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OPEN Cytotoxicity, mutagenicity

and genotoxicity of electronic
cigarettes emission aerosols
compared to cigarette smoke:
the REPLICA project
Rosalia Emma 1,2,13, Virginia Fuochi 2,3,13, Alfio Distefano 3, Konstantinos Partsinevelos 3,
Sonja Rust 4, Fahad Zadjali 5, Mohammed Al Tobi 5, Razan Zadjali 5, Zaina Alharthi 5,
Roberta Pulvirenti 3, Pio Maria Furneri 2,3, Riccardo Polosa 1,2,4, Ang Sun 6, Massimo Caruso 2,3*,
Giovanni Li Volti 2,3 & the Replica Project Group *

Concerns have recently increased that the integrity of some scientific research is questionable
due to the inability to reproduce the claimed results of some experiments and thereby confirm
that the original researcher’s conclusions were justified. This phenomenon has been described
as ’reproducibility crisis’ and affects various fields from medicine to basic applied sciences. In this
context, the REPLICA project aims to replicate previously conducted in vitro studies on the toxicity
of cigarette smoke and e-cigarette aerosol, sometimes adding experiments or conditions where
necessary, in order to verify the robustness and replicability of the data. In this work the REPLICA
Team replicated biological and toxicological assessment published by Rudd and colleagues in 2020. As
in the original paper, we performed Neutral Red Uptake (NRU) assay for the evaluation of cytotoxicity,
Ames test for the evaluation of mutagenesis and In Vitro Micronuclei (IVMN) assay for the evaluation
of genotoxicity on cells treated with cigarette smoke or e-cigarette aerosol. The results showed high
cytotoxicity, mutagenicity and genotoxicity induced by cigarette smoke, but slight or no cytotoxic,
mutagenic and genotoxic effects induced by the e-cigarette aerosol. Although the two studies
presented some methodological differences, the findings supported those previously presented by
Rudd and colleagues.

In recent years, there has been a growing interest in electronic cigarettes (e-cigarettes) as a potentially safer alter-
native to traditional tobacco products. A safety review conducted by the Committee on Toxicity of Chemicals in
Food, Consumer Products, and the Environment (COT) concluded that when e-cigarettes are manufactured and
used correctly, the risk of adverse health effects is significantly lower compared to combustible tobacco cigarettes.
However, there remains some uncertainty regarding the potential health risks associated with inhaling flavorings
and thermally-derived products from e-cigarettes1.
The concern for public health and regulatory policies surrounding the toxicological aspects of vapor products
has gained global attention. This was primarily triggered by reported cases of lung injury (referred to as EVALI)
associated with improper use of e-cigarettes for tetrahydrocannabinol (THC) consumption and the use of vita-
min E ­additives2. Several countries, including India, Australia, Oman, Egypt, Colombia, among others, have

1
Department of Clinical and Experimental Medicine, University of Catania, Via S. Sofia, 97, 95123 Catania,
Italy. 2Center of Excellence for the Acceleration of Harm Reduction (CoEHAR), University of Catania, Via S. Sofia,
97, 95123 Catania, Italy. 3Department of Biomedical and Biotechnological Sciences, University of Catania, Via
S. Sofia, 97, 95123 Catania, Italy. 4ECLAT Srl, Spin Off of the University of Catania, Via. S Sofia 89, 95123 Catania,
Italy. 5Department of Clinical Biochemistry, College of Medicine and Health Sciences, Sultan Qaboos University,
P.C 123, P.O. Box 35, Khodh, Oman. 6Department of Biology, College of Science and Technology, Sbarro Institute
for Cancer Research and Molecular Medicine, Temple University, Philadelphia, USA. 13These authors contributed
equally: Rosalia Emma and Virginia Fuochi. *A list of authors and their affiliations appears at the end of the
paper. *email: [email protected]

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banned e-cigarettes, while others have implemented regulations for the marketing of e-cigarettes and e-liquids.
Regulatory bodies such as the U.S. Food and Drug Administration (FDA) and the European Union (EU) have
issued requirements and guidance to regulate premarket tobacco product applications for electronic nicotine
delivery ­systems3,4.
To assess the toxicological potential of e-cigarettes, international guidelines, such as the International Con-
ference on Harmonisation S2(R1) (2011), the UK Committee on Mutagenicity of Chemicals in Food, Con-
sumer Products, and the Environment (2011), Health Canada (2005), and the Cooperation Centre for Scientific
Research Relative to Tobacco (CORESTA) (2004), recommend the use of a battery of in vitro tests as part of the
pre-clinical assessment strategy. These guidelines call for the evaluation of various toxicological endpoints using
multiple assays, including the bacterial reverse mutation (Ames) assay for mutagenicity, in vitro micronucleus
(IVMN) assay for genotoxicity, and the neutral red uptake (NRU) assay for acute cytotoxicity e­ valuation5–7. These
three in vitro toxicity tests are commonly used as standard assays to assess the toxicity of tobacco products and
e-cigarettes8.
Major e-cigarette manufacturers have published studies evaluating their products, including emissions, cyto-
toxicity, genotoxicity, and mutagenicity d ­ ata9–12. Independent replication of these studies is crucial to verify the
findings and establish the credibility of the data, supporting the regulation of electronic cigarettes. Incorrect or
flawed results can misinform policies and have detrimental effects on research practices, eroding public trust
in science and, ultimately, impacting health and social care practices. To address this issue, the multicenter
REPLICA project was initiated to replicate high-profile studies conducted by tobacco companies’ research and
development (R&D) departments, aiming to assess the validity of the original work under scrutiny (https://​repli​
ca.​coehar.​org/).
During the final phase of the REPLICA project, the Italian team (CoEHAR, University of Catania-LAB-A) and
their partner in Oman (Sultan Qaboos University-LAB-B) conducted a replication study of a paper published by
Rudd and colleagues from Imperial Brands ­PLC9. This paper provides a summary of comparative data on aerosol
emissions and in vitro toxicity, utilizing the neutral red uptake (NRU), bacterial reverse mutation (Ames), and
in vitro micronucleus (IVMN) assays. The study focused on a pod-system e-cigarette (myblu; Imperial Brands
PLC, Bristol, United Kingdom) in comparison to 3R4F reference combustible cigarette (University of Kentucky).
The researchers observed that many of the harmful and potentially harmful components present in combustible
cigarette smoke were not detected in e-cigarette aerosol. Through established in vitro biological tests, the e-ciga-
rette aerosol did not exhibit any mutagenic or genotoxic activity under the given test conditions. In contrast, the
3R4F cigarette smoke demonstrated mutagenic and genotoxic activity. Additionally, the e-cigarette aerosol was
found to be 300 times less cytotoxic than combustible cigarette smoke according to the neutral red uptake assay.
In this study, we performed a replication of the in vitro biological tests to examine the cytotoxic, mutagenic,
and genotoxic activity of myblu e-cigarette aerosol compared to 1R6F combustible cigarette smoke (University
of Kentucky). We employed similar methods to those used by Rudd and colleagues to evaluate and validate their
­findings9.

