Lab-tests of e-cigarette show no evidence of cellular harm

April 16, 2016

Vype ePen, a commercially available e-cigarette, did not induce cell stress, DNA damage or in malignant transformation in cell culture tests.

by Dr Marina Murphy, Head of Scientific Media Relations, Research & Development at British American Tobacco

These past few years have seen a huge increase in the popularity of vaping as an alternative to smoking. But we still see a lot of debate as to the safety of e-cigarettes, especially in comparison to regular combustible cigarettes. They haven’t been around long enough for us to use epidemiological data, which looks at health impacts retrospectively. But what we do know is that cigarette smoking causes a number of diseases. We understand some of the mechanisms underlying the development of some of these diseases and we have ways of observing and measuring it. This means that we can use some of these same methods to look at the biological impact of e-cigarettes and compare it to that of cigarettes.

There are several kinds of tests that look at different stages of the disease process – from cellular stress to DNA damage and ultimately the emergence of diseased cells. We have used these tests to compare the impact of a commercially available e-cigarette, Vype ePen, to that of a reference cigarette and found that the vapour produced by these e-cigarettes does not cause cell stress responses, DNA damage or promote the growth of tumours in the cell-based tests that we used.

E-cigarette vapour can contain nicotine, humectants, flavourings and thermal degradation products, so it is important to understand the potential impact on biological systems. The tests described here are just a few in a series of tests being developed for use with electronic cigarettes and other products in development.

Every product is different, so these results apply only to the product tested and the biological impact of other products can only be determined by doing similar tests on them. The use of these tests to assess the biological impact of e-cigarettes was reported in March at the annual general meeting of the Society of Toxicology in New Orleans.

Three types of tests were used to compare the biological impact of cigarettes and e-cigarettes: cell stress tests; a test to assess DNA damage, which can set the scene for cancer; and a transformation assay, which measures the transformation or conversion of normal cells into a cancerous cell type.

Stress

Stress can take many forms, a racing heart, sweaty palms, high temperature. We all feel stress sometimes and stress can seep into our cells. Infections or high temperatures can cause cells to become stressed and interfere with their normal function. Environmental factors like exposure to toxicants from cigarette smoke can have a similar effect. Cells respond to stress in a number of different ways. They can produce compounds themselves or recruit compounds from the immune system to protect the structure of the cell. When stressful conditions become overwhelming, and all else fails, a cell can commit suicide via a pathway called apoptosis. This pathway sidesteps the cell dying from stressful conditions, which could damage or kill nearby healthy cells.

It is therefore possible to determine the levels of cellular stress by using labbased assays or tests that measure the levels of protective compounds produced and or the level of cell apoptosis/death. The assays were tested using smoke from a conventional 3R4F reference cigarette and vapour from Vype ePen, a commercially available e-cigarette. When cells were exposed to the cigarette smoke, all cell stress responses were activated. These same cell stress responses were not activated on exposure to Vype vapour.

Damage

Stressed cells can become damaged. DNA can become damaged and this damage can lead to genetic instability, which in turn can enhance the rate of cancer development. DNA double-strand breaks (DSB) are the most serious type of DNA damage - breakage of both strands of the DNA double helix structure.

Research has made it increasingly clear that DNA-damage and deficiencies in signalling and repair pathways are likely central to the development of most, if not all, human cancers. DSBs are therefore likely precursors to cancer and potentially lethal to the cell. The cell attempts to repair the DNA damage by modifying the histone or protein around which the DNA is wrapped. The changes observed in this histone can be used as an indicator of the level of DSB.

When this test was used to compare the impact of cigarette smoke (3R4F) and e-cigarette vapour (Vype ePen) on DSB, the results showed that cigarette smoke induced significant DNA damage in human lung cells. This was dose dependent, that is, the higher the dose, the more damage was induced. E-cigarette vapour did not induce DSB, even when the dose used was 15 times higher than the equivalent smoke exposure.

Disease

Assay measuring Damaged cells often go on to become cancerous. The cells are transformed from normal cells to abnormal cells that clump together and grow uncontrollably, eventually becoming tumour-like. This process can be mimicked in the lab by using cells that are already damaged and testing the tumour-promoting activities of different compounds.

In this case, the cell culture system was used to test the ability of conventional 3R4F reference cigarette and Vype ePen to promote tumour formation in a specialised cell type called Bhas 42. After exposure to reference cigarettes, the layers of cells were seen to become transformed, clumping together to create colonies, suggesting that the smoke is a tumour promoter. By contrast, the e-cigarette produced no activity. In each test, the e-cigarette produced the same results as an untreated control – there was no activity.

A Revolution in Public Health?

E-cigarettes are a potentially revolutionary product in tobacco harm reduction. Many in the public health community believe that they represent a historical opportunity to save millions of lives and drastically reduce the public health burden of smoking-related diseases. Public Health England, an executive body of the UK Department of Health, recently published a report saying that e-cigarettes are 95% safer than cigarettes. Other groups that recognise the reduced risk potential of e-cigarettes include ASH (Action on Smoking and Health); Cancer Research UK and the British Heart Foundation.

E-cigarettes produce a vapour and as there is no burning of tobacco, the user is not exposed to the toxicants produced by burning dried tobacco leaf. Chemical analyses shows that there are some compounds released into the vapour of e-cigarettes which are toxic at high levels, but these are found at negligible levels in e-cigarette vapour compared to that in smoke. We have rigorously tested our products and have found that the level that tobacco-specific nitrosamines, are present in e-cigarette vapour is comparable with the level found in pharmaceutical nicotine products. Formaldehyde exposures were at levels well below that considered safe by the WHO – indoor air quality guidelines. These chemical profiles are very reassuring, but we need to continually develop new tests to assess the biological impact of e-cigarette vapour compared to that of smoke. We can use these tests to better define and further reduce any residual risks that there may be to as low a level as possible.

Several companies are actively involved in e-cigarettes and next generation products. The key to demonstrating the reduced-risk potential of these products will be agreeing on testing approaches. We think this means that there is now a great opportunity for the industry, regulators and academia to come together and agree on standards and testing protocols.