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Alternatives to Animal Testing

As a responsible manufacturer, we’ve devised a Scientific Assessment Framework to assess the tobacco harm reduction potential of our Next Generation Products (NGPs) compared to combustible cigarettes. When a product shows fewer and lower levels of toxicants compared to combustible cigarettes, we then evaluate the impact of these reduced toxicant levels using a number of in-vitro cellular assays before commencing clinical assessments with adult smokers.

The US National Academy of Science’s landmark blueprint for Toxicity Testing in the 21st Century (TT21C) informs our approach to this biological research.

TT21C advocates replacing traditional animal testing with in-vitro models prioritising human cells. By looking at human cells and cell systems, these innovative methods can provide more human-relevant data, especially when combined with and verified by relevant human clinical studies.

Dr Liam Simms
Principal Toxicologist

The desire to use fewer animals in research is a global one, and Imperial Brands is a proud and active contributor to this scientific endeavour, aligning with our wider corporate position not to test on animals unless required by regulatory authorities. People for the Ethical Treatment of Animals (PETA)’s Science Consortium have commended our research programme for its TT21C-driven approach.

There has been astonishing progress in the development of human-relevant non-animal testing approaches and in the use of these methods to more reliably and rapidly provide information for predicting the potential toxicity of chemicals to humans. We are a proponent of good science and sparing animals from toxicity tests, as Imperial has done. We support the advancement of scientifically sound non-animal testing approaches, and partner with others fostering their advancement.

For more on Alternatives to Animal Testing, see this Q&A with Liam Simms and explore the interactive research table below.

Imperial Brands Science's use of Novel In-Vitro Assays Prioritising Human Cells

Established Regulatory Assays

AMES Mutagenicity

Established regulatory assays
Before any advanced techniques are used, our products are assessed in standardised assays and tests endorsed by regulatory bodies across the world. These assess cytotoxicity, genotoxicity and mutagenicity – all of which are important for understanding the potential toxicity of the product and how it compares to combustible cigarettes.

Ames: Uses bacteria strains to assess whether a test article can cause changes to the genetic material (mutagenicity). This event can cause cellular dysfunction and potentially lead to cancer susceptibility.

IVM Genotoxicity

Established regulatory assays
Before any advanced techniques are used, our products are assessed in standardised assays and tests endorsed by regulatory bodies across the world. These assess cytotoxicity, genotoxicity and mutagenicity – all of which are important for understanding the potential toxicity of the product and how it compares to combustible cigarettes.

In-vitro Micronucleus (IVM): The IVM assay detects damage to the structure of cellular DNA (genotoxicity) following test article exposure. Genotoxicity has been mechanistically linked with the formation of cancer.

NRU Cytotoxicity

Established regulatory assays
Before any advanced techniques are used, our products are assessed in standardised assays and tests endorsed by regulatory bodies across the world. These assess cytotoxicity, genotoxicity and mutagenicity – all of which are important for understanding the potential toxicity of the product and how it compares to combustible cigarettes.

Neutral Red Uptake (NRU): This cell-based approach assesses the uptake of red dye to quantify the number of living cells in culture. The amount of cell damage or death (cytotoxicity) is assessed following exposure to increasing concentrations of test article. Cytotoxicity is considered a potential step in several chronic disease processes associated with smoking, including cancer, cardiovascular disease and emphysema.

We’ve published numerous pieces of research using these established assays, all indicating NGPs have a different scientific profile compared to combustible cigarette smoke and significant harm reduction potential for adult smokers.


- Chemical Composition and In Vitro Toxicity Profile of a Pod-Based E-Cigarette Aerosol Compared to Cigarette Smoke (2020), K Rudd, M Stevenson, R Wieczorek, J Pani, E Trelles-Sticken, O Dethloff, Applied In Vitro Toxicology 6 (1), 11-41
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- A comparative in vitro toxicity assessment of electronic vaping product e-liquids and aerosols with tobacco cigarette smoke (2020), R Wieczorek, G Phillips, L Czekala, ET Sticken, G O'Connell, L Simms, Toxicology in Vitro, 104866
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Pre-Clinical Assessment of a Dual-Temperature Operated Heated Tobacco Product
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Next Generation Products induce lower biological activity than combusted cigarettes: A comparison of aerosol chemistry & in vitro toxicity
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Imperial Brands Science's use of Novel In-Vitro Assays Prioritising Human Cells