Results
Cytotoxicity: effect of cigarette whole smoke and myblu whole aerosol on cell viability.
After exposure to whole smoke from 1R6F reference cigarettes, BEAS-2B cell viability drastically decreased as
early as 2 puffs until complete cell death at 4 puffs with an ­EC50 value of 1.71 puffs (Fig. 1A). Instead, unlike Rudd
and colleagues, the E ­ C50 value could not be calculated for myblu exposure due to low cytotoxicity at 140 puffs
(Fig. 1B). We observed a reduced cell viability starting from 80 to 140 puffs, which did not decrease below the
80% of viability. Particularly, significant decrease of cell viability was observed for 80 (P = 0.003), 100 (P = 0.008),
120 (P = 0.038) and 140 puffs (P = 0.004) compared to the AIR control.
In addition to Rudd and colleagues, we observed microscopically the cells exposed to both 1R6F smoke and
myblu aerosol at 24, 48, and 65 h. Exposure of BEAS-2B cells to 1R6F smoke induced morphological changes
of cells with alterations in cell volume, nucleus volume, and cell sphericity at all the time-points (Fig. S1 of sup-
plementary material). Similar morphological changes of BEAS-2B cells were observed after exposure to myblu
aerosol starting from 80 puffs at 24 h. But a reversal of morphological changes was observed from 48 h to com-
plete recovery at 65 h (Fig. S2 of supplementary material).

Mutagenicity effect of 1R6F cigarette smoke and myblu aerosol

The negative controls (Solvent control) were in the normal range based on our laboratory experience and to
literature ­data3. Also, the positive controls (i.e., Chem Controls; Sodium Azide, Daunomicyn, and 2-Aminoan-
thracene) were in the range reported in the manufacturing manual (Trinova Biochem GmbH-Germany). No
significant differences were observed between Solvent controls and AIR controls for all the tested conditions.
Instead, significant differences were observed between Solvent controls or AIR controls and the respective Chem
Controls (P < 0.0001).
Exposure to 1R6F combustible cigarette smoke induced significant increase in revertants in a dose-dependent
manner without S9 metabolic activation. Indeed, significant dose-dependent increase in revertants was observed
in both TA98 (up to approximately 3.5-fold change to AIR control; P < 0.0001) and TA100 (up to 45-fold change
to AIR control; P < 0.0001) without S9 metabolic activation after exposure to 1R6F cigarette smoke. The exposure
to myblu aerosol did not induce any significant increase in revertants in both strains (Fig. 2). The linear regression
results of mutagenic activity in Salmonella typhimurium (TA98 and TA100) without S9 metabolic activation are
reported in Table S2 (supplementary material).
Similar results were observed when the Ames assay was performed for both TA98 (up to nearly fourfold
change to AIR control; P < 0.0001) and TA100 (up to onefold change to AIR control; P = 0.002) with S9 metabolic

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BEAS-2B

A 1R6F B

100

80
Cell Viability (% of AIR control)

60
EC50= 1.71 puffs

40

20

0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

Log puff number

Figure 1.  Cytotoxicity evaluation of BEAS-2B cells after exposure to 1R6F combustible cigarette smoke and
myblu aerosol. (A) Dose response curve of BEAS-2B cells exposed to 1R6F smoke showed an E ­ C50 value of 1.71
puffs (Log ­EC50 = 0.23 puff). The optical density (OD) means ± standard deviation (SD) of negative controls
(AIR, INC and ALI) were 0.138 ± 0.03, 0.129 ± 0.02 and 0.117 ± 0.01, respectively. (B) Barplot representing the
BEAS-2B cell viability results after exposure to myblu aerosol. The OD means ± SD of negative controls (AIR,
INC and ALI) for myblu were 0.077 ± 0.02, 0.116 ± 0.04 and 0.102 ± 0.01, respectively. All data are reported as
percentage of the respective AIR control and displayed as mean ± SD from 9 replicated wells from triplicate
Transwell inserts of two independent experiments (LAB-A and LAB-B). *P < 0.05; **P < 0.01; ****P < 0.0001.

A TA98 S9- B TA100 S9-

5 60
1R6F 1R6F
myblu myblu
4
Fold Change (to AIR control)

Fold Change (to AIR control)

40

2 20

1
0
50 100 150 200 250 300 350
0
50 100 150 200 250 300 350
Puff number
-1 Puff number -20

Figure 2.  Mutagenicity evaluation by Ames test in Salmonella typhimurium TA98 (A) and TA100 (B) without
S9 metabolic activation after exposure to 1R6F combustible cigarette smoke (violet round dot) or myblu aerosol
(blue square dot). The means ± standard deviation (SD) of revertants (N) in solvent controls for TA98 and
TA100 of 1R6F experiments were 39.46 ± 35.7 and 104 ± 67.7, respectively. The means ± standard deviation
(SD) of revertants (N) in solvent controls for TA98 and TA100 of myblu experiments were 9.5 ± 2.7 and 85.17
± 13.3, respectively. Data are reported as Fold change to AIR control. Each data point represents the mean ± SD
from triplicate Petri dishes. The dashed lines represent the 95% confidence interval of the regression line.

activation. No significant increase in revertants (linear slope did not differ from zero; P = 0.91) was observed for
TA98 strain with S9 metabolic activation after exposure to myblu aerosol. Instead, a slight increase of revertants
for TA100 S9 + (up to approximately 0.2-fold change to AIR control) was observed after myblu aerosol exposure,
with a linear slope significantly different from zero (P = 0.005) (Fig. 3). The linear regression results of mutagenic
activity in Salmonella typhimurium (TA98 and TA100) with S9 metabolic activation are reported in Table S1
(supplementary material).

