INTRODUCTION:

Benzimidazoles and their derivatives are known for their many therapeutic applications and represent one of the most biological active classes. Literature data report that these molecules are associated with many biological activities, including antimicrobial, antiviral, antifungal^1-3, anthelmintic⁴ and Anxolytic⁵. Moreover, several studies have investigated Benzimidazoles compounds for various pharmacological effects, including antioxidant⁶, Antitubercular⁷, antineoplastic and anti-inflammatory⁸, analgesic⁹, antiulcer¹⁰, anticonvulsant¹¹, anticancer¹²and Gamma-Aminobutyric Acid Agonist¹³.

At chemical level, benzimidazoles have two-ring systems that could bear different substituent, affecting the physicochemical characteristics of the molecules and generating various derivatives with different metabolic and pharmacokinetic properties. The basic rings of benzimidazoles are considered as a main pharmacophore and are widely used in modern drug discovery^14-15.

During the last decades, a great interest was given to synthesize different Benzimidazole derivatives with promising biological activities. In this field, Thiabendazole and Flubendazole have been produced and are widely used as anthelmintic compounds, Omeprazole and Lansoprazole as antiulcerative, and Astemizole as antihistamine products¹⁶. Recently, we have developed a new Benzimidazole compound, (3-[2-(1h-benzimidazol- 2-ylsulfanyl) ethyl]-1, 3-oxazolidin-2- one) (OXB₁), with a potential pharmacologic activity. Its chemical structure is reported in Figure 1.

Figure 1: Chemical structure of OXB₁

It’s widely accepted that assessment of biological and pharmacological activities of OXB₁ will depend on the toxicological parameters and evaluation of sub-toxic doses that could be used in animal experiments. Therefore, the present study was planned to evaluate the acute toxicity of OXB₁ compound in the Wistar rat by determining the Median Lethal Dose (LD50), No-Observed-Adverse-Effect Level (NOAEL) and serum biomarkers values.

MATERIAL AND METHODS:

1. Description of OXB₁:

OXB₁ is a novel derivative of Benzimidazole (Oxazolo-Benzimidazole). In this molecule an Oxazolidin-2-one unit is fixed on the Benzimidazole core. OXB₁ was already characterized by Nuclear Magnetic Resonance (NMR) NMR¹H, Mass Spectrometry, Melting Point, Solubility and Boiling Point, Fourier Transform Infrared Spectroscopy (FTIR) and the 3D structure identified by Monocrystal/X-ray techniques^17-18.

2. Animals and treatment:

Three-month old male Wistar rats were obtained from the Genetic, Neuro-endocrinology and Biotechnology Laboratory breeding (Faculty of Sciences, Ibn Tofail University, Morocco). All rats had access to water and food (standard diet) ad libitum and were acclimated for at least five days before the start of the experiment. All rats were, as recommended, in a range of 250g ± 5% of the average weight¹⁹. Rats were then divided in separate metabolic cages and maintained at a temperature of 25–27°C and humidity between 60 and 70%.

The experimental protocol was carried out according to the Organization for Economic Cooperation and Development (OECD) guide-lines²⁰. All experimental procedures were performed 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 OXB₁:

The toxicity testing was performed as per the OECD-423 guidelines, acute toxicity class method²⁰. After acclimation, healthy rats were randomly divided into 6 groups of 6 rats each. OXB₁ was dissolved in 10% Dimethyl sulfoxide (DMSO) giving a stock solution of 120mg/ml then it was diluted and tested at different concentrations ranging from 500mg/kg and 1200mg/kg of the body weight (bw) which were given by intraperitoneal (i.p.) injection in constant volume (regardless concentration: 1ml per 100g bw). Control animals were injected by the same route with 10% DMSO. All rats were observed for 14 days. During this observation period, behaviour changes (e.g. loss of balance, posture, scraping, appearance and fall of hairs), behavioural responses to external stimuli (e.g. variation of light intensity, noise), and some physiological parameters, including breathing and heart rate, were monitored. Moreover, death rate at different doses and changes in bw of alive’ remaining rats were also monitored.

At the end of the experiment (day 14), rats of the group receiving the limit dose with no observed adverse effect (NOAEL group) were fasted overnight and were anesthetized with sodium pentobarbital (6.5%) at a dose of 0.1ml per 100g of bw. A puncture of the abdominal aorta was performed to collect blood in Ethylenediaminetetraacetic acid (EDTA) blood collection tubes for complete blood count (CBC) and in dry blood collection tubes for biochemical analysis. The liver, kidneys, heart, lungs and brain were carefully removed, rinsed with the normal saline solution then weighed.

