INTRODUCTION:

The advancement of the sustainable material is very important topic of concern for today and future. The production of energy and plastic from fossil fuel has several limitations. It will nearly exhaust soon. For the ecology concerns, as oil resources are depleting, the interest in the development of eco-friendly materials from renewable natural resources is increased.¹ Rosin is the one of most important renewable substance. Rosin or colophony is a natural and abundantly available polymeric material derived from pine tree. It was widely used in the paper, coating, printing ink, polymer, food industries etc. It acts as a precursor for flux in soldering.² The rosin contains about 90% of monocarboxylic acid (of alkylated hydrophenanthrene), rosin acid (molecular formula C₂₀H₃₀O₂).

The major rosin acids include abietic acid which contains tertiary carboxylic acid (-COOH) and conjugated double bonds and pimaric acid with non-conjugated double bonds. The rosin acid is containing two chemically reactive centers namely -COOH group and double bond. They are considered as safe, biodegradable and biocompatible.^3,4 These are soluble in alcohol, benzene, chloroform and ether. It is insoluble in water. Polyethylene glycol (PEG) or poly (ethylene oxide) is a petroleum base polymer represents water-soluble monohydroxy alcohols. These are used as a base in ointments and suppositories, as a plasticizer in film coating, as a auxiliary emulsifier, as flux vehicle in WSFs for electronic industrial etc.^4-6 But the natural polymers remains a choice of matter because of their low cost, quick availability, capacity of undergoing several chemical transformations and biological safety.

The rosin and its derivatives were employed for microencapsulating model drug.^7-9 They were used as an anhydrous binder and matrix former in conventional and sustained release formulations respectively.^10-12 They were exploited as a pharmaceutical aid in chewing gum and a base in cosmetics. The in-vivo study proved that, the glyceryl ester of rosin is biodegradable in-vivo.¹³ Several articles show their in paints and varnishes due to their film forming abilities. They have been successfully employed for enteric and delayed release coating of dosage forms.^14-16

Assessment of the safety and toxicity of any substance before its use in pharmaceuticals is very important. Therefore, the present investigation was executed with the objective of synthesis and assessment of sub-acute toxicity of novel rosin esters prepared from natural rosin and polyethylene glycol 200 using Swiss Albino mice as an animal model.

MATERIALS AND METHODS:

Materials:

Rosin N grade was purchased from Modern Chemicals, India. Polyethylene glycol 200 and maleic anhydride (MA) were purchased from Qualigens, India, and S.D. Fine Chemicals, India, respectively. Corn oil was purchased from local market, Amravati, India. All the other chemicals used were of synthesis grade and used as received.

Methods:

Synthesis of rosin esters:

Accurately weighed rosin was added to a glass reactor and warmed to 220-240°C with heating mantle. The required amount of PEG 200 was added drop wise in the molten mass of rosin and mixed with stirrer at 500rpm. The temperature of the mixture was maintained about 220-240°C till the completion of reaction. The zeolite modified nickel was used as a catalyst at 2% of rosin level.¹⁷ The melt was allowed to cool to 140-160°C. The required quantity of maleic anhydride was added to the melt. The reaction was allowed to proceed further for 1 h. The molten mass was poured on tar plates. The resultant rosin ester (RE) was dried, powdered and washed completely with distilled water. The various rosin esters were synthesized by varying the molar ratios of PEG: rosin as 9:1, 11:1, 13:1 and 15:1. The resultant rosin esters were air-dried and used for further study.^18,19

Experimental animals:

The young healthy Swiss Albino mice of both sexes (age of 7-8 weeks) and weighing around 25-30 g were selected. The selected female mice were nulliparous and non-pregnant.²⁰ The mice were housed in autoclaved standard polypropylene IVC cages. Autoclaved paddy husk were used as bedding material and changed at least twice a week. The six mice of either sex were housed per cage. Every day, the floor of the experimental room was mopped with disinfectant solution. The mice were kept in a clean environment and maintained 12 h light/dark cycle. The air was conditioned at 25±2°C, 45-60% relative humidity (RH) with 100 % exhaust facility. The mice were feed using pelleted rodent feed (procured from Amrut feeds, Pune, India) and fresh water ad libitum in polypropylene bottles with a stainless-steel sipper tube during the study. They were allowed to acclimatize the conditions of laboratory for two weeks before begin of experiment.^21,22 The experiments were conducted in accordance with ethical norms and the protocols of toxicity study were reviewed and approved by Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) and Institutional Animal Ethical Committee (IBBSCOP/ IAEC/06/2018, dated: 01.09.2018).

