Clinical Oral Investigations (2018) 22:2553–2558

https://doi.org/10.1007/s00784-018-2352-y

ORIGINAL ARTICLE

Evaluation of the genotoxic effects of formocresol application in vital

pulp therapy of primary teeth: a clinical study and meta-analysis
Arnoldo Vasconcelos de Alencar Filho 1 & Valdeci Elias dos Santos Junior 1 & Merilane da Silva Calixto 2 & Neide Santos 3 &
Monica Vilela Heimer 1 & Aronita Rosenblatt 1

Received: 25 July 2017 / Accepted: 22 January 2018 / Published online: 6 February 2018
# Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract
Objectives This in vivo research investigated whether pulp treatments using formocresol for 7 days would cause mutagenic
changes in children’s lymphocytes.
Materials and methods The mutagenicity was tested in lymphocyte cultures established from the peripheral blood of children
living in Brazil. The samples consisted of 2000 cells from teeth undergoing formocresol pulpotomies in which the formocresol
pellet was sealed in the primary tooth for 7 days. It was removed on the seventh day, the base was placed, and the tooth was
restored. Two venous blood samples (6–8 ml) were collected from each child; the first was prior to pulp therapy, and the second
was 7 days later. Two thousand metaphases were analyzed. The level of significance adopted for the statistics was P < 0.05, and a
random effects meta-analysis was performed combining this and two previous studies.
Results There was no significant difference found in the metaphase analysis between the blood samples taken before and after the
pulpotomy treatment (Wilcoxon signed rank test); however, the meta-analysis showed a significant difference between the
combined studies.
Conclusions This study did not reveal any mutagenic effects, but based on the combined meta-analysis, we recommend the
careful use of formocresol.
Clinical relevance This research helps to bring scientific evidence of the safe use of formocresol in deciduous pulpotomy
treatments.