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A TA98 S9+ B TA100 S9+

5 1.5
1R6F 1R6F
myblu myblu
4

Fold Change (to AIR control)

Fold Change (to AIR control)

1.0

2 0.5

0.0
50 100 150 200 250 300 350
0
50 100 150 200 250 300 350
Puff number
-1 Puff number -0.5

Figure 3.  Mutagenicity evaluation by Ames test in Salmonella typhimurium TA98 (A) and TA100 (B) with S9
metabolic activation after exposure to 1R6F combustible cigarette smoke (violet round dot) or myblu aerosol
(blue square dot). Data are reported as Fold change to AIR control. The means ± standard deviation (SD) of
revertants (N) in solvent controls for TA98 and TA100 of 1R6F experiments were 32.08 ± 13.7 and 79.5 ± 16.5,
respectively. The means ± SD of revertants (N) in solvent controls for TA98 and TA100 of myblu experiments
were 11.58 ± 3.9 and 79.75 ± 10.5, respectively. Each data point represents the mean ± SD from triplicate Petri
dishes. The dashed lines represent the 95% confidence interval of the regression line.

Genotoxic effect of 1R6F cigarette smoke and myblu aerosol

Due to different exposure system, not able to perform smoke dilution, we performed a dose–response curve
in order to establish the ­EC50 dose for the V79 cells exposed to 1R6F combustible cigarette whole smoke: the
calculated ­EC50 value for V79 cells was 3.149 puffs (Fig. S3 of supplementary material). We then performed the
IVMN assay with and without S9 metabolic activation after exposure from 1 to 4 puffs of 1R6F combustible
cigarette smoke. For myblu exposure the same puff numbers by Rudd et al. (2020) were used (20–100 puffs).
No difference was shown among the three negative controls, cells left to grow in the incubator submerged in
the culture medium (INC), cells left to grow in the incubator with the apical side exposed to the air in air–liquid
interface mode (ALI) and cells exposed to puffs of particulate-filtered laboratory air using a Cambridge Filter Pad
(AIR) for both IVMN with and without S9 activation. Instead, the positive controls, including cyclophosphamide
A (for IVMN S9 +) and mitomycin C (for IVMN S9-), were significantly increased compared to the respective
AIR controls (Cyclo A P < 0.0001; Mito C P = 0.002).
The results of IVMN assay without S9 (S9-) are shown in Fig. 4. The micronuclei frequency for the 1R6F
exposure, did not show a dose-dependent increase due to high cytotoxicity (Table S5 of supplementary material)
of undiluted smoke (Fig. 4A). However, all the 1R6F puff numbers (from 1 to 4) induced significant increments
of micronuclei frequency (P < 0.0001) compared to AIR control (Fig. 4B). Instead, no significant increase in
micronuclei frequency was observed after exposure to myblu aerosol until 100 puffs (Fig. 5B and Table S6 of
supplementary material).
When the IVMN assay was performed with the S9 metabolic activation, high cytotoxicity was observed for
1R6F, and less cytotoxicity was observed for myblu (see Tables S3 and S4 of supplementary material). Particu-
larly, marked cytotoxicity was observed for V79 cells exposed to combustible cigarette smoke to the point of
being unable to perform micronuclei counts. The cause of this cytotoxicity was attributed to the S9 mix, which is
known to be c­ ytotoxic4 (see Fig. S4 of supplementary material). Especially for the IVMN assay with S9 of 1R6F,
the cytotoxic effect of both S9 mix and undiluted combustible cigarette smoke have added up, making the assay
unfeasible. Whereas, despite the S9 cytotoxicity, we were able to perform micronuclei quantification in order
to complete IVMN assay for myblu. The regression slope was not different from zero (P = 0.5) (Fig. 5), and all
the micronuclei frequencies corresponding to each myblu puff number were not different from AIR control.
Because of the high cytotoxicity of S9 mixture and since the OECD n. 487 guideline reported that IVMN
assay can be performed with or without S9 activation, we performed this assay without S9 mixture in addition
to what was done by Rudd and colleagues.

Discussion
In recent years the problem of the replicability crisis has been raised in various sectors of science. For a sensitive
topic such as the regulation of products intended for human consumption, such as e-cigarettes, this issue is par-
ticularly relevant. For this reason, the Replica project set itself the objective of replicating in vitro studies that led
to conclusions of significant interest, to refute or confirm their ­validity5,6. This study replicated the work by Rudd
and ­colleagues7, which compared the in vitro toxicity of the myblu e-cigarette aerosol with that of combustible
cigarette smoke. They performed a standard toxicological battery of three assays used for product assessment
and regulatory applications: the NRU assay to assess c­ ytotoxicity8, the bacterial reverse mutation (Ames) assay
to evaluate ­mutagenicity9, and the in vitro micronucleus assay to measure ­genotoxicity10. Their results indicated

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A B
0.30
0.30
Controls
1R6F ****
myblu 1R6F
0.25 0.25 ****
myblu
****

Micronuclei Frequency
Micronuclei Frequency
0.20 0.20

0.15 0.15
****
0.10 0.10
**
0.05 0.05

0.00 0.00
0.0 0.5 1.0 1.5 2.0 2.5

LI

m IR

ffs

ffs

ffs

20 fs

40 fs

60 fs

80 fs

10 ffs

ffs
IN

f
A

yn

Pu

Pu
Pu

Pu

Pu

Pu

Pu

Pu

Pu
Log puff number

ic

0
ito
M
Figure 4.  Genotoxicity evaluation by in vitro micronucleus assay without S9 activation in V79 cells after
exposure to undiluted 1R6F combustible cigarette smoke or myblu aerosol at the air liquid interface. (A) Linear
slopes of the dose–response IVMN results for 1R6F (violet round dot) and myblu (blue square dot). The dashed
lines represent the 95% confidence interval of the regression line. (B) Barplot representing IVMN results,
including both negative (INC; ALI; AIR) and positive (mitomycin C) controls, 1R6F, and myblu results. All data
are reported as micronuclei frequency. Each data point or bar represents the mean ± standard deviation (SD)
from 9 replicated wells from triplicate Transwell inserts. The micronuclei frequency means ± SD of negative
controls (INC, ALI and AIR) were 0.007 ± 0.001, 0.007 ± 0.003 and 0.006 ± 0.002, respectively. **P < 0.01;
****P < 0.0001 compared to AIR control.

