The In Vitro assessment of Respiratory Sensitisation Potential of Electronic Cigarette Liquids

Matthew Stevenson, Lukasz Czekala, Liam Simms, Nicole Tschierske, Henrik Johansson, Tanvir Walele
Imperial Tobacco Ltd, Reemtsma Cigarettenfabriken GmbH, an Imperial Brands PLC Company, SenzaGen AB, Fontem Ventures B.V., an Imperial Brands PLC Company,


Introduction and Objectives

There is a general consensus amongst the scientific and public health community that e-cigarettes constitute a less harmful source of nicotine than combustible cigarettes, and that flavours play a critical role in attracting and retaining smokers into the vaping category. Due to the dynamic nature of innovation with e-cigarettes new assays are required to quickly determine the subtle biological response of these products for product stewardship activities. The size of this task is considerable as recent estimates state that more than 8,000 e-liquid flavours are on the market (Hartung, 2016). One particular toxicological endpoint which is of interest for the Stewardship of e-liquids, is Respiratory Sensitisation.
Respiratory sensitization (RS) is an allergic type I hypersensitivity reaction of the upper and lower respiratory tract caused by an immune response triggered by low molecular weight compounds or other environmental proteins. Clinical symptoms of RS include asthmatic attacks, bronchoconstriction and wheezing upon repeated exposure to the same compound. However, respiratory sensitisers are rare, with around 100 well characterised substances described in the literature.
It is Fontem Ventures policy to screen all novel e-liquid ingredients for Respiratory sensitising activities using published literature and in silico techniques. However, there is a need for alternative techniques to fill data gaps and add to a weight of evidence. Several in vitro assays have been described and validated to assess skin sensitisation, however for respiratory sensitization there are no validated predictive assays. It is of note that not all skin sensitizers are also respiratory sensitizers. In 2015, Basketter and Kimber concluded that “…airborne fragrance materials, including skin sensitising fragrance materials, do not pose a risk of the induction or elicitation of allergic reactions consequent upon exposure via the respiratory tract”. Therefore, it is critical that any assays developed to determine the sensitising properties of a chemical can distinguish between dermal and respiratory activity.
The objective of this study was to assess experimental and commercial e-liquids in GARDair™; an assay which claims to detect respiratory sensitisers.
GARDair measures the genomic biomarker signature of a human myeloid leukemia-derived cell line exposed to test substances; making this technology in keeping with the 3Rs (Reduce, Replace and Refine) and Toxicity Testing in the 21st Century principles. Gene expression analysis is performed using Affymetrix microarray technology and a prediction model is used to classify each sample according to its respiratory sensitizing potential.



• From the Benchmark Control data it was estimated that GARDair™ had a sensitivity and specificity of 71% and 100% respectively; with an overall predictive accuracy estimated as 89%.
• Extensive validation of this assay is ongoing, however, the lack of well characterised Chemical Respiratory Sensitisers may limit this.
• None of the experimental or commercial samples were classified as respiratory sensitisers.
• Further exploration of this assay is required, particularly its ability to detect low concentrations of sensitiser in complex mixtures and to ensure that the e-liquid matrix does not interfere with the detection of activity.

The In Vitro assessment of Respiratory Sensitisation Potential of Electronic Cigarette Liquids_British ToxSocCong 2018 Imperial