INTRODUCTION:

Heterocyclic structures, whether of synthetic or natural origin, appear to be a particularly interesting support in various fields, notably the pharmaceutical field. Benzimidazole and oxazoline derivatives have attracted considerable interest due to their antimicrobial, antiviral, antifungal^1-3, anthelmintic⁴, and anxiolytic⁵ biological activities. In addition, several studies have shown a particular interest in the benzimidazole family for its diversified pharmacological effects, such as antihypertensive⁶, analgesic⁷, anticonvulsant⁸, antiulcer⁹ and anticancer¹⁰, but above all that antioxidant¹¹, antineoplastic and anti-inflammatory¹², and GABA agonist^13-14.

Their chemical structure consists of a two-ring system that can carry different substituents, which gives them the quality of having a multitude of combinations resulting in various molecules with different physicochemical characteristics and characterized by different pharmacological and pharmacokinetic and metabolic properties. These basic rings of benzimidazoles are widely used in modern drug discovery and are considered a primary pharmacophore1^5-16. Consequently, interest has been focused on the synthesis of new derivatives in order to find new ones with promising biological activities such as thiabendazole and flubendazole which are pharmaceutical products with anthelmintic effects, omeprazole and lansoprazole which are drugs with antiulcer effect and astemizole as antihistamine pharmaceuticals¹⁷. Hence the interest in synthesizing new derivatives which can have an additional pharmacological effect and reinforce the therapeutic arsenal deriving from this family. In this sense, we designed and synthesized (3-[2-(2-Amino-1H-benzimidazol-1-yl)ethyl]-1,3-oxazolidin-2-one) (OXB₂), a new derivative of Oxazolo-Benzimidazoles, with potential pharmacological activity (fig1).

Figure 1: Chemical structure of OXB₂

It is generally accepted that before carrying out in vivo studies of a newly synthesized molecule to evaluate these biological and pharmacological activities, to go through a toxicity study in order to define these toxicological parameters; essentially the LD₅₀ and the NOAEL in the animal model¹⁸. Therefore, this study aimed to determine these OXB₂ parameters and serum biomarkers in Wistar rats.

MATERIAL AND METHODS:

1. Description of OXB₂:

The molecule studied (OXB₂) is a new derivative of Benzimidazole (Oxazolo-Benzimidazole) resulting from the alkylation of Oxazolidin-2-one on the Benzimidazole ring of 2-Aminobenzimidazole. Its melting point, solubility and boiling point have been identified. Its 3D structure was also identified using single crystal/X-ray techniques while Fourier transform infrared spectroscopy (FTIR), NMR¹H nuclear magnetic resonance (NMR) and mass spectrometry techniques were used for the characterization^19-20.

2. Animals and treatment:

The animal model used in this study is the Wistar Rat from the Genetics, Neuroendocrinology and Biotechnology Breeding Laboratory (Faculty of Sciences, Ibn Tofail University, Morocco). All Wistar rats used were male, three months old and weighed 250 g ± 5% of the recommended average weight²¹. At the start of the experiment, the rats were acclimatized for a period of 5 days. These rats were then placed in separate metabolic cages and maintained at 25-27°C and 60-70% humidity and fed a standard diet with access to water and food ad libitum. The experiments were carried out in accordance with the internationally recognized principles for the use and maintenance of European Community laboratories (EEC Directive 1986; 86/609/EEC) to minimize animal suffering. All experimental procedures were performed also according to the National Institutes of Health guide for the care and use of Laboratory animals and were authorized by the Doctoral Study Center at the university.

3. Acute toxicity study.

Experimental acute toxicity of OXB₂:

Preparation of animals: Six groups of six rats each randomly distributed healthy rats were acclimated to a room dedicated to the study in the animal facility.

Preparation of the OXB₂ injection solution: The stock solution of OXB₂ at a concentration of 120 mg/ml was prepared based on the molecule dissolved in DMSO (Dimethylsulfoxide) in 10% distilled water. A dilution series was carried out in order to prepare concentrations corresponding to 5 doses ranging from 500 mg/kg and 1000 mg/kg of body weight. A vehicle solution devoid of OXB₂ was prepared for the control group.

