E+04 g/m3 MachinCentral nervous system

depressionCal EPA

Phenol NOAEL 2.00E+04 g/m3 Rat Neuropathy, hepatopathy Cal EPA

NH3 NOAEL 3.28E+03 g/m3 Human Respiratory system injury US EPA

Crotonaldehyde NOAEL 1.08E+00 mg/kg-day Rat Death US EPA

B[a]P BMDL10 7.00E-02 mg/kg-day mouse

Tumors of the anterior

stomach, esophagus and

tongue

European Food

Safety

Authority,2009

NNK BMDL10 5.20E-03 mg/kg-day Rat Lung cancer Naufal et al. 2009

POD data (for MOE calculation)

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3. QRA-based prioritization of mainstream cigarette smoke toxicants

Exposure assessment

➢ Population exposure parameters

➢ Cigarette samples: 163 commercial brands

➢ Exposure cncentrations: mainstream cigarette smoke analysis results

➢ Smoking regimen: ISO

➢ Lifetime Average Daily Intake (LADI):

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3. QRA-based prioritization of mainstream cigarette smoke toxicants

Exposure assessment

Parameter Unit Mean value Data source

Daily Cigarette Consumption (CpD) cig/d 16.4 2006 CHNS

Exposure Duration (ED)years 61.7 WHO 2006 for China

Exposure Frequency (EF) d/years 319.6NHANES 1999-2008

（NCHS 2010）

Body Weight (BW) kg 63.7 2006 CHNS

Average Time (AT) dayscancer：(70 y x 365 d/y)

non-cancer：365 d/yEPA,1989

Daily Inhalation Rate (DIR) L/（d*kg） 271.8 Cal EPA, 2003

Population exposure parameters

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3. QRA-based prioritization of mainstream cigarette smoke toxicants

Exposure assessment

ToxicantCO

(mg/cig)

HCN

(µg/cig)

Phenol

(µg/cig)

NH3

(µg/cig)

Crotonaldehyde

(µg/cig)

B[a]P

(ng/cig)

NNK

(ng/cig)

Yield 12.8 7.7 120.2 16.1 17.6 9.5 6.7

The yields (weighted mean) of 7 representative toxicants*

* The yields of 7 representative toxicants in mainstream cigarette smoke from domestic market in 2010

Lifetime Average Daily Intake for 7 representative toxicants, (LADIi)

ToxicantCO

(µg/m3)

HCN

(µg/m3)

Phenol

(µg/m3)

NH3

(µg/m3)

Crotonaldehyde

(mg/kg-d)

B[a]P

(µg/m3)

/(mg/kg-d)

NNK

(mg/kg-d)

LADI 1.06E+04 9.97E+01 1.34E+01 6.39E+00 3.97E-036.95E-03/

1.89E-061.00E-06

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3. QRA-based prioritization of mainstream cigarette smoke toxicants

Risk Characterization

➢ Cancer risk: Incremental Lifetime Cancer Risk (ILCR)

ILCR = CPF (or IUR) x LADI

➢ Non-cancer risk: Hazard Quotient (HQ)

ADI

RfC/RfD

➢ Margin of Exposure for Constituent (MOE)

POD

LADI

HQ =

MOE =

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3. QRA-based prioritization of mainstream cigarette smoke toxicants

Risk Characterization

Toxicant ILCR

B[a]P 7.64E-06

NNK 2.41E-05

Σ ILCR 3.17E-05

Hazard share

➢ ILCR can quantify the carcinogenic risk of each toxicant

➢ Σ ILCR can reflect the total hazard of carcinogenic toxicants

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3. QRA-based prioritization of mainstream cigarette smoke toxicants

Risk Characterization

Toxicant HQ

CO 0.46

HCN 0.29

Phenol 0.07

NH3 0.06

Crotonaldehyde 0.37

Σ HQ 1.25

Hazard share

➢ HQ can quantify the non-carcinogenic risk of each toxicant

➢ Σ HQ can reflect the total hazard of non-carcinogenic toxicants

NH3 (5.10%)

Phenol (5.33%)

HCN (23.41%)

Crotonaldehyde (29.32%)

CO (36.84%)

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3. QRA-based prioritization of mainstream cigarette smoke toxicants

Risk Characterization

Toxicant MOE

CO 2.17

HCN 341.04

Phenol 1497.74

NH3 513.59

Crotonaldehyde 272.20

B[a]P 37082.50

NNK 3905.92

➢ MOE < 10,000: target for risk mitigation

➢ MOE > 10,000: probability that exposure not of

concern

➢ The MOE value of each toxicant cannot be added

to the sum

➢ The MOE value of each toxicant varies greatly,

which is not conducive to hazard comparison

among them

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Hazard Index

Research strategy

Non-cancer risk（HQ）

Cancer risk（ILCR）

Hazard index

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Hazard Index

➢Setting two hazard indexes

✓ Cancer risk index

HC = ILCRB[a]P + ILCRNNK

✓ Non-cancer index

HNC= HQCO + HQHCN + HQPHE + HQNH3+ HQCRO

0.8

0.85

0.9

0.95

1

1.05

Hc Hnc H0 焦油

2008

2009

2010

2011

HNCHC H0 Tar

2008-2011

National cigarette census sample hazard index

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Summary

➢ The screening and validation of representative toxicants

showed that 7 representative toxicants can be used to

characterize the harmfulness of mainstream cigarette smoke.

➢ Based on the risk assessment framework developed by US

NRC, a risk assessment method for harmful constituents of

cigarette smoke was established.

➢ The risk of representative toxicants was evaluated by ILCR,

HQ and MOE, which provided scientific basis for quantitative

regulation of seven representative toxicants.

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Thank you for your attention!

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