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Genotoxic effects in oral mucosal cells caused by the use of orthodontic fixed
appliances in patients after short and long periods of treatment

Article  in  Clinical Oral Investigations · January 2019

DOI: 10.1007/s00784-018-02795-8

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Clinical Oral Investigations
https://doi.org/10.1007/s00784-018-02795-8

ORIGINAL ARTICLE

Genotoxic effects in oral mucosal cells caused by the use of orthodontic

fixed appliances in patients after short and long periods of treatment
María Gabriela Flores-Bracho 1 & Catarina Satie Takahashi 2 & Willian Orlando Castillo 2 &
Maria Conceição Pereira Saraiva 1 & Erika Calvano Küchler 1 & Mírian Aiko Nakane Matsumoto 1 &
José Tarcísio Lima Ferreira 1 & Paulo Nelson-Filho 1 & Fabio Lourenço Romano 1

Received: 5 May 2017 / Accepted: 20 December 2018

# Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract
Objective This study aimed to evaluate the genotoxic effects in the oral epithelial cells of patients undergoing fixed orthodontic
treatment and to compare these to a control group without treatment. The null hypothesis to be tested is that corrective orthodontic
treatment at different periods does not cause genotoxic effects in patients.
Material and methods An observational cross-sectional study including 74 patients enrolled in corrective orthodontic treatment
and 21 control patients, between 11 and 35 years of age, of both genders, participated in the research. Patients undergoing
treatment were divided into four treatment groups differentiated by treatment periods: G1, n = 21 (1 month to 12 months); G2,
n = 21 (13 to 24 months); G3, n = 23 (25 to 48 months); and G4, n = 9 (over 48 months). Cells were collected by scraping the
internal side of the cheek and subsequently placed in tubes containing 0.9% sodium chloride solution. The sample underwent
evaluation for genotoxic effects by means of the micronucleus test (MNT). Bivariate analyses were performed using parametric
tests (t test or ANOVA) and nonparametric tests (Chi-square test, Kruskal-Wallis test, Dunn post-test). The adopted level of
significance was 5%.
Results Statistically significant differences for any of the genotoxic abnormalities (binucleated, trinucleated, karyolysis, piknosis,
nuclear buds) were not found except for karyolysis, which was higher in the control group than in G4 (p < 0.05).
Conclusions This study did not demonstrate evidence of genotoxic effects even after long periods of corrective orthodontic
treatment.
Clinical relevance This study explores genotoxic effects in fixed orthodontic patients.

Keywords Orthodontic appliances . Genetic . Cytogenetic . Test of micronucleus

Introduction inflammatory response and cause alterations in buccal tissues

Orthodontic appliances are made of different metal alloys,
with nickel and chromium as their main components. The
corrosion of orthodontic appliances can induce an
* Fabio Lourenço Romano
fabioromano@forp.usp.br
1
Department of Pediatric Clinic, School of Dentistry of Ribeirão
Preto, University of São Paulo, Av. do Café, s/n Monte Alegre,
Ribeirão Preto, SP 14040-904, Brazil
2
Mutagenesis and Cytogenetics Laboratory, Department of Biology,
Faculty of Philosophy, Science and Letters of Ribeirão Preto,
University of São Paulo, Ribeirão Preto, SP, Brazil
[1–4]. Scientific evidence has demonstrated that orthodontic
treatments are not associated with hypersensitivity to nickel,
even in sensitive individuals, because the amount of nickel
released by orthodontic appliances is small compared to the
daily amounts released by food [1–4]. However, the degree of
hypersensitivity in oral tissues caused by these metals when
they are in contact with the mucosa for a long time remains
controversial.
The effects of orthodontic appliances can be evaluated by
well-established biomarkers, such as the micronuclei test
(MNT). MNT is an effective marker of DNA damage in human
buccal epithelial cells and can be applied to both in vivo and
in vitro studies [5, 6]. The micronuclei (MN) are cytoplasmic
bodies of nuclear-genetic material improperly incorporated
during cell division, showing errors during chromosomal