Procedure of the Manipulation:

The injection: The vehicle solution and the 5 doses were administered intraperitoneally (i.p.) to rats in the control group and the other 5 groups, respectively. A constant volume was used (whatever the concentration: 1 ml per 100 g of body weight).

Monitoring: In addition to behavioral responses to external stimuli such as variation in light intensity and noise, behavioral changes such as posture, appearance of hair loss, scratching and loss of balance were monitored during an observation period that lasted 14 days. Also, certain physiological parameters, notably breathing and heart rate, were observed during this period. This period was also marked by daily monitoring of the body weight of the rats and by counting mortality in the different groups studied.

Blood samples, rat sacrifices, organ samples and biochemical analyses: After 14 days, the rats receiving 600 mg/kg bw of OXB₂ (NOAEL group) were weighed after fasting for one night to sacrifice them. They were injected with pentobarbital sodium at 6.5% at an equivalent volume of 1 ml/kg bw for the anesthetized then a puncture of the abdominal aorta was carried out in order to take blood and collected it in tubes of dry blood collection and EDTA blood collection tubes to perform biochemical analysis and complete blood count (CBC) respectively. After sacrificing the animals, the vital organs (heart, liver, kidneys, brain and lungs) were carefully removed and then rinsed with normal saline before weighing them.

NB: The entire study was carried out in accordance with the acute toxicity class method according to the OECD-423 guidelines²².

Acute poisoning effect and determination of LD₅₀, MLD and NOAEL:

The experiment consists of administering increasing doses of the substance to groups of animals until 100% mortality is achieved after 14 days, according to OECD guidelines²². The and the NOAEL is the dose without observed adverse effects while the MLD is the dose which can kill at least one treated animal. Doses giving 50% and 100% mortality correspond to LD₅₀ and LD100, respectively. and the LD₅₀ value is determined according to the OECD harmonized general classification system²².

4. Haematological and Biochemical analysis:

Haematology (CBC):

Cell counting (on Tomas cells) using a light microscope at 10 × 40 magnification was used to assess total erythrocytes while the Drabkin colorimetric method was used to assess hemoglobin status. some samples. blood collected in EDTA blood collection tubes.

Biochemical Parameters:

A centrifugation was carried out to obtain the serum which underwent biochemical analyzes to determine glutamic oxaloacetic transaminase (GOT), glutamino-pyruvic (TGP), bilirubin, serum triglycerides, total proteins, creatinine, urea, glucose and total cholesterol using a biochemical analyzer.

NB: These biochemical analyzes were carried out by Laboratoires Ibn Sina (Kenitra, Morocco).

5. Data analysis (statistical tools):

A comparison of the mean ± standard deviation (SD) of the results of the groups injected with OXB₂ with those of the control group was carried out with a statistical analysis based on the ANOVA test with a level of significance considered at P < 0.05.

RESULTS:

1. Effect of OXB2 on rat mortality:

Reduced motor activity was recorded in rats 1 hour after OXB₂ injection at doses >900 mg/kg with an intensity proportional to the administered dose accompanied by disturbance in physical appearance and behavior showing signs obvious toxicity. Also note that series of diffuse and irregular muscular contractions, with blockage of breathing followed by death was recorded for rats having received the high doses.

Table 1: The mortality rate in rats injected with different doses of OXB₂:

Group no	Dose (mg per kg b.w.)	Dead rats
Group no	Dose (mg per kg b.w.)	Number	Percentage
1	0 (control)	0 / 6	0 %
2	500	0 / 6	0 %
3	600	0 / 6	0 %
4	700	1/6	16,66 %
5	800	2/6	33,33%
6	1000	6/6	100%

The administration of OXB₂ caused in the rats of the groups receiving the doses of 700 mg/kg, 800 mg/kg and 1000 mg/kg, various clinical signs including crises of agitation and the intensity increase proportionally to the dose injected. until it causes, at a high dose (1000 mg/kg), a seizure similar to that caused by epilepsy. The most characteristic signs are a decrease in motor activity. Also, no twisting, straightening of the hairs or diarrhea were observed.

