Safety Assessment of Ayurvedic Formulation “Annabedi chendooram” in Experimental Animals

Safety evaluation of flexirubin from Chryseobacterium artocarpi CECT 8497: Acute, sub-acute toxicity and mutagenicity studies

Wuzhuyu decoction (WZYD) is a well-known classic traditional Chinese medicine prescription and has been widely used to treat headache, nausea, vomiting, insomnia, etc. However, little published information is available about its safety. Our aim was to investigate the acute and subacute oral toxicity of WZYD extract in rats following the technical guidelines from China’s National Medical Products Administration (NMPA) for single and repeated doses toxicity studies of drugs. Acute oral toxicity was assessed in rats via oral administration of WZYD extract at 4 g/kg three times within a day followed by a 14-day observation period. To evaluate the subacute toxicity, rats were orally administered with WZYD extract at doses of 0, 0.44, 1.33, and 4 g/kg for 28 days. The items examined included clinical signs, body weight, food consumption, hematological and biochemical parameters, bone marrow smear, organ index, and histopathology. After the rats were administered with 12 g/kg (3 × 4 g/kg) WZYD extract, no mortality and toxic effects were observed during the observation period. In the subacute toxicity study, WZYD extract did not cause any significant treatment-related abnormality in each examined item of rats, so the no observed adverse effect level (NOAEL) of WZYD extract for 28 days orally administered to rats is considered to be 4 g/kg, which is approximately 80-fold of its clinical proposed dosage.

Objective: This study aimed to evaluate acute and sub-acute oral toxicity of ethanol extract of Clinacanthus nutans leaves in Swiss mice.
 Methods: Acute oral toxicity study was performed as per OECD-423 guidelines. Sub-acute oral toxicity study was performed as per OECD-407 guidelines. The extract was dissolved in 10% dimethyl sulfoxide and administered orally, while the control group received only the vehicle.
 Results: The acute oral toxicity test on mice showed that this extract was well tolerated up to LD50 5000 mg/kg body weight/day oral dosage level and non-toxic to mice under the present experimental conditions. The sub-acute toxicity study was carried out on mice with the oral dosage of the extract from 100 mg/kg–500 mg/kg body weight/day and 5000 mg/kg body weight/day for 28 d. The results showed that this extract did not induce death or adverse effects in activity, feed consumption or body weight gain. There were not significant changes in heamotological and biochemical parameters between control and experiment groups.
 Conclusion: Thus, Clinacanthus nutans leaf has a very low toxicity value.

The REACH Regulation requires information on acute oral toxicity for substances produced or imported in quantities greater than one ton per year. When registering, animal testing should be used as last resort. The standard acute oral toxicity test requires use of animals. Therefore, the European Chemicals Agency examined whether alternative ways exist to generate information on acute oral toxicity. The starting hypothesis was that low acute oral toxicity can be predicted from the results of low toxicity in oral sub-acute toxicity studies. Proving this hypothesis would allow avoiding acute toxicity oral testing whenever a sub-acute oral toxicity study is required or available and indicates low toxicity. ECHA conducted an analysis of the REACH database and found suitable studies on both acute oral and sub-acute oral toxicities for 1,256 substances. 415 of these substances had low toxicity in the sub-acute toxicity study (i.e., NO(A)EL at or above the limit test threshold of 1,000 mg/kg). For 98% of these substances, low acute oral toxicity was also reported (i.e., LD50 above the classification threshold of 2,000 mg/kg). On the other hand, no correlation was found between lower NO(A)ELs and LD50. According to the REACH Regulation, this approach for predicting acute oral toxicity needs to be considered as part of a weight of evidence analysis. Therefore, additional sources of information to support this approach are presented. Ahead of the last REACH registration deadline, in 2018, ECHA estimates that registrants of about 550 substances can omit the in vivo acute oral toxicity study by using this adaptation.

