Laboratory research from Imperial Brands, published in the journal Mutagenesis, shows that “undiluted vape e-liquids and their aerosol extracts exhibited entirely absent or vastly reduced indications of DNA damaging potential in cells” compared to cigarette smoke.
This material was published by Imperial Brands on 6 April 2021. Note: Imperial Brands, formerly Imperial Tobacco, set up Fontem Ventures in 2012, through which it has developed and sold e-cigarettes since 2014.
Imperial Brands scientists have utilised Toxys' ToxTracker suite of stem-cell based in-vitro assays, which provide mechanistic insight into the potential DNA damaging properties of chemicals, comparing vape e-liquid samples and their aerosols to combustible cigarette smoke.
Imperial scientists are the first to publish results using the ToxTracker system for the assessment of vape e-liquids and aerosols, and it forms part of the company's continuing research into the tobacco harm reduction potential of Next Generation Products such as vapes.
The assays help assess how product samples may impact on cellular functioning across six reporter cell lines, picking up the tell-tale molecular signs of potential harm in the form of oxidative stress, DNA and protein damage, as well as activation of the p53 gene that has a role in cell cycle regulation and tumour suppression.
The results, peer reviewed and published in the journal Mutagenesis, showed that under the conditions of test, undiluted vape e-liquids and their aerosol extracts exhibited entirely absent or vastly reduced indications of DNA damaging potential in cells, compared to smoke from combustible cigarettes.
"Overall, the data from our latest study adds to the weight of scientific evidence demonstrating [that] vape products offer significant harm reduction potential compared to combustible cigarettes," says Lukasz Czekala, Senior Pre-Clinical Toxicologist at Imperial Brands and lead author of the paper.
After calibrating and validating the system to ensure the principal e-liquid components propylene glycol (PG) and vegetable glycerine (VG) were compatible with the ToxTracker suite, a selection of neat myblu vape e-liquids and their aerosol extracts were compared to smoke samples from the reference 1R6F combustible cigarette. Results showed that:
- Vape aerosols trapped in a buffer solution did not induce responses in any of the six cell lines.
- Undiluted flavoured e-liquids (tested up to 1%) did induce both oxidative stress reporters, but this was considered an effect of osmolarity (a measure of solution concentration) caused by PG/VG in an in-vitro testing environment.
- Nicotine content did not affect responses: tobacco flavour e-liquid at either 1.6% freebase nicotine, 1.6% nicotine salt or nicotine-free produced the same results.
- In addition, nicotine (tested alone) only produced an oxidative stress response at levels that would be a concentration of more than 40,000 times higher than found in the blood from typical smoking.
Dr Fiona Chapman, Pre-Clinical Toxicologist and corresponding author, added: "ToxTracker is quick, sensitive and can provide a greater mechanistic resolution than existing Next Generation Product stewardship DNA damage tests like the micronucleus and Ames assays".
She continued: "Our adoption of this cutting-edge in-vitro suite reinforces our commitment to using advanced cellular assays which adhere to Toxicity Testing in the 21st Century (TT21C) principles.
“This also contributes to reducing industry reliance on in-vivo (animal) experiments." (Imperial does not test any products on animals: see our Position here.)
"This paper adds to the established body of scientific data that shows vaping products, when manufactured to high quality and safety standards, have significant tobacco harm reduction potential relative to continued cigarette smoking," says Dr Grant O'Connell, Head of Tobacco Harm Reduction Science at Imperial Brands.
"We appreciate society's ongoing concerns about the health risks of smoking, and are committed to undertake high quality research on potentially less harmful nicotine product alternatives to combustible tobacco for adult smokers."
The in vitro ToxTracker and Aneugen Clastogen Evaluation extension assay as a tool in the assessment of relative genotoxic potential of e-liquids and their aerosols
Lukasz Czekala, Fiona Chapman, Liam Simms, Kathryn Rudd, Edgar Trelles Sticken, Roman Wieczorek, Lisa Maria Bode, Jutta Pani, Nynke Moelijker, Remco Derr,
Inger Brandsma, Giel Hendriks, Matthew Stevenson, and Tanvir Walele
Published in the journal Mutagenesis on 18 March 2021
In vitro (geno)toxicity assessment of electronic vapour products (EVPs), relative to conventional cigarette, currently uses assays, including the micronucleus and Ames tests. Whilst informative on induction of a finite endpoint and relative risk posed by test articles, such assays could benefit from mechanistic supplementation.
The ToxTracker and Aneugen Clastogen Evaluation analysis can indicate the activation of reporters associated with (geno)toxicity, including DNA damage, oxidative stress, the p53-related stress response and protein damage.
Here, we tested for the different effects of a selection of neat e-liquids, EVP aerosols and Kentucky reference 1R6F cigarette smoke samples in the ToxTracker assay.
The assay was initially validated to assess whether a mixture of e-liquid base components, propylene glycol (PG) and vegetable glycerine (VG) had interfering effects within the system.
This was achieved by spiking three positive controls into the system with neat PG/VG or phosphate-buffered saline bubbled (bPBS) PG/VG aerosol (nicotine and flavour free). PG/VG did not greatly affect responses induced by the compounds.
Next, when compared to cigarette smoke samples, neat e-liquids and bPBS aerosols (tobacco flavour; 1.6% freebase nicotine, 1.6% nicotine salt or 0% nicotine) exhibited reduced and less complex responses.
Tested up to a 10% concentration, EVP aerosol bPBS did not induce any ToxTracker reporters. Neat e-liquids, tested up to 1%, induced oxidative stress reporters, thought to be due to their effects on osmolarity in vitro. E-liquid nicotine content did not affect responses induced. Additionally, spiking nicotine alone only induced an oxidative stress response at a supraphysiological level.
In conclusion, the ToxTracker assay is a quick, informative screen for genotoxic potential and mechanisms of a variety of (compositionally complex) samples, derived from cigarettes and EVPs. This assay has the potential for future application in the assessment battery for next-generation (smoking alternative) products, including EVPs.
Mutagenesis journal article – The in vitro ToxTracker and Aneugen Clastogen Evaluation extension assay as a tool in the assessment of relative genotoxic potential of e-liquids and their aerosols (Open access)
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