doi: https://doi.org/10.52973/rcfcv-e34310
Received: 19/08/2023 Accepted: 26/09/2023 Published: 01/01/2024
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Revista Científica, FCV-LUZ / Vol. XXXIV, rcfcv-e34310
ABSTRACT
Metronidazole is an imidazole group bactericidal antibiotic used
against anaerobic bacteria and some protozoa. There is no detailed
information about the embryotoxic effect of Metronidazole. This study
aims to determine the embryotoxic activity of Metronidazole using
an in ovo method. A total of 210 fertile chicken eggs were placed in
an incubator, divided into seven equal groups of 30. The rst group
was considered as the control group. On the seventh day of the study,
Metronidazole was administered to the other six groups within 50 µL
saline solution at doses of 250, 125, 62.5, 31.25, and 15.62 µg·egg
-1
(5;
2.5; 1.25; 0.625; 0.312 mg·kg
-1
). At the end of the incubation period,
the eggs hatched, and the number of live and dead embryos was
determined. There were no signicant differences in deaths between
the groups (P>0.05 in all cases). No anomaly was detected in the
macroscopic morphology of the embryos. As a result, it can be stated
that Metronidazole may be safe for use during pregnancy. However,
it is necessary to conduct molecular and histopathological studies
to investigate the effects of this drug on organogenesis, especially
in mammalian embryos.
Key words: Metronidazole ; embryotoxicity; in ovo
RESUMEN
El metronidazol es un antibiótico bactericida del grupo de los
imidazoles que se utiliza contra las bacterias anaerobias y algunos
protozoos. No hay información detallada sobre el efecto embriotóxico
del metronidazol. Este estudio tiene como objetivo determinar la
actividad embriotóxica del metronidazol mediante un método in
ovo. Se colocaron en una incubadora un total de 210 huevos fértiles
de gallina, divididos en siete grupos iguales de 30. El primer grupo
se consideró como grupo control. En el séptimo día del estudio, se
administró metronidazol a los otros seis grupos dentro de 50 µL de
solución salina en dosis de 250; 125; 62,5; 31,25 y 15,62 µg·huevo
-1
(5;
2.5; 1.25; 0.625 y 0.312 mg·kg
-1
). Al nal del período de incubación, los
huevos eclosionaron y se determinó el número de embriones vivos y
muertos. No hubo diferencias signicativas en las muertes entre los
grupos (P>0.05 en todos los casos). No se detectó ninguna anomalía
en la morfología macroscópica de los embriones. Como resultado,
se puede armar que el metronidazol puede ser seguro durante el
embarazo. Sin embargo, es necesario realizar estudios moleculares
e histopatológicos para investigar los efectos de este fármaco sobre
la organogénesis, especialmente en embriones de mamíferos.
Palabras clave: Metronidazol, embriotoxicidad, in ovo
Determination of the embryotoxic effect of Metronidazole using an
in ovo model
Determinación del efecto embriotóxico del Metronidazol mediante un modelo in ovo
Muhittin Uslu
1
* , Rahmi Canbar
2
, Mustafa Sedat Arslan
3
, Harun Kızılay
4
1
Yozgat Bozok University, Department of Laboratory and Veterinary Health, Sefaatli Vocational College. Yozgat, Türkiye.
2
Necmettin Erbakan University, Faculty of Veterinary, Department of Pharmacology and Toxicology. Konya, Türkiye.
3
Selcuk University, Faculty of Veterinary Medicine, Department of Anatomy. Konya, Türkiye.
4
Selcuk University, Faculty of Pharmacy, Department of Pharmacology. Konya, Türkiye.
Corresponding Author: muhittin.uslu@bozok.edu.tr
Determination of the embryotoxic effect of Metronidazole / Uslu et al. _____________________________________________________________
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INTRODUCTION
Metronidazole is an imidazole group bactericidal antibiotic used
to treat anaerobic and some protozoan infections (e.g., giardiasis,
amoebiasis, histomonas, trichomoniasis). Although its use in animals
with nutritional value is prohibited in Veterinary Medicine [1, 2, 3] it is
frequently used in Human Medicine. Commercial preparation for pet
clinics is licensed in Veterinary Medicine, where it is used mainly in
pyometra, pyoderma, dental abscesses, and bite injuries [4].
