INTRODUCTION:

Eggshells are composed of abundant calcium carbonate (CaCO3), approximately 98.2%, and calcium about 28% of the total shell weight¹. The eggshell powder has inhibitory activity against bacteria in the mouth, so it is used as dental cement^2,3. Eggshells, as a source of calcium, can be used as a supplement to bone grafts and toothpaste^4,5,6. Calcium carbonate in the form of nanoparticles is used as a carrier for anticancer drugs and has the potential for targeted delivery^7,8.

Supplements and traditional medicines must be produced using ingredients that meet quality standards and safety requirements⁹. The incorporation of eggshell as a constituent in pharmaceutical formulations is a feasible application. Therefore, it is necessary to ensure its safety and quality through a toxicity test. It is because of the potential toxicity of the eggshells is founded, which contain lead and bacteria. Eggshells have around 10.000 – 20.000 pores that can adsorb chromium, lead, methyl orange, and cadmium^10,11,12,13. Lead content was detected in eggshells at concentrations of 0.075-1.8 μg/g sample¹⁴. Some bacteria, such as Pseudomonas sp., Alcaligenes sp., and Salmonella sp. were also found to be able to penetrate the eggshell^15,16. An acute eggshell membrane toxicity test in mice with a single oral dose yielded an LD₅₀ of more than 2000mg/kgBW, and there were no signs of toxicity symptoms during the 14 day observation¹⁷. The subchronic toxicity test carries out because it was a follow-up test from the acute toxicity test, which saw the hematologic test, blood biochemistry, and organ histology¹⁸.

Oral acute toxicity evaluation determines toxic effects that manifest shortly after administering a substance in a single dose or in repeated doses within 24 hours.Observations were made by looking at the toxic effects and deaths. Data on animal mortality as stated by Lethal Dose 50 (LD₅₀). The subchronic toxicity test had a longer duration than the acute toxicity test, 28 days with repeated doses. The hematological analysis parameters comprised the following: mean corpuscular hemoglobin (MCH), hematocrit (HCT), red blood cell count (RBC), gray blood cell count (WBC) (including distribution of lymphocytes, monocytes, basophils, eosinophils, and neutrophils), and mean corpuscular volume (MCV) and platelet, meanwhile blood biochemical profile which included total cholesterol, triglycerides, HDL (High Density Lipoprotein), LDL (Low Density Lipoprotein), glucose, urea, creatinine, SGOT (Serum Glutamic Oxaloacetic Transaminase), and SGPT (Serum Glutamic Pyruvic Transaminase), also on histology examination use five organs, which are heart, kidneys, liver, lungs, and spleen¹⁸. Toxicity testing is essential in affirming and evaluating traditional medicines according to the quality standards of the World Health Organization (WHO)¹⁹. The recommended test method is a method based on OECD guidelines²⁰. The results of the study are presented herein.Consequently, the objective of this research endeavor was to examine the acute and sub-chronic oral toxicity of eggshell powder in rodents in accordance with the OECD's recommended guidelines for safety or dose-dependent toxicity.

MATERIALS AND METHODS:

Materials:

Preparation of Eggshell Powder:

Chicken eggshells are obtained from Pontianak, West Borneo, then washed using running water to separate the membrane layer and remove the dirt, then soaked in hot water. The eggshells are then dried in the sun for one day. The dried eggshells then ground using a blender until they become a fine powder and sieved with a 100 mesh sieve²¹, randomized according to weight at ± intervals of 20% of the mean. Prior to commencing the study, the rats underwent a seven-day acclimatization period. They were securely confined in individual enclosures, which were maintained under standard experimental conditions including a 12-hour light/dark cycle, 30-70% humidity, and a temperature of 22 ± 3^O C. The rats were provided with a standard rat diet (Charon Phokpan®, Indonesia) and ranoff water. The eggshell that matched the specified quality standards and was free from any impurities was deemed acceptable based on measurements of its water content, heavy metal content, and ash content.

