INTRODUCTION:

The use of dermal preparations has increased globally in the recent years which exhibit potential health hazards along with hypersensitivity and lethal intoxication. As a result, the basic requirement for any dermal preparation including cosmetic formulations is to determine its toxicity. Many of the dermal formulations are also available over-the-counter. For this reason, the safety profile of these preparations is necessary which can be performed by preclinical models like animals or cell lines.¹

Greater than 80% of World’s population depends on herbal products for their basic ailments. The medicinal values of plants are attributed to the secondary metabolites present in them such as alkaloids, tannins, phenolic compounds, flavonoids, etc. In many developing nations, herbal or traditional medicine is widely used for treatment of various diseases.²Pharmaceuticals manufactured from synthetic compounds are prone to adverse reactions. Moreover, it is the nature of human body to reject some chemical compounds which do not occur naturally.³ One of the major reasons for the popularity of the natural products is because of the belief that they are safe. As a result, assurance of quality, safety and efficacy of herbal medicine needs to be addressed which will also strengthen the acceptance of natural products by scientific community.⁴

Ziziphus oenoplia plant, belonging to the family Rhamnaceae is generally found in the hotter parts of India such as Topical Asia, Ceylon and Australia. It is used widely in Ayurveda for the treatment of various diseases like skin infections, diarrhoea, digestive disorders, diabetes, ulcer, wounds, liver problems, and others.⁵

In medical and pharmaceutical field, hydrogels have a variety of applications. They promote wound healing by producing a moist environment and also absorb the exudates from wounds. Thus, hydrogels are very suitable novel formulations for wound dressings.⁶The ethanolic and aqueous leaf extract of Ziziphus oenoplia is found to possess wound healing and anti-inflammatory activities which may be due to the presence of phytoconstituents like phenolic compounds and flavonoids.⁷In this research work, dermal toxicity studies are performed on formulated PVA-gelatin hydrogel sheet containing Ziziphus oenoplia leaf extract meant for enhanced wound healing activity in order to ensure that the formulation is safe to use on the skin and do not produce any skin reactions.

MATERIALS AND METHODS:

Animals:

The experiments were carried out on 15 healthy adult New Zealand white rabbits from Saha Enterprise (Register breeder CPCSEA). Single animal was housed per cage in standard cages (Size: 1½ ft x 1 ½ ft x 1 ft) having facilities for holding pellet feed in bowl and drinking water in water bottle fitted with stainless steel sipper tube or stainless-steel bowl. Animals were housed under standard laboratory conditions, air-conditioned with adequate fresh air supply (Air changes 12-15 per h), room temperature 17-23^oC, relative humidity 30- 70 %, with 12h light and 12 h dark cycle. Relative humidity and temperature were observed once daily. Animal feeding was done ad libitum throughout the period of acclimatization and experiment. Ghosh rabbit feed (Manufactured by M/S Rita Ghosh Private Ltd. Kolkata, West Bengal, India) was provided. Acclimatization was done for 7 d in laboratory conditions and was observed daily for any clinical signs and symptoms. Veterinary examination of all the animals was recorded on the day of receipt and randomization. Water was given ad libitum during acclimatization and experimental period. Water was purified by Pureit water purifiers made by Hindustan Unilever and was provided in polypropylene water bottles with stainless steel sipper tubes. All animal tests were carried out according to OECD Principles of Good Laboratory Practices, Schedule Y requirements of Drugs and Cosmetic Act with approval from the Institutional Animal Ethics Committee (IAEC) registration no. 1938/PO/Rc/S/17/CPCSEA.

Materials:

Solvents and reagents of analytical grade were used in the experiment.