Context: Amorphophallus paeoniifolius tuber is an important constituent of Ayurvedic system of medicine. The tuber of this plant has high medicinal value and is consumed as a food. It is associated with acridity (itchy sensation in mouth and throat) upon oral consumption and presence of high oxalates raphides. Aims: To evaluate the acute and subacute oral toxicity studies of methanolic (APME) and aqueous (APAE) extracts of Amorphophallus paeoniifolius tuber in Swiss albino mice according to OECD guidelines. Methods: In acute oral toxicity study, the mice were orally administered a single dose of APME or APAE (2000 mg/kg) and clinical signs and mortality were observed for 14 days. In subacute (repeated dose) oral toxicity study, the mice were administered once daily, orally with APME or APAE (1000 mg/kg) up to 28 days. The parameters assessed were behavior, clinical signs, body weight, feed and water consumption, urinary, biochemical, hematological and major organ weights and histology. Results: In acute toxicity study, there was no treatment related mortality and morbidity in any of the group. In subacute toxicity study, there were no significant changes in behavior, body weight, feed and water consumption, urinary, biochemical, hematological and organ weight and histological parameters compared to vehicle treated group. There was no treatment related mortality or morbidity. Conclusions: Administration of methanolic and aqueous extracts of Amorphophallus paeoniifolius tuber, individually in acute and 28 days repeated dose in mice, did not exhibit any toxicity or adverse effect at the doses used.

In 2022, approximately 2.3 million new cases of female breast cancer and 670,000 related deaths worldwide despite significant advancements in conventional treatments. BobyGuard C (BGC) is a novel polyherbal nutraceutical formulated from five plants, selected for their antioxidant, anticancer, anti-inflammatory and nutritional properties to be used for breast cancer management. This study aimed to characterize its physicochemical, nutritional, and phytochemical properties as well as assess its safety through acute and sub-acute oral toxicity studies in Wistar rats. Thecomposition of BGC was analyzed for macronutrients, minerals, and phytochemicals using standard methods. Antioxidant activity was assessed through DPPH, TAC and FRAP assays, while antiproliferative activity was evaluated using the MTT assay on MDA-MB 231 and MCF-7 breast cancer cell lines. Acute (single 5,000mg/kg dose with 14days observation) and sub-acute oral (daily administration of 784, 1,568, and 3,136mg/kg for 28days) toxicity studies in female Wistar rats followed OECD guidelines. BGC was found to be rich in proteins (38.36g/100g), carbohydrates (59.70g/100g), and essential minerals such as magnesium (60,066.67µg/100g), and it was free from toxic heavy metals. Several bioactive compounds, including diosgenin, diosbulbin H, β-carotene, Bafoudiosbulbin G and catechin were identified in BGC. Phytochemical analysis revealed high levels of phenols (9,783.48mg GAE/100g), flavonoids (47.72mg QuE/100g), and alkaloids (106.14mg berberine eq/100g), contributing to its strong antioxidant activity (DPPH inhibition: 90.39%). BGC exhibited significant antiproliferative effects on MDA-MB 231 cells, highlighting its potential anticancer activity. Acute toxicity tests showed no mortality at 5,000mg/kg, with an LD50 exceeding this dose. In the sub-acute 28-day repeated-dose oral study, doses up to 3,136mg/kg/day resulted in some dose dependent hematological and biochemical changes but no histopathological abnormalities were observed indicating its safety at lower doses. BGC is a nutritionally rich formulation with potent antioxidant and anticancer potential, demonstrating a favorable safety profile at lower dose (784mg/kg).

Safety evaluation of a polyherbal formulation containing hydroalcoholic extracts of Hippophae salicifolia, Nyctanthes arbor-tristis, Ocimum tenuiflorum, and Reinwardtia indica in rodents anti-oxidant properties [5] .Reinwardtia indica (family Linaceae) contains saponins, which could potentially help in the management of hyperglycemia [6][7] .We conducted acute and sub-acute oral dose toxicity studies of the test formulation in Swiss albino mice and albino Wistar rats, respectively.Single doses (10 to 5,000 mg/kg) were administered orally to mice.No treatment related deaths or clinical signs of toxicity were recorded at any of the doses at two weeks after drug administration and the lethal dose 50% of the test drug was greater than 5,000 mg/kg.For the sub-acute toxicity assessment, the doses employed ranged from 100 to 800 mg/kg•day (and vehicle as the control), which, in most cases, is acceptable as the limit dose for toxicity studies [8] .The formulation was administered orally to rats for either 14 or 28 days during which food intake and body weight were monitored.At the end of the treatment period, organ weights and haematological and biochemical parameters were measured along with a histopathologic examination.No treatment-emergent toxicities or mortality was observed.Additionally, no treatment related changes in the behaviour of the rats were observed.There was a small and insignificant reduction in body weight and food consumption of the rats in the treatment groups compared with the control group (Table 1), suggesting that sub chronic administration of the test formulation did not affect the normal growth of rats.Similarly, there were no significant changes in the weight of the organs (brain, liver, kidney, heart) following either 14 or 28 days of treatment at any of the doses compared to the controls.Haematological parameters including haemoglobin, red blood cell count, white blood cell count, packed cell volume, mean corpuscular volume, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration were found to be all within the normal range in both the control and treatment groups (Table 2), with

