Investigation of TSNA Formation in Electronic Cigarette ...

Xiaohong Cathy Jin, Karen C. Avery, Georgios D.

Karles, William P. Gardner, Matt S. Melvin,

Donna C. Smith, Willie J. Mckinney, and Karl A.

Wagner

Investigation of TSNA Formation in Electronic Cigarette Liquids and Aerosols

Altria Client Services | Regulatory Sciences | Xiaohong Cathy Jin | October 9, 2017 | 2017 Coresta -ST10 | 1

2017

_ST

10_J

inC

athy

.pdf

SS

PT

2017

- D

ocum

ent n

ot p

eer-

revi

ewed

by

CO

RE

ST

A

Introduction


Preliminary data indicated that TSNA formation was

possibly occurring during the generation of aerosols1

TSNAs in tobacco are formed through the reaction of

nitrite with nicotine and minor alkaloids2

E-liquids typically contain tobacco derived nicotine and

may also contain other tobacco constituents 3,4

No studies on TSNA formation in e-liquids and aerosol in

literature

2017

_ST

10_J

inC

athy

.pdf

SS

PT

2017

- D

ocum

ent n

ot p

eer-

revi

ewed

by

CO

RE

ST

A

Objective


Investigate the formation of TSNAs in e-liquids and

aerosols

•Role of nitrite and minor alkaloids

−temperature, time, and concentration effects

Evaluate TSNA levels in e-liquids and aerosols of

commercial cigalike e-cigarette products

2017

_ST

10_J

inC

athy

.pdf

SS

PT

2017

- D

ocum

ent n

ot p

eer-

revi

ewed

by

CO

RE

ST

A

Hoffmann et al. (1994)

TSNA Formation Pathway


Nicotine Nornicotine Anabasine Anatabine Nitrosation

NNK NNN NAB NAT

NO2 NO2 NO2 NO2 NO2

2017

_ST

10_J

inC

athy

.pdf

SS

PT

2017

- D

ocum

ent n

ot p

eer-

revi

ewed

by

CO

RE

ST

A

Investigation of the TSNA Formation in E-

liquids and aerosols


2017

_ST

10_J

inC

athy

.pdf

SS

PT

2017

- D

ocum

ent n

ot p

eer-

revi

ewed

by

CO

RE

ST

A

Role of Nitrite in TSNA Formation: E-liquids


Lab fortified e-liquid (82.5% PG/Gly (50:50),15%

water, 2.5% nicotine) was fortified with nitrite (2 µg/g -

50 µg/g)

Temperature

Storage time (ambient)

TSNAs were analyzed by UPLC/MS/MS1

2017

_ST

10_J

inC

athy

.pdf

SS

PT

2017

- D

ocum

ent n

ot p

eer-

revi

ewed

by

CO

RE

ST

A

TSNA in e-Liquids at Different Temperatures E-liquid (82.5% PG/Gly (50:50), 15% water, 2.5% nicotine) fortified with 2

µg/g nitrite, heated for 5 min


0

500

1000

1500

2000

2500

3000

NNK NAB NAT NNN

TS

NA

(n

g/g

e-l

iqu

ids

) Rm Temp

80°C

150°C

200°C

250°C

2017

_ST

10_J

inC

athy

.pdf

SS

PT

2017

- D

ocum

ent n

ot p

eer-

revi

ewed

by

CO

RE

ST

A

TSNA in E-liquids Over Time E-liquid (82.5% PG/Gly (50:50), 15% water, 2.5% nicotine) fortified with 3 levels of

nitrite, stored at room temp, n=3


0

10

20

30

40

50

60

0 50 100

NN

K (

ng

/g)

Days after nitrite fortified

NNK

2µg/g NO2

10µg/g NO2

50µg/g NO2

S=0.58

S=0.16

S=0.03 0

1

2

3

4

0 50 100

NN

N (

ng

/g)

Days after nitrite fortified

NNN

2µg/g NO2

10µg/g NO2

50µg/g NO2

S=0.04

S=0.01

2017

_ST

10_J

inC

athy

.pdf

SS

PT

2017

- D

ocum

ent n

ot p

eer-

revi

ewed

by

CO

RE

ST

A

Role of Nitrite in TSNA Formation: Aerosols


Aerosol generation • Cartridges filled with the same fortified e-liquids

• Aerosol generation regimen: 55mL puff volume, 5s duration,

30s intervals

Comparison of TSNAs in e-liquids and aerosols

2017

_ST

10_J

inC

athy

.pdf

SS

PT

2017

- D

ocum

ent n

ot p

eer-

revi

ewed

by

CO

RE

ST

A

E-liquid (82.5% PG/Gly (50:50), 15% water, 2.5% nicotine) fortified with 2 levels of

nitrite (n=3); cartridges filled with the fortified e-liquids, 50 puffs aerosols collected (n=4)

TSNAs in E-liquids and Aerosols


Elevated TSNA levels in aerosol compared to e-liquid when nitrite is present.