Experimental design:

In this study the safety profile of the formulated hydrogel was observed by the parameters such as mortality/morbidity, skin reactions, change in body weight and food consumption according to the Organization for Economic Co-operation and Development guidelines following modern Good Laboratory Practices. The test item was applied topically on skin of dorsolateral trunk of Rabbits. The dermal route was selected to assess the toxic potential of the test item during dermal exposure. The hydrogel sheet was formulated by a method used by Pal in 2007. Briefly, required amount of HCl and gelatin was added to a 10% aqueous solution of PVA and stirred for 30 mins for esterification of PVA and gelatin. After sonication, glycerol was added and Ziziphus oenoplia leaf extract was mixed with it uniformly. The films were formed by conventional solvent casting method, washed with distilled water and stored for further studies.^[8]The pH of the formulation was measured by the method used by Khan in 2020. The pH of the freshly prepared formulation and that of the formulation after 30 d, 60 d and 90 d were determined.⁹ The pH was found to be stable and in accordance with the skin pH in all the cases (data not shown). This also ensured stability of the formulation.

Acute Dermal Toxicity Study:

On the day of the test, the fur on the animal’s back was clipped closely on both sides of the spinal column over a

size of 2/2 or 3/3inch large test area (approximately 10% of the skin surface) by taking care of mechanical irritation and trauma to the animals. Prior to dermal contact of the test item the lose hairs are removed; the skin was swabbed with dilute ethanol and dried. A total of 15 animals were divided into 5 dose groups (Control, Hydrogel film without extract, Hydrogel film with extract low, middle, and high) of rabbits as in Table 1. The test sample was applied over shaved area on the Rabbit’s skin (Fig 3). On the control group 0.9% NaCl solution was applied. Dermal application was performed once over a period of 3 d. After 72 h dermal contact site was thoroughly observed for evidence of any tissue reaction such as Erythema, edema and necrosis. The skin was swabbed lightly with dilute ethanol to facilitate reading of dermal contact sites. The animals were observed during the time intervals of 24 h, 48 h, and 72 h after dermal contact of the formulations for evaluating the skin reaction, food consumption and body weight variations. The observations were based on a numerical scale for the sample and for the blank. Fur was reclipped during the observation period. The average erythema and edema scores for sample and blank sites were determined at every scoring interval for each rabbit. All the animals were observed once daily for clinical signs and twice daily for mortality/morbidity till the termination of the experiment. Individual animal body weights and food consumption were recorded on 0 d to 3 d of every 24h periods of the study.

Table 1: Allocation of Animals to Various Groups

Group No.	Dose	Sex	No. of Animals in each group	Animal Nos.
I	Control	Male and Female	3	1 - 3
II	Hydrogel without extract 4g/kg	Male and Female	3	4 - 6
III	Hydrogel with extract Low Dose 1g/kg	Male and Female	3	7 - 9
IV	Hydrogel with extract Middle Dose 2g/kg	Male and Female	3	10 - 12
V	Hydrogel with extract High Dose 4g/kg	Male and Female	3	13 - 15

The following observations were undertaken during the study.

Clinical Signs and Mortality of rabbits:

Animals were observed once daily for clinical signs and twice daily for mortality/morbidity till the termination of the experiment. The skin reactions were observed and recorded according to Draize (1959) method. The method of evaluation of the skin reactions is presented below in Table 2. Edema formation scoring was identified as in Table 3.

Table 2: Skin Reactions Scoring

Erythema and Eschar Formation:	Scoring
No erythema	0
Very slight erythema (barely perceptible)	1
Well-defined erythema	2
Moderate to severe erythema	3
Severe erythema (beet redness) to slight eschar formation	3
Injuries in Depth	4
Maximum possible	4

Table 3: Edema Formation Scoring

Edema Formation	Scoring
No edema	0
Very slight edema (barely perceptible)	1
Slight edema (edges of area well defined by definite raising)	2
Moderate edema (raised approximately 1 mm)	3
Severe edema (raised more than 1 mm and extending beyond Area of exposure)	4
Maximum possible	4

Body Weight of rabbits:

The weight of each rabbit was recorded time to time throughout the course of the study. The group mean body weights were calculated.

Food Consumption of rabbits:

The quantity of food consumed by groups consisting of three rabbits each was recorded in g/kg body weight/d.

RESULTS:

1. Clinical Signs and Mortality:

No clinical signs of irritation such as edema or erythema were observed for both the control as well as treated groups of rabbits throughout the study. The animals did not show any morbidity or mortality, or any signs of toxicity as represented in tables 4, 5 and 6.