Toxicity study:

The sub-acute toxicity study was executed according to Organization for Economic Co-operation and Development (OECD) 407 and 423 guideline for testing of chemicals.²³ The Swiss Albino mice model was employed for study due to ease in extrapolating data with human. Exclusive literature can support the use of rodents in the toxicity study. The mice were grouped into 5 groups. Each group was containing 6 male and 6 female mice which were devoid of any disease symptoms. The first group was kept as control group, where the mice were given the vehicle (corn oil) orally. The group II, III, IV and V were treated with RE-I, -II, -III and -IV respectively. They were numbered 1, 2, 3, 4, 5 and 6 by marking head, body, tail, body-tail, leg and no marked respectively with picric acid. After fasting period over, the body weights of all the mice was recorded. The sufficient water was provided to mice during fasting period.²⁴ The group number, cage number, gender and animal number were labelled as in Table 1.

Table 1: Animal grouping, identification and treatment

Group	Cage	Gender	Animal number	Treatment	Route of administration
I	1	Male	1‒6	Corn oil	Oral, 28 d
I	2	Female	7‒12	Corn oil	Oral, 28 d
II^†	3	Male	13‒18	RE-I + Corn oil	Oral, 28 d
II^†	4	Female	19‒24	RE-I + Corn oil	Oral, 28 d
III^†	5	Male	25‒30	RE-II + Corn oil	Oral, 28 d
III^†	6	Female	31‒36	RE-II + Corn oil	Oral, 28 d
IV^†	7	Male	37‒42	RE-III + Corn oil	Oral, 28 d
IV^†	8	Female	43‒48	RE-III + Corn oil	Oral, 28 d
V^†	9	Male	49‒54	RE-IV + Corn oil	Oral, 28 d
V^†	10	Female	55‒60	RE-IV + Corn oil	Oral, 28 d

^†Each group was treated with 25 mg/kg of RE in constant volume of corn oil.

The dose was administered orally because rosin and rosin esters have been used as pharmaceutical aid in oral drug delivery systems. The dose was calculated in accordance to body weight.²⁵ The rosin esters were administered orally using an intubation needle (stainless steel, 16 G) fitted into a polypropylene disposable syringe. The dose of rosin ester was adjusted according to its weekly body weight. The dose volume was not exceeding 10 ml/kg of its body weight.²⁶ The food was provided to mice after an hour of administration of oral dose.²⁷ After study period, all animals were sacrificed on the day 29.

Study observations:

Body weight (BW):

The body weight of mice was recorded a day before the initiation of treatment. At the end of study, the final body weight of mice was recorded a day prior to blood collection. The body weight of mice was monitored weekly.²⁸

Food and water consumption:

The quantity of the feed and volume of water offered based on the requirement of the each animal in each cage per week were recorded. The leftover weight of feed and volume of water were recorded weekly. The weight of feed and volume of water consumed i.e., per animal/week were calculated by subtraction of the left over feed from the total quantity of feed and water provided during the week.^29,30

Clinical signs:

All the mice were observed daily for any abnormal clinical signs and behavioral changes. The appearance, changes and disappearance of clinical signs were recorded for 28 d. The mice showing signs of pain and other severe signs were humanly killed. The cage side changes were recorded related to skin, fur, eye and mucous membrane. The mice were observed for autonomic changes like lacrimation, piloerection, pupil size, abnormal respiratory pattern, posture, gait, response to handling, presence of tonic or clonic movement, stereotype (excessive grooming and repetitive circling) or bizzared behavior (self-mutilation, walking backwards etc.).^31‒33

Mortality and morbidity:

All the mice were observed daily for mortality and morbidity during the study period.³⁴