Evaluation of the acute and sub-acute toxicity of the ethanolic extract of Pericampylus glaucus (Lam.) Merr. in BALB/c mice

Background: Acute and subacute toxicity screenings of medicinal plants are the fastest way to evaluate the toxicological profiles of medicinal plants. It helps to have a quick idea of the harm or safety potency of drugs. This study aims to conduct an acute and subacute oral toxicity study on the Cyperus Papyrus (CP) ash in Wistar albino rats. CP is traditionally used for managing painful spasms, eye diseases, ulcers, fever, diarrhea, and inflammations. Further study is required to reflect the safety profile of this plant ash. Methods: Acute oral toxicity study of the CP ash was evaluated by using a dose of 2000 mg/kg body weight on rats according to the Organization for Economic Co-operation and Development (OECD) guidelines using the Aot425 software. Subacute oral toxicity was carried out on 4 groups of 6 rats, at doses of 175 mg/kg, 550 mg/kg, 1750 mg/kg, and 1750 mg/kg. The recovery group (control group) received water according to OECD 407 guidelines for consecutive 28 days. At the end of the experiments (day 29), all rats were weighed and sacrificed except the rats of the recovery group which were continued without ash administration for extra 14 days and then weighed and sacrificed. Subsequently, the hematological parameters, plasma biochemical parameters, and histopathological examination were carried out. Results: Upon completion of the acute toxicity, no deaths or signs of acute oral toxicity were observed. Hematologic observations after oral subacute toxicity included a decrease in hematocrit percentage, mean corpuscular volume, and lymphocyte percentage, while there was a slight increase in the mean corpuscular hemoglobin concentration, however, it returned to the normal range in the recovery group. Biochemical tests showed a mild increase in the serum creatine and aspartate aminotransferase levels but they also returned to their normal range in the recovery group. No morphological changes in the kidney, liver, lung, spleen, heart or small intestine were observed after the histopathological examinations. Conclusion: Dried ash of CP is considered to be safe and non-toxic at acute exposure and as the lethal dose 50% value exceeds 2000 mg/kg body weight, it has a mild reversible effect on some hematological and biochemical parameters while no morphological changes of vital organ histopathology occur.

Introduction:Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2. No drug has been generally approved as safe and effective for the treatment of COVID-19. Several therapeutic agents such as COVID Organics® (CVO) have been explored as treatment options. CVO is an herbal tea composed of 62% of Artemisia annua and 38% of other plants. There is presently no existing scientific report and data on the safety and efficacy of CVO herbal drug. Thus, acute and subacute toxicity studies were undertaken to evaluate the safety and toxicity of CVO on short- and long-term usage in animal models.Materials and Methods:Phytochemical and nutritional compositions of CVO were determined using standard methods. Acute oral toxicity was investigated using female Swiss albino mice (three per group). While subacute oral toxicity was done using female and male Swiss albino rats (five per group). The animals were administered 2000 mg/kg, 5000 mg/kg, therapeutic dose; 5500 mg/kg and supratherapeutic dose; 11,000 mg/kg of CVO herbal product. The control group received water ad libitum. The oral toxicity studies were done in accordance with Organization for Economic Corporation and Development guidelines. The experimental protocol was approved by the Institutional Animal Care and Use Committee, Nigerian Institute of Medical Research (Ethics No. IRB/17/043).Results:CVO is rich in antioxidants: flavonoids (10.3%), tannins (29.1%), and phenolics (434.4 mg). It contains proteins (33.8%), carbohydrates (34.5%), fat (6.8%), and fiber (0.5%). In the acute toxicity study, no mortality was recorded in all the treated and untreated groups. The lethal dose of CVO is >5000 mg/kg body weight. The hematological, biochemical, lipid profile, and histologic parameters were all normal at therapeutic doses when compared to the control group.Conclusion:The acute and subacute oral toxicity studies revealed that CVO is not toxic. The specific organ toxicity evaluations also indicated that CVO has no toxic effects on blood parameters and vital organs structure and function at therapeutic dose. Thus, CVO is safe for short- and long-term usage. We recommend that CVO should be subjected to efficacy studies to investigate whether it is effective for COVID-19 treatment as claimed by the manufacturer.