DNA replication and subsequent division [7–9]. The MN in
exfoliated mitotic cells are formed in the basal layer of the
epithelium. During the processes of proliferation and cell divi-
sion of the daughter cell, which may or may not contain MN,
differentiate and cross the epithelial layers to reach the surface
layer to then be exfoliated within the oral cavity [7, 10–13].
The frequency of MN in buccal cells may be influenced by
age, gender, lifestyle, systemic diseases, and diet [10, 14–21].
Studies report that the frequency of MN in healthy subjects
varies from 1.9 to 4.0 per 1000 cells with a standard deviation
of 0.99. These values increase with age in both sexes but are
higher for females. Regardless of lifestyle, MN frequency is
higher in smokers and individuals with systemic diseases such
as diabetes and cancer [13, 22–26].
Recent studies did not find significant levels of DNA dam-
age caused by orthodontic appliances [27–31]. This may be
due to the individuals’ ability to repair, as well as the adapt-
ability during orthodontic treatment and reversal of deleteri-
ous effects after device removal [6, 27–31]. However, all pre-
vious studies were performed during short time periods.
Therefore, it is necessary to conduct studies in patients with
prolonged periods of orthodontic treatment. Thus, the aim of
our study was to analyze, the MNT, the genotoxic effects on
exfoliated cells from buccal epithelium in patients undergoing
corrective orthodontic treatment at different periods. The null
hypothesis to be tested is that corrective orthodontic treatment
at different periods, primarily long-term treatment, does not
cause genotoxic effects in patients.
Materials and methods
The Research Ethics Committee (CAAE 35905914.1.0000.5419)
approved this project. The patients agreed to participate in the
study and they, or their guardians, signed an informed consent.
Selection of patients
The study was performed according to the STROBE report
[32]. Sample size was estimated based on linear trend that was
expected to be observed among five groups, including control
group with analyses of variance. For MN cells, estimation
from previous study showed 1.06 with standard deviation of
0.6 (Heravi et al., 2013). We hypothesized a linear increase
from 1.0 to 1.75 for the group over 48 months, with standard
deviations from 0.5 to 1.0. Then, the sample size varied from 5
to a maximum of 21 depending on the variance with the actual
power of 0.819 for 21 individuals.
This is an observational, cross-sectional study in which
patients were recruited from the Postgraduate Orthodontic
Clinic at the School of Dentistry of Ribeirão Preto from
February to June of 2014. The inclusion criteria were patients
with permanent dentition without amalgam restorations,
Clin Oral Invest
without previous orthodontic treatment, and with absence of
reported hypersensitivity to metals such as nickel and chrome.
Not included in the study were patients with syndromes or
systemic diseases such as diabetes, anemia, or debilitating
diseases such as cancer or diseases related with genomic in-
stability. Also not included were smokers, alcoholics, drug
addicts, those exposed to radiation or chemicals, those cur-
rently being treated with antibiotics or some type of steroids,
patients using mouthwashes with alcohol, and patients with
oral diseases such as caries and periodontitis. This information
was collected through anamnesis with the patients and their
caregivers.
A total of 95 patients from both genders, between the ages
of 11 and 35 years, were selected. Seventy-four patients were
undergoing corrective orthodontic treatment in the
Postgraduate Orthodontic Clinic (experimental group), while
21 patients were not receiving any orthodontic treatment (con-
trol group). Patients in the experimental group were
subdivided into three groups which differed by treatment time:
G1 with 21 patients (1 month to 12 months); G2 with 21
patients (13 to 24 months); G3 with 23 patients (25 to
48 months); and G4 with 9 patients (more than 48 months of
treatment). All patients had 20 bonded brackets (Morelli,
Sorocaba, SP, Brazil) from second premolar to second premo-
lar in the maxillary and mandibular arches, and four fixed
tubes in the upper and lower second molars. All brackets
and tubes, and four bands in the first molars of both arches,
were bonded with composite Transbond XT (3M Unitek,
Monrovia, CA, USA). All brackets followed the Edgewise
standard system: 0.022″ × 0.028″ slot and are composed of
stainless steel (17.0 to 20.0% chromium, 8.0 to 10.5% nickel,
molybdenum 0.60% max) with stainless steel wires (0.016″,
0.018″, 0.020″,or 0.019″ × 0.025″). The wire thickness, tips,
and holds were used in accordance with the treatment need.
Sample collection
The patients were asked to rinse their mouth with saline solu-
tion (sodium chloride solution 0.9%—Arboretum, Juiz de
Fora, MG, Brazil) for 2 min before the collection of the cells.
This procedure was performed twice to ensure the elimination
of the exfoliated dead cells. Then, the epithelial cells from oral
mucosa were collected by gentle scraping of the internal side
of the right and left cheeks with a toothbrush for 30 s. The cells
were transferred to 15-mL plastic tubes (Falcon, LMP, Kasui,
China) containing 5 mL of 0.9% sodium chloride solution and
then properly identified. The tubes were kept refrigerated in
ice coolers (approximately 4 °C) and then immediately
transported to the laboratory for analysis.
The MNT followed the protocols described by Tolbert et al.
(1992) [7] and Thomas et al. (2009) [10]. First, the tubes con-
taining the cells in 0.9% sodium chloride solution were centri-
fuged (Fanem, São Paulo, SP, Brazil) at 1000 rpm for 5 min.
Clin Oral Invest
Then, part of the supernatant was removed and discarded using a