Determination of LLD, LD50, LD100, NOAEL and observation of acute intoxication manifestations:

According to OECD guidelines, the experiment consists in administering to batches of animals increasing doses of the substance until reaching a mortality of 100% after 14 days²⁰. In this case the absolute lethal dose (LD100) is the lowest dose of a substance that under defined conditions is lethal for 100% exposed animals while the Middle Lethal Dose (LD50) is the dose giving 50% of mortality. On the other hand, the Lowest Lethal Dose (LLD) is the dose that can kill at least one treated animal and the the no-observed-adverse-effect level (NOAEL) denotes the level of exposure of animal, found by experiment or observation, at which there is no biologically or statistically significant increase in the frequency or severity of any adverse effects of the tested protocol. The LD50 value was determined according to the Globally Harmonized Classification System of OECD²⁰.

3. Haematological and Biochemical analysis:

Haematology (CBC):

Blood samples were collected in EDTA blood collection tubes. Haemoglobin status was evaluated using the colorimetric method of Drabkin. Total erythrocytes were assessed by simple cell counting on Tomas cells (magnification: 10 × 40) using an optical microscope.

Biochemical parameters:

Sera were obtained after centrifugation and then assayed using a biochemical analyser to determine bilirubin, total protein, Aspartate aminotransferase (AST) or Glutamic Oxaloacetic Transaminase (GOT), Alanine transaminase (ALT) or Glutamic-Pyruvic Transaminase (GPT), serum triglycerides, glucose, total cholesterol, creatinine and urea. The biochemistry analysis was completed by Ibn Sina Laboratories (Kenitra, Morocco).

4. Statistical analysis:

In this study, all data are expressed as means±Standard Deviation (SD). Statistical analyses were conducted by comparing the treatment groups with the control group using the Analysis of variance (ANOVA) test. The significance level was considered at P < 0.05.

RESULTS:

1. Assessment of acute toxicity:

In the acute toxicity testing with the highest doses (> 900 mg/kg), the disruption of physical appearance and behavior becomes more important and shows clear signs of toxicity. Rats have also shown a decrease of motor activity that occurred 1h after the start of the study, and the intensity was proportional to the administered dose. At high doses, an epileptic seizure was observed just before death of rats.

Table 1: Death rate after i.p. administration of OXB₁ at different doses

Group no	Dose (mg per kg bw)	Dead rats
Group no	Dose (mg per kg bw)	Number	Percentage
1	0 (control)	0 / 6	0 %
2	500	0 / 6	0 %
3	700	0 / 6	0 %
4	900	0 / 6	0 %
5	1000	1 / 6	16.7 %
6	1200	6 / 6	100 %

All rats receiving 1200mg/kg have died [20 – 90] minutes after the drug injection, which can be considered as the end point of toxicology tests. The number of dead rats during the study is reported in Table 1 and Figure 2. Accordingly, the minimal lethal dose (MLD) of OXB₁ to rats is 1000mg per kg bw and LD50 was 1084mg per kg bw.

Figure 2: Change in percentage of death after i.p injection of OXB₁ in normal Wistar rats

Of particular interest, no clinical signs of the other dose groups were observed. Thus, the injection of OXB₁ at this dose is the threshold from which both physiological and pathological states of behavior begin to appear in the rat. We can therefore assume that administration of OXB₁ at 900mg/kg bw is a NOAEL dose and consequently behavior states could be assessed at OXB₁ concentration not exceeding 900mg/kg bw.

2. Impact of OXB₁ administration on rats’ body weights:

The bw of rats of the 6 groups was followed during 14 days after drug administration and results are reported in Figure 3. At the beginning of the intervention (day 0), the weight of all groups fluctuated between 240 and 260g. Rats receiving i.p. injection of OXB₁ at doses (500, 700 and 900mg/kg) didn’t exhibit any modification in bw, during the follow-up period, as compared to control (p > 0.05). However, injection of 1000mg/kg bw of OXB₁ induced a highly significant decrease in bw compared to the control group (*** P < 0.001).

Figure 3: Evolution of bw in the 5 rats’ groups during 14 days. Values are presented as mean ± SD.