Keywords Pulpotomy . Formaldehyde . Mutagenesis . Formocresol . Genotoxicity

Introduction effects on cellular fixation and protein binding. In addition,

Formocresol was developed by Buckley in 1904 [1, 2]. In its
original formula, it contained 19% formaldehyde, 35% cresol,
15% glycerin (as a vehicle), and 100 cm3 of distilled water
sufficient for the preparation. Buckley introduced formocresol
to pulp therapy to control pulp degeneration because of its
* Arnoldo Vasconcelos de Alencar Filho
arnoldofilho@gmail.com
1
Department of Preventive and Social Dentistry, Faculty of Dentistry,
University of Pernambuco, Av. Gal. Newton Cavalcanti, 1650,
Tabatinga, Camaragibe, PE 54756-220, Brazil
2 Although the current literature reports several studies indi-
Department of Genetics, Federal University of Campina Grande,
Campina Grande, Brazil
3 concerns about the continued use of formocresol during pri-
Department of Genetics, Federal University of Pernambuco,
Recife, Brazil
formocresol has good bactericidal properties due to its high
alkalinity [3]. It is the most widely used medication for primary
tooth pulpotomies in public health care in many countries [4].
A pulpotomy procedure is indicated if removing caries
causes pulp exposure in a primary tooth diagnosed with vital
reversibly inflamed pulp. The coronal pulp is removed, and
medication, such as formocresol, is placed on the radicular
pulp stump to maintain vitality [5]. Although this procedure
has been used for many years, the use of formocresol in pri-
mary tooth pulpotomies remains controversial due to its bio-
compatibility. The International Agency for Research on
Cancer (IARC) has classified formaldehyde as a human car-
cinogen [6].
cating the clinical efficacy of formocresol [7, 8], there are still
mary tooth pulpotomy [9, 10]. In addition, formaldehyde
2554
exposure has been proven to be potentially carcinogenic [11].
Due to its low cost and ease of use, the formocresol-based
pulpotomy procedure has been performed in various ways.
The typical formocresol-based pulpotomy technique employs
a formocresol cotton pellet applied for 5 min prior to the
placement of a reinforced zinc-oxide eugenol base and the
final restoration [12]. An alternate method for hyperemic pulp
is to leave the formocresol pellet in the pulp chamber for
7 days covered by a temporary filling. The pulpotomy is com-
pleted after this time period [13].
Zarzar et al. [14] tested the following mutagenic changes:
chromatid gap, chromatid break, chromosome gap, chromo-
some break. Those changes were analyzed after 5 min of
formocresol application, in which they used lymphocyte cul-
tures before and 24 h after the formocresol-based pulpotomy.
Their results reported that one patient exhibited mutagenic
changes.
Understanding the relationship between exposing and in-
ducing chromosomal abnormalities is a highly important fac-
tor when considering the risk of adverse effects on human
health. However, it should be noted that mutagenic tests do
not necessarily provide definitive answers, but may serve as a
contemporary supplement to the rationale for the use of certain
drugs because of their association with cancer [17, 21, 28].
The aim of the present study was to investigate in vivo
whether a pulp treatment with formocresol for 7 days may
cause chromosomal changes in a children’s lymphocytes as
an indicative biomarker of systemic genotoxicity.
Materials and methods
This study was approved by the National Research Ethics
Committee (CONEP) and the Fernando Figueira Integral
Medicine Institute (IMIP) Ethics Committee in Brazil (proto-
col: 748/2006). Written informed consent was obtained from
the parents of the children, who were advised about the risks
and benefits according to the Declaration of Helsinki. Blood
samples were collected at the IMIP (Recife, Pernambuco,
Brazil) from a group of ten children of both genders ranging
from 5 to 7 years of age.
The patient criteria for inclusion in this study were the
following: not exposed to previous endodontic treatment,
not recently exposed to X-rays, not taking any medication,
and no periodontal disease.
The genotoxic effects of the formocresol were evaluated in
children treated with a formocresol pellet (Buckley’s full
strength). In this study, approximately 0.025 ml of the drug
was applied using a microliter pipette, as recommended by
Wesley, Marchal, and Rosen [15]. The first blood sample
was taken from the patient prior to treatment, which was then
compared to the sample collected 1 week after treatment. The
same researcher (AF) performed all of the pulpotomies. This
Clin Oral Invest (2018) 22:2553–2558
procedure included evaluating the medical history of each
patient. Local anesthesia was applied to the affected area,
which was isolated with a rubber dam, and the carious tissue
was removed with a high-speed handpiece to allow access to
the pulp chamber. The coronal pulp was removed using an
excavator until the root canal was accessible. This was follow-
ed by the application of a sterile cotton pellet moistened with
formocresol (Maquira Industry Dental Products S.A.,
Maringá, Brazil). The tooth was filled using a layer of zinc
oxide (Biodinâmica Química e Farmacêutica LTDA, Ibiporã,
Brazil) and Vitrebond glass ionomer (3M ESPE, Sumaré,
Brazil), followed by a last layer of Filtek Z250 resin (3M
ESPE, Sumaré, Brazil).
Bloodletting samples were taken before and after the
formocresol treatment, with 6 to 8 ml of peripheral venous
blood being collected and stored in a heparin Vacutainer blood
tube (Becton Dickinson S.A., Juiz de Fora, Brazil). The blood
samples were used to prepare the lymphocyte cultures and
slides for analysis. Each blood smear slide was evaluated by
two previously calibrated examiners (AF, MC). A kappa of
0.89 was obtained.
The cytogenetic method for evaluating cellular metaphases
was developed by Moorhead et al. [16] and modified by the
Laboratory of Genetics and Animal Cytogenetics at the
Federal University of Pernambuco in Brazil. This was the
same methodology used in the Zarzar et al. [14] and Leite
et al. [24] studies. It is important to emphasize a widespread
and consolidate use of this methodology to evaluate chromo-
some aberrations in cell culture [17].
In this study, 200 metaphases per culture per patient (100
metaphases before treatment and 100 metaphases after treat-
ment, for a total of 2000 metaphases in ten patients) were
analyzed from the blood samples.
The culture medium consisted of 78% Roswell Park
Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich
Brazil LTDA, São Paulo, Brazil) supplemented with 20%
fetal bovine serum (FBS) (Sigma-Aldrich Brazil LTDA,
São Paulo, Brazil) and 2% phytohemagglutinin (PHA)
(Sigma-Aldrich Brazil LTDA, São Paulo, Brazil). The cul-
tures were individually incubated at 37 °C and harvested
at 48 h. One and one-half hours before the end of the total
culture time, 0.1 ml of 0.016% colchicine (Sigma-Aldrich
Brazil LTDA, São Paulo, Brazil) was added to each cul-
ture. Following a hypotonic KCl treatment (5 ml of
0.075 M KCl for 5 min), the cells were fixed in a 3:1
mixture of methanol and acetic acid. The slides were
stained for 5 min with a Giemsa solution of 1.5 ml dye
and 30 ml buffer (0.06 M Na2HPO4 and 0.06 M KH2PO4,
pH 6.8).
The cytogenetic analysis was performed according to the
method of Albertini et al. [18] using a light microscope with
an immersion lens (100×). The position of the centromere was
noted, along with any chromosomal structural aberrations,
Clin Oral Invest (2018) 22:2553–2558 2555