IVM S9+ (myblu)

0.25

0.20
Micronuclei Frequency

0.15

0.10

0.05

0.00
1.2 1.4 1.6 1.8 2.0 2.2
Log puff number

Figure 5.  Genotoxicity evaluation by in vitro micronucleus assay with S9 activation in V79 cells after exposure
to myblu aerosol (from 20 to 100 puffs) at the air liquid interface. Data are reported as micronuclei frequency.
Each data point represents the mean ± standard deviation (SD) from 9 replicated wells from triplicate Transwell
inserts. The dashed lines represent the 95% confidence interval of the regression line. The micronuclei
frequency means ± SD of negative controls (INC, ALI and AIR) were 0.159 ± 0.03, 0.183 ± 0.05 and 0.177 ± 0.04,
respectively.

that e-cigarette aerosol was low cytotoxic, and it did not show any mutagenic or genotoxic activity unlike the
3R4F cigarette smoke, which showed high cytotoxic, mutagenic and genotoxic activity.
Despite some different methodological aspects in our study, we obtained results similar to those obtained by
Rudd and colleagues. The main methodological differences were: (i) the use of 1R6F reference cigarette in place
of the 3R4F reference cigarette because the latter is no longer produced by the Center for Tobacco Reference
Products (University of Kentucky); (ii) the different smoking and vaping apparatus. Our laboratory is equipped
with separate machines, the LM1 smoking machine and the LM4E vaping machine (Borgwaldt, Hamburg,
Germany). Instead, Rudd and colleagues used the SAEIVS five-port smoking/vaping machine for generation of
both smoke and aerosol. Also, the SAEIVS machine is able to perform dilution of smoke/aerosol with air while
the LM1 and LM4E machines were not designed to perform dilutions; (iii) the different smoke/aerosol ALI
exposure in vitro system. The smoke/aerosol exposure in vitro system (SAEIVS) used by Rudd and colleagues was

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designed to expose cells in 96 and 24 multi-well plates, only the latter with Transwell inserts. Instead, our in vitro
ALI exposure system (described in the “methods” section) allows the cell exposure with Transwell inserts of all
diameters by the use of a dedicated exposure chamber. All these differences have been filled by implementing
some modifications to the protocols used in the original work as described in the “methods” section.
NRU assay was performed both in LAB-A and in LAB-B. Our results confirmed the higher cytotoxicity of
1R6F cigarette smoke compared to the e-cigarette aerosol as showed by Rudd and colleagues. However, the cal-
culated ­EC50 for the 1R6F smoke (1.71 puffs) was different from that obtained in the original work (0.236 puffs).
In addition, we did not observe the same cytotoxic effect for the myblu aerosol. Indeed, the low cytotoxicity
induced by myblu aerosol did not allow us to calculate the value of E ­ C50. But we observed only a reduced cell
viability, around the 80% of viability, starting from the 80 puffs to 140 puffs. These differences in results may
be ascribed to the different ALI exposure apparatus. Rudd and colleagues exposed BEAS-2B cells seeded in the
96-well plate, but the cells do not have medium in the basal face of cells by this type of exposure. Then it is not a
real ALI exposure because the cells are dry as the apical medium is taken to perform an air-interface exposure.
As a result, part of the cytotoxicity observed by Rudd and colleagues could be due to conditions that are not
optimal for normal cell health. Conversely, we exposed BEAS-2B cells using Transwell inserts placed into the
exposure chamber filled with culture medium at the basal compartment that provides nutrition for cells through
the Transwell membrane. This exposure apparatus provides the optimal environment to avoid the cells to dry
out, especially when performing long exposures (10 to 77 min, in this case). The same ALI-exposure system
combined with cytotoxicity evaluation was successfully used in our previous w ­ orks11–14, and other published
­works15,16. Moreover, several in vitro toxicity studies used the ALI exposure with cell cultures by using appropriate
apparatus developed by dedicated manufactures, such as the VITROCELL and CULTEX system, that simulate
real in vivo exposure ­conditions17,18. Furthermore, we also added a morphological evaluation of cells during the
recovery period at 24, 48 and 65 h (Figs. S1 and S2 in supplementary material). Thise set of experiments showed
a good recovery of cells over time even though we observed some discrepancies with NRU assay results when
compared to the morphological data. However, our previous study showed that NRU may present some limita-
tion in detecting apoptotic c­ ells14 especially during the early phase of this process.
The Ames test was performed only in the LAB-A, as reported by Rudd and colleagues, with Salmonella typh-
imurium TA98 and TA100 strains, which are particularly relevant for tobacco products since they have been
shown to be sensitive to combustion p ­ roducts19,20. Unlike the original work, we conducted the Ames test with and
without S9 metabolic activation. Our results showed that neither TA98 nor TA100 with and without S9 showed a
mutagenic response after myblu aerosol exposure even at high doses (from 60 to 300 puffs), as opposed to what
has been observed for 1R6F cigarette smoke with minor doses (from 9 to 45 puffs). These results are aligned
with what Rudd and colleagues showed in their work, even though we used different exposure machines. The
mutagenicity evaluation of e-cigarette aerosol by Ames assay has been also reported in literature with similar
results to those reported in this w ­ ork21–24.
Genotoxicity evaluation was conducted by LAB-A in a similar way to those reported by Rudd and colleagues,
with the following exceptions: (i) we added the genotoxicity evaluation without S9 metabolic activation to
improve their results; (ii) the exposure of 1R6F cigarette smoke was performed undiluted. Though, we expe-
rienced some methodological issues following their protocol. Indeed, they reported the use of S9 mix at 10%,
but using the same concentration we observed a massive cell mortality (more than 50%) especially for 1R6F
cigarette smoke that affects the observation of micronuclei in V79 cells. Instead, we were able to perform the
micronuclei evaluation with S9 for the V79 cells exposed to myblu aerosol, showing no genotoxic effect. Based
on literature data, we found that the S9 mix has a full set of liver metabolic enzymes, but it displays high cyto-
toxicity in cell-based ­assays4. Consequently, we performed a dose–response curve with different concentrations
of S9 mix (from 1 to 5%), and we observed that cell viability decreased with the increment of S9 enzymatic mix
percentage (Fig. S4). Probably, the high cytotoxicity levels observed in the IVMN assay with S9 are due to the sum
of undiluted whole smoke cytotoxicity in addition to the S9 enzymatic mix cytotoxicity. A limitation of IVMN
assay is that higher cytotoxicity levels may induce chromosome damage as a secondary effect of cytotoxicity, then
it is suggested not to exceed 50% ­cytotoxicity10. Indeed, the IVMN assay without the S9 metabolic activation
allowed us the micronuclei count for both 1R6F cigarette smoke and myblu aerosol. A high genotoxicity (exceed-
ing the positive control) was demonstrated for 1R6F cigarette smoke at 1 puff, although no clear dose response
was observed due to the cytotoxic effect of 1R6F cigarette smoke. Therefore, the results of the genotoxicity for
1R6F smoke can not be considered reliable due to the high cytotoxicity detected. However, no genotoxicity was
observed for all myblu exposure conditions. In line with our results there is the work by Thorne et al. (2019),
which showed the highest responsivity of V79 cells to combustible c­ igarette25. Another study investigated the
genotoxic activity of aerosolized e-liquids in three immortalized cell lines not included in the OECD g­ uidelines26,
observing that in certain conditions, and with some chemical flavors, e-cigarette liquids could be able to induce
genotoxicity. Moreover, the researchers reported that at the lowest dose recommended the S9 caused considerable
death with the cell lines used in the study. In another study investigating the mutagenic and genotoxic potential of
different chemical flavors used in e-liquids27, for some of them a certain mutagenic activity was observed at high
concentrations on S. typhimurium TA100 and TA98 strains, but not for most of them. Otherwise, the IVMN test
in CHO–K1 cells evidenced a certain increase in micronuclei for a low number of chemical flavors and at high
concentration, with or without S9 fraction. However, all these studies were conducted with homemade systems
and without following a standardized validated exposure regime, making them difficult to be replicated by other
groups. All these scientific evidence on the mutagenic and genotoxic effects of e-cigarette aerosols highlight the
lack of clarity still existing in this area and the need for further studies conducted following the official guidelines
and that are replicable, to verify the results.
In conclusion, our findings confirmed the results on low toxicity profile of myblu e-cigarette obtained by Rudd
and colleagues, despite some differences in methodology. Moreover, our study covered some methodological