pipette, leaving approximately twice the volume of the cells.
Subsequently, methanol/acetic acid (3:1) was added to
complete 5 mL with an additional of 5 drops of dimethyl
sulfoxide (DMSO). The suspension was stirred and centri-
fuged again as previously described. The supernatant was
discarded. This step was repeated twice. The suspension was
dropped (2 to 3 drops) onto pre-cleaned slides (Knittel Glass,
Starfrost, Germany) at a height of 3–4 cm and left to dry at
room temperature for 24 h.
The Feulgen/Fast Green method was used for the staining
of slides. Slides were placed in a vessel containing hydrochlo-
ric acid (HCl 5 N, Labsynth-Diadema, SP, Brazil) for 20 min
and subsequently washed three times with distilled water. The
slides were placed in another vessel, wrapped in aluminum
foil, and then the Schiff reagent (1 g Basic Fuchsin, 200 mL
distilled H2O, 300 mL 1 N HCl, 3 g potassium metabisulfite
(K2S2O5), and 0.5 g of decolorizing carbon) was added for
90 min. After this, the Schiff reagent was removed, and dis-
tilled water was added for 10 min. The slides were dried at
room temperature. Finally, the slides were counterstained with
3% Fast Green (95% ethanol and 0.25 g Fast Green FCF,
Merck Darmstadt, Germany), diluted in 100% methanol, and
dried again at room temperature.
The slides containing the cells were analyzed in a light
microscope (binocular optical microscope, Carl Zeiss,
Göttingen, Germany) at 400× magnification. For each patient,
two coded slides were analyzed. A single trained and calibrat-
ed investigator (MGFB) performed blinded analysis and
scored a total of 2000 cells. All evaluations were independent-
ly confirmed by another experiment (MCT) with high inter-
examiner agreement. The first 1000 cells were evaluated for
the characterization of cell types (differentiated or basal cells)
and assessment of nuclear abnormalities (mononucleated, bi-
nucleated cells, karyolysis, piknosis, nuclear buds, and broken
eggs). In this first analysis, MNs and nuclear buds were count-
ed in differentiated cells. In the second stage, the remaining
differentiated cells were analyzed to verify the presence of
micronuclei and nuclear buds.
The inclusion criteria for the counting of differentiated cells
and abnormal nuclear buds followed the recommendations
described by Tolbert et al. (1992) [7] and Katarkar et al.
(2014) [26]. Criteria for inclusion in the total cell count were
the following: intact cytoplasm lying relatively flat; little or no
overlap with adjacent cells; little or no debris; normal and
intact nucleus; and smooth and distinct nuclear perimeter.
Micronuclei identification and structures to consider were de-
fined as micronuclei that are 1/3–1/6 diameter of the main
nucleus; the same plane of focus as the main nucleus; the same
color; texture and refraction as the main nucleus; a smooth
oval or round shape; and the nuclear boundary of the micro-
nucleus should be clearly distinguishable from that of the
main nucleus (Fig. 1).
Statistical analysis
Descriptive analyses were performed for checking the distri-
bution of each variable. These distributions were tested using
Anderson-Darling statistics in Minitab. All of the other anal-
yses were performed using SAS/STAT software Version 9.3
considering an alpha of 0.05. Since the study is observational,
distribution of age and sex among each group was evaluated
using Chi-square and ANOVA, respectively. Also, explorato-
ry analysis of age for both males and females was tested using
t test, 95% confidence limits were calculated whenever nec-
essary. Genotoxic abnormalities followed an unknown distri-
bution and therefore descriptive analysis of these variables
included median and 95% confidence limits for free distribu-
tion. Comparisons among groups were performed using
Kruskal-Wallis test, followed by Dunn’s post-test. The pres-
ence of certain genotoxic abnormalities (binucleated and
trinucleated broken eggs) was analyzed with Fisher’s exact
test for contingency tables. Comparisons of genotoxic abnor-
malities between males and females were performed using the
Mann-Whitney test.
Results
Age and sex distributions for each group are presented in
Table 1. Thirty-four males and 61 females participated in the
study for a total of 95 individuals. Heterogeneity in the distri-
bution of age (p < 0.01) and sex (p = 0.0127) among the
groups was observed. On average, the time of treatment in
months for G1 to G4 was, respectively, 8.1 (SD, standard
deviation = 2.4), 20.3 (SD = 3.7), 38.3 (SD = 7.2), and 71.2
(SD = 13.7).
Table 2 demonstrates that there was no significant statisti-
cal difference for any genotoxic abnormalities, except for
karyolysis (p = 0.0166), with increased levels in the control
group in comparison to G4 (p < 0.05), which underwent the
longest fixed treatment time. Binucleated cells were present in
greater quantities in all evaluated groups in comparison with
other cellular abnormalities. However, statistically significant
differences were not observed (p > 0.05).
In the comparison of trinucleated cells with the other ab-
normal cells, the Fisher’s exact test for contingency tables was
used. There were no statistically significant differences be-
tween groups, although in G3, there was an apparent increase
in these anomalies. Broken eggs only showed values of 0.5
and 1, with eight individuals presenting values of 0.5 and two
individuals with the value of 1. Therefore, the values were
classified as present or absent, so the Fisher’s exact test for
contingency tables resulted in a p value of 0.6526. This
showed that there were no statistically significant differences
between groups. The binucleated cell count was also consid-
ered to be present or absent (Table 3). Note that in Table 2 the
 Clin Oral Invest