Table 1 Differences in the

chromosome alterations before Variable Evaluation P value
and after the 7-day formocresol
treatment Before formocresol treatment After formocresol treatment
Mean ± SD (median) Mean ± SD (median)

Chromatid gap 1.00 ± 1.41 (0.50) 0.50 ± 0.97 (0.00) P1 = 0.102

Isochromatid gap 0.00 ± 0.00 (0.00) 0.10 ± 0.32 (0.00) P1 = 0.317
Chromatid break 1.00 ± 1.05 (1.00) 0.90 ± 0.99 (1.00) P1 = 0.773
Isochromatid break 0.10 ± 0.32 (0.00) 0.10 ± 0.32 (0.00) P1 = 1.000
Simple acentric fragment 0.10 ± 0.32 (0.00) 0.00 ± 0.00 (0.00) P1 = 0.317
Double acentric fragment 0.20 ± 0.42 (0.00) 0.40 ± 0.84 (0.00) P1 = 0.414
Ring chromosome 0.00 ± 0.00 (0.00) 0.00 ± 0.00 (0.00) P1 = 1.000
Other chromosome alterations 0.10 ± 0.32 (0.00) 0.30 ± 0.67 (1.00) P1 = 0.414
Total 2.50 ± 2.17 (2.00) 2.30 ± 1.89 (2.00) P1 = 0.609
1
Wilcoxon signed rank test. Significant difference at 5%