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gaps and limitations in the original work, including the non-optimal ALI exposure for the cytotoxicity evalua-
tion and improved mutagenicity and genotoxicity results by adding experiments without S9 metabolic activation
as recommended in the OECD guidelines. Overall, this replication study supports the tobacco harm reduction
strategy as having the potential to substantially reduce exposure to toxic combustion agents in adult smokers.
Future studies are needed to advance in vitro methods in order to evaluate the long-term effects of electronic
nicotine delivery systems.

Methods
Test products
Unlike Rudd and colleagues, we used the 1R6F reference combustible cigarette (University of Kentucky, Center
for Tobacco Reference Products, Lexington, KY, USA), since the 3R4F are no longer produced by the University
of Kentucky. 1R6F and 3R4F reference combustible cigarettes are very similar and only slight differences were
reported regarding smoke chemistry and in vitro ­assays28. Prior to every experimental session, the 1R6F com-
bustible cigarette were conditioned for a minimum of 48 h at 22 ± 1 °C and 60 ± 3% relative humidity, according
to ISO 3402:199929.
The same electronic cigarette used by Rudd and colleagues, myblu (Imperial Brands PLC, Bristol, United
Kingdom), was used for this replication study. The myblu is a "closed pod-system” e-cigarette consisting of two
elements: a rechargeable battery (battery capacity, 350 mAh) and a replaceable e-liquid pod with 1.5 mL volume
and a coil resistance of 1.3 Ω (cartomizer). The tobacco flavored e-liquids with 1.6% (w/w) nicotine were used
for the experiments. All the myblu e-cigarettes and myblu pods were purchased from Italian retailers.

Smoke and vapour exposure

The selected products were tested on standardized equipment simulating smoking topography: the LM1 Smok-
ing Machine (Borgwaldt, Hamburg, Germany) (Fig. S6A) was used to smoke the 1R6F combustible cigarettes
following the Health Canada Intense (HCI) regime (puff volume, duration and frequency of 55 mL, 2 s and 30 s
(55/2/30), with bell shaped profile, 27.5 ml/s puff velocity, and hole vents blocked), accredited under ISO/TR
19478-2:201530.The LM1 is a direct exposure system and does not perform smoke dilution unlike the smoking
machine used by Rudd and colleagues. The LM4E Vaping Machine (Borgwaldt, Hamburg, Germany) (Fig. S6B)
was used to vape myblu following the “CORESTA Reference Method n. 81” (CRM81) regimen (55 ml puff vol-
ume, drawn over 3 s, once every 30 s with square shaped profile, with a puff velocity of 18.3 ml/s), accredited
into ISO 20,768:201831. The standard exhaust time for both LM1 and LM4E was 0.7 s with a flow rate in the
exposure chamber of 78.57 ml/s.
An air–liquid exposure system different from the original paper by Rudd et al. (2020) was used to expose
BEAS-2B to perform NRU assay and V79 cells to perform IVMN assay. The BAT (British American Tobacco)
aerosol exposure chambers (Fig. S7) were used to expose in vitro cells at the air–liquid interface (ALI) to combus-
tible cigarette smoke and aerosols from e-cigarettes. In particular, cells cultured in Transwell inserts were deprived
of the apical medium and placed in the exposure chambers containing the medium in the lower compartment,
keeping wet the basal face of the cells, and then connected to the smoking or vaping machine to deliver undiluted
whole smoke or whole aerosol to the apical face of cells (Fig. 6)13.
For the cytotoxicity evaluation of BEAS-2B, 1R6F cigarette smoke was delivered undiluted from 1 to 8 puffs
(exposure time from 00:02 to 03:46 mm:ss). The myblu aerosol was delivered undiluted from 20 to 140 puffs
(exposure time from 10:30 to 76:30 mm:ss), as reported by Rudd et al. (2020). Cytotoxicity evaluation was also
performed to establish the ­EC50 of V79 cells exposed to 1R6F undiluted smoke prior to the IVMN assay. In that
case, V79 cells were exposed to 1R6F smoke from 2 to 30 puffs (exposure time from 00:34to 15:30 mm:ss). Based
on the EC50 previously calculated for the V79 cells on the results obtained in the NRU assay, we performed the
1R6F smoke exposure for the IVMN assay delivering from 1 to 4 puffs to the cells (exposure time from 00:02 to
01:38 mm:ss). The myblu aerosol was delivered undiluted from 20 to 100 puffs for the IVMN assay (exposure
time from 10:30 to 54:30 mm:ss), as reported by Rudd et al. (2020). The experiments on cytotoxicity were rep-
licated by the laboratory from Italy (CoEHAR, University of Catania, subsequently referred as LAB-A) and by
the laboratory from Oman (Sultan Qaboos University, referred as LAB-B).
For the Ames assay, 1R6F cigarette smoke and the relative AIR control, or myblu aerosol and the relative
AIR control, were delivered to the bacterial suspensions contained into their corresponding impingers at room
temperature under protection from direct light. A puff with filtered ambient air was applied between smoke or
aerosol puffs. The number of puffs is reported in Table S7 (supplementary material) for both the 1R6F exposure
and for the myblu exposure. In particular, the impinger inlet for the 1R6F smoke exposure was connected to
LM1 smoking machine (1R6F smoke) and to a LM4E channel equipped with a 44 mm Cambridge Filter Pad
(CFP) (AIR interpuff) by means of a double one-way valve, whereas the impinger inlet for the myblu e-cigarette
aerosol exposure was connected to two different LM4E channels, one of which equipped with e-cigarette and
the other with a 44 mm CFP (AIR interpuff).