Cardio Endpoints
Scratch Wound

This assay evaluates the impact of product samples on human endothelial cells (the single layer-thick cells that line all human blood vessels) that are essential for healthy cardio-vascular function. In this assay a scratch is made in the cell layer and the time taken for the normal cell response of filling – or ‘healing’ – the ensuing gap is measured. Cigarette smoke has been shown to delay wound healing, inhibiting endothelial cell normal migratory behaviour.

In marked contrast, our research shows exposure e-vapour aerosol has no notable inhibiting impact and was near identical to the control conditions – even at the highest concentrations – under the test conditions. Likewise, diluted heated tobacco aerosol similarly showed significantly lower inhibiting impact to endothelial cells when compared to those exposed in cigarette smoke.


Next Generation Product Aerosols Induce Lower Biological Activity than Combusted Cigarettes: A Comparison of In Vitro Cell Migration in the Scratch Wound Assay
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Cancer Endpoints
Cellular Transformation

CTAs can detect potential carcinogens (substances capable of causing cancer). These complex assays look at certain traits observed in cancer cells, such as assessing cells’ ability to detach from their container (normal behaviour), or remove contact inhibition (cells keep multiplying when they would normally stop growing when they reach the edge of the container they are in).

Our research using this method shows combustible cigarettes exhibited a tumour promotion response. In stark comparison, NGPs demonstrated drastically reduced bioactivity in terms of cellular promotion, with no statistically significant increase in tumour promotion response following exposure to heated tobacco and blu aerosols.”


Next Generation Product (NGP) Aerosols Induce Lower Biological Activity Than Combusted Cigarettes: A Comparison of Aerosol Bubbled Extract Chemistry and in Vitro Toxicity
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Mechanistic Genotoxicity

This suite of stem-cell based assays provides insight into the mechanisms by which chemicals can interact with cells, using mechanisms that potentially cause DNA damage compared to those such as protein folding that do not interact with DNA. These assays help assess how product samples may impact cellular functioning across various reporter cell lines, picking up the tell-tale molecular signs of potential harm. These indicators may include 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.

Our published research using this methodology shows that undiluted vape e-liquids, and their aerosol extracts, exhibited entirely absent or vastly reduced indications of potential DNA damage in cells, compared to smoke from combustible cigarettes. Our 2021 ToxTracker study was the first to publish results using the assay for the assessment of vape e-liquids and aerosols.


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, in Mutagenesis, (2021)
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Lung Toxicity Endpoints
3D Lung Model

3D Lung Model: A 3D organotypic model (characteristic of the bronchiole tissue in humans) is exposed to cigarette smoke/NGP aerosols, and various endpoints measured to observe any effects. Assays include examining mucus production and measuring the beat frequency of cilia – the mobile, hair-like structures that line the larger airways clearing them of mucus and dirt and trapped particles.

We’ve published several studies using 3D lung models, and all provide promising evidence of harm reduction potential of NGPs for lung health compared to combustible cigarettes. Following exposure e-vapour aerosols in our research, there was no recorded decrease in either the number of cilia, or the number of ciliated cells. The observations were in notable contrast to cigarette smoke, which negatively impacted ciliated lung cells at much lower concentrations. When tested in an equivalent assay, diluted heated tobacco aerosol showed marked reductions in cellular damage when compared to the combustible cigarette comparator.