Fig. 1 Images showing differentiated cells with different types of one micronuclei (arrow); d cell with two micronucleus (circle); e nuclear
abnormalities found in MNT. The alterations are identified by circles buds (circle); f pyknosis; g broken eggs (arrow); h karyolitic
and arrows: a differentiated cell; b binucleated cell (circle); c cell with

analysis was performed considering the data as continuous,
and the p value is similar. Since there was an imbalance of
males and females among the groups, further stratified explor-
atory analysis was required to elucidate possible effect of this
potential confounder in the study. Tables 3, 4, and 5 reveal that
no statistical association could be observed between males
and females.
Discussion
Different steps in DNA repair are affected by the diverse
metals; however, in our study, we were not able to observe
genotoxic effect differences between the four evaluated exper-
imental groups in patients submitted to corrective orthodontic
treatment. It is possible that the release and exposure of the
metal ions from the orthodontic fixed appliance are not clini-
cally significant enough to cause a damage to the patient. In
fact, our results are in agreement with Westephalen et al.
(2008) [27] which found no significant alterations in cytoge-
netic damage. On the other hand, Natarajan et al. (2011) [29]
observed higher cytogenetic damage during installation of or-
thodontic appliances when compared to the control group, but
these effects decreased after 30 days of treatment. It is worth
mentioning that unlike other studies, this work evaluated pa-
tients in long-term treatments. However, in our results, the
orthodontic appliances were not associated with genotoxic
effects in patients regardless of the treatment duration, either
due to DNA repair capacity or adaptability of the patient.
Tintenko-Holland et al. (1994) [33] found an MN frequen-
cy of 0.14% in healthy patients. Hevari et al. (2013) [30]
found frequency of micronuclei before the device installation

Table 1 Age and sex distribution among individuals with and without orthodontic appliances (n = 95)

Time of orthodontic appliance use in months

Control n = 21 G1 (1–12) n = 21 G2 (13–24) n = 21 G3 (25–48) n = 23 G4 (> 48) n = 9

Age (years)
(CL 95%) 24.5 (22.3–26.7) 16.4 (14.2–18.6) 16.7 (14.5–18.8) 16.9 (14.9–19.0) 18.2 (14.9–21.5)
Sex (%)
Male (n = 34) 2 (9.5) 8 (38.1) 7 (33.3) 14 (60.9) 3 (33.3)
Female (n = 61) 19 (90.5) 13 (61.9) 14 (66.7) 9 (39.1) 6 (66.7)

95% IC 95% confidence limits

Groups: different periods of treatment (months): G1–1 and 12 months; G2–13 and 24; G3–25 and 48; G4-over 48
Clin Oral Invest

Table 2 Genotoxic abnormality distribution among patients without (control) and with and orthodontic appliance