Metronidazole causes mild to moderate gastrointestinal side effects
such as diarrhea, nausea, and abdominal pain, but is well tolerated
[2]. In the central nervous system, it is reported that it commonly
causes dysarthria, mental changes, and ataxia, and more rarely,
neurotoxicity, optic neuropathy, encephalopathy, and peripheral
neuropathy [5, 6, 7]. It has been reported that the drug, which is
in the IARC 2B class, has genotoxic and carcinogenic properties in
animals. However, more studies are needed to reveal its genotoxic
and carcinogenic properties in humans [2, 8].
Fertile chicken embryos are often preferred to mammals in
investigating the embryotoxic and teratogenic effects of chemicals
and drugs [9, 10, 11, 12, 13]. For this purpose, the Chick Embryotoxicity
Screening Test (CHEST) test was developed [14]. Reasons for using
chicken embryos in the CHEST method include the cheapness of
chicken embryos, ease of application, well–known developmental
stages, reproducible results, and statistical superiority over
mammalian species due to the availability of large numbers of fertile
chicken eggs in research. In addition, factors such as being able to
carry out studies with low–tech laboratory equipment, obtaining
results quickly, and similarity between the morphological development
of chicken embryos and that of mammals have also been expressed as
advantages [14, 15, 16]. The disadvantages are the lack of a maternal–
fetal relationship typical of mammals, the possibility of false positives,
and the pharmacokinetic differences caused by differences in the
nature of chicken eggs. In addition to its disadvantages, species
diversity is also encountered in other tests, and the closest data to
the target species should be sought [15, 16].
There is no detailed information about the use of Metronidazole
in either Veterinary or Human Medicine. It is dened as categoryB
in pregnancy by the Food and Drug Administration in Human
Medicine [17]. Contrarily, it is advised against using metronidazole
during pregnancy due to the risk of congenital abnormalities or
embryotoxicity [18]. As a result of these studies, it was hypothesized
that the embryotoxic and teratogenic effects of Metronidazole would
be dose dependent, using the in ovo model.
MATERIAL AND METHODS
It was obtained 210 fertile chicken eggs from a commercial
enterprise (Anadolu Damizlik, Konya, Türkiye). The Selçuk University
Faculty of Veterinary Experimental Animals Production And Research
Center Ethics Committee approved the research procedure (2022/93).
Fertile eggs were divided into seven equal groups of 30 and placed in an
incubator (Imza Teknik, Konya, Türkiye). During incubation, eggs were
housed under optimal maintenance conditions (37.8°C, 65% humidity,
and a rotation time of 2 hours). Fertility was checked under light on the
seventh day of incubation and non–fertile eggs were removed from
the groups. Fertile eggs were substituted for non–fertile eggs and
treatment groups consisted of 30 eggs each. On the seventh day of the
study, the blunt ends of the eggs (containing the air sacs) were cleaned
with an appropriate disinfectant, a hole was made with the help of an
egg pricker (Bai Shun, Zhejilang, China), and 50 µL of saline solution
and drug applications were performed. The rst group of the study
was evaluated as a negative control, and no application was made.
The second group of the study was administered physiological
saline in a volume of 50 µL; this was another control as the saline
solution was the vehicle used as the carrier for the drug. The other
ve groups were administered Metronidazole (Fladazole 0.5% Solution
for Injection, Istanbul, Türkiye) at a dose of 250, 125, 62.5, 31.2, and
15.6 µg·egg
-1
(5, 2.5, 1.25, 0.625, 0.312 mg·kg
-1
) within 50 µL saline.
After these applications, the holes in the eggs were closed with liquid
paran. Following treatment, no rotation was made for the rst
hour, but eggs were otherwise provided with optimum conditions
in the incubator to ensure drug absorption from the air sac. At the
end of the incubation period, the eggs hatched and embryotoxicity
and teratogenicity rates were evaluated.
The actual mortality rate was determined using the Abbott
formulation over the embryonic mortality rates [10, 11, 12]. Embryonic
mortality rates between groups were evaluated using the Chi–square
test (SPSS 22.2, IMD SPSS, Armonk, USA). A value of 0.001 was
accepted as the threshold for statistical signicance in the tests.
RESULTS AND DISCUSSION
The embryonic death rates following Metronidazole administration
to fertile eggs are presented in TABLE I. No anomalies were detected
in the macroscopic morphology of the embryos.