0

10

20

30

40

50

60

70

liquid aerosol liquid aerosol liquid aerosol liquid aerosol

NNK NAB NAT NNN

TS

NA

(n

g/g

e-l

iqu

id o

r a

ero

so

l)

0 µg/g NO2

2 µg/g NO2

10 µg/g NO2

2017

_ST

10_J

inC

athy

.pdf

SS

PT

2017

- D

ocum

ent n

ot p

eer-

revi

ewed

by

CO

RE

ST

A

Contributions of Minor Alkaloids When Nitrite is

Present


Minor Alkaloids:

• nornicotine, anabasine, anatabine

E-liquid (85% PG/Gly (50/50)

+15% water, without nicotine)

fortified with 2 µg/g nitrite and

10 µg/g minor alkaloids

Evaluate effects of temperature

and storage time

NO2 NO2 NO2

2017

_ST

10_J

inC

athy

.pdf

SS

PT

2017

- D

ocum

ent n

ot p

eer-

revi

ewed

by

CO

RE

ST

A

E-liquids fortified with 2 µg/g nitrite and 10 µg/g minor alkaloids, heated at 200 oC for

5 minutes (n=3)

TSNA in E-liquids at Elevated Temperature

When Minor Alkaloids and Nitrite are Present


Minor alkaloids

fortified

NNK

(ng/g)

NAB

(ng/g)

NAT

(ng/g)

NNN

(ng/g)

Nornicotine <0.2 ND ND 49.2

Anabasine ND 7.1 ND ND

Anatabine ND ND 8.4 ND

*ND: not detected

2017

_ST

10_J

inC

athy

.pdf

SS

PT

2017

- D

ocum

ent n

ot p

eer-

revi

ewed

by

CO

RE

ST

A

E-liquids (85% PG/Gly (50/50) +15% water) fortified with 2 µg/g nitrite and 10 µg/g

minor alkaloids, stored at room temperature (n=3)

TSNA in E-liquids Over Time When Minor

Alkaloids and Nitrite are Present


0

1

2

3

4

NNN NAB NAT

Nornicotine fortified Anabasine fortified Anatabine fortified

TS

NA

(n

g/g

)

initial

2 weeks

4 weeks

8 weeks

2017

_ST

10_J

inC

athy

.pdf

SS

PT

2017

- D

ocum

ent n

ot p

eer-

revi

ewed

by

CO

RE

ST

A

TSNAs in Liquids and Aerosol When Minor

Alkaloids and Nitrite are Present


E-liquids (85% PG/Gly (50/50) +15% water) fortified with 2 µg/g nitrite and 10 µg/g

minor alkaloids (n=3); Aerosols were collected 150 puffs (n=4)

0

2

4

6

8

10

12

NNN NAB NAT

Nornicotine fortified Anabasine fortified Anatabine fortified

TS

NA

(n

g/g

) Liquid

Aerosol

2017

_ST

10_J

inC

athy

.pdf

SS

PT

2017

- D

ocum

ent n

ot p

eer-

revi

ewed

by

CO

RE

ST

A

Evaluation of TSNA in Commercial

Cigalike e-Cigarette Products


2017

_ST

10_J

inC

athy

.pdf

SS

PT

2017

- D

ocum

ent n

ot p

eer-

revi

ewed

by

CO

RE

ST

A

Evaluation of TSNA in Commercial

e-Cigarette Products


8 commercial cigalike e-cigarette products were

evaluated

• TSNA in e-liquids and aerosols

• Nitrite and minor alkaloids in e-liquids

2017

_ST

10_J

inC

athy

.pdf

SS

PT

2017

- D

ocum

ent n

ot p

eer-

revi

ewed

by

CO

RE

ST

A

TSNAs in e-Liquids of Commercial Products


0

5

10

15

20

25

30

35

A B C D E F G H

TS

NA

(n

g/g

)

Products

NNK

NAB

NAT

NNN

E-liquids were removed from e-cig cartridges and analyzed on the same day 2017

_ST

10_J

inC

athy

.pdf

SS

PT

2017

- D

ocum

ent n

ot p

eer-

revi

ewed

by

CO

RE

ST

A

TSNAs in Aerosols of Commercial Products


150 puffs collected or until battery exhaustion (n=4)

