Comments on Scheffler et al. Cytotoxic Evaluation of E-Liquid Aerosol using Different Lung Derived Cell Models. Int. J. Environ. Res. Public Health, 2015, 12, 12466-12474/Reply

Abstract

There is merit in considering a simple toxicological screening method that evaluates the total cytotoxic potential of e-liquids or electronic cigarettes (ECs) aerosol emissions in one single testing. However, there is growing confusion, with several researchers endorsing their personal solution to the problem. Here, we discuss as an example the recent paper by Scheffler and colleagues, in which the authors suggest that more relevant and well differentiated cell lines from human airways could be the most suitable candidates for toxicological evaluation of ECs aerosol emissions. We advance recommendations for validated protocols and advocate for an international coordinated effort aimed at establishing consensus on methodology.

Headnote

Abstract: There is merit in considering a simple toxicological screening method that evaluates the total cytotoxic potential of e-liquids or electronic cigarettes (ECs) aerosol emissions in one single testing. However, there is growing confusion, with several researchers endorsing their personal solution to the problem. Here, we discuss as an example the recent paper by Scheffler and colleagues, in which the authors suggest that more relevant and well differentiated cell lines from human airways could be the most suitable candidates for toxicological evaluation of ECs aerosol emissions. We advance recommendations for validated protocols and advocate for an international coordinated effort aimed at establishing consensus on methodology.

Keywords: vapour emission; electronic cigarette; cytotoxicity; tobacco product directive

According to the tobacco product directive (TPD) of the European Union, electronic cigarettes (ECs) will be regulated from May 2016 [1]. In particular, the TPD obliges manufacturers and importers of ECs and refill containers to submit a pre-marketing notification to the competent authorities of the Member States of any such products, which they intend to place on the market. The notification shall contain some information, including toxicological data regarding aerosol emissions of the product. It requires the manufacturers to submit what toxicological data they have (e.g., a compilation of the literature data publicly available), but it does not require them to do and submit with the notification any specific toxicological testing, as there is no explicit mention in either the TPD or in the draftof Implementing Act. However, the pre-marketing notification can be supplemented by any toxicological testing with the product the manufacturers may have gathered for themselves.

In relation to this, it is worth noting that there is merit in considering a simple toxicological screening method that evaluates the total cytotoxic potential of e-liquids or ECs aerosol emissions in one single testing, rather than presenting a long list of toxicological risk assessment of several dozens of chemicals tested in isolation. An additional advantage is that toxicological findings from such in vitro cellular cytotoxicity system may also detect toxicological potential attributable to unknown contaminants/by-products in the ECs emissions.

Although it should not be too difficult to put this in practice, there is growing confusion with several researchers endorsing their personal solution to the problem. As an example, we discuss the recent paper by Scheffler and colleagues [2]. The authors try to address the need for suitable cytotoxicity models to test e-liquids or ECs aerosol emissions, by suggesting that their in-house immortalized human bronchial epithelial cell line (i.e., CL-1548) would be the most suitable candidate for toxicological evaluation.

Their working hypothesis is that it is important to consider the anatomical site of primary impact of aerosols (i.e., the conducting zone of the respiratory tract) in order to establish a more relevant cell culture model for a toxicological evaluation of ECs emissions. Based on the assumption that the most of these emissions impact on the respiratory tract (and not on the alveolar lining), they conclude that the human bronchial epithelial cell culture is the most suitable model and propose their in-house immortalized human bronchial cell line as a candidate (i.e., CL-1548). The main problem with this approach is that fully characterized human bronchial epithelial cell lines are already available from ATCC (e.g., BEAS-2B, 16HBE) and generally used for regulatory purposes by FDA [3,4] and that cell differentiation is not an essential requirement for cytotoxicity testing.