Control (n = 21) G1 (n = 21) G2 (n = 21) G3 (n = 23) G4 (n = 9) p*

Md (95% CLfd*) Md (95% CLfd*) Md (95% CLfd*) Md (95% CLfd*) Md (95% CLfd*)

1 MN 2 (0–2.5) 1.5 (0.5–2.0) 1.5 (1.0–2.0) 0.5 (0.5–2.5) 1.0 (0–2.0) 0.9016
2 MN 0 (0–0.5) 0 (0–0) 0 (0–0) 0 (0–0) 0 (0–0) 0.0723
Binucleated 15 (12–21) 9 (8–16) 14 (9–21) 11 (9–16) 9 (7–15) 0.4257
Pyknosis 1 (0–7) 1 (0–4) 0 (0–1) 2 (0–3) 3 (0–4) 0.3467
Karyolysis 5 (2–10) 4 (0–9) 2 (0–4) 2 (0–5) 0 (0–4) 0.0166**
Nuclear Buds 1 (0–1) 0 (0–1) 0 (0–1) 0 (0–0) 0 (0–3) 0.6480
Total 28 (18.5–36.5) 19 (11.5–36.5) 19.5 (14.0–30.0) 18 (15.5–24.0) 17.5 (7.0–21.0) 0.1470

*p = value Kruskal-Wallis test

Md median, 95% CLfd 95% confidence limits for free distribution
MN micronuclei
**Statistical significance difference
Groups: different periods of treatment (months): G1–1 and 12 months; G2–13 and 24; G3–25 and 48; G4-over 48

(1.06%) and after 9 months of treatment (0.92%) with no
statistically significant results. In our study, no statistically
significant differences between the groups were observed.
Toy et al. (2014) [6] found that the number of binucleated
cells significantly increased in all groups of patients undergo-
ing orthodontic treatment and cells with karyolysis increased
during the 6 months of treatment. However, genotoxic effects
were absent because of the ability of epithelial cells to renew
and regenerate in the short time span of 7–14 days. In our
study, the number of binucleated cells in the experimental
groups was lower than in the control group; however, the
difference was not statistically significant. Regarding the
karyolysis count, similar results were obtained in this study
with higher frequency of karyolysis cells in the control group
and the reduction of these cells in the group with long-term
orthodontic treatment. According to Ramirez and Saldanha
(2002) [34], increments of karyolysis cells occur in the kera-
tinization process, representing the adaptive response of inju-
ries that occurred in the oral epithelium, and also related to
necrotic cells and cytotoxic effects. This anomaly is normal in
squamous epithelia, especially for the effects caused by
chewing. However, the constant action of mutagenic agents
such as alcohol, tobacco, radiotherapy in cancer patients, and
orthodontic appliances could increase the rate of cell death as
indicated by a significant increase of this anomaly. This adap-
tive response was not observed in our study.
Binucleated cells are associated with neurodegenerative
diseases and cancer cells, suggesting that high proportions of
binucleated cells may be associated with these diseases [33,
35]. This study showed high values of binucleated cells when
compared to other evaluated nuclear alterations, but with nor-
mal distribution between the groups and no statistically sig-
nificant values, demonstrating that corrective orthodontic
treatment and the time of the treatment did not increase the
number of these cells.
The literature reports that there is a tendency for females to
have higher frequencies of MN and other cell abnormalities
[13, 22–25]. However, our results did not show this tendency.
Bloching et al. (2008) [22] found a higher number of MN in
older patients presenting oral problems, such as missing teeth
and periodontal disease. Hevari et al. (2013) [30] concluded
that advanced age, systemic diseases, and risk factors such as
tobacco and alcohol consumption may aggravate the harmful
effects of corrective orthodontic appliances. In our study,
smokers, alcoholics, and individuals with systemic or oral
diseases were not included. There were no statistically signif-
icant differences in any of the genotoxic abnormalities when
comparing the nuclear alterations with the age of the patients
in all groups.
Our results should be interpreted with caution because
DNA damage caused by the use of orthodontic appliances
can be repaired, but any decrease in repair capacity or changes

