Preclinical safety assessment of glycosides based standardized fenugreek seeds extract : Acute , subchronic toxicity and mutagenicity studies

Article history: Received on: 29/03/2016 Revised on: 07/07/2016 Accepted on: 08/08/2016 Available online: 26/09/2016 Objective: To evaluate acute oral toxicity (AOT), subchronic toxicity, and mutagenic potential of glycosides based standardized fenugreek (Trigonella foenum graecum L.) seeds extract (SFSE-G). Materials and Methods: The AOT, subchronic (90-day repeated dose) toxicity and mutagenicity (reverse mutation test) of oral administration of SFSE-G were evaluated using Sprague-Dawley (SD) rats as per OECD guideline no. 423, No. 408 and 471 respectively. Results: The SFSE-G did not show mortality or treatment-related adverse signs during acute (limit dose of 2000 mg/kg) and subchronic (90-days repeated dose of 250, 500 and 1000 mg/kgwith 28 days of recovery period) administration. The SFSE-G showed oral median lethal dose (LD50) more than 2000 mg/kg during AOT study. The no-observed adverse effect level (NOAEL) of SFSE-G was 1000 mg/kg in male rats and 500 mg/kg in female rats during subchronic toxicity study. Furthermore, SFSE-G did not show mutagenic potential in vitro. Conclusions: SFSE-G was found safe for acute and subchronic (90 days repeated dose) administration in rats with no mutagenic potential.


INTRODUCTION
There is increasing awareness and general acceptability of the use of herbal drugs in today's medical practice (Upton et al., 2016) Most of the natural products or herbal medicines contain a diverse variety of phytoconstituents because of variation in growth pattern, geographical location, time of harvesting and storage (Yau et al., 2015).Correct identification of composition of herbal medicine is essential to ensure consistent quality, safety and efficacy (Upton et al., 2016).The process of standardization is used to prescribe a set of standards or inherent characteristics with definitive qualitative and quantitative values thatare indicative of consistent quality and reproducibility.Apart from consistent quality, all medicines need to fulfill the basic requirements of safety.However, the safety of herbal medicines has become a major concern to both regulatory health authorities and the general public (Kalaiselvan et al., 2015, Starr, 2015).Therefore, safety or toxicological studies on herbal medicines needs to be conducted using well accepted international guidelines to ensure safe use for long-term human consumption.One of the potential natural sources of nutritional and medicinal applications is fenugreek (Trigonella foenum-graecum L.) seed.Fenugreek seeds are being used by people in Asia, Africa and Mediterranean countries as one of the ingredients in daily diets (Ulbricht et al., 2008).
Past reports on fenugreek seeds highlighted a variety of medicinally active phytoconstituents namely alkaloids and polysaccharides (Petropoulos, 2003), flavonoids (Huang andLiang, 1999, Wagner et al., 1973), triterpenoids (Shang et al., 1998) and steroidal sapogenins (Sauvaire et al., 1991).Safety of fenugreek seeds in powder form in human has been established in many clinical trials and reviews (Basch et al., 2003).Fenugreek seeds are certified as GRAS (Generally recognized as safe) item under clause §182.20 (Essential oils, oleoresins and natural extractives including distillates) by US Food and Drug Administration (US FDA).
In order to explore clinical potential of SFSE-G for longterm human consumption, toxicology testing in laboratory animals using well-accepted international guidelines is required.Therefore, the present study aimed to evaluate acute oral toxicity (AOT), repeated dose (90-days, subchronic) toxicity in laboratory rats and mutagenicity testing using OECD guidelines

Animals
Male and female Sprague-Dawley (SD) rats were used for the acute and subchronic toxicology studies.The rats were obtained from the animal house of Indian Institute of Toxicology, Pune, India.The animals were acclimatized to laboratory conditions for 7 days prior to the experiments.The rats were maintained at a room temperature of 22-24°C, relative humidity between 30% and 70%; 10-15 air changes per hour with a 12 h light/dark cycle.
During acclimatization, the animals were housed in polypropylene cages provided with a standard pellet diet (M/s.Nav Maharashtra Chakan Oil Mills Ltd., Pune) and water ad libitum.All protocol was approved by Institutional Animal Ethics Committee of Indian Institute of Toxicology, Pune, India.
All the animal experimentations were performed as per Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines in accordance with "Principles of Laboratory Animal Care" (NIH publication no.85-23, revised 1985).