The authors compared cell viability of ECs aerosol emission to that of cigarette smoke (positive control) and clean air (negative control) 24-h post exposure of normal human bronchial epithelial (NHBE) cells, immortalized human bronchial epithelial (CL-1548) cells, and adenocarcinoma human alveolar basal epithelial (A549) cells. After 24-h incubation with aerosol emission, cell viability was reduced in CL-1548 much more than A549 and less than NHBE and based on this observation the authors conclude that it is best to use CL-1548 for testing of ECs aerosol emissions by virtue of its heightened cytotoxic sensitivity. The sensitivity of a cell line is a relative concept and it is not surprising that different cell types expressing different grade of differentiation may exhibit different sensitivity. Therefore, conclusions about the appropriateness of a cell line compared to another, as the most "suitable" candidate for toxicological evaluation, requires justification beyond simplistic considerations about the anatomical site of primary impact of aerosols. What if after exposure ECs aerosol emissions, cell viability was not reduced in alternative bronchial epithelial cell lines (e.g., BEAS-2B, 16HBE)? These cell lines should be included for cross checking purposes and to support these authors working hypothesis. Nonetheless, cell differentiation is not an essential requirement for regulatory cytotoxicity studies, it may be a valid scientific approach to when addressing other aspect concerning bronchial epithelial health (e.g., reduction in cilia beating frequency, electrophysiological studies for establishing dysfunctional tight junctions, etc.).

It is also unjustified to select a specific cell lines just because generates a predefined response. It is very common among researchers to go for those cell lines that generate responses of interest. Thus, appropriateness of a cell line compared to another as the most "suitable" candidate can be also dictated by evidence of positive responses and not by rational choices.

When analyzing positive responses in term of cell toxicity, Scheffler and coll. [2] paid great attention to the anatomical site of primary impact of aerosols, but failed to recognize that the aerosol and smoke generation protocols are the most important factor that influences cytotoxicity. For this reason, it is more important to establish the correct exposure protocols (time, dose) in relation to the culture model utilized. There is no justification for exposing cell cultures to 200 puffs for ECs and only to 60 puffs for conventional cigarettes. This choice is arbitrary and will introduce bias when comparing cytotoxicity between ECs aerosol emissions and tobacco smoke. These methodological problems often arise when not using validated protocols.

In conclusion, when assessing potential cytotoxicity effects of ECs aerosol emissions, it is mandatory to compare them with those resulting from the exposure of cigarette smoke. In the absence of clearly defined ECs aerosol generation methods and exposure protocols, it is recommended to perform an ISO 10993-5 [5] study on a human bronchial epithelial cell lines available from ATCC (e.g., BEAS-2B, 16HBE). The ISO 10993-5 protocol has pre-determined toxicity end-points (i.e., <70% cell survival), defines the level of exposure (extract of 1% concentration) and is used for approval of medical devices or products. It is critical that future evaluation of the harm potential of ECs aerosol emissions gets away from the controversial toxicological debate that has been generated in recent laboratories studies because of experimental protocols that do not mimic realistic condition of use [6]. Last but not least, given the concerning lack of uniformity in methods used to generate ECs aerosol emissions [7], it is crucial to set up an internationally coordinated effort aimed at establishing technical consensus if we wish to advance science and better inform regulators.

Author Contributions: Riccardo Polosa, Massimo Caruso, and Francesca Guarino contributed equally to this work.

Conflicts of Interest: Riccardo Polosa has received lecture fees and research funding from Pfizer and GlaxoSmithKline, manufacturers of stop smoking medications. He has also served in the past as a consultant for Pfizer and Arbi Group Srl, an Italian distributor of e-Cigarettes. Riccardo Polosa is currently scientific advisor for LIAF, Lega Italiana Anti Fumo (Italian acronym for Italian Anti Smoking League). Massimo Caruso and Francesca Guarino have no relevant conflict of interest to declare in relation to this work.

Referring to the comments of Polosa and colleagues [1] on our latest in vitro e-liquid aerosol testing publication, we would like to give our statements about some points.

In general, we are in agreement with Polosa et al. that validated protocols should be the basis for an international test strategy for e-liquids and their aerosols. Due to the upcoming enforcement of the tobacco product directive for e-cigarettes and e-liquids in May 2016, attempts have been made to first establish testing protocols to obtain toxicological data. However, these efforts are limited to chemical data, which might not be sufficient to ensure complete consumer protection in the future. The complexity of the problem cannot be solved by a simple toxicological screening method and should be based on different assays addressing the cytotoxic spectrum of e-liquids and/or their aerosols. In our opinion, it is also necessary to analyze the effects of e-liquid aerosols in vitro, since the generated inhalable vapor represents the actual hazardous compounds interacting directly with the epithelium of the respiratory tract.