Blood sample collection and processing:

After the treatment period of 28 d, the blood samples were collected from all the mice by providing ether anesthesia on the next day. About 5ml of blood sample was collected from the retro-orbital sinus or cardiac puncture of each mouse. About 2.5ml blood was transferred to 5ml capacity micro-centrifuge tube containing sodium citrate solution as an anticoagulant. The remaining 2.5ml blood was transferred to the tube without citric acid. The former blood samples were utilized for haematological analysis. The tube without citric acid containing blood samples were used for the biochemical analysis.³⁵

Haematological studies:

The haematological parameters were estimated using automated hematic analyser (ADVIA-120, Siemens, India). The haematological parameters like Red Blood Cells (RBC), White Blood Cells (WBC), Hemoglobin (Hb), Haematocrit (HCT), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Total Leukocyte Count (TLC) and Differential Leukocyte Count (DLC) in both sexes of all treatment groups and control group were determined and compared.³⁶

Biochemical studies:

The biochemical parameters were estimated using automatic analyser (Hitachi 901, Japan). The biochemical parameters like glucose, creatinine, albumin, total protein, urea, total bilirubin, cholesterol, triglycerides, Aspartate Amino Transferase (AST), Alanine Amino Transferase (ALT) and Alkaline Phosphatase (ALP) were estimated in study animals of both sexes. The results of all treatment groups were compared with control group.^37-39

Necropsy and organ weight:

All living male and female mice of all treatment groups were euthanized on day 29 using carbon dioxide gas. After the blood collection, mice were sacrificed by cervical dislocation and the vital organs (liver, kidney, heart, spleen and lung) removed through a midline incision in the mice abdomen. The organs were cleaned of fat and blotted with clean tissue paper and then weighed on balance. Necropsy and related findings were determined for all mice including the dead and moribund animals. The relative organ weight (ROW) of brain, spleen, thymus, pancreas, lungs, adrenals, lungs, heart, liver, kidneys, testies, ovaries and uterus were calculated in each animal. The paired organs were weighed together.⁴⁰

Absolute organ weight

Relative organ weight =------------------------------ × 100

Body weight at sacrifice

Histopathological recordings:

The organs from all the dead, moribund or sacrifices or terminally sacrificed animals belonging to control and treatment groups were preserved in 10% formalin solution. The histopathological examination of these organs was performed for animals belonging to control and treated groups. The organs were fixed in 10% buffered formalin, routinely processed and embedded in paraffin wax. The paraffin sections (5μm) was cut on glass slides and stained with hematoxylin and eosin. The sections were analyzed by an experienced pathologist without any idea about the experimental study. The slides were examined under a light microscope (Nikon E50i, Nikon Corporation, Japan) as mentioned elsewhere.^41-44

Statistical analysis:

All the study data were processed to get group mean and standard deviation. The statistical analysis was performed using Student’s t-test for the comparison of treated groups with the control group for different parameters.⁴⁵

RESULTS AND DISCUSSION:

The present study was performed as per the CPCSEA and OECD (Organization for Economic Co-operation and Development) guidelines 423 and 407 for testing of chemicals. The mice in the control and treated groups were administrated with vehicle and rosin esters respectively and observed for various parameters.

Body weight:

The body weight of each mouse was recorded a day before the treatment and at the interval of week throughout the study. The final body weight of each mouse was also recorded a day prior to blood collection. No treatment related adverse effects on weekly body weight were found in the mice of both sexes. Increase in body weight and growth of treated animals were observed in the similar pattern as that of control group. The body weight of the mice was found to increased, indicates the rosin esters does not show any adverse effect on the growth of the animals (Table 2).

Food and water consumption:

The determination of food intake and water consumption by mice is important to understand the safety of products. The food and water consumption in all study groups were increased in similar pattern with control group. No gender-based changes in food and water consumption were observed. The increased food and water consumption was matches with the growth and increase in body weight of those particular groups (Table 2).

