24 February 2021
 

Toxicological Assessment of Flavored e-liquids in Sprague Dawley Rats in an OECD Subchronic Inhalation Study Complemented by Systems Toxicology Endpoints

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Hi, I'm Jenny Ho. Today I would like to present the poster entitled Toxicological Assessment of Flavored e-liquids in Sprague Dawley Rats in an OECD Subchronic Inhalation Study Complemented by Systems Toxicology Endpoints. It is important you note that Philip Morris international is the sole source of funding and sponsor of this project. 

Smoke-free products have the potential to replace cigarettes in the near future. An important aspect for the success of these products is the use of flavoring substances. However, very little is known about the safety of these substances when used in inhalations. In this work, we assess the toxicity of flavored e-liquids in a 90-day inhalation study in accordance with the OECD test guideline 413.

 
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The test item was an e-liquid containing nicotine, PG, and VG at the defined concentrations and flavor mixture with 28 flavoring substances, which were selected using a combination of predictive software and extensive literature review to represent different chemical and metabolic classes. We had six different groups of rats, three of them were tested at three different flavor mixture concentrations. The results were compared with a control, which is PBS and two comparator groups, which exposed to non-flavored e-liquids and PG/VG with flavor mixture at medium concentrations to gain better understanding of possible synergistic effects between nicotine and flavoring substances. Aerosols were generated by using 6-jet Collison nebulizers while the rats were restrained and exposed in flow-past nose-only exposure chambers. Aside from classic OECD endpoints, we also assessed a series of biomarkers of exposure, pulmonary inflammation in rats, as well as global transcriptomics changes in tissues. Test atmosphere characterizations revealed that aerosol generation was under control since studying means of daily nicotine, PG and VG at the breathing zone of each exposure chamber fall within plus minus 10% of the target concentrations. 

Moreover, the L-carvone quantifiers, representative of the flavor mixture in the test atmosphere, was found to be within the expected concentrations. Correct aerosol uptake was confirmed by measuring different biomarkers of exposure in blood to measure concentration of cotinine a metabolic of nicotine and L-carvone confirmed the consistent uptake of aerosols by the rats. 

Pulmonary inflammation was assessed by evaluating the free lung cells and inflammatory mediators in the broncheoalveolar lavage fluid of rats exposed to test and reference items. The results showed no significant increase in the number of recovered cells and minimal changes in the inflammatory mediators. Organ weights show the typical changes associated with a procedure related stress response, including higher weights of adrenal glands, and lower weights of thymus and spleen, mainly in groups exposed to nicotine-containing aerosols. Nicotine exposure also resulted in higher liver weight.

Histopathology of the respiratory organs revealed low incidence changes in the test item-exposed rats and mainly sporadic findings observed in a few rats across groups, including the vehicle control. Only in the larynx, which is the most sensitive site, we observe a minimal to mild squamous metaplasia in all groups, and all the effects were fully reversed after the recovery period. The quantitative, more formal metric analyses were in agreement with the histopathological assessment and highlighted a slight trend in increase of thickness following nicotine exposure, with the majority being not statistically significant.

The transcriptome analysis of the nasal epithelium, lung and liver revealed no or limited differentially expressed genes following exposure to flavoring substances.  In contrast, the effect of nicotine on liver transcriptome was evident, with few hundreds of genes up and downregulated.  

In conclusion, the results confirmed that the aerosols were well generated and taken up by the rats. The biological effects related to test item exposure were limited and in line with our previous findings following nicotine exposure. Analyses of broncheoalveolar lavage fluid suggested the absence of pulmonary inflammation in agreement with histopathology of respiratory organs, which revealed on these subtle, adaptive, and reversible changes. The flavoring substances did not induce a measurable biological response at the transcriptome levels. Finally, no apparent additive or synergistic toxic effects were observed among the flavoring substances at the tested levels. 

Thank you very much for your attention.

Important: This presentation is for the purpose of publishing and disseminating scientific information about Philip Morris International’s efforts to develop and assess products that have the potential to reduce individual risk and population harm associated with tobacco use. This presentation is for audiences of scientists, public health and regulatory communities, and other stakeholders with an interest in tobacco policy. The purpose is not advertising or marketing. It is not intended for use by consumers.

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