Animal models of toxicology:

Research Methods:

Tanjungpura University's Pharmacy Faculty Laboratory in Pontianak is where this investigation was carried out. The Ethics Committee of Research at Tanjungpura University has granted ethical approval for this research procedure under the reference number 6496/UN22.9/TA/2020 for Acute oral toxicity test; 6447/UN22.9/TA/2020 for Haematological assays; 6281/UN22.9/TA/2020 for Blood Biochemical Tests and 6337/UN22.9/TA/2020 for histology examination. Utilizing a post-test control group design, the research employed an experimental methodology.

Test Subjects:

The animals involved in this research were Wistar rats, both male and female, ranging in weight from 150 to 250 grams and aged from 6 to 8 weeks. The Department of Food, Agriculture, and Fisheries in Pontianak City, West Borneo officially documented the weight and age of the rodents. The individual possesses distinct attributes, including lucid eyes, vibrant hair, immobility, and the absence of anatomical flaws. While the rodents were confined in cages, we supplied them with ample water and Charon Phokpan 551® (CP 551) pellets.

Acute oral toxicity in rats:

Testing was performed by the Laboratory of Pharmacology, Faculty of Medicine, University of Tanjungpura (Pontianak, Indonesia) based on Up and Down Procedure, title 425, Code Document Guidelines for Organization Economic Co-Operation and Development. To achieve a dose volume of 1ml/100 gBW, we suspended the eggshell powder in 1% carboxymethylcellulose in distilled water at concentrations of 200mg/mL and 500mg/mL. After a fasting period of 16 hours, eggshell powder suspension is given as a single dose at 2000 or 5000mg/kgBW body weight (bb) by oral administration to female Wistar rats for a maximum of 10 animals (a maximum of five animals per group). We assessed mortality, changes in body weight, and clinical signs of toxicity (including lacrimation, catalepsy, flexion, hafner, grooming, and respiration) by observing the rats daily for 14 days after administering the test. On day 15, we terminated all animals by using cervical dislocation.

Subchronic Oral Toxicity In Rats:

Adjustments Made to Animal Models:

Five male and five female rats comprised each of the six categories into which we arbitrarily categorized the animal models of rats. The duration of the investigation was twenty-eight days. The BPOM (Badan Pengawas Obat dan Makanan) guidelines govern the dosage determination for oral toxicity tests for osteoporosis. Although fatalities and severe symptoms of toxicity were not reported in response to the highest doses of this test preparation, it did induce a toxic effect. Milder toxic symptoms were observed at medium doses, while the lowest dosages returned no toxic effects. The grouping of rats is divided into six groups. Group 1 was given a solution of CMC (carboxymethyl cellulose), while groups 2, 3, and 4 were given eggshell suspension with doses of 100, 400, and 1000mg/kgBW, respectively. Groups 5 and 6 were satellite groups, in which group 5 was given a solution of CMC (carboxymethyl cellulose), while group 6 was given eggshell preparations at a dose of 1000mg/kgBW. The administration of all these substances was done orally. On the 29th day following administration of the combined botanicals for a duration of 24 hours, blood samples were collected. A cardiac aneurism was procured, and the blood was subsequently gathered within microtubes. Approximately 2mL of blood were extracted. Blood-filled tubes were maintained at a temperature of 2° – 6°C in containers fabricated with ice crystals. The organs, including the lung, heart, liver, and spleen, were soaked in 10% formalin for a period of time before being transferred to a container and allowed to cool at room temperature.

Haematological assays:

We analyzed 1mL of blood taken from the heart and placed in an EDTA-containing vial as an anticoagulant for hematological testing on the same day. Hematological analysis was conducted using the following parameters: platelet count, mean corpuscular volume (MCV), white blood cell count (WBC) (including distribution of lymphocytes, monocytes, basophils, eosinophils, and neutrophils), hematocrit (HCT), amyloid-α, and mean corpuscular hemoglobin (MCH).