Toxicological comparison of cigarette smoke and e-cigarette aerosol using a 3D in vitro human respiratory model, L Czekala, L Simms, M Stevenson, N Tschierske, AG Maione, T Walele, (2019), Regulatory Toxicology and Pharmacology 103, 314-324
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Acute Electronic Vapour Product whole aerosol exposure of 3D human bronchial tissue results in minimal cellular and transcriptomic responses when compared to cigarette smoke (DOI: 10.1177/2397847320988496/ ID: TOR-20-0063.R2) in Toxicology Research and Application, (2021) [Pre-Print]
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E-cigarettes induce lower biological responses than conventional cigarettes: A comparison of in vitro toxicity following repeated whole aerosol exposure to human bronchial tissue for 4 weeks
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The use of human 3D reconstructed bronchial tissue to study the effects of cigarette smoke and e-cigarette aerosol on a wide range of cellular endpoints
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Czekala L et al (2021) Multi endpoint analysis of human 3D airways epithelium following repeated exposure to whole electronic vapour product or cigarette smoke Current Research in Toxicology Current Research in Toxicology PRE PROOF
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Systems Toxicology
Omics Techniques

These approaches observe the interaction between different system biology networks, including genomics, transcriptomics, proteomics and metabolomics. By studying the relationship between genes and molecules, these approaches shed light on how molecular interactions influence events such as disease initiation and progression.

Our research using this technique shows vape aerosol elicits significantly less biological response to human lung cells in vitro than the equivalent exposure to combustible cigarette smoke.


Phillips G et al (2021) Acute electronic vapour product whole aerosol exposure of 3D human bronchial tissue results in minimal cellular and transcriptomic responses when compared to cigarette smoke Toxicology Research and Application 5(1) 1-19
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High Content Screening

This assay combines automated imaging and quantitative data analysis to provide rich systems biology information, detecting cellular events in human cell lines in response to test materials.

In numerous pieces of research using this methodology, we’ve shown that e-vapour e-liquids elicit lower biological activity when compared to cigarette smoke.


High Content Screening in NHBE cells shows significantly reduced biological activity of flavoured e-liquids, when compared to cigarette smoke condensate (2019), L Czekala, L Simms, M Stevenson, E Trelles-Sticken, P Walker, T Walele, Toxicology in Vitro 58, 86-96
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Next generation product aerosol bubbled extracts show little to no effect in high content screening endpoints when compared to cigarette smoke bubbled extracts
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The use of High Content Screening (HCS) in human primary lung cells to assess e-liquids
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Other Endpoints
Developmental Toxicity

These assays assess the potential for substances to interfere with normal pre-natal development using metabolomics.

To asses developmental toxicity potential, and critically if and at what concentration this occurs, we use a human cell model assay looking at both cytotoxicity and the disruption of key metabolites in the cell.

Our research so far suggests NGPs show significantly reduced developmental toxicity potential compared to combusted cigarettes.


The use of human induced pluripotent stem cells to screen for developmental toxicity potential indicates reduced potential for non-combusted products, when compared to cigarettes, L Simms, K Rudd, J Palmer, L Czekala, F Yu, F Chapman, ET Sticken, Current Research in Toxicology, (2021) 1, 161-173
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Next generation products predicted to have no potential to induce developmental toxicity in the devTOX quickPredict assay
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Skin and Respiratory Sensitisation

Using transcriptional profiles of genomic biomarkers (molecules that indicate normal or abnormal processes that may be a sign of an underlying condition or disease), these techniques can identify the respiratory and skin sensitisation potential of various substances.

In the first ever vape-related application of this method, our published research shows how Genomic Allergen Rapid Detection assays may effectively differentiate and broadly classify e-liquids based on their sensitisation potential. This suggests such methods could, in the future, be applied to e-liquid testing to raise industry standards and improve product quality for adult smokers.


The use of Genomic Allergen Rapid Detection (GARD) assays to predict the respiratory and skin sensitising potential of e-liquids
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Multi-Organ Screening

These assays use a selection of human primary cells from different body organs to test the actions of different chemicals on biomarkers in each cell system.

Our research using this method suggests various NGPs, including e-vapour, are likely less toxic than combustible cigarette smoke, that caused toxicity signatures for thrombosis, as well as immune suppression and skin irritation at higher doses. In contrast, none of the NGPs caused any toxicity signatures during the same testing regime.


Next Generation Products Induce Lower Biological Activity than Combusted Cigarettes Using BioMAP® System of Human Primary Cell Based Co-Cultures
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The use of in vitro human biomarkers from relevant primary cell systems, to assess the effects of experimental and commercial e-liquids
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