such as achromatic lesions, gaps, chromatid and chromosome
breaks, and dicentric chromosomes.
The International Atomic Energy Agency (IAEA) [19]
criteria were used for the chromosomal aberration analysis
and scores. The unstained or faintly stained regions found in
one or both chromatids in apparently identical loci with a
width lower than the thickness of the chromatid were consid-
ered to be gaps. The widths higher than the thickness of the
chromatid were considered to be breaks.
In order to reduce the variations between the scorers,
each one analyzed the same number of cells from the
slides of all of the subjects (the same cells were not
scored twice), rather than one scorer analyzing all of the
cells from some subjects and another scorer analyzing all
of the cells from the other subjects. An annotator codified
the slides, and the scorers did not know which group they
were analyzing (blind test). Only those metaphases con-
taining 45–47 chromosomes were analyzed, and the gaps
were recorded separately. This was in accordance with the
International Program on Chemical Safety (IPCS) guide-
lines for monitoring the genotoxic effects of carcinogens
in humans [19].
The Statistical Package for the Social Sciences (SPSS)
version 20 (IBM Corporation, Armonk, NY, USA) was
used for the statistical calculations. The descriptive and
inferential statistical analyses were performed using a
comparative test between the two groups. The Wilcoxon
signed rank test was used for the following variables:
number of chromatid gaps, isochromatid gaps, chromatid
breaks, isochromatid breaks, other chromosome alter-
ations, and total alterations (obtained from the sum of
all of the gaps, breaks, and others alterations).
A random effects meta-analysis model was used to estimate
the risk ratio of the chromosome alterations for this study and
the other two previously published studies in Brazilian
populations (Zarzar et al. [14] and Leite et al. [24]). A hetero-
geneity test was included in the meta-analysis using the
Review Manager (RevMan) 5.3 software (The Nordic
Cochrane Centre, Copenhagen, Denmark). We measured the
heterogeneity for all three studies combined [I2 = 71% (P =
0.03), P = 0.01] (Fig. 4).
Results
Table 1 shows the statistical analyses of the chromatid gaps,
isochromatid gaps, chromatid breaks, isochromatid breaks,
simple acentric fragments, double acentric fragments, ring
chromosomes, and other chromosome alterations. A total of
200 metaphases were analyzed before and after the pulpotomy
treatments for each patient, for a total of 2000 examined cells.
Even so, at a 5% significance level, no statistical significance
was observed between those intervals.
The cytogenetic analyses performed are represented in
Figs. 1, 2, and 3. As part of the methodology, those pictures
summarize part of the chromosome alterations found in this
study. In Fig. 1, it is possible to observe a chromatid gap in one
chromosome after formocresol treatment in 7 days. Also,
Fig. 2 represents a normal metaphase before formocresol treat-
ment. A chromatid break is represented in Fig. 3; this chro-
mosome alteration is highlighted on B.
Figure 4 represents a meta-analysis of the selected studies.
A number of cells evaluated were 18,000. Next, two groups
were created: 9000 before and 9000 post intervention illustrat-
ing the main outcomes of each group. The heterogeneity test
[I2 = 71% (P = 0.03), P = 0.01] confirms the random effects
meta-analysis used to estimate the risk ratio. The varying sizes
of the filled squares and the filled diamond represent the
weight for the random effects model in the meta-analysis.
2556 Clin Oral Invest (2018) 22:2553–2558

Discussion

In this study, a total of 2000 cells from blood samples of ten

systematically health children before and after formocresol-
based pulpotomy treatment were analyzed regarding
genotoxic effects in pediatric deciduous teeth. This technique
was an alternative to the one-visit pulpectomy technique used
for the evaluation of the effects of formocresol and for the
second collection of blood samples [20].
In this study, approximately 0.025 ml of the drug was ap-
plied to all patients as recommended by Wesley, Marchal, and
Rosen [15] and McClellan [21], emphasizing the need to de-
velop a methodology for evaluating the mutagenic effects of
formocresol. However, this study did not focus on measuring
the amount of formaldehyde in the circulating blood. That
topic has already been the subject of previous studies that
showed that the amount of formaldehyde converted into car-
bon dioxide in the liver is 22 mg/min in the blood stream
during the first 24 h [22]. Moreover, formaldehyde has been
blamed for both exogenous mutagenicity in pulp therapy and
Fig. 2 This figure represents a normal metaphase before formocresol
endogenous damage resulting from its metabolism. treatment
Recent investigations have reported the possible genotoxic
and mutagenic effects of formocresol and have led to a remark-
able reduction in its clinical use in several countries [4, 23]. The statistical significance. These findings are not in agreement
current literature indicates that the peak of the chemical activity with Albertini et al. [18], which indicated that the clastogenic
of formocresol in the peripheral blood stream occurs 24 h after effects of formulations containing formaldehyde depend on
use [18], which explains why previous studies examined blood the level, duration, and frequency of exposure.
samples collected within this time interval [14, 24, 25]. The blood samples were analyzed, and the cytogenetic
The results of this study showed that the number of chro- method was chosen to evaluate the metaphases based on the
matid gaps and breaks decreased after treatment, compared to protocol developed by Moorhead et al. [16]. This method
the sample examined before treatment, however, with no accurately evaluates chromosome abnormalities and is useful
in determining whether a particular substance is clastogenic.
This method also identifies the types of chromosome damage,
unlike the comet and the micronucleus assays [26].