Table 5: Mortality

Group No.	Dose	Mortality
Group No.	Dose	Absolute	Relative
I	Control	0/3	0
II	Hydrogel without extract 4g/kg	0/3	0
III	Hydrogel with extract Low Dose 1g/kg	0/3	0
IV	Hydrogel with extract Middle Dose 2g/kg	0/3	0
V	Hydrogel with extract High Dose 4g/kg	0/3	0

Table 6: Skin Reactions

Gr. No.	Dose	Hours(h)
Gr. No.	Dose	24	48	72
I	Control	-	-	-
II	Hydrogel without extract 4g/kg	-	-	-
III	Hydrogel with extract Low Dose 1g/kg	-	-	-
IV	Hydrogel with extract Middle Dose 2g/kg	-	-	-
V	Hydrogel with extract High Dose 4g/kg	-	-	-

- Means no reaction occur, + Means reaction occur.

Table 7: Group Mean Body Weight (g)

Gr. No.	Dose		Hours (h)
Gr. No.	Dose		24	48	72
I	Control	Mean	2182.90	2188.63	2192.80
I	Control	± SD	46.95	43.89	44.19
II	Hydrogel without extract 4g/kg	Mean	2114.60	2118.90	2156.63
II	Hydrogel without extract 4g/kg	± SD	46.41	47.10	14.15
III	Hydrogel with extract Low Dose 1g/kg	Mean	2121.70	2125.23	2129.77
III	Hydrogel with extract Low Dose 1g/kg	± SD	48.08	48.08	46.63
IV	Hydrogel with extract Middle Dose 2g/kg	Mean	2176.43	2206.67	2236.70
IV	Hydrogel with extract Middle Dose 2g/kg	± SD	56.56	59.15	60.24
V	Hydrogel with extract High Dose 4g/kg	Mean	2221.40	2251.27	2277.27
V	Hydrogel with extract High Dose 4g/kg	± SD	25.39	28.82	31.21

2. Body Weight:

Table 7 and Figure 1 shows body weight gain of all animals of Control, Hydrogel Film without extract and Hydrogel Film with extract dose groups throughout the dosing period of 24 h, 48 h, 72h. After reviewing the body weight of different dose groups of animals it was observed that all the animals gained normal body weight throughout the study period and there was no significant difference in the body weight gain of any dose group of animals. From this it can be observed that the body weights of the male and female rabbits were not affected after the application of the hydrogel formulations or control.

Fig 1: Graphical representation of group mean body weight between different doses of dermal toxicity patch test in rabbit.

3. Food Consumption:

The mean food consumption (g/kg body weight/d) is represented in Table 8 and Figure 2. There was no significant difference in food consumption in all the five groups of rabbits throughout the study.

Table 8: GROUP MEAN FOOD CONSUMPTION (g/ animal)

Gr. No.	Dose		Hours(h)
Gr. No.	Dose		24	48	72
I	Control	Mean	21.7	22.2	22.6
II	Hydrogel without extract 4g/kg	Mean	23.6	24.1	24.5
III	Hydrogel with extract Low Dose1g/kg	Mean	27.2	27.9	28
IV	Hydrogel with extract Middle Dose 2g/kg	Mean	25.3	25.8	26.4
V	Hydrogel with extract High Dose 4g/kg	Mean	21.7	22.2	22.6

Fig 2: Graphical representation of group mean food consumption between different doses of dermal toxicity patch test in rabbits

Fig 3: Application of hydrogel on dorsolateral trunk of New Zealand white rabbit

DISCUSSION:

In the present research work skin irritation studies, mortality and morbidity, change in body weight and food intake of the New Zealand white rabbits treated with hydrogel sheet without extract, hydrogel with extract (low, middle and high doses) and control were observed. Similar toxicity studies were carried out by Chakrabarty in 2018 for silver sulfadiazine-bFGF incorporated hydrogel formulation for partial thickness burn wounds. Skin irritation, sensitization, dermal toxicity, terminal body weight, food and water consumption and eye irritation tests were carried out to confirm the safety profile of the hydrogel formulation.¹⁰