Clinical signs:

The mice were monitored on daily until day 28 for any toxic signs and mortality. During study, the mice which were orally administrated with rosin esters at 25 mg/kg does not showed overt signs of distress or any toxicity signs. The mice were devoid of pain or other severe signs. No cage side changes associated with skin, fur, eye, and mucous membrane were recorded. No autonomic changes related to lacrimation, piloerection, pupil size, abnormal respiratory pattern, posture, gait, response to handling, presence of tonic or clonic movement, stereotype (excessive grooming and repetitive circling) or bizzared behavior (self-mutilation and walking backwards) were recorded in any animal.

Mortality and morbidity:

The treatment group and control group mice were observed for physical changes. After daily observation in life phase revealed no death or treatment related abnormal clinical signs either in control or in treatment groups of both sexes (Table 2).

Table 2: Weight gain, feed-water intake, clinical sign and mortality and morbidity observed during study

Parameter	Gender	Control I	Group II	Group III	Group IV	Group V
Initial body weight (g)	Male	26.12 ±1.28	26.08 ±0.63	25.94 ±1.03	25.17 ±0.73	25.97 ±1.36
Initial body weight (g)	Female	25.67±0.54	25.52±0.61	25.33±0.37	25.07±0.18	25.64±0.51
Final body weight (g)	Male	28.36 ±1.03	28.14±1.11	27.98 ±0.83	27.05 ±0.14	28.21 ±1.28
Final body weight (g)	Female	27.44±0.84	27.39±0.92	26.89±0.57	26.79±0.61	27.13±1.04
Food consumed per animal (g/week)	Male	18.03±0.18	17.65±0.64	17.93±0.73	18.12±1.08	18.81±0.19
Food consumed per animal (g/week)	Female	16.38±0.26	16.67±1.02	16.46±1.12	17.45±0.32	17.19±0.77
Water consumed per animal (ml/week)	Male	23.68±1.11	23.87±0.98	23.88±0.84	24.23±1.07	23.68±1.13
Water consumed per animal (ml/week)	Female	24.45±0.83	24.98±0.56	24.83±0.67	25.12±1.13	25.07±0.32
Clinical sign	Male and Female	No sign	No sign	No sign	Itching nose	No sign
Mortality and morbidity	Male and Female	Not observed	Not observed	Not observed	Not observed	Not observed

All the values were reported as mean ± SD for (n=6) determination.

Table 3 A: Haematological parameters of mice after treatment of rosin esters on the day 29

*Group*	M/F	*RBC’s* *(10⁶/µl)*	*HCT* *(%)*	Hb *(mg/dl)*	*MCV* *(fl)*	*MCH* *(pg)*	*MCHC* *(g/dl)*	*Platelets* *(10⁶/µl)*
I	M	10.51±0.13	48.1±0.19	14.3±0.21	47.91±0.66	14.25±0.35	29.75±0.42	1308±49.71
I	F	9.53±0.12	46.33±0.41	13.63±0.13	48.55±0.48	14.35±0.26	28.54±0.3	1262±44.63
II	M	9.64±0.14	46.66±0.24	13.54±0.24	48.41±0.69	14.01±0.33	28.2±0.39	1154±93.19
II	F	8.93±0.13	45.75±0.49	13.66±0.16	51.21±0.63	15.33±0.32	29.51±0.38	1170±54.53
III	M	9.37±0.09	45.1±0.91	13.03±0.31	48.06±0.56	13.83±0.22	28.85±0.16	1154±65.58
III	F	9.05±0.25	45.35±1.23	13.41±0.35	50.13±0.73	14.63±0.22	29.85±0.15	1067±89.81
IV	M	9.43±0.21	46.04±1.25	13.48±0.35	48.73±0.25	14.26±0.12	29.3±0.15	1157±83.78
IV	F	8.99±0.12	44.75±0.64	13.45±0.19	49.57±0.52	14.23±0.22	29.3±0.28	1210±62.3
V	M	9.51±0.12	45.12±1.02	13.02±0.32	48.71±0.21	13.84±0.11	24.5±0.12	1149±81.23
V	F	9.02±0.13	43.21±0.52	13.16±0.21	49.76±0.75	14.23±0.21	29.45±0.21	1230±65.21

All the values were reported as mean ± SD for (n=6) determination.