Cell cultures
Normal bronchial epithelial cells (BEAS-2B / ATCC-CRL-9609) were cultured in collagen coated flasks using
the bronchial epithelial growth medium supplemented with Lonza Bullet Kit (BEGM, Lonza CC-3170), as
described by ATCC culture instructions. Hamster lung fibroblast cells (V79 / ICLC-AL99002) were cultured
using Dulbecco’s Modified Eagle Medium–high glucose (DMEM-hg, Thermo Fisher Scientific) with 10% FBS,
2 mM L-Glutamine, 50 U/mL penicillin, and 50 μg/mL streptomycin, as described by ICLC (Interlab Cell Line
Collection; http://​bioin​forma​tics.​hsanm​artino.​it/​iclc/) instructions.

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Figure 6.  Air–liquid interface (ALI) exposure systems used by Rudd et al. (2020) and by this replication study
in order to perform NRU assay. (A) Rudd et al. (2020) used the 96-well plate and they removed the culture
medium from each well to expose BEAS-2B cells at the ALI. (B) In this study, the culture medium is removed
from the upper part of the Transwell inserts and then placed in the exposure chambers on a plastic support that
allows the cells to remain basally wet with medium and to be exposed to the smoke/vapor apically by the LM1/
LM4 machines. The BAT exposure chamber allows a symmetrical aerosol distribution by the disc and ensures
uniform cellular ALI exposure avoiding the accumulation of aerosol inside the system.

Cytotoxicity evaluation: NRU assay

Cytotoxicity evaluation was performed using the BEAS-2B cells by using the NRU ­assay8,32. Moreover, cytotoxic-
ity evaluation was performed with 1R6F whole smoke for the V79 cells, prior to genotoxicity evaluation, in order
to establish the number of puffs to be used in the IVMN assay.
Prior to exposure, 300 μl of BEAS-2B cell suspension (BEGM supplemented with Lonza Bullet Kit and with
20 mM of HEPES buffer) was seeded in 24-well Transwell inserts at density of 150.000 cells/well and incubated for
20 ± 3 h. After incubation, the apical cell culture medium was removed, and the Transwell inserts were transferred
into the corresponding exposure chamber filled with 25 ml of DMEM-hg supplemented with 50 U/mL penicil-
lin and 50 μg/mL streptomycin in order to proceed to the smoke/vapor ALI exposure. After ALI exposure, each
insert was transferred in a new 24-well plate filled with 500 μl and 300 μl of fresh BEGM (supplemented with
Lonza Bullet Kit + 20 mM of HEPES buffer) respectively at the basal and apical compartments. Next, the cells
were incubated for a recovery period of 65 ± 2 ­h7. The day before NRU assay, the NRU solution was prepared in
BEGM medium at ratio 1:65 (0.05 g/L) plus HEPES buffer at 20 mM and placed in incubator at 37 °C 5% ­CO2.
The day of NRU assay, the NRU solution was filtered prior to use. The culture medium was removed from the
apical and basal compartments of each culture insert. The cells were washed twice with pre-warmed PBS, then
incubated with neutral red solution (500 μl at the bottom and 300 μl at the top) for 3 h at 37 °C in 5% ­CO2 and a
humidified atmosphere. After incubation, cells were washed twice with pre-warmed PBS to remove unincorpo-
rated dye. The incorporated solution was eluted from the cells by adding 330 μl of destain solution (50% ethanol,
49% distilled water, 1% glacial acetic acid v:v:v) to each insert and incubated for 10 min at 300 rpm on a plate
shaker. Extracts were transferred to a 96-well plate in duplicate (100 μl per well) and optical density of neutral
red extracts was read with a microplate spectrophotometer at 540 nm using a reference filter of 630 nm. Blank

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inserts (without cells) were used to assess how much neutral red solution stained the Transwell membranes and
the mean of background values was subtracted from each measurement.
The same procedure was used for the cytotoxicity evaluation of V79 cells. In brief, 300 μl of V79 cell suspen-
sion (DMEM-hg supplemented with 10% FBS, 2 mM L-Glutamine, 50 U/mL penicillin, 50 μg/mL streptomycin,
and 20 mM HEPES) was added in 24-well Transwell inserts at density of 100.000 cells/well and incubated for 24 h.
The day of ALI exposure, the Transwell inserts (without apical cell culture medium) were transferred into the
corresponding exposure chamber filled in the basal compartment with 25 ml of DMEM-hg supplemented with
50 U/mL penicillin and 50 μg/mL streptomycin. After ALI exposure, each insert was transferred in a new 24-well
plate filled with 500 μl and 300 μl of fresh DMEM-hg (supplemented with 10% FBS, 2 mM L-Glutamine, 50 U/mL
penicillin, 50 μg/mL streptomycin, and 20 mM HEPES) respectively at the basal and apical compartments. Next,
the cells were incubated for a recovery period of 24 h. The NRU solution was prepared in DMEM-hg at ratio 1:65
(0.05 g/L) plus HEPES buffer at 20 mM. The next steps of NRU assay for V79 cells were the same as BEAS-2B
and are described above. Three negative controls were performed for this assay: (i) cells were maintained in the
incubator with both the basal and apical culture medium (INC), (ii) the cells were maintained in the incubator
without the apical medium to reproduce the air–liquid interface exposure (ALI) and (iii) the cells were exposed
to puffs of particulate-filtered laboratory air (AIR).

