1 of 5 Received: 21/01/2026 Accepted: 20/03/2026 Published: 24/04/2026 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Revista Cienﬁca, FCV-LUZ / Vol. XXXVI hps://doi.org/10.52973/rcfcv-e362904 Determinaon of Embryotoxic eﬀects of Ketoprofen at diﬀerent phase and doses using an In ovo model Determinación de los efectos embriotóxicos del Ketoprofeno en diferentes fases y dosis ulizando un modelo In ovo Harun Kizilay* , Seyma Tetik-Rama Selcuk University, Faculty of Pharmacy, Department of Pharmacology. Konya, Türkiye. *Corresponding author: harunkizilay@gmail.com ABSTRACT Ketoprofen is a nonsteroidal an-inﬂammatory drug with analgesic, an-inﬂammatory, and anpyrec acvies. This study aimed to evaluate the potenal embryotoxic eﬀects of ketoprofen, on chicken embryos using the in-ovo method. The LD 50 value, embryotoxic dose limit, and macroscopic ﬁndings in the embryos were examined. A total of 420 ferlized chicken eggs were randomly divided into 2 groups and both groups were placed in the incubator at the same me. Group-1 (n = 210): The group treated on the 14th day of embryonic development. and group-2 (n = 210): Group-1 and group-2 were divided into 7 subunits, each consisng of 30 ferlized eggs. Diﬀerent doses of ketoprofen (100, 50, 25, 12.5, and 6.25 mg/kg) were injected into the air-sacs of ferlized eggs in both groups. At the end of the 21-day incubaon period, embryotoxicity was evaluated in all groups. As a result, a stascally signiﬁcant diﬀerence in mortality increase was detected between the control group and the 100 mg/kg and 50 mg/kg groups following the 7th day injecon (P < 0.05). No stascally signiﬁcant diﬀerences were found in the other groups (P > 0.05). Similarly, no stascally signiﬁcant diﬀerence was detected among all experimental groups as a result of the 14th day injecon (P > 0.05). In conclusion, the ﬁndings obtained in this study demonstrate that ketoprofen causes a dose-dependent embryotoxic eﬀect in the early embryonic period, but this eﬀect is not evident in the late period. Key words: Embryotoxicity; in ovo model; ketoprofen; teratogenicity. RESUMEN El ketoprofeno es un fármaco aninﬂamatorio no esteroideo con acvidades analgésicas, aninﬂamatorias y anpirécas. El objevo de este estudio fue evaluar los posibles efectos embriotóxicos del ketoprofeno, en embriones de pollo ulizando el método in ovo. Se examinaron el valor LD 50 , el límite de dosis embriotóxica y los hallazgos macroscópicos en los embriones. Se dividieron aleatoriamente 420 huevos de gallina ferlizados en dos grupos y ambos grupos se colocaron en la incubadora al mismo empo. Grupo 1 (n = 210): El grupo tratado el sépmo día de desarrollo embrionario y el grupo 2 (n = 210): El grupo tratado en el decimocuarto día de desarrollo embrionario. El grupo 1 y el grupo 2 se dividieron en 7 subunidades, cada una de las cuales constaba de 30 óvulos ferlizados. Se inyectaron diferentes dosis de ketoprofeno (100, 50, 25, 12,5 y 6,25 mg/kg) en los sacos aéreos de los huevos ferlizados de ambos grupos. Al ﬁnal del periodo de incubación de 21 días, se evaluó la embriotoxicidad en todos los grupos. Como resultado, se detectó una diferencia estadíscamente signiﬁcava en el aumento de la mortalidad entre el grupo de control y los grupos de 100 mg/kg y 50 mg/kg tras la inyección del sépmo día (P < 0,05). No se encontraron diferencias estadíscamente signiﬁcavas en los demás grupos (P > 0,05). Del mismo modo, no se detectaron diferencias estadíscamente signiﬁcavas entre todos los grupos experimentales como resultado de la inyección del día 14 (P > 0,05). En conclusión, los resultados obtenidos en este estudio demuestran que el ketoprofeno causa un efecto embriotóxico dependiente de la dosis en el período embrionario temprano, pero este efecto no es evidente en el período tardío. Palabras clave: Embriotoxicidad; modelo in ovo; ketoprofeno; teratogenicidad.

