The smoking laboratory is equipped with smoking machines for the collection and analysis of emissions from cigarettes, RYO, e-cigarettes, heated-tobacco products, and waterpipes. A pyrolysis-GC-MS machine is available for the analysis of thermal decomposition products of single ingredients or mixtures of ingredients. The following five machines are used:

• A 10-port linear smoking machine (SM410, Cerulean, Milton Keynes, UK)
• A 20-port rotary smoking machine (RM20H, Borgwaldt, Hamburg, Germany)
• A 4-port e-cigarette/heated tobacco product smoking machine (LM4E, Borgwaldt, Hamburg, Germany)
• A single port waterpipe (shisha) smoking machine (SH1, Borgwaldt, Hamburg, Germany)
• A pyrolysis module(Gerstel) coupled to a GC-MS (Agilent 7890B/5977A)

To mimic the behaviour of smokers more closely, our SM410 smoking machine has been customised to allow it to ‘play back’ puffs from actual smokers that have been recorded previously, using a CReSSmicro topography flowmeter (Plowshare Technologies, Baltimore, MD, USA).

The filter design of many cigarettes currently on the market includes several very small perforations in the wrapping paper. To allow measurements of the extent of filter ventilation of different products, a QUANTUM SOLO/ Cerulean (Milton Keynes, UK) is available in the smoking laboratory at RIVM.

To investigate the effect of additives, a machine is available for spiking additives into existing (commercial) cigarettes. It injects a solution of an additive along the length of the tobacco plug using a thin, long needle that is slowly retracted while the cigarette is rotating. The machine was originally designed and built by the University of Minnesota.

The following instruments/techniques are available for analytical chemical analyses. This equipment is used to measure a range of different compounds and compound classes including aldehydes, organic compounds (such as flavourings, VOCs, PACs and nicotine), metals, sugars and nitrosamines. New methods are developed if necessary to support new research.

• ICP-MSMS (Thermo Fisher scientific)
• NMR, 60 MHz tabletop instrument (Magritek))
• GC-MS, including headspace, SPME, pyrolysis (Gerstel), Twister/stir bar (SBSE), TD (Agilent)
• GC-FID (Shimadzu)
• GC-TCD (Shimadzu)
• GC-EI-MS (Agilent)
• LC-MSMS (ABI sciex)
• LC-QTOF (Waters)
• LC-orbitrap (Thermo Fisher scientific)
• LC-ELSD
• LC-DAD (Shimadzu)
• LC-IC (Shimadzu)

Emission from tobacco and nicotine products contain harmful compounds. RIVM uses cell models of the airways to assess the hazard. These cell models are cultured on the barrier between air and medium, so-called Air-Liquid-Interface or ALI. This allows the cells to be exposed to airborne compounds, which closely resembles human inhalation. We use airway epithelial cells that form the inner lining of the lungs, and macrophages, a type of immune cell, that plays an important role in the effects of particle exposure. In addition to the cell model, the exposure method should resemble human exposure as closely as possible. That is why we have connected a smoking machine to our exposure units, which allows continuous exposure of cells with a chosen topography.

Hazard can be characterised by measurement of general parameters such as cell viability or barrier function in addition to cellular responses, such as an inflammatory response. In addition, ALI-cultured cells can be used to study the translocation of nicotine or other compounds, which gives insight into the uptake of compounds and thereby the exposure of other organs in the body.
 

RIVM is developing a zebrafish embryo (ZFE) model for screening potentially addictive neuroactive compounds, such as nicotine. To do this, we study behavioural changes after exposure to neuroactive substances using a ZebraBox (Viewpoint, Lyon, France) following a light-dark transition protocol. A whole-organism tool such as the ZFE model can be of particular value for first-tier screening purposes to decide whether to proceed with e.g., in vivo studies. Alternatively, ZFE could be included in a battery of new approach methodology (NAM) tools, to build and inform toxicological pathways describing exposure to substances leading to addiction in humans.
The use of the zebrafish as a research model to study neuroactive drugs is emerging, as it has multiple advantages over traditionally applied animal models. This includes ease of reproduction, fast development and a high level of conservation between zebrafish and humans on the level of brain function, including neurotransmitter signalling and receptor function. Moreover, complex endpoints such as behaviour can be studied in ZFE. In addition, the ZFE until 120 h post-fertilization is not protected under animal experimentation laws, making their use in line with the 3R principle (Replacement, Reduction, and Refinement of animal experiments). Collectively, this makes ZFE an attractive research model for the assessment of the addictive potential of tobacco components and additives.

RIVM uses quantitative and qualitative studies to investigate behaviour and perceptions of TNP users. We conduct questionnaires, for example, to examine prevalence of use and reasons for use of TNP. We also conduct focus groups to obtain more in depth insights into users’ attitudes and views on their products. Experimental studies are also conducted, for example to assess how products are used and to determine exposure to substances in smoke or vape. More information about RIVM’s behavioural research of TNP users can be found on the webpage Research line: Product use, perception and exposure.

In the European Union, manufacturers and importers are legally obliged to provide information about the composition and other properties of tobacco and nicotine products they market in each EU European Union (European Union ) country. This is done using a secure database called the European Union Common Entry Gate (EU-CEG). RIVM uses EU-CEG data to analyse product characteristics and trends of products on the Dutch market, for example the number and type of ingredients added to the tobacco or changes in e-liquid flavouring over time. RIVM also analyses in-house produced lab data, for example to determine the influence of tobacco ingredients and smoking method on emission compositions. The analyses described here use methods such as multivariate statistics, pattern recognition, prediction modelling, and machine learning.