Effects of Product Format on Nicotine and TSNA Extraction ...

Effects of Product Format on Nicotine and TSNA

Extraction from Snus Pouches

Nathan Gale, Graham Errington and Kevin McAdam

Group Research & Development, British American Tobacco, Southampton, UK

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67th Tobacco Science Research Conference, Williamsburg, VA, 15-18 September 2013

Introduction

� Background and Objective

� Methods- Approach for estimating exposure

- Study design

- Study protocol

- Chemical & statistical analysis

� Results- Mass and percentage extracted

- Effects on extraction of nicotine, NNN and NNK

- Comparison with literature

� Summary

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Background and Objective

Understanding Exposure

� Understanding quantity of constituents extracted during snus use is an important step in estimating exposure

- STPs, NNN and NNK identified as Group 1 carcinogens by IARC

- Nicotine, NNN and NNK feature on FDA HPHC list

� A number of factors are likely to affect extraction- Product format, human use behaviours, constituents

� Our previous studies of pouched snus have concentrated on a single product

- To allow evaluation of effects of human and constituent factors on extraction

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Methods

Approach for Estimating Exposure

� Constituent levels in used snus portions compared with levels in unused portions

� Amount and extent of extraction calculated:

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Amount Extracted = Quantity in Unused Snus – Quantity in Used Snus

% Extracted = (Amount Extracted / Quantity in Unused Snus)*100

Methods

Study Design

� Study designed to assess effects of pouch mass (3 levels), moisture content (2 levels) and flavour (3 levels) on extraction

- Balanced incomplete block design

� 18 test pieces manufactured specifically for this study- No effect of flavour level on nicotine, NNN or NNK extraction, therefore data

for products with same pouch size and moisture content combined here

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ProductPouch mass

(g ± tolerance)Moisture content(% ± tolerance)

Product ‘style’

1 0.5 ± 0.05 52 ± 2 Brown

2 1.0 ± 0.10 52 ± 2 Brown

3 1.5 ± 0.15 52 ± 2 Brown

4 0.5 ± 0.05 45 ± 2 White

5 1.0 ± 0.10 45 ± 2 White

6 1.3 ± 0.13 45 ± 2 White

Methods

Study Protocol

� Central Location study in Stockholm, Sweden

� 18 male snus users aged 19-65 completed the study- Each volunteer provided informed consent and was reimbursed for time

and travel

� Controlled intake before and during study- No food or tobacco product consumption 1 hour prior to attendance

- No food or drink during session, other than option of water between samples

- Snus pouch held in mouth by subjects for 60 minutes1

� Control (unused) pouches taken from same tin as test sample at same time

� Post-use, snus samples transported frozen in individual glass vials

61 Digard, H., Errington, G., Richter, A. and McAdam, K. Nicotine and Tobacco Research 2009 11:1175-1181

Methods

Chemical and Statistical Analysis

� Validated multi-analyte extraction method1 used to examine multiple constituents from same snus pouch

- Each pouch extracted in methanol and centrifuged

- Aliquot analysed for nicotine by GC-MS

- Second aliquot analysed for TSNAs by LC-MS/MS

� Extraction values calculated for each pair of pouches- General Linear Model used to assess effects of pouch mass and moisture

content

71 Digard, H., Gale, N., Errington, G., Peters, N. and McAdam, K. Chemistry Central Journal 2013 7:55

Results

Mass Extracted

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Meanpouchmass

(g ± SD)

Meanmoisturecontent(% ± SD)

Meannicotine

extracted(mg ± SD)

MeanNNN

extracted(ng ± SD)

MeanNNK

extracted(ng ± SD)

0.5 ± 0.03 55 ± 1.6 1.5 ± 0.6 91.6 ± 30.5 29.7 ± 9.7

1.0 ± 0.03 55 ± 0.9 2.6 ± 0.8 160.2 ± 42.3 50.9 ± 13.3

1.5 ± 0.04 54 ± 0.5 3.7 ± 1.2 226.2 ± 67.1 74.2 ± 20.7

0.5 ± 0.04 45 ± 0.5 1.3 ± 0.4 66.5 ± 24.8 20.9 ± 8.1

1.0 ± 0.05 45 ± 0.5 2.7 ± 1.2 149.4 ± 71.6 47.1 ± 23.4

1.3 ± 0.05 45 ± 1.4 3.1 ± 0.9 168.4 ± 59.4 50.7 ± 16.7

N = 50-54 due to sample administration error and laboratory losses, except for

moisture content where N=6 and determined on tobacco removed from pouches

Results

Percentage Extracted

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Meanpouchmass

(g ± SD)

Meanmoisturecontent(% ± SD)

Meannicotine

extracted(% ± SD)

MeanNNN

extracted(% ± SD)

MeanNNK

extracted(% ± SD)