Embryotoxic eﬀects of Ketoprofen using an In ovo model / Kizilay and Tek-Rama UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION Ketoprofen is a nonsteroidal an-inﬂammatory drug (NSAID) with analgesic, an-inﬂammatory, and anpyrec acvies [1]. Ketoprofen is widely used in the treatment of inﬂammatory rheumatological diseases such as juvenile idiopathic arthris, rheumatoid arthris, and ankylosing spondylis, as well as post-operave and traumac pain. Due to its rapid absorpon, potent an-nocicepve acvity, and rapid passage through the blood-brain barrier, ketoprofen is commonly preferred in clinical pracce [2 , 3]. Common adverse eﬀects associated with ketoprofen use include headache, peripheral edema, platelet dysfuncon, increased liver enzyme levels, and photosensizaon and skin sensivies associated with topical use. Adverse eﬀects developing with chronic ketoprofen use are generally related to inhibion of the cyclooxygenase enzyme. Ketoprofen’s inhibion of renal prostaglandin synthesis can reduce renal blood ﬂow, leading to renal failure, electrolyte imbalance, and hypertension. Ketoprofen’s inhibion of gastric prostaglandin synthesis creates a risk of ulceraon and bleeding, along with gastric and duodenal irritaon [4 , 5]. Similarly, chronic use of diclofenac, another important NSAID, has been found to be associated with serious complicaons such as hypertension, stroke, acute myocardial infarcon, and liver damage [6]. In a case study, it was found that ketoprofen administraon to a pregnant woman led to fetal ductus arteriosus stenosis, a condion that persists even aﬅer disconnuaon of the drug. This ﬁnding was supported by pharmacokinec/pharmacodynamic modeling predicng similar fetal and maternal drug concentraons [7]. Addionally, it is thought that ketoprofen inhibits melanin producon, causing phototoxic eﬀects due to drug accumulaon in pigmented ssues [8]. Ketoprofen exhibited higher acute toxicity in the embryonic stages of zebraﬁsh compared to juveniles, and the lethal concentraon 50 (LC 50 ) for embryos was determined to be 6.44 mg/L. This increased sensivity has been aributed to the underdeveloped enzymac systems in embryos [9]. Another study reported that ketoprofen exhibited concentraon-dependent toxic eﬀects in zebraﬁsh (Danio rerio) embryos, including edema, spinal curvature, slow heart rate, delayed hatching, and death [10]. Chicken (Gallus gallus domescus) embryos are frequently preferred in the invesgaon of the embryotoxic, teratogenic, mutagenic, and genotoxic eﬀects of various drugs. Chicken embryos are a reliable model for embryotoxicity studies due to their well-deﬁned developmental stages, faster results, and greater ethical acceptance [11]. Addionally, in poultry, the drug is administered directly into the egg using the in ovo method [12]. The ability to use a large number of eggs provides a stascal advantage over other studies. The in ovo method is more cost- eﬀecve than mammalian pregnancy models and is suitable for objecve evaluaon in terms of faster assessment of observaonal endpoints (such as mortality, malformaon, and growth parameters) [13]. Addionally, the literature indicates that the in-ovo method is used not only for vaccines and biological materials but also for anbiocs, hormones, nutrional supplements, and environmental toxins, and that it provides a good basis for morphological and histopathological evaluaon of the embryos [14 , 15 , 16]. Adverse reacon (mortality, developmental delay, organ-speciﬁc malformaons) idenﬁed in other embryo models such as zebraﬁsh are also standard endpoints in chicken embryos [17]. The present study was conducted to evaluate the potenal embryotoxic eﬀects of ketoprofen on chicken embryos using the in-ovo method. Therefore, the LD 50 value, embryotoxic dose limit, and macroscopic ﬁndings in the embryos were examined. Determining these parameters is crucial for establishing the safety proﬁle of NSAIDs during diﬀerent developmental stages. This research provides reliable data on ketoprofen-induced embryotoxicity, addressing a gap in the current literature and strengthening the evidence regarding the developmental risks associated with prostaglandin synthesis inhibitors [13 , 14 , 15 , 16 , 18 , 19 , 20 , 21 , 22 , 23 , 24]. MATERIALS AND METHODS Experimental design This study was designed as a prospecve, controlled, and dose-escalaon in ovo experimental study. The study was conducted in two phases: drug administraon on day (d) 7 and d 14. Incubaon periods were completed in an egg incubator (Imza Teknik, Konya, Türkiye). The device was set to 55 % humidity and 37.8 ± 0.2 °C temperature. Additionally, the eggs