3. Impact of OXB₁ administration on rats’ ingestions (Food ingestion and water intake):

During the follow up period, rats receiving 500, 700 or 900mg/kg of OXB₁ didn’t show any significant changes in the food ingestion (g/day/rat) and water consumption (ml/day/rat) compared to the control group. However, food ingestion and water consumption were seriously reduced in rats receiving 1000mg/kg b.w. of OXB₁ (*** P < 0.001) (Figures 4 and 5).

Figure 4: Assessment of food ingestion (g day/rat) in the 5 rats’ groups during 14 days. Values are represented as mean ± SD. *** P < 0.001

Figure 5: Assessment of Water intake (ml/ day / rat) in the 5 rats’ groups during 14 days. Values are represented as mean ± SD. *** P < 0.001

3.1 Impact of OXB₁ administration on haematological parameters:

All obtained haematological results were compared to normal values already reported in rats Wistar²¹. As shown in Table 2, treatment-related hematology changes found that the mean of leukocytes and monocytes were higher in both 900mg/kg OXB₁ treated rats and control group as compared to normal values. However, the difference between OXB₁ treated and control groups is not statistically significant.

Table 2: Hematology analysis of rats treated with OXB₁

Parameters	Normal value of Wistar rats *(Descat Fleur, 2002)*	Control (Vehicle solution)	NOAEL (900 mg/kg of OXB₁)
Erythrocytes (10¹²/l)	5,79 – 9,72	7,06 ± 0.2	7,9 ± 0.3
Hematocrit HCT (%)	32 – 48	41,25 ± 0.7	41,8 ± 0.6
Hemoglobin (Hgb) (g/dl)	12,2 – 19,3	13,95 ± 0.9	15,4 ± 0.5
Mean corpuscular volume (MCV) (fL)	47 – 59	58,49 ± 0.3	53,03 ± 0.9
Mean corpuscular hemoglobin MCH (pg)	17 – 23	19,77 ± 1.31	19,51 ± 0.92
Mean corpuscular hemoglobin concentration (MCHC) (g/dl)	33 – 45	33,88 ± 0.59	36,9 ± 1.21
Platelets (10⁹/l)	700 - 2122	859 ± 20	740 ± 13
Leukocytes (10⁹/l) **	4,19 – 13	18,45 ± 1.27 **	21,25 ± 1.1 **
Neutrophils (%)	1 – 40	12,8 ± 0.9	16,5 ± 1.5
Eosinophils (%)	0 - 6	1.95 ± 0.7	3,75 ± 0.3
Basophils (%)	0 -2	1,55 ± 0.13	1,65 ± 0.21
Lymphocytes (%)	59 - 99	72,8 ± 1.6	65,65 ± 0.8
Monocytes (%)**	0 – 6	10,9 ± 1.8*	12,8 ± 1.2 **

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

Table 3: Biochemical analysis of rats treated with OXB₁

Parameters	Normal values of the Wistar rat (Lazare, 2011)	Control (Vehicle solution)	NOAEL (900 mg/kg of OXB₁)
Urea (g/l)	0,12 – 0,51	0,22	0,44
Glucose (g/l)	0,55 – 1,3	1,01	1,13
Creatinine (mg/l)*	4,2 – 8,3	4,02 *	5,54
AST [TGO] (UI/l)	< 303	240	300
ALT [TGP] (UI/l)	< 198	90,7	131
Total bilirubin (mg/l)	2,73 – 8,27	2,8	3,6
Total cholesterol (g/l)	0,5 –2,3	0,52	0,63
Triglyceride (g/l)	0,28 – 1,02	0,4	0,64
Total protein(g/l)	63,19 – 82,31	61,64	75,68

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

For the other parameters, Erythrocytes, Hematocrit, Hemoglobin, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelets, Neutrophils, Eosinophils, Basophils and Lymphocytes, no significant difference was obtained between treated and control rats (p < 0.05) and all results were in the ranges of references values²¹.

3.2 Impact of OXB₁ administration on biochemical parameters.

As shown in Table 3, there were no significant differences in the mean values of the urea, glucose, AST /TGO, ALT/TGP, total bilirubin, total cholesterol, triglyceride and total protein between the rats group injected by 900mg/kg of OXB1 and control group (p < 0.05).

Moreover, all obtained results were in the ranges of reference values²². Of particular interest, the mean value of creatinine in the 900mg/kg OXB₁ treated rats was in the normal ranges of reference values, but was significantly higher than that obtained with DMSO control, that was slightly lower than normal values.