Blood Biochemical Tests:

Blood taken from the rat's heart was then put into 2 Eppendorf tubes (± 3mL), centrifuged for 10 minutes at a speed of 1400rpm to get blood serum, and stored in the refrigerator until measurements of the blood biochemical profile, which included total cholesterol, triglycerides, HDL (high-density lipoprotein), LDL (low-density lipoprotein), glucose, urea, creatinine, SGOT (serum glutamic oxidase), and SGPT (serum glutamic pyruvic transaminase).

Histology Examination:

The organs selected to examine histologically are the heart, liver, kidneys, lungs, and spleen. The stages in making histological preparations are surgery, organ harvesting, fixation, cutting organs, imprinting with paraffin, cutting tissue blocks, and staining using Hematoxylin-Eosin. Observations were qualitative. Qualitative analysis was carried out by comparing the histological images of cells assisted by a light microscope using a 40-fold magnification, then comparing one group to another.

Statistical analysis:

Mean±standard deviation (SD) was presented, and we used the statistical program IBM SPSS Statistics 26 to perform a one-way analysis of variance (ANOVA) and a post hoc test to identify significant differences. Statistical significance was determined for differences at a p-value of less than 0.05.

RESULT:

Eggshell Powder:

The chicken eggshell powder has brownish-white physical characteristics because the eggshell contains more protoporphyrin IX pigments, in the form of a fine powder, distinctive odor, and has no taste^22,23. The yield of eggshell powder was 83.56% through the yield calculation. This was due to the reduction in mass in the form of separated shell membranes and the remaining egg whites that were cleaned and the drying process resulting in a reduction in weight in the yield.

Toxicological animal Model:

Rats exhibiting acute oral toxicity:

The 14-day evaluation period following treatment did not yield any fatalities. Figure 1 illustrates the body masses of all treated animals. Body weight did not change significantly in response to the various concentrations of treatment. The group that received eggshell treatment showed reduced motor activity, which was reversible after 24 hours. According to the criteria of acute toxic classifications, the LD50 (lethal dose 50) for the single oral dose was greater than 5000 mg/kgBW; therefore, the substance is nontoxic as defined by the GHS.

Figure 1. Average body weight during a 14-day single oral dose of eggshell. Data are shown as means ± standard deviations (n = 5).

Table 1. Effect of Eggshell on behaviour of rats in acute toxicity studies

Parameters	Observations of Eggshell treated groups
	2000 mg/kgBW					5000 mg/kgBW
	30min	1h	2h	24h	14 days	30 min	1h	2h	24h	14 days
Motor activity	↓	↓	↓	N	N	N	N	↓	N	N
Straub	A	A	A	A	A	A	A	A	A	A
Piloerection	A	A	A	A	A	A	A	A	A	A
Ptosis	A	A	A	A	A	A	A	A	A	A
Pineal reflex	N	N	N	N	N	N	N	N	N	N
Cornea reflex	N	N	N	N	N	N	N	N	N	N
Lacrimation	A	A	A	A	A	A	A	A	A	A
Catalepsy	A	A	A	A	A	A	A	A	A	A
Posture	N	N	N	N	N	N	N	N	N	N
Hanging	N	N	N	N	N	N	N	N	N	N
Retablismen	N	N	N	N	N	N	N	N	N	N
Flexion	N	N	N	N	N	N	N	N	N	N
Hafner	N	N	N	N	N	N	N	N	N	N
Mortality	A	A	A	A	A	A	A	A	A	A
Defecation	N	N	N	N	N	N	N	N	N	N
Urination	N	N	N	N	N	N	N	N	N	N
Respiration	N	N	N	N	N	N	N	N	N	N
Salivation	A	A	A	A	A	A	A	A	A	A
Vocalization	A	A	A	A	A	A	A	A	A	A
Tremor	A	A	A	A	A	A	A	A	A	A
Seizures	A	A	A	A	A	A	A	A	A	A
Writhing	A	A	A	A	A	A	A	A	A	A