were turned by the incubator every 90 minutes (min) at a 45-degree angle. On the 7th and 14th d of the incubaon period, ferlity checks were performed using an ovoscope under light, and inferle eggs were removed from the groups and replaced with ferle eggs. The number of eggs (n) in each experimental group was set at 30. A commercial ketoprofen formulaon (Ketojezik 100 mL ﬂk. TeknoVet, Istanbul, Türkiye) was used, and all doses were administered in a volume of 50 μL. Animal experiments A total of 420 ferlized eggs from Babcock White breed hens (Anadolu Entegre Damızlık, Konya, Türkiye) were obtained for the study. The eggs were randomly divided into two groups and both groups were placed in the incubator at the same me. Group-1 (n = 210): The group treated on the 7th d of embryonic development Group-2 (n = 210): The group treated on the 14th d of embryonic development Firstly, the 210 ferle eggs in group-1 were randomly divided into 7 equal groups (n = 30) and placed in the incubator. Group-1A (n = 30): No intervenon was performed (control group). Group-1B (n = 30): Saline (physiological serum) without ketoprofen was administered. 2 of 5

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Group-1C (n = 30) : Ketoprofen was administered at a dose of 100 mg/kg. Group-1D (n = 30) : Ketoprofen was administered at a dose of 50 mg/kg. Group-1E (n = 30) : Ketoprofen was administered at a dose of 25 mg/kg. Group-1F (n = 30) : Ketoprofen was administered at a dose of 12.5 mg/kg. Group-1G (n = 30) : Ketoprofen was administered at a dose of 6.25 mg/kg. The procedures performed on group-1 were applied to group-2 on the 14th d of embryonic development in the same order. The 210 ferlized eggs in group-2 were randomly divided into 7 equal groups (n = 30) and placed in an incubator. Group-2A (n = 30) : No intervenon was performed (control group). Group-2B (n = 30) : Saline (physiological serum) without ketoprofen was administered. Group-2C (n = 30) : Ketoprofen was administered at a dose of 100 mg/kg. Group-2D (n = 30) : Ketoprofen was administered at a dose of 50 mg/kg. Group-2E (n = 30) : Ketoprofen was administered at a dose of 25 mg/kg. Group-2F (n = 30) : Ketoprofen was administered at a dose of 12.5 mg/kg. Group-2G (n = 30) : Ketoprofen was administered at a dose of 6.25 mg/kg. The drug doses administered to all experimental groups were injected into the air-sacs of ferlized chicken eggs. No turning was performed during the 1 h following drug applicaon to allow the drug to diﬀuse. All eggs were kept under opmal condions in the incubator to ensure drug absorpon from the air sac. At the end of the 21-d incubaon period, the eggs were hatched and embryotoxicity was assessed [25 , 26 , 27]. Stascal analysis To assess the independence between natural death rates and drug-related death rates in determining embryotoxicity, it is important to express actual death rates using Abbo’s method. For this reason, in this study the actual death rate was determined using the Abbo method on embryonic mortality rates [25 , 26 , 27]. Embryonic mortality rates between groups were evaluated using the chi-square test (SPSS 22). The embryonic lethal dose 50 (LD 50 ) value was determined using the probit test (SPSS 22). P < 0.05 value was considered stascally signiﬁcant. RESULTS AND DISCUSSION The mortality rates following ketoprofen administraon on the 7 d and 14 d are presented in Table I. TABLE I MORTALITY RATES OF CHICKEN EMBRYOS ON DAYS 7 AND 14 FOLLOWING IN OVO ADMINISTRATION OF VARIOUS DOSES OF KETOPROFEN. Groups Dose (μg.egg -1 ) N NAE EED LED Death Rate (%) Survival Rate (%) Actual Death Rate (Abbo method) Control 30 30 0 0 0.0 100 - Saline 30 30 0 0 0.0 100 - 100 30 13 10 7 76.7* 23.3 76.7 Group-1 50 30 25 3 2 16.7* 83.3 16.67 (Day 7) 25 30 28 2 0 6.7 93.3 6.67 12.5 30 28 0 2 6.7 93.3 6.67 6.25 30 30 0 0 0 100 0 Control 30 29 0 1 3.3 96.7 - Saline 30 28 0 2 6.7 93.3 - 100 30 29 0 1 3.3 96.7 -3.57 Group-2 50 30 29 0 1 3.3 96.7 -3.57 (Day 14) 25 30 29 0 1 3.3 96.7 -3.57 12.5 30 28 0 2 6.7 93.3 0 6.25 30 28 0 2 6.7 93.3 0 N: Total number of ferle eggs (embryos) per group, NAE: Number of alive embryos at the end of the 21-day incubaon period, EED: Early embryonic death (mortality occurring between 0-7 d of incubaon), LED: Late embryonic death (mortality occurring between 8-21 d of incubaon), Abbo method: A formula used to calculate the actual death rate by correcng for natural mortality in the control group. *P < 0.05: Indicates a stascally signiﬁcant increase in mortality compared to the control group, determined using Chi-square and Probit analysis. 3 of 5