The test compound, SFSE-G
The test compound, SFSE-G, is a standardized extract of fenugreek seeds (containing 89.42% of glycosides), prepared and standardized by HPLC using earlier reported procedure (Kandhare et al., 2015)and provided for the study by Indus Biotech Private Limited (Pune, India).The solution of SFSE-Gwas freshly prepared in distilled water to obtain dose volume of 10 ml/kg body weight of rats.SFSE-G solution in sterile water for injection was used for mutagenicity evaluation.

Acute oral toxicity (AOT) study
The acute toxicity study of SFSE-G was performed according to OECD guideline 423 (Organisation for Economic Cooperation and Development, (Organisation for Economic Cooperation and Development, 2002).The rats were grouped in 2 groups (5 rats/sex/group) and treated with a single dose as follows:G1 -Vehicle control (VC) (double distilled water, 10 mL/kg, oral), and G2 (SFSE-G, 2000 mg/kg, oral.).The rats were observed daily for 14 days following administration for mortality and clinical signs of toxicity.On day 15, all rats were euthanized and underwent gross pathological examination for signs of toxicity via necropsy.

Subchronic (90-day repeated dose) toxicity study
The study complies with the OECD Guideline for the testing of Chemicals No.408 (Organisation for Economic Cooperation and Development., 1998).The separate vehicle control (VC) group was maintained on distilled water as designated as G1.Based on median lethal dose (LD 50 ) (2000 mg/kg) obtained from AOT study, animals were orally administered daily once with following treatments: G1 -VC (Distilled water, 10 mL/kg, 90 days) -15 animals per sex G2 -SFSE-G-250 (SFSE-G, 250 mg/kg, 90 days) -15 animals per sex G3 -SFSE-G-500 (SFSE-G, 500 mg/kg, 90 days) -15 animals per sex G4 -SFSE-G -1000 (SFSE-G, 1000 mg/kg, 90 days) -15 animals per sex G1R -VC reversal group -VC-R -(Distilled water, 10 mL/kg, 119 days) -10 animals per sex G4R -SFSE-G -1000-Reversal group (SFSE-G-1000-R) (SFSE-G, 1000 mg/kg for 90 days) followed by (distilled water, 10 mL/kg (from day 91 to day 119) -10 animals per sex All treated rats were observed daily for mortality and clinical signs during the 90-day study period whereas reversal groups (G1R and G4R) were observed for upto day 119 (Reversal period of 28 days).The eyes of control and all the treated dose group animals were examined prior to the initiation of the dosing and on day 91 and 119 (for G1R and G4R) of the study.Eye examination was carried out using an ophthalmoscope (mini 2000, HEINE Optotechnik, Herrsching, Germany) after induction of mydriasis with 0.5% solution of tropicamide.The body weight and feed consumption of each rat were recorded at weekly intervals throughout the study period.Towards the end of the exposure period (day 91), functional observations (by grading different sensory reactivity to stimuli of different types (auditory, visual and proprioceptive stimuli), assessment of grip strength (digital grip strength meter (Columbus Instruments International, Columbus, OH, USA) and motor activity measurement were performed.The animals were placed in metabolic cages overnight and urine excreted by each animal was collected on day 91 and on day 119 (G1R and G4R) of the study for urinalysis.The parameters for urinalysis included: specific gravity, pH, occult blood, protein, bilirubin, ketones, glucose, nitrite, and urobilinogen.Using whole blood, hematological and coagulation analyzes were carried out.The parameters for hematological analysis included: red blood cell count (RBC), reticulocyte count, hemoglobin (Hb), hematocrit (HCT), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV) and total leukocyte count (TLC), % cells in differential leukocyte count (DLC) (including lymphocyte (L), monocyte (M), basophils (B), eosinophils (E), and Prothrombin time (PT).Additionally, clinical chemistry was evaluated including Blood Urea Nitrogen (BUN), Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), Alkaline Phosphatase (ALP), Gamma Glutamyl Transferase (GGT), Creatine Phosphokinase (CK), Lactate Dehydrogenase (LDH), Fasting plasma glucose (FPG), Calcium (Ca), Phosphorus (P), Bilirubin, Albumin, creatinine (CR), Sodium (Na), Potassium (K), Chlorine (Cl), Cholesterol, and Triglycerides (Trig).On day 91, all animals were euthanized and underwent gross pathological examination for signs of toxicity via necropsy.
All organs, mucosa, body cavities, etc. were examined for gross pathological changes.Major organs and major endocrine glands (pituitary, adrenal, thymus, thyroid, sex, etc.) were weighed and absolute and relative weights (i.e.percent of the body weight) were calculated.Tissue samples from selected organs (heart, kidney, liver, lung, spleen, stomach, pancreas and skeletal muscle) from the VC (G1 and G1R) and 1000 mg/kg (G4 and G4R) were preserved, fixed, and stained for histopathological evaluation via light microscopy.The remaining tissues/organs were preserved in 10% neutral buffered formalin from control and different dose groups.