Fig. 3 This figure represents a metaphase with chromosome alteration

Fig. 1 This figure represents a metaphase with chromosome alteration (A) after formocresol exposure in 7 days. Highlighted (B) a chromatid
after formocresol exposure in 7 days. The arrow indicates a chromatid gap break in one chromosome
Clin Oral Invest (2018) 22:2553–2558
Fig. 4 Summary of meta-analysis results of all three available studies (Zarzar et al. [14]; Leite et al. [24]; Filho et al. present study). Total random effects
P = 0.01
To investigate whether the formocresol in Buckley’s
original formula produced mutagenic effects in vivo in
human lymphocytes, Zarzar et al. [14] conducted a study
in which the authors took two blood samples from each
child before and 24 h after the pulpotomy. The
formocresol embedded cotton pellet only remained in
contact with the pulp tissue for 5 min [5], and the second
blood sample was taken 24 h later. Their results showed
that the formocresol did not produce genotoxic effects on
the target cell samples. Leite et al. [24] also studied the
genotoxic effects of formocresol by testing blood samples
before and 24 h after the removal of the cotton pellet
embedded in formocresol. Their results showed that the
cell samples had significant chromosomal alterations after
the formocresol treatment. By comparing our results
concerning the type of chromosomal aberrations with
those found by Leite et al. [24], it is clear that they are
not in agreement. However, this study is in agreement
with that of Zarzar et al. [14].
The IARC has classified formaldehyde as a potential hu-
man carcinogen. The concerns for pediatric dentistry are
linked to the presence of formaldehyde in the formocresol
formula. Moreover, mutagenicity tests have shown the po-
tential of certain drugs to induce genotoxicity and confirm
the link between certain drugs and cancer [6]. Mutations can
contribute to the occurrence of cancer by activating the pro-
tein function of oncogenes or by inactivating the function of
tumor suppressors. Additionally, genes, such as p53, are
linked to carcinogenic effects. Conversely, the BRCA1
and BRCA2 genes are related to the prevention of the dele-
terious effects caused by formaldehyde [27, 28].
Human cells have a specialized DNA repair system that
can prevent DNA damage, and there are more than 10,000
attacks a day from either endogenous or exogenous agents.
These repair mechanisms include base excision repair (BER),
mismatch repair (MMR), nucleotide excision repair (NER),
homologous recombination (HR), and non-homologous end
joining (NHEJ) [29, 30].
The meta-analysis results suggest that the studies exhibited
a high degree of heterogeneity and that the chromosome alter-
ations may be relevant when all of the studies were combined,
but when considering the multifactorial aspect of exposure to
2557
the lymphocytes, it was possible to confirm that the
formocresol could be directly responsible for the alterations
seen in the combined studies. Thus, it is important to reinforce
the fact that mineral trioxide aggregate (MTA) has shown
better clinical results than formocresol, according to recent
systematic reviews. It has the advantage of being biocompat-
ible and could be a good substitute for formocresol in a routine
pulpotomy procedure. However, the cost of MTA remains a
prohibitive barrier in many countries, and there remains the
huge problem of widespread dissemination between pediatric
dentists in routine clinical practice [31–33].
The results of this research did not support the hypothesis
that formocresol has a high mutagenic effect, but the meta-
analysis did show the potential of formocresol to be a harmful
substance.
Conclusion
The mutagenic effects of formocresol were not proven in this
study. However, based on the results of the combined meta-
analysis, dentists should use formocresol carefully in routine
clinical practice.
Acknowledgments The authors would like to thank the Brazilian
Ministry of Education (Coordination for the Improvement of Higher
Education Personnel, CAPES) for their support during the development
of this study.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval All procedures performed in studies involving human
participants were in accordance with the ethical standards of the institu-
tional and/or national research committee and with the 1964 Helsinki
Declaration and its later amendments or comparable ethical standards.
The ethical committee of Medical Center Fernando Figueira Institute
(IMIP – Brazil – IRB 748/2006) approved the study.
Informed consent For all participating children, parents and/or guard-
ians provided written informed consent.
2558
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