A- Absent; N- Normal; ↓- Decrease

Table 2. Effect of eggshell powder on hematological paramaters of female rats

Parameters	Control	100 mg/kgBW	400 mg/kgBW	1000 mg/kgBW	Satellite Control	Satellite Dose of 1000 mg/kgBW
RBC (x10⁶/µl)	6.88±1.06	7.72±1.08	7.96±0.84	6.66±0.27	7.68±0.28	6.95±0.44
HGB (g/dl)	13.01±1.35	12.43±2.14	12.06±0.75	11.49±0.84	12.81±0.49	11.62±1.10
HCT (%)	39.04±4.05	37.30±6.42	35.78±3.06	34.46±2.51	38.44±1.49	34.86±3.31
MCV (fl)	57.38±4.74	48.00±4.51	45.32±5.96*	51.85±4.74	50.05±0.57	50.092±2.08
MCH (pg)	19.12±1.58	16.11±1.46	15.28±1.78	17.27±1.59	16.67±0.18	16.70±0.69
MCHC (%)	33.64±0.47	33.25±1.20	33.16±0.29	32.66±0.57	33.54±0.27	33.104±0.36
WBC (x10³/µl)	7.24±3.20	6.56±1.29	8.06±1.56	7.14±1.17	7.06±1.89	4.90±0.99
BAS (%)	0	0	0	0	0	0
EOS (%)	0.60±0.55	0.80±0.45	0.80±0.45	0.60±0.55	1	0.60±0.55
NEUT Stab (%)	1.8±0.84	1.8±0.83	1.2±0.45	2.2±0.84	2.8±0.84	3.2±0.84
NEUT Segmented (%)	35.20±7.15	27.60±6.50	27.60±9.02	36±4.74	30.60±9.15	26.60±9.34
LYMPH (%)	64±5.48	69.2±7.19	69.8±8.41	61±4.64	65±9.46	69±9.57
MONO (%)	0.40±0.55	0.60±0.55	0.60±0.55	0.20±0.45	0.60±0.55	0.60±0.55
Platelet (x10³/µl)	186±27.02	154±27.02	166±19.49	152±13.04	194±24.08	183±27.06

RBC = red blood cell count, HGB = hemoglobin, HCT = hematocrit, MCH = mean corpuscular hemoglobin, MCHC = mean corpuscular hemoglobin concentration, MCV = mean corpuscular volume, WBC = white blood cell count, LYMPH = lymphocytes, MONO = monocytes, BAS = basophils, EOS = eosinophils, NEUT = neutrophils. Data are shown as means ± standard deviations (n = 5). * = p < 0.05

Table 3. Effect of eggshell powder on hematological paramaters of male rats

Parameters	Control	100 mg/kgBW	400 mg/kgBW	1000 mg/kgBW	Satellite Control	Satellite Dose of 1000 mg/kgBW
RBC(x10⁶/µl)	7.29±0.46	7.68±0.72	7.60±0.76	7.33±0.90	7.52±0.73	7.46±0.58
HGB g/dl	12.62±0.69	12.68±0.31	11.44±1.46	11.61±1.01	12.23±1.47	12.53±1.28
HCT (%)	36.85±2.31	37.04±1.51	34.83±3.48	35.14±2.65	36.70±4.41	37.60±3.84
MCV (fl)	51.45±2.68	49.89±5.04	45.83±1.20^*	48.33±4.85	48.77±2.76	50.32±1.36
MCH (pg)	17.15±0.89	16.63±1.68	15.18±0.53	16.11±1.62	16.25±0.92	16.77±0.45
MCHC (%)	33.48±0.59	33.60±0.36	32.97±0.53	33.14±0.18	33.46±0.59	33.42±0.46
WBC (x10³/µl)	10.19±2.74	8.56±4.19	8.7±2.24	5.8±1.68	12.28±0.65	9.7±2.95
BAS (%)	0	0	0	0	0	0
EOS (%)	0.8±0.45	0.8±0.45	0.6±0.55	0.6±0.55	0.6±0.55	0.8±0.45
NEUT Stab (%)	2.2±0.84	3±1	2.6±1.34	1.6±0.89	2.6±1.14	2.2±1.30
NEUT Segmented (%)	35.8±7.95	35.6±7.27	31±8.49	32.2±10.94	34.4±13.35	25.8±9.88
LYMPH (%)	60.4±8.08	60±6.78	65±9.51	64.8±10.47	61.6±13.46	70.6±9.45
MONO (%)	0.8±0.45	0.6±0.55	0.8±0.45	0.8±0.45	0.8±0.45	0.6±0.55
Platelet (x10³/µl)	176±18.17	182±31.14	166±27.02	168±19.24	190±38.08	178±27.75