0

5

10

15

20

25

30

35

40

45

50

A B C D E F G H

TS

NA

(n

g/g

)

Products

NNK

NAB

NAT

NNN

TSNAs in aerosol per puff ranged from ND to 0.16 ng/puff

2017

_ST

10_J

inC

athy

.pdf

SS

PT

2017

- D

ocum

ent n

ot p

eer-

revi

ewed

by

CO

RE

ST

A

Nitrite and Minor Alkaloids in E-liquids

of Commercial Products


Products

Nitrite

(µg/g)

Nornicotine

(µg/g)

Anabasine

(µg/g)

Anatabine

(µg/g)

A <0.5 3.2 ND ND

B <0.5 11.8 ND 2.6

C <0.5 <2.5 ND ND

D <0.5 4.0 ND ND

E <0.5 5.7 8.3 9.3

F <0.5 <2.5 ND ND

G 0.8 <2.5 ND ND

H 9.9 8.9 9.3 2.5

2017

_ST

10_J

inC

athy

.pdf

SS

PT

2017

- D

ocum

ent n

ot p

eer-

revi

ewed

by

CO

RE

ST

A

Product B: TSNAs in e-Liquids and Aerosols


0

5

10

15

20

25

30

35

NNK NAB NAT NNN

TS

NA

(n

g/g

) Liquid

Aerosol

No significant differences between TSNA levels in aerosols and liquids most

likely due to no quantifiable levels of nitrite (< 0.5 µg/g)

2017

_ST

10_J

inC

athy

.pdf

SS

PT

2017

- D

ocum

ent n

ot p

eer-

revi

ewed

by

CO

RE

ST

A

Product H: TSNAs in e-Liquids and Aerosols


0

5

10

15

20

25

30

35

40

45

50

NNK NAB NAT NNN

TS

NA

(n

g/g

)

TSNA

Liquid

Aerosol

TSNA levels elevated in aerosol compared to liquid most likely due to high levels of

nitrite and minor alkaloids.

2017

_ST

10_J

inC

athy

.pdf

SS

PT

2017

- D

ocum

ent n

ot p

eer-

revi

ewed

by

CO

RE

ST

A

Conclusions


Low levels of TSNAs were observed in e-cigarette liquids and

aerosols.

Nitrite in e-liquids could react with nicotine or minor alkaloids to

form TSNAs: • TSNA increased with time under normal storage when nitrite is present.

• TSNA formation is accelerated by elevated temperature when nitrite is

present

TSNA could form during aerosol generation when nitrite is

present.

This study provides useful information that may be used to

develop and evaluate electronic cigarette products.

2017

_ST

10_J

inC

athy

.pdf

SS

PT

2017

- D

ocum

ent n

ot p

eer-

revi

ewed

by

CO

RE

ST

A

References


1. Jin, X. C, et al, Determination of TSNAs in e-Cigarette Liquids and

Aerosols by UPLC-MS/MS, www.altria.com/ALCS-Science, 2016

2. Hoffmann D. et al., Tobacco-Specific N-Nitrosamines and

ARECA-Derived N-Nitrosamines: Chemistry, Biochemistry,

Carcinogenicity, and Relevance to Humans; J. of Toxi. And Envir.

Health, 1994, 41, 1-52

3. Farsalinos, K.E; et al, Int. J Envir. Res. Public Health, 2015 Apr,

12 (4): 3439-3452

4. CORESTA e-Cigarette Task Force Reference Report, Electronic

Cigarettes: Assessment of Analytical Literature, May 2014

2017

_ST

10_J

inC

athy

.pdf

SS

PT

2017

- D

ocum

ent n

ot p

eer-

revi

ewed

by

CO

RE

ST

A

Acknowledgement


Yezdi Pithawalla

Tammy Blake

2017

_ST

10_J

inC

athy

.pdf

SS

PT

2017

- D

ocum

ent n

ot p

eer-

revi

ewed

by

CO

RE

ST

A

Acknowledgement


Yezdi Pithawalla

Tammy Blake

www.altria.com/ALCS-Science

2017

_ST

10_J

inC

athy

.pdf

SS

PT

2017

- D

ocum

ent n

ot p

eer-

revi

ewed

by

CO

RE

ST

A

Investigation of TSNA Formation in Electronic Cigarette ...

Documents

Transcript of Investigation of TSNA Formation in Electronic Cigarette ...