In our publication [2], we did not state that the immortalized human bronchial epithelial cell line CL-1548 would be the most suitable candidate for in vitro testing of e-liquid aerosols in general. However, due to the fact that the primary impact site of e-liquid aerosol is the respiratory tract, cells from this anatomical region are the most suitable ones. In our opinion, primary cells from healthy human lung tissue would be the most relevant cell model, but due to donor-dependent variations, limited lifespan and limited availability, those cells have their limitations for standard routine testing. Here, immortalized cell lines offer an alternative, because they have unlimited availability and allow testing procedures with comparable cell populations. In our opinion, cytotoxic studies should also not be limited to acute toxicity testing with undifferentiated cells of the respiratory tract, but should also include long-term (chronic) studies on differentiated 3D constructs with all characteristic cell types to address cell-specific cytotoxic effects relevant for the in vivo situation. In this context, it is of great importance to have one cell line, which can be used to perform both acute and long-term toxicity studies, in order to obtain a broad spectrum of toxicological data.

Polosa et al. [1] mentioned that different fully characterized human bronchial epithelial cell lines are available from ATCC like BEAS-2B and 16HBE14o- cells. Here, it has to be mentioned that the virus-transformed BEAS-2B cells do not exhibit a differentiation comparable to that of their parent cells, lack tight junctions [3] and become malignant after several passages [4]. The also named 16HBE14o- cells, also virus-transformed, are not able to differentiate into a pseudostratified airway epithelium under submersed as well as air-liquid interface conditions [5]. In our studies, we integrated a cell line which has been immortalized at SIRION BIOTECH GmbH (Germany) using lentiviral constructs containing cyclin-dependent kinase (CDK4) and human telomerase reverse transcriptase (hTERT), which shows comparable morphological characteristics of the donor cells (ciliated and mucus-producing as well as progenitor cells).

We compared the cellular effects (viability and the production of reactive oxygen species) after e-liquid aerosol and mainstream smoke exposure on freshly isolated primary bronchial epithelial cells, the immortalized cell line CL-1548 and the alveolar cell line A549 [2]. Our experiments demonstrated that A549 cells exhibit a significantly lower susceptibility to cellular damage than the primary cells and also the reaction pattern between the different exposure groups is not comparable with them, whereas it is comparable for freshly isolated bronchial epithelial and immortalized CL-1548 cells. These results clarify that A549 cells have a different response characteristic to that of the primary cells. Based on the assumption that primary cells should be set as the "gold standard" for cytotoxic evaluation, a cell line used for routine tests should give results as close as possible to this standard.

Regarding Polosa et al.'s [1] notes about the experimental design, we would like to explain our study approach. Since there are no standard protocols for e-cigarette testing as yet, we decided to work according to ISO 3308 and compared the toxicity of cigarette mainstream smoke to e-liquid vapor. In order to be able to compare the results of both exposures (e-liquid aerosol/mainstream smoke), we used the same smoking protocol for e-cigarettes as for combustible cigarettes, generated dose-response curves dependent on the number of puffs during the exposure and chose for our experiments a "dose" of 200 puffs for e-liquid aerosols. However, a decrease in cell viability was seen already after the exposure to 50 puffs. In the case of mainstream cigarette smoke, 60 puffs induced a strong cytotoxicity (about 80%) and a further increase in the number of puffs resulted in complete cell death. Accordingly, only the consideration of 60 puffs or less allows a theoretical comparison of the results, presented on a puff-to-puffcomparison. Such an adjustment of the results was possible due to the linear dose-response relationships in both cases (cigarette and e-cigarette exposure).