For doses greater than 600 mg/kg, the symptoms of toxicity appeared 24 hours after injection of OXB₂, whereas doses equal to or less than 600 mg/kg showed no signs of toxicity.

Figure 2: Change in percentage of deaths of rats (Wistar) according to the injected dose of OXB₂

The results show that an injection of OXB₂ at a dose of 700 mg/kg constitutes the point at which the physiological and mental states of the animal deviate from normal. From this dose, disturbances in physical appearance, behavior and temperature become more and more important and denote an obvious toxicity. Therefore, the minimum lethal dose (MLD) of OXB₂ for rats is 800 mg/kg bw, while the LD₅₀ of OXB₂ was 854 mg/kg bw. At the dose of 1000 mg/kg, all the animals are moribund, which constitutes the endpoint in the toxicological tests.

2. Effect of OXB₂ on the evolution of body weight in the Wisat rat:

Body weight range of the different rats was between 215 and 235 g at the start of the study (day 0). According to the results of monitoring the body weight of rats in the 6 groups during the monitoring period (14 days after administration of the molecule), a very significant decrease in body weight compared to the control group (*** P < 0.001) was observed in rats injected with 700 and 800 mg/kg bw of OXB₂, while rats injected with 500 and 600 mg/kg bw of OXB₂, showed no significant changes (p> 0.05). The results are presented in Figure 3.

Figure 3: Evolution of the average body weight of rats from the different groups studied, during the 14 days (mean ± SD).

3. Efect of OXB₂ on food ingestion and water intake:

Food intake (g/day/rat) and water consumption (ml/day/rat) decreased over 14 days, significantly in rats receiving 700 and 800 mg/kg bw of OXB₂ (***P < 0.001), compared to the control group, during the 14 days of follow-up while no significant change was observed in rats receiving 500 or 600 mg/kg bw of OXB₂ (Figures 4 and 5).

Figure 4: Evolution of mean rats food ingestion (mg/day/rat) during 14 days (mean ± SD. *** P < 0.001).

Figure 5: Evolution of mean rats water intake (ml/day/rat) during 14 days (mean ± SD. *** P < 0.001).

3.1 Impact of OXB₂ on hematological parameters of the Wistar rat.

The hematological results obtained are presented in Table 2 and they were compared to the normal values of the Wistar rat²³. OXB₂ related hematological changes showed that mean leukocytes and lymphocytes were higher in rats of the control group, compared to normal values, while means of all parameters of treated rats at 600 mg/kg OXB₂ was within the normal range.

Table 2: The results of hematological analysis of rats treated with OXB₂

Parameters	Normal value of Wistar rats (Descat Fleur, 2002)	Control (Vehicle solution)	NOAEL (600mg/kg of OXB₂)
Erythrocytes (10¹²/l)	(5,79 – 9,72)	7,06	6,93
Hematocrit HCT (%)	(32 – 48)	41,25	34,95
Hemoglobin (Hgb) (g/dl)	(12,2 – 19,3)	13,95	13,65
Mean corpuscular volume (MCV) (fL)	(47 – 59)	58,49	50,63
Mean corpuscular hemoglobin MCH (pg)	(17 – 23)	19,77	19,8
Mean corpuscular hemoglobin concentration (MCHC) (g/dl)	(33 – 45)	33,88	39,01
Platelets (10⁹/l)	(700 - 2122)	859	693,5
Leukocytes (10⁹/l) **	(4,19 – 13)	18,45	13,05
Neutrophils (%)	(1 – 40)	12,8	31,52
Eosinophils (%)	(59 - 99)	72,8	56,5
Basophils (%)	(0 - 6)	1.95	1,4
Lymphocytes (%) **	(0 – 6)	10,9	1,0
Monocytes (%)	(0 -2)	1,55	0,6

Values are represented as mean ± SD. *p< 0.05; **p< 0.01

The results obtained from the different hematological parameters analyzed in the rats injected with OXB₂ and the control rats were within the reference value ranges²³ and no significant difference was recorded (p < 0.05) compared to the normal values.