Table 3 Percentage of
individuals with genotoxic Control (n = 21) G1 (n = 21) G2 (n = 21) G3 (n = 23) G4 (n = 9) p*
abnormal cells among those with n (%) n (%) n (%) n (%) n (%)
and without orthodontic
appliance Tetra nucleated 2 (9.5) 2 (9.5) 1 (4.8) 4 (17.3) 1 (11.1) 0.7671
Broken eggs 2 (9.5) 4 (19.1) 1 (4.4) 1 (4.4) 1 (11.1) 0.6526
Binucleated 7 (33.3) 2 (9.5) 1(4.8) 1 (8.7) 1 (11.1) 0.0957

*p value for Fisher’s exact test. Groups: different periods of treatment (months): G1–1 and 12 months; G2–13 and
24; G3–25 and 48; G4-over 48
 Clin Oral Invest

Table 4 Distribution of cells with genotoxic abnormalities according to Conclusion

the sex

Male (n = 34) Female (n = 61) The null hypothesis was accepted. Corrective orthodontic
Md (95% CLfd*) Md (95% CLfd*) p* treatment did not cause genotoxic effects in patients, regard-
less of the time of treatment.
1 MN 1 (0.5–2) 1.5 (1–2) 0.2399
2 MN 0 (0–0) 0 (0–0) 0.3199 Authors’ contributions María Gabriela Flores Bracho conducted a review
Binucleated 13.5 (10–15) 12 (10–16) 0.8800 of literature, wrote the manuscript, collected the data, and did the exper-
Pyknosis 2 (0–4) 1 (0–4) 0.4328 imental procedures of the project.
Catarina Satie Takahashi supervised all the experimental procedures of
Karyolysis 4 (0–6) 2 (0–5) 0.4564
the project.
N. Buds 0 (0–1) 0 (0–1) 0.5660 Willian Orlando Castillo supervised all the experimental procedures of
Total 19.7 (18 - 25.5) 19.5 (16–26) 0.9690 the project.
Maria Conceição Pereira Saraiva designed the study and performed
*p value for Wilcoxon rank test the statistical analyses.
Md median, 95% CLfd 95% confidence limits for free distribution Erika Calvano Küchler organized the data and wrote the manuscript.
Mírian Aiko Nakane Matsumoto collaborated in the selection of ex-
MN micronuclei perimental subjects.
Groups: different periods of treatment (months): G1–1 and 12 months; José Tarcísio Lima Ferreira collaborated in the selection of experimen-
G2–13 and 24; G3–25 and 48; G4-over 48 tal subjects. Fabio Lourenço Romano and Paulo Nelson-Filho conceived
the study and supervised all aspects of its implementation.
All authors contributed to the interpretation of results and manuscript
in the immune system may allow the DNA damage to cause review.
future mutations in the genome [30].
Our study had some limitations. The power of the study can Funding The work was supported by CAPES-Coordination for the
Improvement of Higher Education Personnel-Brazilian federal
be seen as a limitation, in spite of estimation that 21 individ- government.
uals per group would be enough to reach a power greater than
0.80. However, in spite of having only nine individuals in G4, Compliance with ethical standards
the actual power of the study, assuming a standard deviation of
about 1.1 and only nine individuals in each group resulted in a The Research Ethics Committee (CAAE 35905914.1.0000.5419) ap-
relatively reasonable power of 0.714. When we considered the proved this project. The patients agreed to participate in the study and
actual number for the other groups (n = 21) with the exception they, or their guardians, signed an informed consent.
of the last one, the power was greater than 0.80. However, we
Conflict of interest The authors declare that they have no conflict of
cannot rule out the possibility of false negative results by interest.
chance. The evaluation mechanism for the slides that was
conducted through optical light microscope is also a concern. Ethical approval The Research Ethics Committee (CAAE
This limited the ability to capture the complete image gallery 35905914.1.0000.5419) approved this project. All procedures performed
for future reanalysis and quality control. In addition, it in- in studies involving human participants were in accordance with the
ethical standards of the institutional and/or national research committee
creases the risk of obtaining heterogeneous values, regardless and with the 1964 Helsinki declaration and its later amendments or com-
of the evaluator’s level of training [36]. Orthodontic treat- parable ethical standards.
ments did not cause significant cytogenetic alterations, even
in patients undergoing long-term treatments. Further studies Informed consent Informed consent was obtained from all individual
should be performed to evaluate the genotoxic effects of or- participants that were included in the study.
thodontic materials as self-ligating brackets, mainly aligners,
Publisher’s Note Springer Nature remains neutral with regard to jurisdic-
that are made of composite or plastics. tional claims in published maps and institutional affiliations.

Table 5 Occurrence of genotoxic abnormal cells among males (n = 34)

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