RBC = red blood cell count, HGB = hemoglobin, HCT = hematocrit, MCH = mean corpuscular hemoglobin, MCHC = mean corpuscular hemo- globin concentration, MCV = mean corpuscular volume, WBC = white blood cell count, LYMPH = lymphocytes, MONO = monocytes, BAS = basophils, EOS = eosinophils, NEUT = neutrophils. Data are shown as means ± standard deviations (n = 5). * = p < 0.05.

Table 4. Effect of eggshell powder on blood biochemical paramaters of male rats.

Parameters	Control	100 mg/kgBW	400 mg/kgBW	1000 mg/kgBW	Satellite Control	Satellite Dose of 1000 mg/kgBW
CHOL (mg/dl)	112.70±5.53	111.91±10.33	109.28±4.99	114.38±12.73	107.66±6.60	106.70±6.86
TG(mg/dl)	167.73±2.53	169.28±5.13	171.38±3.43	171.42±2.66	169.08±1.01	167.41±2.39
HDL (mg/dl)	60.2±1.48	54.06±4.74	58.22±3.11	61±2.45	56.26±5.91	57.34±5.059
LDL (mg/dl)	18.95±3.75	23.99±11.20	16.84±6.3	19.1±13.73	17.59±3.2	15.88±11.34
GLU (mg/dl)	117.3±27.76	76.46±24.35	102.64±24.83	97.68±30.54	95.25±28.26	113.75±12.09
UREA (mg/dl)	36.862±5.65	37.17±5.96	40.33±6.75	41.62±6.72	49.52±12.91	42.89±3.31
CREA (mg/dl)	0.92±0.039	0.77±0.05	0.81±0.07	0.84±0.13	0.9±0.08	0.77±0.07
SGOT (U/L)	40.14±9.79	65.00±16.02	34.31±9.54	63.45±12.98	57.12±11.74	54.91±15.46
SGPT (U/L)	25.13±6.53	25.39±2.68	19.41±475	35.36±23.98	32.75±10.61	25.64±9.33

GLU = glucose, CREA = creatinine, CHOL = cholesterol, TG = triglycerides, HDL =high density lipoprotein, LDL = low density lipoprotein, SGOT= serum glutamic oxaloacetic transaminase, and SGPT = serum glutamic pyruvic transaminase, Data are shown as means ± standard deviations (n = 5).* = p < 0.05.

Table 5. Effect of eggshell powder on blood biochemical paramaters of female rats. Data are shown as means ± standard deviations (n = 5).