In our case, we did not work according to the standard protocol (ISO 10993-5) for testing substance extracts in vitro, because testing extracts under submersed culture conditions does not reflect the situation after vaping/smoking in vivo. In the lung, the cells are not coveredwith a liquid layer as found during submersed cultivation, but are exposed directly to the surrounding atmosphere. Furthermore,water-soluble and volatile vapor components cannot be trapped in the extracts and are therefore not analyzed during the testing.

In summary, we are convinced that direct exposure studies with normal human bronchial epithelial cells or relevant immortalized cell lines in an undifferentiated as well as differentiated stage will contribute to the evaluation of the cytotoxic potency of e-liquid vapor. Such investigations should be included in a validated research protocol accepted by international regulatory authorities.

References

References

1. Official Journal of European Union. Available online: http://ec.europa.eu/health/tobacco/docs/ dir_201440_en.pdf (accessed on 17 November 2015).

2. Scheffler, S.; Dieken, H.; Krischenowski, O.; Aufderheide, M. Cytotoxic evaluation of e-liquid aerosol using different lung-derived cell models. Int. J. Environ. Res. Public Health 2015, 12, 12466-12474. [CrossRef] [PubMed]

3. Lechner, J.F.; LaVeck, M.A. A serum-free method for culturing normal human bronchial epithelial cells at clonal density. J. Tissue Culture Meth. 1985, 9, 43-48. [CrossRef]

4. Ramirez, R.D.; Sheridan, S.; Girard, L.; Sato, M.; Kim, Y.; Pollack, J.; Peyton, M.; Zou, Y.; Kurie, J.M.; DiMaio, J.M.; et al. Immortalization of human bronchial epithelial cells in the absence of viral oncoproteins. Cancer Res. 2004, 64, 9027-9034. [CrossRef] [PubMed]

5. ISO 10993-5 Standard. Biological Evaluation of Medical Devices-Part 5: Tests for in vitro Cytotoxicity. 2009. Available online: http://www.iso.org/iso/home/store/catalogue_tc/catalogue_detail.htm?csnumber=36406 (accessed on 17 November 2015).

6. Farsalinos, K.E.; Polosa, R. Safety evaluation and risk assessment of electronic cigarettes as tobacco cigarette substitutes: A systematic review. Ther. Adv. Drug Saf. 2014, 5, 67-86. [CrossRef] [PubMed]

7. Cheng, T. Chemical evaluation of electronic cigarettes. Tob. Control 2014, 23, 11-17. [CrossRef] [PubMed]

References

References

1. Polosa, R.; Caruso, M.; Guarino, F. Comments on Scheffler et al. cytotoxic evaluation of e-liquid aerosol using different lung derived cell models. Int. J. Environ. Res. Public Health 2015, 12, 12466-12474. Int. J. Environ. Res. Public Health 2016, 13. [CrossRef]

2. Scheffler, S.; Dieken, H.; Krischenowski, O.; Aufderheide, M. Cytotoxic evaluation of e-liquid aerosol using different lung derived cell models. Int. J. Environ. Res. Public Health 2015, 12, 12466-12474. [CrossRef] [PubMed]

3. Steward, C.E.; Torr, E.E.; Mohd Jamili, N.H.; Bosquillon, C.; Sayers, I. Evaluation of differentiated human bronchial epithelial cell culture systems for asthma research. J. Allergy 2012. [CrossRef] [PubMed]

4. Ramirez, R.D.; Sheridan, S.; Girard, L.; Sato, M.; Kim, Y.; Pollack, J.; Peyton, M.; Zou, Y.; Kurie, J.M.; Dimaio, J.M.; et al. Immortalization of human bronchial epithelial cells in the absence of viral oncoproteins. Cancer Res. 2004, 64, 9027-9034. [CrossRef] [PubMed]

5. Pohl, C.; Hermannc, M.I.; Uboldi, C.; Fuchs, M.; Dei-Anang, J.; Mayer, E.; Kehe, K.; Kummer, W.; Kirkpatrick, C.J. Barrier functions and paracellular integrity in human cell culture models of the proximal respiratory unit. Eur. J. Pharm. Biopharm. 2009, 72, 339-349. [CrossRef] [PubMed]