The aim of the study is to investigate different in vitro toxicity methods for the assessment of ENDs toxicity. According to the Italian researchers, the most used assay, the neutral red uptake, should be combined with different tools (enlisted in the study) to better identifies the toxicity of the products and can avoid approximate and incorrect evaluation.

Catania, 28th July 2021– Appraising the potential ENDS toxicity and comparing it with cigarette smoke is becoming the most important challenge in tobacco harm reduction studies. So far, the alternative tobacco products are tested following the standard approach use for tobacco products.

However, the rapid evolution of these products, and the availability of new products in the market, make it difficult to develop standard protocols for toxicity assessment.

According to CoEHAR researchers: “There’s a need of specific standard for lab tests to evaluate the toxicity of ENDS in order to develop safer products”.

Published in the magazine Regulatory Toxicology and Pharmacology, entitled “Screening of different cytotoxicity methods for the assessment of ENDS toxicity relative to tobacco cigarettes, the CoEHAR study, compared the neutral red uptake, the most used assay for cytotoxicity evaluation, to other tests.

“The key point is to understand how much the aerosol of electronic cigarettes differs from traditional smoking. It is necessary to have established parameters available that obviate the natural technological evolution in the field of ecigs” – explained the director of CoEHAR, prof. Giovanni Li Volti. The researchers studied different methods of assessing the potential toxicity of  ENDS. “It has been confirmed that electronics are safer than other devices on the market”.

THE STUDY

Under different exposure regimes, the researcher analyzed the smoke exposure with a reference cigarette, the vapour exposure thanks to two different ecigs and two heated tobacco products.

Nicotine, due to its chemical properties, tends to increase the intracellular pH, thus affecting the effective evaluation of toxicity in routine laboratory methods.

The researchers decided to combine the neutral red uptake with other cytotoxicity tests that would not be affected by the pH change.

The first test allows evaluating in addition to cell toxicity also death by apoptosis, or programmed cell death, which neutral red cannot measure. The second test is based on the ability of healthy cells to stick to the bottom of the devices used for cell culture.

This last test has the great advantage of being able to carry out multiple measurements resolved over time on the same sample and therefore being able to identify any toxicity in a short or longer time than 24 hours, a time that is instead used as a standard also for products.

“The results of the study – added Li Volti – confirmed that the neutral red uptake is an excellent test for the evaluation of toxicity, giving results comparable to those of cytofluorimetric. The technology instead made it possible to define the timing with which toxicity is observed and showed different profiles for the tested devices”.

The conclusion of the study suggests to combined the neutral red uptake with a time-resolved test to evaluate precisely the toxicity and avoiding over or underestimating the toxicity of the products.