Parameters	Control	100 mg/kgBW	400 mg/kgBW	1000 mg/kgBW	Satellite Control	Satellite Dose of 1000 mg/kgBW
CHOL (mg/dl)	126.94±4.23	111.56±4.48	123.91±4.09	120.48±10.41	132.09±7.45	112.49±10.91
TG(mg/dl)	153.09±16.12	141.35±13.52	152.70±12.45	177.72±7.68	134.60±16.82	161.30±12.70
HDL (mg/dl)	56.92±1.97	51.36±3.02	54.1±2.37	50.98±5.15	57.96±2.36	51.68±2.27
LDL (mg/dl)	39.40±2.6	31.93±5.02	39.27±1.4	33.95±7.16	47.2±9.187	28.55±10.60
GLU (mg/dl)	88.66±32.34	109.10±34.72	134.94±2.89	121.15±19.27	115.42±15.96	88.43±34.25
UREA (mg/dl)	44.41±2.7	41.73±3.8	41.78±2.5	43.27±4.17	40.33±3.27	40.42±2.35
CREA (mg/dl)	0.73±0.07	0.69±0.14	0.68±0.06	0.69±0.07	0.93±0.035	0.70±0.06
SGOT (U/L)	53.11±12.26	61.77±15.29	51.08±12.06	41.76±14.3	51.44±12.64	48.16±10.26
SGPT (U/L)	21.93±4.43	19.88±6.93	22.42±6.74	17.44±3.09	29.26±7.77	20.52±5.71

DISCUSSION:

The results of the acute toxicity study of eggshell showed that no mortality and no treatment-related clinical signs of toxicity were recorded in albino rats in two weeks after a single oral dose up to 5000mg/kgBW body weight. In this study, the RBC index (MCV) mostly changed significantly in female and male rats (Tables 2 and 3). MCV, or mean erythrocyte volume, is an index used in determining the size of red blood cells obtained from erythrocyte and hematocrit data. Iron deficiency, thalassemia, and secondary anemia are thought to cause anemia when MCV levels decrease below the normal range. Researchers believe that the decrease in MCV levels, which is significant, is caused by a reduction in hematocrit levels at a dose of 400 mg/kgBW in female and male rats. A decreased hematocrit indicates anemia, hemolytic reactions, leukemia, cirrhosis, profuse blood loss, and hyperthyroidism. A hematocrit that has decreased by 30% indicates moderate to severe anemia. Increased hematocrit can occur in erythrocytosis, dehydration, chronic lung damage, polycythemia, and shock. Hematocrit levels <20% can cause heart failure and death, whereas > 60% are associated with spontaneous blood clotting. Researchers believed that the decrease in MCV in female rats in the 400mg/kgBW dose group and male rats in the 400mg/kgBW dose group resulted from blood cell lysis caused by prolonged handling and storage time, rather than being less than 20%, indicating its safety.

The body weight weighing of the test animals was done to observe changes in body weight during the test. The change in body weight is an early indicator of the toxic effect of the test sample given. Test animals that experience toxicity generally lose weight due to decreased appetite. The daily feeding of test animals per animal is 10% of the animal's body weight. The test animals were fed Charoen Pokphand (CP 551), which has the highest level of protein at 18.50% - 20.50%, followed by water, fiber, fat, calcium, and phosphorus. The results of feeding can have an impact on increasing the body weight of female and male test animals for 28 days. The increase in body weight of the test animals did not have a significant difference. Therefore, exposure to eggshell powder for 28 days does not affect the body weight of the test animals.

The blood biochemical profile test showed a significant difference in the statistical test, but the parameters whose average exceeded the normal limit were cholesterol, LDL in female rats, and creatinine. This increase is thought to have been caused not purely by giving eggshells alone but due to the effect of high-fat feeding, namely Charon Phokpan 551. The biochemical profile of blood is very important to know or detect the toxic effects of eggshells due to cholesterol, HDL, LDL, and triglycerides being used as risk parameters for cardiovascular disease. The biochemical profile is to see liver function based on levels of serum glutamate oxidase and serum glutamate pyruvate transaminase, while kidney function is seen from the values of creatinine and urea. These results indicate that eggshells can still be considered safe for consumption as a calcium supplement.

