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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