Mutagenicity evaluation: Ames assay

The in vitro mutagenic effect of fresh 1R6F smoke and myblu aerosols was determined using Ames ­test33 as
described by Rudd et al.7 with some modifications, and it was conducted only by LAB-A. The Ames screen
was employed using only S. typhimurium TA98 and TA100 strains (Trinova Biochem GmbH) ± S9 treatment,
which are the most sensitive to combustion products. The Ames assay was conducted in accordance with OECD
(Organization for Economic Cooperation and Development) test guideline 4­ 719.
Briefly, bacterial cultures of the TA98 and TA100 strains were prepared in 25 mL Nutrient Broth No.2
(OXOID) by inoculating one bacterium- followed by incubation overnight at 37 °C with shaking at 120 rpm.
Then, bacterial suspensions were prepared by centrifugation of 25 mL cultures at 1800 g for 20 min at 4 °C,
and the pellet was resuspended in 12 mL of C ­ a2+, ­Mg2+-free Dulbecco’s phosphate buffered saline (PBS). Next,
10 mL of the bacterial suspensions were placed in the corresponding impingers and exposed to test aerosols/
smoke and filtered ambient air (negative control of exposure) as described above (exposed Bacterial Suspension
or eBS). For each experiment, an aliquot of PBS with untreated PBS bacterial suspension (for the assay without
S9 mix) and S9 mix with untreated PBS bacterial suspension (for the assay with S9 mix) as internal negative
controls (Solvent control).
After each exposure, the bacterial suspensions were immediately used for Ames screening by following manu-
facturer’s protocol (Salmonella Mutagenicity Test Kit, MOLTOX®). Briefly, an aliquot of bacterial suspensions
and relative reagents were added to sterile 15 mL test tubes as described in Table S8 (supplementary material).
The solution was thoroughly mixed and then decanted on top of a minimal glucose agar plate, covered and set
aside to solidify. When the top agar was solidified, the plates were inverted and placed in an incubator at 37 °C.
After 48–72 h of incubation, the number of revertant colonies growing on the plates was counted manually.
Controlchem™ Mutagens were used as positive controls for both S. typhimurium stains TA98 and TA100 (see
Table S9 of supplementary material). Each concentration of test aerosols or smoke and positive controls was
tested in triplicate.

Genotoxicity evaluation: IVMN assay

The IVMN assay was performed in concordance with OECD test guideline no. 487 10 with and without S9 meta-
bolic activation, and it was conducted only by LAB-A. Genotoxicity evaluation of whole fresh tobacco smoke and
e-cigarette aerosol was performed using the hamster lung V79 cell line (Interlab Cell Line Collection (ICLC)-
AL99002). The day before cell exposure, V79 cells were seeded in 24-well Transwell inserts (0.4 µm pore mem-
brane) at density of 10 × ­104 with 200 µl of DMEM-hg supplemented with 10% of FBS. The inner wells of each
24-well plate were filled with 500 µl of DMEM-hg supplemented with 10% of FBS. The V79 cells were incubated
for 24 h at 37 °C and 5% ­CO2. After 24 h of incubation, the apical medium was removed, and the inserts were
transferred into the exposure chamber filled with 25 ml of DMEM-hg with the addition of HEPES buffer (20 mM
final concentration) in the basal compartment. The exposure to 1R6F whole smoke and myblu e-cigarette whole
aerosol is described in the previous section on “Smoke and vapour exposure". After the exposure, each insert was
transferred in a new 24-well plate filled with 500 µl of DMEM-hg supplemented with HEPES buffer (20 mM).
For the IVMN with S9, each insert containing exposed V79 cells, was filled with 300 µl of S9 mix at 10% in the
apical compartment, and then incubated for 3 h at 37 °C. After incubation, the apical S9 medium was removed,
the V79 cells were covered with DMEM-hg supplemented with HEPES buffer (20 mM) and incubated for 24 h
to allow for at least one cell division cycle. For the IVMN without S9, inserts with exposed V79 cells were filled
with DMEM-hg supplemented with HEPES buffer (20 mM) and incubated for 24 h at 37 °C and 5% ­CO2. Three
negative controls were performed for both IVMN with and without S9 activation: (i) cells were maintained in
the incubator with both the basal and apical culture medium (INC), (ii) the cells were maintained in the incu-
bator without the apical medium to reproduce the air–liquid interface exposure (ALI) and (iii) the cells were
exposed to puffs of particulate-filtered laboratory air (AIR). Positive controls, including cyclophosphamide A
(CAS 6055–19-2) for the S9 fraction and mitomycin C (CAS 50–07-7) for the IVMN without S9, were used. All
the tested conditions were assessed in triplicates.
After 24 h of recovery, the cells were detached and counted using the Muse® Cell Analyzer using the Muse®
Count & Viability Kit (Luminex Corp.). The V79 cells were then seeded in 96-well plate (CellCarrier Ultra-96
Black, Optically Clear Bottom—PerkinElmer) at density of 10 × 1­ 03 per well and incubated for 24 h. Next, the

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cells were fixed with 4% (PFA) (paraformaldehyde) for 20 min at room temperature. After fixation, the cells were
washed once with PBS and then stained with DAPI (1 μg/mL). Micronuclei assessment was performed by using
Harmony® High-Content Imaging and Analysis Software (PerkinElmer).

Statistics
All raw data were collected and processed using Excel software (Microsoft, Redmond, WA, USA). R version 3.4.3
(2017-11-30) was used to assess reproducibility of NRU data between LAB-A and LAB-B. Reproducibility of
NRU data obtained by LAB-A and LAB-B were evaluated by linear regression analysis of cell viability percent-
ages (to AIR controls) between the two laboratories. Moreover, the mean differences and the limits of agreement
(95% confidence interval) were calculated to assess the agreement between LAB-A and LAB-B and visualized
by Bland–Altman plots. A 1-tailed sample T test was also performed to assess the mean differences between the
two laboratories from zero (Table S10 and Fig. S5 of supplementary material).
For the cytotoxicity evaluation (NRU), data were expressed as percentage to the AIR control. The E
­ C50 values
for each exposure (1R6F and myblu) were calculated by fitting a sigmoidal dose–response curve with a variable
slope to determine the best fit values for the 1R6F log E
­ C50 of 7 parameter nonlinear regression model and for
the myblu log ­EC50 of 7 parameter nonlinear regression model. Moreover, comparison of myblu results and AIR
control was carried out by ANOVA followed by Dunnett’s post hoc multiple comparison test.
Data from Ames assay (mutagenicity evaluation) were reported as fold change relative to AIR ­control3, and
calculated as follow:
 
revertant number relative to puff number −(mean of revertant number relative to AIR control)
/mean of revertant number relative to AIR control.
Linear regression analyses for each strain were performed to evaluate the mutagenic activity. Moreover,
comparisons among 1R6F and respective controls and among myblu and respective controls were performed by
using mixed-effect model or ANOVA followed by Tukey’s post hoc multiple comparison test.
Genotoxicity data were analyzed by linear regression of 1R6F or myblu dose–response slopes with comparison
between slopes. Comparisons among the different dose of smoke or aerosol and respective controls were per-
formed by ANOVA followed by Tukey’s (IVMN without S9 activation) or Dunnett’s (IVMN with S9 activation)
post hoc multiple comparison tests.
All analyses were considered significant with a p value < 0.05. GraphPad Prism 8 software was used for data
analysis and generation of graphs unless otherwise stated. Raw data were shared by Zenodo repository (https://​
doi.​org/​10.​5281/​zenodo.​83352​01).