Metronidazole, a drug defined as categoryB by the Food and
Drug Administration (FDA) in pregnancy, is an effective drug against
anaerobic bacteria and some protozonoal infections that can cause
embryotoxic or congenital malformations [1, 2, 3, 17, 18].
The drug was injected into the fertile chicken eggs on the seventh
day when the liver detoxication mechanism was functional, in a
volume of 50 μL saline, into the air chambers located at the blunt end
of the eggs. Air chambers are preferred because of their advantages,
such as low infection risk compared to other regions, rapid diffusion
of the test solution, minimal mechanical damage compared to other
regions, and ease of application [14, 19]. With these applications,
Metronidazole's possible embryotoxic and/or teratogenic effects
could be observed and measured.
TABLE I
Death rates from Metronidazole administration.
Doses
(µg·egg
-1)
NAE NDE N
Death
rate* (%)
Survival
rate %
Actual death rate
(Abbott method)
Chest–1
Control
28 2 30 6.7 93.3 –
Saline control
29 1 30 3.3 96.7 –
250
28 2 30 6.7 93.3 3.44
125
29 1 30 3.3 96.7 0
62.5
30 0 30 0 100 -3.44
31.25
29 1 30 3.3 96.7 0
15.62
29 1 30 3.3 96.7 0
NAE: Number of alive embryos, NDE: Number of dead embryos. *No statistical
dierence was determined in death rates (
P>0.05)
_____________________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIV, rcfcv-e34310
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This study found no statistical difference between the experimental
groups (P>0.05, TABLE I). In addition, no macroscopic anomaly was
observed in any group that received Metronidazole. Previous work has
shown that teratogenic effects cannot be observed when Metronidazole
is administered intragastrically at a daily dose of 2mg·kg
-1
during
organogenesis in pregnant mice (Mus musculus) [20]. It is stated that
as a result of controlled studies in pregnant mice, rats and rabbits, no
teratogenic effects of Metronidazole were observed [21]. It is stated that
fetal and obstetrical side effects are not observed when Metronidazole
is administered at a dose of 500 mg·kg
-1
twice a day or at a dose of 250
mg·kg
-1
three times a day for seven days to pregnant women with genital
system infection [22]. In humans, it was reported that 597 pregnant
women (62 in the rst trimester, 284 in the second trimester, and 251 in
the third trimester) with trichomoniasis were treated at a dose of 200
mg three times per day for 7–10 days; no differences in prematurity,
birth weight, or teratogenicity were found [23].
Neither was any congenital malformation observed in the babies
of women who were administered Metronidazole at a dose of 200 mg
three times per day for seven days at different times of pregnancy
[24]. Two meta–analyses also reported no relationship between the
teratogenic effects of Metronidazole use in the rst trimester of
pregnancy [25, 26]. Based on these studies, it has been stated that
Metronidazole administered to treat trichomoniasis in women does
not harm the fetus in the rst trimester of pregnancy. However, it is
still recommended to delay the treatment with Metronidazole until
the second trimester [27].
It has also been reported that Metronidazole and Miconazole did not
cause axial skeletal disorders in mice when either Metronidazole or
Miconazole was administered at a dose of 60 mg·kg
-1
intraperitoneally
on the 8th, 9th, and 10th days of pregnancy. However, when these
drugs were administered in combination, as is often done in the
treatment of vaginal infections, there were teratogenic effects in
mice; thus, there is potential for such effects also in pregnant humans
[28]. In a study in mice, it was reported that when Metronidazole and
ethyl alcohol were administered simultaneously, ethyl alcohol caused
an increase in embryotoxicity and teratogenicity of Metronidazole
[29]. When the literature reviews are evaluated together with the
study data, it can be stated that Metronidazole alone does not cause
lethal or morphological changes directly in the embryo.
CONCLUSION AND IMPLICATIONS
As a result, it can be stated that Metronidazole does not have any
embryotoxicity and teratogenicity effects at the treatment doses
it was used here. Therefore, its use during pregnancy can be safe.
Due to the interaction of Metronidazole with many drugs, combined
drugs are not recommended, especially during pregnancy. It can be
suggested that this study should be supported by studies investigating
the ecacy of the drug, with histopathological and/or molecular
analyses on organogenesis especially in mammalian embryos.
ACKNOWLEDGEMENT
No financial support was received from any institution or
organization for this study. We would like to thank Prof. Dr. Enver
Yazar for his academic contributions.
Conict of interest statement
The authors declare there is no conict of interest.
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