BackgroundEchinops kebericho is widely used for treatment of a variety of diseases including infectious, non-infectious disease and fumigation during child birth. Antibacterial, antimalarial, anti-leshimania, anti-diarrheal and insect repellent activities have been elucidated. Its toxicity profile is not yet investigated and thus this study was to investigate acute and sub-acute toxicity of E. kebericho decoctions.MethodsAcute toxicity study was performed in female Wistar albino rats with single oral dose and followed up to 14 days. The sub-acute oral dose toxicity studies were conducted in rats of both sexes in accordance with the repeated dose 28-day oral toxicity study in rodent OECD guidelines. Physical observations were made regularly during the study period while body weight was measured weekly. Organ weight, histopathology, clinical chemistry and hematology data were collected on the 29th day. Results were presented as mean ± standard deviation. One-way analysis of variance (ANOVA) was performed if assumptions were met; otherwise Kruskal-Wallis analysis was performed.ResultOral administration of E. kebericho decoction showed no treatment-related mortality in female rats up to the dose of 5000 mg/kg. In sub-acute toxicity studies, no significant treatment-related abnormalities were observed compared to negative controls. Food consumption, body weight, organ weight, hematology, clinical chemistry, and histopathology did not show significant variation between controls and treatment groups. However, creatinine, relative lung weight, triglycerides, and monocytes were lower in treated compared to control groups. Significant variations between male and female groups in food consumption, relative organ weight, hematology, clinical chemistry were observed. Histolo-pathology of high-dose treated groups showed fatty liver.ConclusionEchinops kebericho showed LD50 of greater than 5000 mg/kg in acute toxicity study and is well tolerated up to the dose of 600 mg/kg body weight in sub-acute toxicity study.

Toxicological studies are essential for developing novel medications in pharmaceutical industries including ayurvedic preparation. Hence, the present study is aimed to evaluate acute and 28-days repeated dose oral toxicity of anti-obesity polyherbal granules (PHG) in Sprague Dawley rats by OECD guidelines No 425 and 407, respectively. In an acute oral toxicity study, a single dose of 2 g/kg PHG was administered to rats and mortality, body weight, and clinical observations were noted for fourteen days. However, in the subacute oral toxicity study, the PHG was administered orally at doses of 0.3, 0.5 and 1 g/kg daily for 28 days to rats. Food intake and body weight were recorded weekly. On the 29th day, rats were sacrificed and subjected to haematological, biochemical, urine, necropsy, and histopathological analysis. In an acute oral toxicity study, no treatment-related, mortality, behavioral changes, and toxicity were found throughout fourteen days. Likewise, in the sub-acute toxicity study, no mortality and toxic effects were found in haematology, biochemical, urine, necropsy and histopathological analysis in rats for 28 days of treatment with PHG. Based on these results, the LD50 of PHG was found to be greater than 2 g/kg and the no-observed-adverse-effect level (NOAEL) of PHG for rats was found to be 0.5 g/kg/day. Thus, anti-obesity polyherbal granules showed a good safety profile in animal studies and can be considered an important agent for the clinical management of obesity.

BackgroundEquisetum diffusum D. Don commonly known as ‘Himalayan horsetail’, has been traditionally used in the treatment of back pain, bone fracture and dislocation, and arthritis by various tribal communities of India. Our previous study confirmed the anti-inflammatory efficacy of the plant through in silico, in vitro, and in vivo model studies. Therefore, the current research is focused on safety dose evaluation for the first-time of the whole-plant methanol extract (EDME) of E. diffusum through appropriate in silico, in vitro, and in vivo approaches.MethodThe whole plant, along with its rhizomes, was collected, and the methanol extract was prepared. The in silico ADMET study was performed to predict the pharmacokinetics profile and toxicity of all the identified phyto-compounds of EDME previously screened by GC–MS study. In vitro cytotoxicity study of EDME was performed using two cell lines: kidney (HEK293) and liver (Huh7) cell lines. The in vivo toxicity study of EDME was validated by the acute toxicity (OECD 423, 2002) and sub-acute toxicity assays (OECD 407, 2008) in the Wistar Albino rat model.ResultsThe in silico ADMET study of all 47 bioactives predicted good pharmacokinetic and low toxicity profiles. In vitro cytotoxicity showed higher IC50 values of EDME viz., 672 ± 15.7 μg/mL and 1698 ± 6.54 μg/mL for both kidney (HEK293) and liver (Huh7) cell lines, respectively, which were considered as low-toxic. Based on acute oral toxicity, the LD50 value of the extract was considered “non-toxic” up to a feeding range of 2000 mg/kg of body weight. The regular consumption of the extract for an extended period (28 days) was also qualified as safe based on the body and organ weight, hematological, biochemical, and histoarchitecture results in the sub-acute toxicity assay.ConclusionThe detailed in silico, in vitro, in vivo (acute and sub-acute oral toxicity) studies gave us a new insight to the safety dose evaluation of Equisetum diffusum, which may serve as a reliable documentation for undertaking the experimental validation of the ethnobotanical uses of the plant which would help in the field of drug development for the treatment of inflammation related complications.