Mutagenicity study
Mutagenicity was evaluated by bacterial reverse mutation test (AMES test) was performed in full compliance with the OECD guidelines for mutagenicity testing namely Test No: 471 (Organisation for Economic Co-operation and Development, 1997).As no significant cytotoxic effect was observed, the five highest doses were then used in the subsequent mutagenicity evaluation.
To evaluate mutagenicity, five strains of histidinedependent Salmonella typhimurium (TA97a, TA98, TA100, TA1535 and TA102) were tested in triplicate at the five highest doses (5000.00, 1666.67, 555.55, 185.18 and 61.72 µg/plate) of SFSE-G.Rat liver homogenate tested with mutagen 2aminofluorene before use.Metabolic activation was performed using a cofactor-supplemented post-mitochondrial fraction (S9 fraction).Positive controls (2-aminofluorene, 2-aminoanthracene, methyl methanesulfonate, 4-Nitroquinolene-N-Oxide, danthron and sodium azide) with and without S9 activator and negative controls (VC and phosphate buffer) with and without S9 activator were included in the evaluation.This was done to ensure the test system was functioning properly (positive controls) and to obtain baseline revertant frequencies for the various strains of bacteria used in the study (negative controls).The plates were counted after 48 h of incubation at 37 °C.The mutagenic activity of the test substance was considered for positive in case of increased concentration over the range tested and a reproducible increase at one or more concentrations in a number of revertant colonies per plate in at least one strain with or without metabolic activation system.
The test substance was considered to be toxic if there was a decrease in the number of revertants and/or thinning or absence of background lawn.

Statistical analysis
Statistical analysis was performed using SPSS analysis program for windows Version 17.0 (SPSS Inc, USA).All the data were checked for normality and values were represented as mean ± standard deviation (SD).Data of body weight and food intake was analyzed by two-way ANOVA followed by unpaired 't' test.Remaining parameters were analyzed by separate One way ANOVAs.
Dunnett's test was used to analyze the differences between treated groups with respective VC groups (G2, G3, G4 v/s.G1 and G4R v/s.G1R).The value of P < 0.05 was considered to be statistically significant.

Acute oral toxicity (AOT)
None of the rats died during the observation period.No clinical signs of toxicity, such as ill health or abnormal behavior, occurred during the study.There were no abnormal observations in body weight of rats.No abnormalities or pathological changes were observed in the necropsy.Under the experimental conditions, the results indicated that the median lethal dose (LD 50 ) of SFSE-G is greater than 2000 mg/kg of body weight.