0.5 ± 0.03 55 ± 1.6 45 ± 15 40 ± 12 43 ± 12

1.0 ± 0.03 55 ± 0.9 40 ± 11 35 ± 9 36 ± 8

1.5 ± 0.04 54 ± 0.5 37 ± 11 32 ± 9 33 ± 9

0.5 ± 0.04 45 ± 0.5 31 ± 8 23 ± 8 24 ± 9

1.0 ± 0.05 45 ± 0.5 33 ± 13 26 ± 12 26 ± 13

1.3 ± 0.05 45 ± 1.4 27 ± 7 22 ± 8 22 ± 7

� Changes to pouch mass and moisture content appear to affect NNN and NNK extraction in the same way as they do nicotine extraction

Results

Percentage Extracted

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Meanpouchmass

(g ± SD)

Meanmoisturecontent(% ± SD)

Meannicotine

extracted(% ± SD)

MeanNNN

extracted(% ± SD)

MeanNNK

extracted(% ± SD)

0.5 ± 0.03 55 ± 1.6 45 ± 15 a 40 ± 12 a 43 ± 12 a

1.0 ± 0.03 55 ± 0.9 40 ± 11 a 35 ± 9 b 36 ± 8 ab

1.5 ± 0.04 54 ± 0.5 37 ± 11 a 32 ± 9 b 33 ± 9 ab

0.5 ± 0.04 45 ± 0.5 31 ± 8 a 23 ± 8 b 24 ± 9 b

1.0 ± 0.05 45 ± 0.5 33 ± 13 a 26 ± 12 b 26 ± 13 b

1.3 ± 0.05 45 ± 1.4 27 ± 7 a 22 ± 8 b 22 ± 7 b

� Changes to pouch mass and moisture content appear to affect NNN and NNK extraction in the same way as they do nicotine extraction

- One-way ANOVA shows NNN and NNK extracted to a lesser extent than nicotine (p<0.05) for 45% moisture content pouches

Results

Percentage Extracted

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Meanpouchmass

(g ± SD)

Meanmoisturecontent(% ± SD)

Meannicotine

extracted(% ± SD)

MeanNNN

extracted(% ± SD)

MeanNNK

extracted(% ± SD)

0.5 ± 0.03 55 ± 1.6 45 ± 15 a 40 ± 12 a 43 ± 12 a

1.0 ± 0.03 55 ± 0.9 40 ± 11 a 35 ± 9 b 36 ± 8 ab

1.5 ± 0.04 54 ± 0.5 37 ± 11 a 32 ± 9 b 33 ± 9 ab

0.5 ± 0.04 45 ± 0.5 31 ± 8 a 23 ± 8 b 24 ± 9 b

1.0 ± 0.05 45 ± 0.5 33 ± 13 a 26 ± 12 b 26 ± 13 b

1.3 ± 0.05 45 ± 1.4 27 ± 7 a 22 ± 8 b 22 ± 7 b

� Changes to pouch mass and moisture content appear to affect NNN and NNK extraction in the same way as they do nicotine extraction

- One-way ANOVA shows NNN and NNK extracted to a lesser extent than nicotine (p<0.05) for 45% moisture content pouches

Results

Effects on Extraction of Nicotine

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� Increase in pouch mass = increase in mass of nicotine extracted

� …and a decrease in percentage nicotine extracted

� Increase in moisture content = increase in mass of nicotine extracted

� …and an increase in percentage nicotine extracted

Results

Effects on Extraction of NNN and NNK

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Results

Comparison with literature

� Lunell & Lunell1 reported extraction of nicotine from four different pouched snus products

141 Lunell, E. and Lunell, M. Nicotine and Tobacco Research 2005 7:397-403

Mass / Moisture / StyleMean Nicotine Extraction (%)

This BAT study Lunell & Lunell

0.5g / 52% / Brown 45 ± 15 (n=50) 44 (n=12)

1.0g / 52% / Brown 40 ± 11 (n=54) 31 (n=12)

1.5g / 52% / Brown 37 ± 11 (n=54) -

0.5g / 45% / White 31 ± 8 (n=52) 22 (n=12) [0.3g, 25%]

1.0g / 45% / White 33 ± 13 (n=53) 22 (n=12)

1.3g / 45% / White 27 ± 7 (n=54) -

� Supported by the findings of the current study- Decrease in moisture content results in lower percentage extraction

- Increase in pouch mass leads to lower percentage extraction

Summary

� Extraction of nicotine, NNN and NNK from pouched snus is incomplete (<50% of unused pouch content)

� Both pouch mass and moisture content measurably influence extraction of nicotine, NNN and NNK from snus during use

- Increase in pouch mass results in decreased extraction efficiency but in increased total exposure

- Increase in moisture content results in increased extraction efficiency and total exposure

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Acknowledgements

� GfK Sverige AB – Fieldwork

� Research Support Services staff at GR&D, British American Tobacco

- Tim John Wheeler – Product manufacturing

- Nicola Peters – Method development and sample analysis

- Donna Byrom – Method development and sample analysis

- Paul Blachford – Sample analysis

- Margaret Spence – Sample analysis

- Cheryl Johns – Sample analysis

- Maria Carradus – Sample analysis

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