Microscopic observations of heart preparations showed hypertrophy in several female rats and necrosis in several male rats. Hypertrophy can be caused by oxidative stress in mice, which increases reactive oxygen species (ROS), an essential factor in heart hypertrophy, or calcium in eggshells, which can make the blood vessels thicken and harden, thereby reducing the heart's elasticity^24,25. Necrosis is a common lesion in infarction in the heart organ, or can be caused by free radicals which will attack the mitochondria^26,27. The necrosis caused can be suspected because calcium from eggshells has entered the intracellular space and accumulates in damaged myocytes. Besides that, it can be suspected that there is a buildup of calcium salts in serum^27,28. The histological results of Liver showed hepatocyte necrosis, karyolysis, and hydropic degeneration in several female and male rats. Hepatocytes have several metabolic functions, such as the storage and regulation of intracellular calcium levels found in the endoplasmic reticulum. This section is susceptible to changes in its various functions. Besides that, in the mitochondrial part, physiological stress conditions can occur in excess calcium concentrations. This condition can be used as a suspicion for necrosis and homeostatic disorders that cause hydropic degeneration^29,30. The results of microscopic histology observations of kidney organs showed glomerular atrophy in the female and male rats. The changes are expected to have an adaptation mechanism to compensate for the lack of blood flow. Additionally, the disturbance may be attributed to the test animals' tendencies or habits. Their tendencies or habits are marked by the shrinking of the glomerulus in the bowman's capsule^31,32. The histology results show that there are no changes that occur in the spleen and lung organs of female and male rats in this study. Spleen’s histology is observed on the red and white pulp of all tested animal groups, and there’s no proliferation and an increase in these organs immune activity³³. The alveoli are visible still intact with endothelial cells around them, and the distance between the alveoli is still relatively tight^34,35,36.

CONCLUSION:

The research findings indicated that the acute and subchronical administration of eggshell for a duration of 14 days at doses of 2000 mg/kgBW and 5000mg/kgBW produced LD50 values exceeding 5000mg/kgBW in male and female Wistar rats. Furthermore, subchronically administering 100mg/kgBW, 400 mg/kgBW, and 1000mg/kgBW for 28 days did not induce any toxicological alterations in body weight, hematological, biochemical, and histological parameters.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

This study received financial support from Tanjungpura University through the DIPA Faculty of Medicine.

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35. Prashant KG. Chandradeo N. Kamal S. Arvind K. Brij MS. In vitro analysis of synergistic effect of honey against chemically induced hepatic insult in Balb/c mice. Research Journal of Pharmacy and Technology. 2021; 14(12): 6309-4. doi: 10.52711/0974-360X.2021.01091

36. Haider N. Yahya, Zahraa A. Okhti, Ruqaya M. Al-Ezzy, Ahmed A. Mhawesh, Afnan A. Alsaed. Protective activity of Ruta chalepensis methanolic extract against nephrotoxicity and testicular damage induced by Carbon tetrachloride on albino male mice. Research Journal of Pharmacy and Technology. 2021; 14(12): 6340-4. doi: 10.52711/0974-360X.2021.01096

Received on 01.01.2024 Revised on 11.05.2024

Accepted on 12.08.2024 Published on 24.12.2024

Available online on December 27, 2024

Research J. Pharmacy and Technology. 2024;17(12):5680-5688.

DOI: 10.52711/0974-360X.2024.00865

No.	Organ	Female	Male
1.	Heart	Dose of 1000 mg/kgBW Satellite Dose of 1000 mg/kgBW	Dose of 100 mg/kgBW Dose of 400 mg/kgBW	Dose of 1000 mg/kgBW
2.	Liver	Dosage of 400 mg/kgBW Dosage of 1000 mg/kgBW	Satellite Control Dose of 100 mg/kgBW Dose of 400 mg/kgBW	Dose of 1000 mg/kgBW Satellite Dose of 1000 mg/kgBW
3.	Kidney	Dose of 100 mg/kgBW Dose of 1000 mg/kgBW	Dose of 100 mg/kgBW Dose of 1000 mg/kgBW