Subchronic (90-day repeated dose) toxicity
Daily oral administration of SFSE-G for 90 days did not induce any obvious symptom of toxicity in rats of any sexes, up to the highest tested dose (1000 mg/kg).No deaths or obvious clinical signs were found in any groups throughout the study period.Body weights of male and female rats did not show significant difference during treatment period except for SFSE-G-1000 group in female rats on 90 days period and SFSE-G-1000-R group (v/s.G1R group) (Table 1).No gross abnormalities were observed in necropsies of any of the rats.The food and water consumptions were measured throughout the study and did not significantly change as compared to the VC rats (Figure 1).Ophthalmoscopic examination and functional observation tests revealed no abnormalities attributable to the treatment.Hematological (Table 2 and Table 3) and biochemical observations (Table 4 and Table 5) and urinalysis were recorded in male and female rats.All the values hematological and biochemical values were within normal biological and laboratory limits and the differences between the values were not consistent between doses or periods of observation (treatment period / reversal period) and considered incidental.For example, hematology values in SFSE-G treated groups of rats were significantly different from corresponding VC groups in terms of Hb, RBC, HCT, MCHC, TLC during 90 day treatment period and   Hb, RBC, HCT and platelets during the recovery period (Table 2 and Table 3).Biochemical parameters related to kidney function test (CR, CK and BUN) did not show significant change as compared to respective VC group in female rats whereas BUN values show significant (P < 0.05) decrease in G2 (SFSE-G-250 mg/kg treated) group (Table 4).
Similarly, all liver function related biochemical parameters (ALT, AST, ALP, GGT, Bilirubin, Albumin) were unchanged in female rats whereas ALT values in G2 (SFSE-G-250 treated group) showed significant (P <0.05) increase in male rats (Table 4 and Table 5).The electrolyte concentrations (Ca, P, Na, K and Cl) levels in male rats did not show significant changes as compared to respective VC groups except Ca levels (SFSE-G-1000 group, P < 0.01) and Cl levels (SFSE-G-250, P < 0.01).However, female rats showed significant changes in electrolyte concentration related parameters such as Na levels (SFSE-G-250 and SFSE-G-500 but not SFSE-G-1000 group), Ca and K levels (SFSE-G-500 and SFSE-G-1000 group), Cl levels (all SFSE-G treated groups) with no significant change in P levels.The plasma lipid-related parameters (FPG, cholesterol, triglyceride, and LDH levels) in SFSE-G treated male and female rats remained except G2 (SFSE-G-250 treated) group which showed significant (P < 0.05) decrease in triglyceride levels in female rats.0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 Data was represented as Mean ±Standard Deviation, Data was analyzed by unpaired 't' test, * = P < 0.05, as compared with respective parameter value of corresponding VC group Hb: Hemoglobin; RBC: Red Blood Corpuscles, HCT : Hematocrit, MCV: Mean Corpuscular Volume, MCH: Mean Corpuscular Hemoglobin, MCHC:Mean Corpuscular Hemoglobin Concentration, TLC: Total leukocyte(White Blood Corpuscles) count, PT.: Prothrombin time, N: Neutrophils, L : Lymphocytes, E: Eosinophils, M : Monocytes, B : Basophils.The data of weights of organs in SFSE-G treated rats during treatment and reversal period in male and female rats is presented in Table 6 and Table 7. None of the SFSE-G treated group show significant difference during treatment or reversal period as compared with corresponding VC group.No treatment-related gross pathological changes were observed in any organs of the test animals during necropsy (Table 8).383.00 ± 19.52 Data was represented as Mean ±Standard Deviation, Data was analyzed by unpaired 't' test, * = P < 0.05, ** P < 0.01 as compared of respective parameter value of corresponding VC group.ALT: Alanine Aminotransferase,AST: Aspartate Aminotransferase, ALP: Alkaline Phosphatase, GGT: Gamma Glutamyl Transferase, CR: Creatinine, CK: Creatine Phospho-Kinase, BUN : Blood Urea Nitrogen, Ca: Calcium, P: Phosphorus, Na: Sodium, K: Potassium, Cl: Chlorine, FPG: Fasting plasma glucose, LDH: Lactate De-Hydrogenase.

Table 6:
Effect of SFSE-G on relative organ weights of during 90 days repeated dose toxicity study (Male rats).The finding from histological examination of sections of organs is presented in Table 9 and representative photomicrographs are presented as Figure 2. Histopathological examination of the VC group and SFSE-G treated rats showed anormal structure with mild changes and absence of any gross pathological lesion in organs.The changes observed in both VC and SFSE-G-1000 treatment groups were similar and comparable in both sexes and hence considered as incidental, congenital, and spontaneous.Based on the results of present subchronic toxicity study of SFSE-G, the no observed adverse effect level (NOAEL) is 1000 mg/kg per day in male rats and 500 mg/kg per day in female rats.
Thus, the test strains fulfilled the quality check criteria.No substantial increases in the revertant colony count in any of the five strains were reported in the SFSE-G treated plates in the presence or absence of metabolic activation (S9 mix).
The spontaneous reversion rates in the negative and positive controls were within the range of historical data.The results of these investigations suggest that under the experimental conditions, SFSE-G did not induce gene mutation by pair changes or frame-shifts in the genome of the strains used.Thus, SFSE-G can be considered as non-mutagenic.

