28-Day repeated dose toxicity study of polyherbal gel formulation in Wistar rats

Introduction

It is well known that a variety of dosage forms, primarily PHF (Polyherbal formation), are used in the preparation of Ayurvedic herbals. Polyherbalism offers certain advantages not found in a single herbal formulation because of its synergistic effects. A single multiconstituent formulation can yield better therapeutic results (Parasuraman et al., 2014) [8] . Gels have been utilized in the current situation to deliver drugs to the body. For the treatment of oral ulcerative lesions, no therapy can be considered fully effective, despite the commercial availability of numerous formulations such as suspensions, ointments, and solutions. The use of polyherbal oral mucosal gel can increase the therapy's effectiveness. To prolong the duration of the medication's stay at the application site, the gel formulation's base must possess a satisfactory level of mucoadhesion. These medication delivery systems create close contact with the oral mucosal membrane, which speeds up the release of drug molecules at the site of absorption and improves the drug's bioavailability (Bhinge et al., 2017) [3] . But, it's crucial to test novel formulations for a specified period to assess potential toxic effects due to phytochemical interactions. Therefore, a Preclinical toxicity study of new drugs through in vivo testing is essential to ensure safety before before being tested in clinical trials on volunteers or patients. Hence, the present study explores the safety profile of the polyherbal gel developed with five herbs. sodium benzoate, triethanolamine and water (q.s). The gel preparation followed the method devised by Mate et al. (2021) [5] . Excepients and preservatives were added following the inclusion levels mentioned by Sheshkey et al.. Calculated amount of soaked gelling polymers, plant extracts and excepients were mixed thoroughly in the mortar and pestle. On addition of triethanolamine, the required bubble-free, homogenous gel consistency was achieved. Temperature and relative humidity were maintained between 22 and 26 o C and 30 and 70 per cent respectively. Light and dark cycles were controlled to give approximately a sequence of 12 hours of light and 12 hours of dark (Abebe, 2023) [1] . Animals were provided with ad libitum supply of feed and purified drinking water during the entire study period. Animals were acclimatized for seven days before the treatment and observed for clinical signs daily. On the day of initiation of the experimental trial, body weights were recorded and clinical examinations of animals were performed. Animals were identified by tail marking throughout the treatment period.

Experimental Animals

Experimental design

The repeated dose 28-day oral (gavage) toxicity study of polyherbal gel was conducted as per the OECD TG 407 (OECD 407, 2008) using 18 male and 18 female Wistar rats. The rats were randomly distributed to six different groups using the RAND function in Excel and it was ensured that the variation in body weight of the animals was not ≥ 20% (Tiwari et al., 2020) [12] . The treatment schedule for each group is detailed in the Table 1. [4] .

Body Weight

Body weight (g) was recorded weekly during the treatment period. On the day 28 of treatment, the animals were kept on water only fasting and on day 29 fasting weights were recorded before necropsy to calculate organ to fasting body weight ratio.

Feed Consumption

Feed consumption (g) was recorded for 24 h once daily for four weeks during the treatment period using an electronic weighing balance. Cage wise animal feed consumption (g) was recorded from the amount of feed offered and feed left over for 24 hours once every week. The feed consumption was normalized to 100g body weight of the animal.

Necropsy

Rats from the control and treatment groups were sacrificed at the end of the treatment period on day 29. The animals were kept for overnight fasting (12 -14 h) and euthanized by intraperitoneal administration of xylazine and ketamine at the dose rate of 10mg/kg b.wt and 80 mg/kg b.wt, respectively (Struck et al., 2011) [11] .

Necropsy was performed on all animals and tissues were collected for histopathology in 10 percent neutral buffered formalin.

Blood Collection

Terminal blood collection was done by cardiac puncture.

For the analysis of haematological parameters blood samples were collected in potassium EDTA-coated vacutainer and were analysed using autoanalyser (Exigo).

For serum biochemistry analysis, blood was collected in plain vacutainer. It was allowed to clot for about 20 min and then serum was separated by centrifuging 2655 x g for 10 min and were analysed using automated biochemical analyser (Selectra PRO XS, Netherland).

Gross Pathology

Gross pathological observations were made on all animals both externally and internally. The external examination included the examination of external surfaces and orifices while the internal examination included the examination of abdominal, thoracic and cranial cavities, various organs and tissues after opening the body.