Table 3B: Haematological parameters of mice after treatment of rosin esters on the day 29

*Group*	*M/F*	*TLC (10³/µl)*	*Neutrophils (%)*	*Lymphocytes (%)*	*Monocytes (%)*	*Eosinophiles (%)*	*Basophiles (%)*
I	M	4.73±0.43	19.53±2.29	76.66±2.11	0.46±0.04	1.25±0.16	0.25±0.042
I	F	4.66±0.56	17.58±2.33	72.69±1.92	0.63±0.14	2.31±0.16	0.15±0.04
II	M	4.83±1.06	22.68±3.75	71.83±1.39	1.31±0.82	1.36±0.28	0.18±0.03
II	F	5.66±0.61	18.23±2.43	71.83±0.93	0.76±0.13	1.51±0.21	0.26±0.03
III	M	4.42±0.53	21.58±0.97	73.78±2.74	0.45±0.04	1.36±0.17	0.25±0.04
III	F	4.79±0.71	17.09±2.11	73.78±1.08	0.40±0.05	3.1±1.22	0.21±0.04
IV	M	5.38±0.54	27.8±1.23	65.31±1.66	1.45±0.99	1.85±0.18	0.2±0.02
IV	F	4.42±0.34	19.25±2.43	65.31±1.45	0.45±0.05	1.81±0.29	0.3±0.03
V	M	5.21±0.58	26.51±2.73	65.24±1.47	1.41±0.04	1.56±0.15	0.21±0.03
V	F	4.54±0.39	19.14±2.45	68.24±2.65	0.77±0.12	2.42±0.32	0.28±0.02

All the values were reported as mean ± SD for (n=6) determination.

Table 4: Biochemical parameters of mice after treatment of rosin esters on the day 29

Parameter	M/F	Group I	Group II	Group III	Group IV	Group V
Glucose (mg/dl)	M	103.62±4.31	104.09 ±3.72	102. 41±4.05	103.17±2.56	103.62±4.31
Glucose (mg/dl)	F	99.64 ±2.63	100.24±2.19	101.11±0.34	99.23±3.33	98.97±3.07
Total protein (g/dl)	M	6.57±1.13	6.48±0.93	6.63±2.09	6.67±0.84	6.58±0.53
Total protein (g/dl)	F	6.13±0.87	6.09±0.76	6.11±0.09	6.23±0.83	6.19±0.44
Albumin (g/dl)	M	3.32±0.06	3.28±0.04	3.34±0.05	3.37±0.06	3.35±0.02
Albumin (g/dl)	F	3.09±0.05	3.11±0.03	3.17±0.04	3.15±0.03	3.13±0.05
Uric acid (mg/dl)	M	4.45 ±0.38	4.48±0.16	4.51±0.13	4.52±0.08	4.44±0.06
Uric acid (mg/dl)	F	4.09±0.26	4.11±0.23	4.18 ±0.11	4.15±0.14	4.17±0.12
Urea (mg/dl)	M	16.50±2.74	15.67±2.71	16.33±2.09	14.93±3.60	15.67±2.16
Urea (mg/dl)	F	15.67±2.87	16.83±3.19	15.66±0.09	18.17±2.86	16.50±3.08
Creatinine (mg/dl)	M	0.87±0.12	0.86±0.02	0.89 ±0.11	0.87±0.07	0.88±0.14
Creatinine (mg/dl)	F	0.81±0.09	0.79±0.08	0.78 ±0.07	0.82±0.06	0.80±0.05
Total bilirubin (µmol/l)	M	0.87±0.05	0.88±0.04	0.89±0.04	0.90±0.03	0.86±0.04
Total bilirubin (µmol/l)	F	0.81±0.03	0.83±0.03	0.84±0.02	0.82±0.01	0.83±0.04
Direct bilirubin (µmol/l)	M	0.73±0.02	0.72±0.04	0.74±0.01	0.75±0.02	0.72±0.03
Direct bilirubin (µmol/l)	F	0.71±0.01	0.70±0.02	0.72±0.02	0.73±0.02	0.71±0.01
Cholesterol (mg/dl)	M	194.87±11.32	196.14±9.19	197.15±10.35	197.02±8.84	194.13±7.43
Cholesterol (mg/dl)	F	196.14±9.67	200.09±6.33	201.84±8.56	208.29±6.27	203.41±6.63
Triglycerides (mg/dl)	M	132.12±0.48	135.56±1.18	133.12±2.48	138.38±1.81	134.12±1.48
Triglycerides (mg/dl)	F	131.46±0.63	133.95±2.42	132.39±3.47	134.25±0.23	133.83±3.02
AST (IU/l)	M	102.25±4.75	103.38±3.23	104.63±4.56	100.98±4.11	101.41±3.75
AST (IU/l)	F	100.56±8.19	99.83±2.26	102.44±3.55	98.97±2.21	100.75±0.78
ALT (IU/l)	M	41.82±4.37	42.70±3.38	42.48±3.41	42.40±4.03	41.52±1.24
ALT (IU/l)	F	38.90±2.32	39.27±4.22	40.50±4.23	39.20±3.95	38.95±0.96
ALP (IU/l)	M	98.67±11.32	99.12±9.87	98.88±10.29	100.07±10.69	99.87±11.65
ALP (IU/l)	F	97.35±9.29	96.83±9.34	97.68±8.73	98.06±7.11	98.44±9.93

