BAT Science - Snus chemical and biological characterisation

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Snus chemical and biological characterisation

There has been relatively little research to develop and validate analytical methods for characterising the levels of constituents found in snus and other smokeless tobacco products. We are currently working with other companies to both develop new analytical methods where necessary and to evaluate the analytical variability of existing measures of constituents in smokeless tobacco products.

Chemical analysis of a range of smokeless tobacco products

We recently analysed a sample of thirty different smokeless tobacco products currently sold in European Union countries [ 1 ].  The products included nasal snuff, chewing tobacco, gutkha and snus.

All of the products were permitted for manufacture and sale in all EU countries except for snus, which can only be sold in Sweden.

We carried out a chemical analysis of these products to determine the levels of several analytes including:

  • water;
  • nicotine;
  • nitrite;
  • benzo(a)pyrene (B(a)P);
  • N-nitrosodimethylamine (NDMA);
  • tobacco specific nitrosamines (TSNAs) measured as the combined amount of N’-nitrosoanabasine (NAB), N’-nitrosoanatabine (NAT), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N’-nitrosonornicotine (NNN); and
  • certain heavy metals - arsenic, cadmium, chromium, lead and nickel. 

Around 85% of the smokeless tobacco products contained at least one toxic constituent at levels above those found in Swedish snus products designed to have lower levels of certain toxic constituents.

Around 20% of the smokeless tobacco products contain toxic constituents at levels above those proposed by the European Smokeless Tobacco Council (ESTOC)  as a standard for smokeless tobaccos.

Swedish snus was the smokeless tobacco product that had some of the lowest levels of toxic constituents of those tested.

Samples of gutkha and gutkha tulsi also had low levels of some of the constituents tested.  But like many of the Asian based products these contain potentially harmful ingredients such as areca nut and slaked lime which have been associated with risks of oral disease.  They also contain a much lower percentage of tobacco than other smokeless tobacco products.

This study found that most of the products examined - all legally permitted for sale within the EU - contained higher levels of toxic constituents than those typically found in Swedish snus.

Effect of storage on chemical composition of snus

The levels of tobacco-specific nitrosamines have been shown to increase over time in some types of smokeless tobacco products - particularly in those products that are high in water content. 

Snus is moist but is heat treated using a process similar to pasteurisation.  We have investigated whether the levels of TSNAs increase in snus during storage by analysing four snus brands stored under various conditions:

  • cold at 4ºC;
  • 22ºC and 60% relative humidity;
  • 34ºC and 20% relative humidity; and
  • 34ºC and 80% relative humidity. 

Samples were analysed every two weeks for a period up to 24 weeks but the TSNA levels did not increase for any of the storage conditions for all brands presented [ 2 ].

Transfer of snus constituents from snus and products to consumers

We are conducting studies to evaluate the transfer of a range of individual snus constituents to Swedish consumers during use [ 3 ].  Studies of this kind will inform a potential risk assessment for ingredients used in snus.

The methodology developed employs “central-location” trials in which snus users are invited to use snus in their usual way for a one hour period, under supervised conditions.  Snus pouches are analysed chemically after use, and the content of constituents is compared to the levels in unused control products.  The difference in these two values provides a measure of the extent of transfer.  Multi-analyte analytical methods have been developed that allow for examination of a range of constituents and ingredients from the same exercise.  The multi-analyte approach helps to build an understanding of the factors underlying the extent of transfer of different compounds from snus. 

The results of pilot trials revealed significant differences in pouch moisture and nicotine content between unused and extracted pouches.  On average, pouch moisture increased from 48 to 63%, primarily due to saliva absorption by the snus pouch.  Nicotine levels fell by approximately 40%, consistent with results observed in previous studies..  Measurements of a range of constituents indicated that the water solubility of a constituent is a strong indicator of the amount likely to be transferred during snus use. 

These results are being used as the foundation for the development of a standardised laboratory measure for the transfer of snus constituents.

In vitro characterisation of snus

There is no standard method for applying snus - or extracts of snus - to in vitro test systems such as the Ames test [ 4 ].

We are currently investigating the effect of various extraction techniques, including extracts with water and with artificial saliva, for use in in vitro tests.

  1. Williamson, J., Massey, E. D., Digard, H. (2007). Constituent comparison in smokeless tobacco products used in Europe. Poster presented at the 9th Annual Society for Research on Nicotine and Tobacco Europe Conference, Madrid, Spain, October 3-6, 2007.  PDF: PDF: Constituent comparison in smokeless... - PDF: Constituent comparison in smokeless... (315 kb)
  2. Proctor, C., Sandi, D. (2007). Presentation to Life Science Research Office Expert Panel. Oral presentation to the LSRO Expert Panel, Bethesda, Maryland, USA, October 3, 2007.  PDF: PDF: Presentation to Life Science… - PDF: Presentation to Life Science… (413 kb)
  3. McAdam, K., Digard, H., Williamson, J., Proctor, C. (2008). Analysis of tobacco constituent extraction by snus users.  Poster presented at the 11th Annual Society for Research on Nicotine and Tobacco European Conference, Rome, Italy, September 23-26, 2008.
  4. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. (2007). Volume 89: Smokeless tobacco and some tobacco-specific N-nitrosamines . Lyon: International Agency for Research on Cancer.
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Methods for measuring constituents in smokeless tobacco products