3.2 Impact of OXB₂ administration on biochemical parameters.

No significant differences in the mean values of total protein ALT, AST, total cholesterol, urea, total bilirubin, glucose, triglycerides, AST and ALT between rats treated with 900 mg/kg bw of OXB₂ and control rats (p < 0.05). The results obtained were within the reference value ranges²⁴.

On the other hand, the creatinine level in rats injected with the vector was noted to be low, which can be explained by a slight effect of DMSO on the kidneys and consequently on the degradation of creatine (muscle protein) which can be explained by mild muscle atrophy or myopathy, then all results of other parameters of liver and kidney function remained normal for this group.

Table 3: Results of analyzes of biochemical parameters of rats injected with OXB₂ and control rats.

Parameters	Normal values of the Wistar rat *(Lazare, 2011)*	Control (Vehicle solution)	NOAEL (600 mg/kg of OXB₂)
Urea (g/l)	(0.12 – 0.51)	0.22	0.49
Glucose (g/l)	(0.55 – 1.3)	1.01	1.3
Creatinine (mg/l)*	(4.2 – 8.3)	2.04 *	3
ASAT (TGO) (UI/l)	Inférieur à 303	240	293
ALAT (TGP) (UI/l)	Inférieur à 198	90.7	117.7
Total bilirubin (mg/l)	(2.73 – 8.27)	2.8	3.1
Total cholesterol (g/l)	(0,5 –2,3)	0,52	0.64
Triglyceride (g/l)	(0.28 – 1.02)	0.4	0.31
Total protein(g/l)	(63.19 – 82.31)	61.64	72.09

Values are represented as mean ± SD.*p<0.05

3.3 Weight of various body organs:

A slight, but statically insignificant difference in the relative weight of vital organs (liver, kidney, spleen, pancreas, heart and brain) was noted in rats treated with 600 mg/kg bw of OXB₂ compared to the control group (p < 0.05). The calculation of the relative weight of the different organs was carried out according to the following formula: [(/weight of the animal) x 100]. It represents the relative weight changes of the organ in relation to body weight.

Table 4: Mean organ weights (g) of rats injected with 600 mg/kg OXB₂ and control rats.

Organs	Brain	Kidneys	Lungs	Heart	Liver
Control (Vehicle solution)	1.58 ± 0.31	1.59 ± 0.12	1.99 ± 0.33	1.40 ± 0.6	7.26 ± 0.3
NOAEL (600 mg/kg of OXB₂)	1.69 ± 0.67	1.41 ± 0.35	2.0 ± 0.21	1.3 ± 0.33	7.1 ± 0.94

Values are represented as mean ± SD.

DISCUSSION:

3-[2-(2-Amino-1H-benzimidazol-1-yl)ethyl]-1,3-oxazolidin-2-one (OXB₂) is of considerable interest due to its potential GABA agonist effect specific to alpha 2 subunit of the GABA-A receptor^{13, 19-20} and the said effect of OXB₂ has been patented²⁵ under the reference MA54635. This pushed us to determine its toxicological profile on the Wistar rat. The LD₅₀ and the NOAEL are among the parameters necessary to identify a molecule toxicologically and which give both an idea of the relatively acute dangers of a chemical product but also an idea of its therapeutic interval for possible use of the molecule in vivo.

We evaluated the acute toxicity of OXB₂, in the present study, and the results showed that its LD₅₀ value was 854 mg/kg bw and is considered a moderately toxic substance, according to the International Toxicity Scale (ITS)²⁶.

On the other hand, the clinical signs are fare basically similar but with increased intensity for a higher dose (1000 mg/kg), whereas the intraperitoneal injection of the 600 mg/kg dose of OXB₂ in rats did not produced no external clinical signs and did not cause any animal deaths. Also, the average body weight and body weight gain of rats treated with a dose equal to or less than 600 mg/kg of OXB₂ evolved in the same way as those of the control group, which explains the similarity of the feeding efficiency of these two groups.