DISCUSSION
Toxicology studies are the platform for hazard identification stage of safety assessment (Wallace, 2011).The preclinical toxicity testing with safe dose level determination is needed to initiate the clinical evaluation of investigational products (Setzer and Kimmel, 2003).Choosing the appropriate tests and dosing regimens that will demonstrate an adequate margin of exposure is a critical step in establishing human safety.Toxicity data from studies following well-accepted international guidelines in animals is required by regulatory agencies throughout the world to support regulatory applications and marketing approval of natural products.
During the evaluation of the safety information of plants derived natural products, the determination of median lethal dose (LD 50 ) is usually an initial step to be conducted.Data from the acute toxicity study may provide initial information on the mode of toxic action of a substance, help in dose determination in animal studies (Ukwuani et al., 2012).In the present study, acute administration of SFSE-G at a dose of 2000 mg/kg, showed no adverse effects up to 14 days and so considered as the median lethal dose (LD 50 ).
Based on results of AOT, subchronic (90-days repeated dose) toxicity study of SFSE-G was conducted in rats.Subchronic studies assess the undesirable effects of repeated long-term exposure of test product over a portion of the average life span of experimental animals, such as rodents.Therefore, they provide useful information on target organ toxicity and help to determine appropriate dose regimens for long-term use.Administration of SFSE-G for 90 days produced no clinical signs of toxicity or mortality in either sex.
The body weight changes serve as a sensitive indication of the general health status of animals (El Hilaly et al., 2004).Loss of appetite is often synonymous with weight loss due to disturbances in carbohydrate, protein or fat metabolisms (Klaassen, 2013).At higher doses, plant extracts may metabolize to a toxic end product, which could interfere with gastric function and decreased food conversion efficiency (Chokshi, 2007).In the present study, SFSE-G treated groups of rats showed body weight gain (although statistically not significant).Furthermore, the food consumption of SFSE-G treated rats remained unaltered during the 90-day treatment period.These results indicated the absence of adverse effects on the normal metabolism of food components during the treatment period and maintained expected nutritional benefits of normal food and water, such as weight gain and stability of the appetite.
Evaluation of hematological parameters can be used to determine the extent of the deleterious effect of SFSE-G on the blood of an animal (Chukwuemeka et al., 2015).Furthermore, such analysis is relevant to risk evaluation as changes in the hematological system have higher predictive value for human toxicity when the data are translated from animal studies (Chanda et al., 2015, Diaz et al., 2016).
A hematology data of SFSE-G treated rats did not show treatment-related changes as compared to VC rats.The results of hematology indicated the safety of SFSE-G on erythropoiesis, morphology or osmotic fragility of the red blood cells (Hall, 2015).Leukocytes are the first line of cellular defense that respond to infectious agents, tissue injury, or inflammatory process.The absence of significant treatment-related changes on TLC or differential leukocyte parameters in SFSE-G treated rats in the present study confirmed the safety of SFSE-G.

Fig. 1 :
Fig. 1: Effect of SFSE-G food consumption in (A) Male and (B) Female rats during 90 days repeated dose toxicity study.Data are expressed as mean ± standard deviation (SD).

Fig. 2 :
Fig. 2: Effects of SFSE-G on histological findings of heart (A-D), kidney (E-H), liver (I-L) and lung (M-P) tissue in rats during 90-days repeated dose toxicity study.Photomicrographs from representative rats from respective groups: VC (A, C, E, G, I, K, M, and O) and SFSE-G-1000 (B, D, F, H, J, L, N and P) (H&E stain) at 40X.

Table 1 :
Effect of SFSE-G on body weights of rats during 90 days repeated dose toxicity study.

Table 2 :
Effect of SFSE-G on hematological parameters during 90 days repeated dose toxicity study (Male rats).

Table 3 :
Effect of SFSE-G on hematological parameters during 90 days repeated dose toxicity study (Female rats).

Table 4 :
Effect of SFSE-G on blood chemistry on during 90 days repeated dose toxicity study (Male rats).Data was represented as Mean ±Standard Deviation, Data was analyzed by unpaired 't' test, * = P < 0.05, ** P < 0.01 as compared of respective parameter value of corresponding VC group.

Table 5 :
Effect of SFSE-G on blood chemistry on during 90 days repeated dose toxicity study (Female rats).

Table 7 :
Effect of SFSE-G on relative organ weights of during 90 days repeated dose toxicity study (Female rats)

Table 9 :
Summary of histopathology findings.