3.3 Weight of various body organs:

The relative weight of different organs [(Organ weight /animal weight) x 100] indicates the relative weight changes of the organ to the bw. Following the injection of 900mg/kg of the OXB₁, the relative weights changes of different organs (liver, kidney, spleen, pancreas, heart, and brain) in OXB₁ treated rats showed a slight, but statically insignificant, difference compared to control group (p < 0.05).

Table 4: Average organ weights (g) in control, vehicle control and OXB₁ (900 mg/kg) treated 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 (900 mg/kg of OXB₁)	1.56 ± 0.36	1.7 ± 0.18	2.1 ± 0.15	1.2 ± 0.62	7.9 ± 1.82

Values are represented as mean ± SD.

DISCUSSION:

In the present study, we have assessed acute toxicity of OXB₁, with promising biological activities, and provided interesting information about the toxicological parameters of OXB₁ and their useful scientific use in ex vivo and in vivo testing.

For newly synthesized chemicals with limited available toxicological data, the evaluation of LD50 is of great interest and gives information on the relatively acute hazards of the chemicals. For OXB₁, LD50 value was 1084mg/kg bw. According to the International Toxicity Scale (ITS), OXB₁, like other benzimidazoles, is considered a moderate toxic substance²³. For a higher dose (1200mg/kg), clinical signs are basically similar but with increased intensity. In this study, intraperitoneal injection of 900mg/kg of OXB₁ in rats did not cause any animal death and didn't produce any external clinical signs.

Our results showed that the mean bw and bw gain of the treated rats with less than 900mg/kg of OXB₁ were similar to those of the control group. Correspondingly, the feed efficiencies of these groups were similar to those of the control group. In toxicological studies, Weight gain is an important indicator for evaluating the overall response of the animal to the administered drug, and the impact of the drug on energy demand, protein synthesis and protein utilization. Accordingly, we can assume that OXB₁ at doses below 900mg/kg don’t affect the global metabolism of the animal and could be considered as NOAEL dose for in vivo explorations.

For all hematological parameters, no significant difference was observed between the rats treated at 900 mg/kg OXB1 and the controls, which means that the physiological mechanisms controlled by these hematological parameters (respiration, immunology, etc.) remain normal. However, increases in leukocytes and monocytes were observed in both treated and control groups, compared to the baseline for Wistar rats²¹. Monocytosis indicates various disease states, such as chronic inflammation, stress response and regeneration of red blood cells²⁴. For these two haematological parameters, it is suggested that this increase is mainly due to the DMSO which was administered to these two groups. This suggestion is explained by the fact that DMSO could cause inflammation in some organs and tissues. Moreover, Willhite and Katz reported that DMSO has a direct effect on all leukocytes, including monocytes, and animals exposed intravenously to DMSO underwent dose-dependent hemodilution as well as fluctuations in certain blood components such as than platelets, leukocytes or blood sugar²⁵.

With regard to clinical and toxicity, no difference was reported in biochemical parameters between treated and control rats. No changes on the rates of glucose, AST, ALT, total bilirubin, total cholesterol, triglyceride and total protein was observed, suggesting that OXB₁ don’t have any effect on the metabolic function of the liver and don’t interfere with macromolecules metabolization and xenobiotic transformation. On the other hand, no change was observed regarding creatinine and urea levels in rats treated with OXB₁ at the NOAEL dose, suggesting a normal renal function after OXB₁ administration. Plasma Creatinine and urea levels are excellent markers of renal function and any modification of their levels can predict a possible dysfunction of the kidney²⁶.

The relative weight of different organs didn’t exhibit any difference between OXB₁ treated rats and controls, suggesting that OXB₁ don’t have any effect on these organs at NOAEL dose.

CONCLUSION:

In conclusion, the study was very informative and, as part of an investigation to assess the toxicological profile of OXB₁, we first evaluated the LD50 to be 1084 mg/kg and NOAEL dose at 900mg/kg and according the International toxicity scale, OXB₁ can be classified as a moderate toxicity substance. The 14-day toxicity study have reported no significant changes in all studied haematological and biochemical parameters, suggesting that OXB₁ at NOAEL dose is safe and could be used for in vivo studies.

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. This paper is dedicated to the memory of the late Pr Ali OUICHOU.

CONFLICT OF INTEREST:

Authors declare that they have no competing interest.

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Annexe: Graphic Abstract:

Received on 23.05.2021 Modified on 20.09.2021

Research J. Pharm. and Tech. 2022; 15(6):2427-2432.

DOI: 10.52711/0974-360X.2022.00404