All the values were reported as mean ± SD for (n=6) determination.

Table 5: Effect of oral administration of rosin esters on relative organ weight (g) of mice

Organ	M/F	Group I	Group II	Group III	Group IV	Group V
Brain	M	1.62±0.02	1.63±0.11	1.59±0.09	1.64±0.05	1.62±0.02
Brain	F	1.71±0.08	1.81±0.05	1.72±0.05	1.76±0.07	1.77±0.08
Spleen	M	0.76±0.04	0.75±0.06	0.76±0.04	0.74±0.02	0.76 ± 0.05
Spleen	F	0.71±0.06	0.79±0.05	0.71±0.03	0.73±0.04	0.74 ± 0.02
Adrenals	M	0.06±0.02	0.06±0.02	0.05±0.02	0.06±0.02	0.06±0.02
Adrenals	F	0.07±0.02	0.07±0.02	0.06±0.02	0.07±0.02	0.07±0.02
Lungs	M	8.74±0.19	8.83±0.06	8.76±0.14	8.74±0.12	8.74±0.19
Lungs	F	7.61±0.08	7.69±0.05	7.65±0.05	7.98±0.16	7.61±0.08
Heart	M	0.79±0.09	0.79±0.06	0.76±0.07	0.81±0.07	0.77±0.07
Heart	F	0.74±0.05	0.73±0.05	0.72±0.06	0.74±0.03	0.73±0.06
Liver	M	7.29±0.54	7.24±0.69	7.32±0.86	7.39±0.64	7.21±0.59
Liver	F	5.73±0.39	6.27±0.18	6.44±0.23	6.16±0.09	6.31±0.34
Kidney	M	2.18±0.10	2.16±0.08	2.19±0.06	2.21±0.07	2.22±0.05
Kidney	F	1.64±0.09	1.67±0.11	1.65±0.05	1.66±0.06	1.68±0.04
Pancreas	M	0.99 ± 0.09	0.99 ± 0.09	0.99 ± 0.09	0.99 ± 0.09	0.99 ± 0.09
Pancreas	F	0.93 ± 0.06	0.93 ± 0.06	0.93 ± 0.06	0.93 ± 0.06	0.93 ± 0.06
Thymus	M	0.31±0.04	0.29±0.03	0.28±0.03	0.30±0.03	0.31±0.01
Thymus	F	0.23±0.03	0.26±0.05	0.24±0.03	0.26±0.04	0.25±0.02
Testies	M	0.75±0.06	0.78±0.07	0.76±0.05	0.79±0.08	0.76±0.05
Ovaries	F	0.21±0.03	0.21±0.04	0.21±0.03	0.20±0.03	0.22±0.03
Uterus	F	0.30±0.03	0.30±0.04	0.30±0.03	0.29±0.06	0.30±0.05

All the values were reported as mean ± SD for (n=6) determination.