Additional Information
All experiments were performed in accordance with relevant guidelines and regulations. No animals or human
tissue samples were used for the experiments. Cells from American Type Culture Collection (Manassas, VA,
USA) were used for the experiments on cytotoxicity: Human normal bronchial epithelial cells (BEAS-2B /
ATCC-CRL-9609). Hamster lung V79 cell line (ICLC-AL99002) from IRCCS Ospedale Policlinico San Martino,
(Interlab Cell Line Collection—ICLC) were used for genotoxicity assessment. The pre-print version of this article
is present on https://​www.​biorx​iv.​org/​conte​nt/​10.​1101/​2022.​10.​28.​51420​5v2. This article is not published nor
is under publication elsewhere.

Data availability
The datasets generated during the current study are available from the corresponding author on reasonable
request.

Received: 24 May 2023; Accepted: 10 October 2023

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Author contributions
Conceptualization: M.C., G.L.V.; Methodology: R.E., V.F., F.Z., M.C., G.L.V.; Formal analysis: R.E.; Investiga-
tion: R.E., V.F., A.D., K.P., M.A.T., R.Z., Z.A., R.Pu.; Resources: S.R.; Data curation: R.E.; Writing – original draft
preparation: R.E., K.P.; Writing – review and editing: P.M.F., R.Po., A.S., M.C., G.L.V., ­RPG#; Validation: A.S.;
Supervision: M.C., G.L.V.

Funding
This investigator-initiated study was sponsored by ECLAT srl, a spin-off of the University of Catania, with the
help of a grant from the Foundation for a Smoke-Free World Inc., a US nonprofit 501(c)(3) private foundation
with a mission to end smoking in this generation. The contents, selection, and presentation of facts, as well as
any opinions expressed herein are the sole responsibility of the authors and under no circumstances shall be
regarded as reflecting the positions of the Foundation for a Smoke-Free World, Inc. ECLAT srl. is a research-
based company from the University of Catania that delivers solutions to global health problems with special
emphasis on harm minimization and technological innovation.

Competing interests
Riccardo Polosa is full tenured professor of Internal Medicine at the University of Catania (Italy) and Medical
Director of the Institute for Internal Medicine and Clinical Immunology at the same University. He has received
grants from U-BIOPRED and AIR-PROM, Integral Rheumatology & Immunology Specialists Network (IRIS),
Foundation for a Smoke Free World, Pfizer, GlaxoSmithKline, CV Therapeutics, NeuroSearch A/S, Sandoz,
Merk Sharp & Dohme, Boehringer Ingelheim, Novartis, Arbi Group Srl., Duska Therapeutics, Forest Laborato-
ries, Ministero dell Universita’ e della Ricerca (MUR) Bando PNRR 3277/2021 (CUP E63C22000900006) and
341/2022 (CUP E63C22002080006), funded by NextGenerationEU of the European Union (EU), and the min-
isterial grant PON REACT-EU 2021 GREEN- Bando 3411/2021 by Ministero dell Universita’ e (MUR) – PNRR
EU Community. He is founder of the Center for Tobacco Prevention and Treatment (CPCT) at the University

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of Catania and of the Center of Excellence for the Acceleration of Harm Reduction at the same university.
He receives consultancy fees from Pfizer, Boehringer Ingelheim, Duska Therapeutics, Forest Laboratories, CV
Therapeutics, Sermo Inc., GRG Health, Clarivate Analytics, Guidepoint Expert Network, and GLG Group. He
receives textbooks royalties from Elsevier. He is also involved in a patent application for ECLAT Srl. He is a pro
bono scientific advisor for Lega Italiana Anti Fumo (LIAF) and the International Network of Nicotine Consum-
ers Organizations (INNCO); and he is Chair of the European Technical Committee for Standardization on
“Requirements and test methods for emissions of electronic cigarettes” (CEN/TC 437; WG4). Giovanni Li Volti
is currently elected Director of the Center of Excellence for the acceleration of HArm Reduction (CoEHAR).
The other authors have no relevant financial interests to disclose.

Additional information
Supplementary Information The online version contains supplementary material available at https://​doi.​org/​
10.​1038/​s41598-​023-​44626-1.
Correspondence and requests for materials should be addressed to M.C.
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© The Author(s) 2023, corrected publication 2024

the Replica Project Group

Giovanni Li Volti2,3, Massimo Caruso2,3, Rosalia Emma1,2, Antonio Giordano6, Ang Sun6,
Vladislav Volarevic7, Ronny Lesmana8,9, Konstantinos Poulas10,11, Alfio Distefano3,
Konstantinos Partsinevelos3, Roberta Pulvirenti3, Aurora Costa6, Aleksandar Arsenijevic7,
Melisa I. Barliana12,8, Konstantinos Mesiakaris10,11, Najwa Albalushi5, Chiara Giardina3 &
Salvatore Furnari3
7
Faculty of Medical Sciences, Center for Molecular Medicine and Stem Cell Research, University of Kragujevac,
Kragujevac, Serbia. 8Center of Excellence for Pharmaceutical Care Innovation, Universitas Padjadjaran, Jl.
Raya Bandung Sumedang KM. 21, Jatinangor 45363, Indonesia. 9Department Biomedical Sciences, Faculty of
Medicine, Universitas Padjadjaran, Jl. Raya Bandung Sumedang KM. 21, Jatinangor 45363, Indonesia. 10Institute
for Research and Innovation, IRIS, Patras Science Park, Patras, Greece. 11Laboratory of Molecular Biology
and Immunology, Department of Pharmacy, University of Patras, Patras, Greece. 12Department of Biological
Pharmacy, Biotechnology Laboratory, Faculty of Pharmacy, Universitas Padjadjaran, Jl. Raya Bandung Sumedang
KM. 21, Jatinangor 45363, Indonesia.

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