Xu and team evaluated the safety profile of Triptolide- loaded hydrogel- thickened microencapsulation meant for treatment of rheumatoid arthritis. Acute toxicity studies and skin irritation tests for the hydrogels have been carried out in rabbits and no obvious toxicity has been observed for all the groups of animals.¹¹Sildenafil Citrate loaded hydrogel was formulated by Kulshrestha in 2019 and wound healing activity and dermal toxicity studies were performed on rats. The studies revealed that the formulations were devoid of any toxic effects and were safe to be used.¹²Zehang L and team of researchers developed a highly stable recombinant human acidic fibroblast growth factor (rh-aFGF) carbomer 940 hydrogel for wound healing. Rh-aFGF carbomer 940 hydrogel was applied topically for 28 d on New Zealand rabbits at a daily dose of 900 IU/cm², 1,800 IU/cm², and 3,600 IU/cm². Results revealed that food intake, body weight, body temperature, heart rate, and eye examinations were all normal, suggesting no obvious toxicity induced by the rh-aFGF hydrogel.¹³

Toxicity studies were performed on herbal formulations of hydrogels too. Lodhi and team performed dermal toxicity study of Basil seed loaded hydrogel on rats and rabbits which revealed safety of the hydrogel formulation from the results for different tests like change in body weight of the animals, food and water consumption, etc. Also, skin irritation tests revealed no allergy or irritation on the rabbit skin ensuring safety of the formulation.¹⁴Acute dermal toxicity study was conducted on Linseed hydrogel formulated by Haseeb and group of researchers in 2018 applied on male albino rabbits. It was found that all the animals survived and no other clinical findings or abnormalities were observed during the total course of study.¹⁵It has also been found in another study that Carbopol hydrogel formulations containing Ziziphus leaf extract was found to produce no dermal toxicity on New Zealand White rabbits.¹⁶ Ziziphus herbal extract and its formulations was observed to have promising pharmacological activities.¹⁷

the present study, no drastic change in body weight, food consumption was observed in all the treated groups of animals and no signs of skin irritation or mortality were visible. Thus, considering all the data obtained it can be inferred that the hydrogel formulations were non-irritating and non-toxic in pre-clinical setting.

CONCLUSION:

In the current research work it was observed that the hydrogel film without extract, extract incorporated films and the control did not show any clinical signs of mortality, skin irritation or dermal toxicity and no significant change in food habits or body weight was found in all the treated groups of rabbits. From the results obtained from the study it can be concluded that the formulated hydrogel films were non-toxic and safe to be used as dermal preparation.

AKNOWLEDGEMENT:

The authors are grateful to Department of Pharmaceutical Technology, JIS University and TAAB Biostudy Services, Jadavpur, Kolkata, India for providing all the facilities, equipment and materials for conducting the study.

AUTHORS CONTRIBUTIONS:

All the authors have contributed equally.

CONFLICTS OF INTERESTS:

The authors report no financial or any other conflicts of interest in this work.

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Received on 18.05.2024 Revised on 08.01.2025

Accepted on 26.05.2025 Published on 01.12.2025

Available online from December 06, 2025

Research J. Pharmacy and Technology. 2025;18(12):5645-5650.

DOI: 10.52711/0974-360X.2025.00815

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Group No.	Dose	Observed Signs	Total Nos. of Animals	Animal Nos.	Period of signs in days from - to	Mortality
I	Control	NIL	3	1 – 3	1 - 3	0/3
II	Hydrogel without extract 4g/kg	NIL	3	4 – 6	1 - 3	0/3
III	Hydrogel with extract Low Dose 1g/kg	NIL	3	7 – 9	1 - 3	0/3
IV	Hydrogel with extract Middle Dose 2g/kg	NIL	3	10 – 12	1 - 3	0/3
V	Hydrogel with extract High Dose 4g/kg	NIL	3	13 – 15	1 - 3	0/3