Trapa natans L. is annual aquatic plant generally kwon as Water caltrp, Water chest nut belonging to the Trapaceae or Lytraceae family. Trapa natans L. is use for the treatment of wide no of disease with out proper toxicity study. The present study was designed to evaluate the acute and sub-acute toxicities of Trapa natans L following the OECD guidelines. In acute oral toxicity, the plant extract was administered orally at the doses of 300 mg/kg, 1000 mg/kg, 2000 mg/kg, and 4000 mg/kg. After the dosing, all animals were kept in close observation for 14 days. In the case of the subacute toxicity study, the total number of animals was divided into four groups, each having ten animals, 5 males, and 5 females in each group. Group I was represented as the control, administered orally with aqueous tween 80 solution 2% (v/v), and group II to group IV received plant extract of doses 400, 600, and 1000 mg/kg, respectively, once a day for 28 days. From the acute and subacute toxicity studies, no sign of toxicity was observed. In the sub-acute toxicity study, no major changes were noted on the biochemical parameters and hematological analysis. The experimental results of the toxicity studies suggested that the LD50 values of the Trapa natans L extract were more than 4000mg/kg and thus, the drug was found to be absolutely safe and nontoxic.

Madhulai Manappagu- a well-known sastric and widely prescribed Siddha herbal syrup formulation indicated for treating Veluppu Noi (Anaemia especially Iron deficiency Anaemia) has been in day today practice in Tamil Nadu for a quite longer decades. The syrup is a herbal preparation which has a sweet pleasant odour and a palatable taste, contain the juice of pomegranate (Punica granatum L.) as the main ingredient. Though the formulation is a fruit juice, the safety profile of the syrup is not established and is being marketed without toxicological evaluation. The study is aimed at ascertaining the acute and sub-acute toxicity assessment of Madhulai Manappagu in Wistar Albino rats. The acute and sub-acute (28day repeated oral) toxicity studies were performed as per the guidelines mentioned in the Organization for Economic Cooperation and Development (OECD) 423 (adopted on December 2001) and TG 407 (adopted on October 2008) with slight modifications respectively. For acute toxicity study, three female rats were randomly selected as control; three female rats were randomly selected and were administered a single dose of 5,000mg/kg body weight per oral route. For sub-acute (28day repeated oral) toxicity studies, three doses of test drug MM of 500mg/kg/day (low dose), 750mg/kg/day (intermittent dose) and 1,000mg/kg/day (high dose) were selected for administration. Both sexes of Wistar Albino rats were randomized into four groups of 10 animals each (five males, five females). Group I was kept as control group. Group II, III and IV served as low, intermittent and high doses of MM respectively. Animals were observed for mortality, morbidity, body weight changes, feed and water intake. Haematology, clinical biochemistry, electrolytes, gross pathology, relative organ weight and histopathological examination were performed. In the acute toxicity study, rats showed no toxicological signs on behavior, gross pathology and body weight of rats when treated with a single dose of 5,000mg/kg body weight per oral route. In the subacute (28days repeated oral) toxicity study, rats have showed no significant changes on behavior, gross pathology, body weight, and hematological and biochemical parameters when treated with Madhulai Manappagu in three different doses. The toxicity studies which include both acute and 28days repeated (subacute) oral toxicity studies, revealed no observed adverse effect level (NOAEL) of Madhulai Manappagu in animals. Thus the safety of the drug in human usage was ensured.