Histopathological studies:

The histopathological alterations in organs were considered as a basic test in the safety assessment of test materials. No abnormalities were observed after gross or histopathological evaluations of organs. The histopathological findings of liver, kidney, heart, lung, pancreas and spleen were normal in all mice. Thus, the alteration in biochemical results may be due to biological variation. The urea levels were found to be increased significantly in case of Group IV as compared to the control group in female. The urea and creatinine are the byproducts of protein metabolism and excreted by the kidney. The marked increased in serum urea was noticed which may functionally damage to the kidney. But the histological examination of kidney revealed no damage in any of the experimental animals. Also significantly increased level of total bilirubin in Group III and Group IV as compared to control in male were observed. Increase in total bilirubin level shows rosin esters may effect on the liver. However, histopathology of liver does not show any damage to liver. The histograms of liver were found to be normal. The histograms of liver and kidney in control and rosin ester treated group were shown in Figure 1.

Figure 1: Histogram of liver and kidney in control group and treated group.

Abbreviations and symbol:

RE: rosin ester; PEG: Polyethylene glycol; ‒COOH: Carboxylic acid group; MA: Maleic anhydride; M: Male; F: Female; RH: Relative Humidity; CPCSEA: Committee for the Purpose of Control and Supervision on Experiments on Animals; IAEC: Institutional Animal Ethical Committee; OECD: Organization for Economic Co-operation and Development; RBC: Red Blood Cells; WBC: White Blood Cells; Hb: Hemoglobin; HCT: Haematocrit; MCV: Mean Corpuscular Volume; MCH: Mean Corpuscular Hemoglobin; MCHC: Mean Corpuscular Hemoglobin Concentration; TLC: Total Leukocyte Count; DLC: Differential Leukocyte Count; AST: Aspartate Amino Transferase; ALT: Alanine Amino Transferase; ALP: Alkaline Phosphatase; ROW: Relative Organ Weight; BW: Body Weight; d: day(s); mg: milligram; kg: kilogram; g: gram; pg: pictogram; mol: mole; IU: International Unit; G: gauge; ml: milliliter; l: liter; fl: femtoliter; dl: deciliter; μl: microliter; μm: micrometer; %: percent; °C: degree Celsius.

ACKNOWLEDGEMENT:

Authors especially thank to Shri. Yogendra Rajendraji Gode, President, Indira Bahudeshiya Shikshan Sanstha, Buldhana, and Prof. (Dr.) V. N. Shrikhadnde, Principal, Dr. Rajendra Gode College of Pharmacy, Malkapur for providing the research facility and support.

CONFLICT OF INTEREST:

No conflict of interest was reported by authors.

FUNDING SUPPORT:

Nil

CONCLUSIONS:

The sub-acute toxicity study of novel rosin esters indicates safe and non-toxic nature of rosin esters at the concentration 25 mg/kg of body weight after oral administration. There were no signs mortality and morbidity observed during study. The biochemical parameters namely total bilirubin and urea were found in higher than normal level, but they does not cause any harm to the kidney and liver which was confirmed by histopathological study. They can be normalized by animal’s own immune system. The haematopoietic and histopathological investigations stated that no abnormalities were seen in serum and tissues. The liver, lungs and spleen were showed normal histology. The increase in lungs weight of group IV was observed due to growth of lungs in female. A small or minute inflammation was observed in one out of 12 mice in terms of kidney histology. According to the findings, the rosin esters at 25 mg/kg were safe and non-toxic after oral administrations repeatedly for 28 d. The biochemical, hematopoietic and histopathological investigations pertaining to toxicity supported the study and confirmed our findings. The present study was a first attempt in terms of the rosin esters made to investigate the toxic nature. The present investigation concluded that the study provides assurance about the safety and non-toxic of novel rosin esters to use as an ingredient in the pharmaceutical formulation at the given daily dose.

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Received on 30.03.2020 Modified on 23.05.2020

Research J. Pharm. and Tech. 2021; 14(4):1859-1866.

DOI: 10.52711/0974-360X.2021.00329