Embryotoxic eﬀects of Ketoprofen using an In ovo model / Kizilay and Tek-Rama UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico The actual death rates (Abbo’s method) were calculated using the following formula: (Saline group’s survival rate % - Drug group’s survival rate %) / (Saline group’s survival rate %)) * 100 According to Abbo’s method, a negave actual death rate indicates that deaths independent of ketoprofen in the saline group during the 14th d of drug administraon, independent of embryotoxicity. As a result of the 7-d applicaons, the embryonic mortality rate in the group administered high-dose ketoprofen (100 mg/ kg) was determined to be 76.7 %, while the survival rate was 23.3 %. At a dose of 50 mg/kg, the mortality rate was determined to be 16.7 %. A stascally signiﬁcant diﬀerence was detected between the control group and the 100 mg/kg and 50 mg/kg groups (P < 0.05). Mortality rates in the 25 mg/kg, 12.5 mg/kg, and 6.25 mg/kg dose groups were not stascally diﬀerent from those in the control and saline groups (P > 0.05) These data indicate that the embryotoxicity caused by ketoprofen is dose-dependent during early embryonic development. This increased sensivity may be explained by the underdevelopment of the embryo’s detoxiﬁcaon mechanisms (such as glutathione peroxidase and catalase) in the early stages. Similar studies using zebraﬁsh embryos have also reported that embryos are more sensive to ketoprofen in the early stages [9 , 10]. Similarly, a study reported that diclofenac administered in early pregnancy caused neural tube closure defects and developmental delays [24]. Addionally, it has been noted that ketoprofen has adverse eﬀects on kidney development in mammalian embryos and increases post-implantaon losses; this ﬁnding further supports the high sensivity of the early developmental stage [23]. As a result of the 14-d applicaons, the mortality rates of the experimental groups ranged between 3.3 % and 6.7 %, and the survival rate in all groups ranged between 93.3 % and 96.7 %. No stascally signiﬁcant diﬀerence was detected between the experimental groups (P > 0.05). The absence of signiﬁcant embryotoxicity in the late period indicates that organogenesis is largely complete by day 14 and that the embryo’s metabolic capacity has developed signiﬁcantly. These results reinforce the importance of ming in the assessment of NSAID-induced toxicity. According to the probit test performed, the LD 50 dose of ketoprofen administered on 7 d was found to be 91.455 mg/kg (78.213 - 111.764). No deaths were observed with ketoprofen doses administered on 14 d, so the LD 50 dose could not be calculated. This mechanism appears consistent with the increased mortality observed during the early-stage ketoprofen administraon in this study. Addionally, ketoprofen is known to elevate liver enzyme levels and reduce renal blood ﬂow [2]; these ﬁndings reinforce the pathophysiological link between systemic toxic eﬀects and embryonic damage. Other studies have shown that ketoprofen causes oxidave damage by increasing reacve oxygen species in environmental exposures [22], and causes a signiﬁcant decrease in anoxidant levels in liver ssue [10]. Finally, this study conﬁrms the eﬀecveness of the in ovo technique previously described in pharmacological toxicity research. CONCLUSIONS The ketoprofen causes signiﬁcant embryotoxicity, parcularly at high doses (100 mg/kg) in the early stages, but this eﬀect decreases in the late stages. Addionally, the LD 50 dose of ketoprofen administered on d 7 was found to be in the range of 78.213–111.764 mg/kg, and further experimental studies are needed on ketoprofen embryotoxicity within this dose range. Current data indicate that ketoprofen causes a dose-dependent embryotoxic eﬀect in the early embryonic period but this eﬀect is not evident in the late period. In addion, this study demonstrates that prostaglandin synthesis inhibitors should be carefully evaluated for developmental toxicity and that the in ovo model is a powerful tool for preliminary tesng of the safety proﬁle of NSAIDs. In further studies, the histopathological ssue eﬀects and eﬀects on oxidave stress of ketoprofen should be invesgated to elucidate the mechanisms of toxicity in detail. Ethical commiee The protocol for this study was approved by the Local Ethics Commiee of Selcuk University (Decision No: 2025/49). Funding Support This study did not receive any ﬁnancial support or funding from instuons, organizaons, or company. Conﬂict of interest The authors declare that they have no conﬂict of interest. BIBLIOGRAPHIC REFERENCES [1] Sehajpal S, Prasad DN, Singh RK. 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