Knowing that a normal evolution of body weight in a study evaluating the toxicological profile of a molecule reveals a normal reaction of the body with the administered molecule and an impact normal on energy demand, protein synthesis and utilization, administration of OXB₂ at doses below 600 mg/kg does not affect the overall metabolism of the animal and could be considered a NOAEL dose for in vivo explorations.

Furthermore, in these rats treated with a dose of 600 mg/kg of OXB₂ the hematological parameters remained normal, and no difference with those of control rats was recorded, knowing that these parameters control physiological mechanisms (respiration, immunology, etc.) and consequently shows a normal physiological state in these rats injected with this dose.

On the other hand, in rats from this same group, but also in control rats, an increase in leukocytes and lymphocytes was recorded compared to the reference value for Wistar rats²³. It has been reported that leukocytosis and lymphocytosis can be indicative of various pathological conditions, such as chronic inflammatory stress response and red blood cell regeneration²⁷. On the other hand, Willhite and Katz reported that intravenous injection of DMSO affects platelet and leukocyte levels²⁸. Which leads us to say that DMSO caused inflammation in rats which induced an increase in lymphocytes and that this increase is mainly due to the use of DMSO as a solvent and not to the molecule studied.

For the clinical and toxic aspects, no differences were recorded in the biochemical parameters between the rats of the two groups compared (600 mg/kg and control), with the exception of the creatinine level which was low compared to normal values in rats from the two groups compared. Given that this decrease was recorded in both groups and that DMSO is the common element in both injections, we suggest that this slight decrease in creatinine level is probably due to the vehicle solution containing DMSO. Also, this reduction is less significant in the presence of OXB₂. Furthermore, it has been reported that the main indicator of normal renal function is not only creatinine tau but also urea²⁹ which was within the normal range for rats from both groups. Creatinine and urea levels did not show, therefore, any significant changes caused by OXB₂ in Wistar rats and the molecule studied did not disrupt renal function at doses equal to or lower than 600 mg/kg bw.

Glucose, AST, ALT, total bilirubin, total cholesterol, triglycerides, and total protein levels showed no changes, suggesting that OXB₂ has no effect on metabolic function of the liver and does not interfere with the metabolism of macromolecules and xenobiotic transformation. In addition, no significant difference was recorded between the relative weight of the different organs of rats treated with OXB₂ compared to that of control rats, which suggests that OXB₂ has no effect on these organs at a dose of 600 mg/kg confirming that this is the NOAEL dose.

CONCLUSION:

The study of the toxicological profile of 3-[2-(2-Amino-1H-benzimidazol-1-yl)ethyl]-1,3-oxazolidin-2-one revealed that its LD₅₀ is equal to 854 mg/ kg and can be classified as a substance of moderate toxicity. During the 14 days of the study, a dose equal to or less than 600 mg/kg did not induce any mortality and did not impact either the behavior or the biochemical and hematological parameters of the rats, which confirmed that the NOAEL dose of OXB₂ is equal to 600 mg/kg. This study identified the acute toxicity of OXB₂ and will lead to the carrying out of a possible study to evaluate its pharmacological effect in vivo.

ACKNOWLEDGEMENTS :

The authors are very grateful to The Ibn Sina Laboratories (Kenitra, Morocco) for the accomplishment of the biochemistry analysis. We would like to thank Miss Rim Bousselham and Mr Miloud Chakit (Laboratory of Genetic, Endocrinology and Biotechnology- Faculty of sciences, Ibn Tofail University, Kenitra, Morocco) for their help in designing.

CONFLICT OF INTEREST:

Authors declare that they have no competing interest.

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Received on 29.05.2024 Revised on 23.12.2024

Accepted on 06.05.2025 Published on 13.01.2026

Available online from January 17, 2026

Research J. Pharmacy and Technology. 2026;19(1):76-82.

DOI: 10.52711/0974-360X.2026.00012

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