Organ Weight

The organs such as liver, kidneys, heart, brain, spleen, ovary and testes were trimmed off to remove any adherent tissue or fat and weighed wet as soon as possible to avoid drying. The organ weights were converted into relative organ weights (Organ weight to body weight ratio). After weighing, the organs were fixed in 10 percent neutral buffered formalin.

Histopathology

The tissue samples were trimmed into a thickness of about 3-4 mm. Tissues were processed, embedded in paraffin, sectioned at 3-5 mm thickness and stained with haematoxylin and eosin (H&E) for histopathological examination (Slaoui and Laurence, 2011). The prepared slides were examined under the microscope to record histopathological findings in different organs. The severity grade of findings was recorded as minimal, mild, moderate, marked and severe. The lesions, if any, were identified to understand the toxic changes in organs.

Results and Discussion

The animals were observed for behavioural and any outward manifestations of the given formulation. From the results, it is evident that there was no observable behavioural and skin morphological changes observed in rats.

Tables 2 and3 present the body weight of male and female rats, respectively, expressed as mean±standard error. No significant (p>0.05) increase in body weight was observed in the high-dose group (1000 mg/kg b.wt). Similarly, no significant changes in body weight were noted in other groups. These findings contribute to the assessment of the repeated dose toxicity, and the observed increase in body weight in the high-dose group may warrant further investigation to understand its implications. Overall, the study provides valuable information on the potential effects of the polyherbal formulation at different dose levels.

Tables 4 display the feed intake (g/day/animal) of the male and female rat treatment groups, respectively. Notably, there were no significant differences in feed consumption among any of the treatment groups. Hematology values viz. hemoglobin, PCV, RBC, WBC, platelet, MCHC, MCH, MCV, MPV, RDW, granulocyte, lymphocyte and monocyte obtained for male and female rats are detailed in tables 5 and 6 indicate no significant differences among the treatment groups, suggesting that the polyherbal formulation had no notable impact on hematological parameters. Similarly, serum biochemistry parameters viz. creatinine, ALT, AST, albumin, globulin, total protein, cholesterol and glucose for males and females are outlined in Tables 7 and8 indicate no significant differences were observed across the treatment groups. The relative organ weight (liver, kidney, heart, lung, spleen, brain, ovary/testes) of the treatment groups and control groups were expressed as mean±SE of both male and females in the tables 9 and 10, respectively. There was no significant changes noticed in the control and polyherbal treated groups. The recovery group revealed no significant differences were observed between the control recovery group and the high dose (1000mg/kg/day) recovery group in hematology, serum biochemistry, body weights and relative organ weights. At the end of the study period, the animals were sacrificed and gross pathological examination exhibited no changes in all the organs. The histopathological evaluations were carried out from the control and high dose groups on both male and female rats. The lesions were showed in plate 1 and 2 and the identified lesions were systematically scored and presented in Tables 11 and12. The histopathology revealed mild changes in the lung and liver, and these changes were unrelated to the administered polyherbal formulation indicating their incidental nature. Transitioning to the recovery period, it becomes apparent that the control recovery group and the 1000mg/kg/day recovery group exhibited no significant differences across a range of key parameters. The determination of LD50 values greater than 2000 mg/kg in various studies underscores the non-toxic nature of polyherbal formulations. Moreover, the establishment of No Observed Adverse Effect Levels (NOAELs) higher than 1000 mg/kg body weight per day aligns with the results of the present study, which demonstrated a similar level of safety reports with the PHG (Tiwari et al. 2020) [12] . The findings from the study conducted revealed no significant treatment-related changes in gross and histopathological examinations (Bhide et al., 2022).

Conclusion

The 28-day repeated dose oral toxicity study with PHG in Wistar rats reaffirmed its safety profile, with no adverse clinical signs or mortality. There were no significant changes in body weight, feed consumption, hematology, clinical biochemistry, organ weight, or histomorphology, aligning with previous polyherbal formulation studies. The recovery period further demonstrated no significant differences between control and recovery groups. No Observed Adverse Effect Level (NOAEL) above 1000 mg/kg body weight was ensured. This means that at or below this dosage, there were no observable adverse effects, indicating a favorable safety profile.

Acknowledgement

AcknowledgementThe authors are thankful to The Dean, Veterinary College and Research Institute, Orathanadu for the financial support.

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