Acute Oral Toxicity Study on

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1 Acute Oral Toxicity Study on SOBEREX CAPSULE OF Sponsor Study Centre: VIRGO UAP PHARMA PVT.LTD. PHARMACOLOGY LABORATORY MAHAGUJARAT IND.ESTATE, SDM CENRE FOR RESEARCH IN PLOT NO. 423/98-B. AYURVEDA AND ALLIED SCIENCES SARKHEJ BAVLA HIGHWAY LAXMI NARAYANA NAGAR, KUTHPADY, VILL MORAIYA, TA SANAND UDUPI DIST AHMEDABAD contact@virgouap.com Web:

2 STUDY REPORT Evaluation of Soberex Capsule for safety profile Acute Oral Toxicity Study Project Director Dr. B. Ravishankar Chief Investigator Mr. Ravi M Pharmacology Laboratory SDM Centre for Research in Ayurveda and Allied Sciences, Kuthpady, Udupi

3 INDEX Title Page No. Index 3 Investigators conclusion 4 Details of experiments 5 Materials and methods 6-9 Observation 10 Result 11 Conclusion 12 Data sheets

4 Acute toxicity study: This study determines acute toxicity of Soberex Capsule an oral formulation prepared with suspension of test drug using gum acacia, in rats was assessed for single dose acute toxicity by employing OECD guidelines 425 using AOT software. The dosed (up down as per requirement) rats were observed for 14 days, for general appearance, cage side behaviour including increased or decreasing motor activity, convulsions, straub s reaction, catatonia, muscle spasm, spasticity, ophisthotonus, hyperesthesia, muscle relaxation, anaesthesia, arching and rolling, lacrimation, salivation, diarrheal, writhing movement, mode of respiration and changes in skin colour etc., with mortality and autopsy finding in case of dead animal. Conclusion: The test drug did not produce any mortality up to the dose of 2000mg/kg per oral which is equivalent to 22.4g total dose for a human being weighing 70 kg man. At the dose level studied the drug also did not produce any observable toxic effect except for mild irritation in animal in the dose550mg/kg and 2000mg/kg. Based on the observation made and recorded it can be concluded that the test drug is without any toxic potential even at the dose of 2000mg/kg in animals equivalent to 22.4g for human being. This dose in human being is 11 fold in comparison to normal human dose 2.04g per day. Mr. Ravi M M.Sc (Medical Pharmacology) Research Officer Pharmacology and Toxicology Lab Dr.B. Ravishankar M.Sc (Med Pharmacology)., Ph.D Director and Professor or Exptl Medicine SDM Centre for Research in Ayurveda and Allied Sciences Udupi Karnataka State 4

5 Acute oral toxicity study of test drug Soberex Capsule on rats Date of experiment : Acclimatization of animals : 7 days Date of treatment : End of experiment : Observation period : 14 days Regulatory guidelines followed : OECD test guideline No. 425 IAEC approved number : IAEC-12/11-SDMCRA SP/04 5

6 2. Materials methods: 2.1 Details of test formulation: 1. Name of the test formulation : Soberex Capsule Soberex Capsule : Information about name and quantity of the ingredients Ingredients name Botanical name Part or form Book Ref Quantity used 1. Ext. Brahmi Bacopa monnieri Whole Plant A.P.I. 50 mg 2. Ext. Jatamansi Nardostychas jatamsnsi Rhizome A.P.I. 30 mg 3. Ext. Shankhpushpi Convolvulus pluricaulis Whole Plant A.P.I. 50 mg 4. Ext. Sarpagandha Rauwolfia serpentina Root A.P.I. 10 mg 5. Vacha Acorus calamus Rhizome A.P.I. 30 mg 6. Ashwagandha Withania somnifera Root A.P.I. 20 mg 7. Arjun Terminalia arjuna Stem Bark A.P.I. 30 mg 8. Akik Bhasma ---- Powder R.T.S mg 9. Uste khudus Lavandula stoechas Flower N.A.VOL mg 10.Jyotismati Seed Celestrus paniculatus Seed A.P.I. 50 mg 11.Khurasani Ajama Hyocyamus niger Seed A.B. 50 mg 12.Excipients Q.S. 2. Batch number : SCA01( Trial batch for product permission and mfg. Licence) 3. Manufacturing date : December Expiry date : November Storage condition : Cool and dry place 6. General use : Stress reliever and memory enhancer. 7. CAS number : APPLIED 9. Country of origin : India 10. Manufacturer s name : VIRGO UAP Pharma Pvt.Ltd. Mahagujarat Ind.Estate Plot no. 423/ 98-B, Sarkhej - Bavala highway, Vill - MORAIYA, Ta - Sanand Dist - AHMEDABAD (GUJARAT) 6

7 2.2- TEST CONDITIONS 1. Animal species : Rats 2. Strain : Wistar albino 3. Source : Animal house attached to SDM Research centre, SDM Ayurveda College Udyavara 4. Selection : A total of 5 healthy either sex of body weight g Rats were selected according to AOT software. 5. Acclimatization period : All the selected animals were kept under acclimatization For 7 days before dosing. 7. Numbering and identification : The animal were marked with saturated Picric acid solution in water for proper identification. The marking within the cages is as follows. Animal Marking number 1 Head 2 Neck 3 Middle of the back 4 Base of the tail 5 No mark 7

8 The group number, animal number and sex of the animal were identified with the help of cage cards, as presented in following table. Sl. no Identification of animals Desired dose (according to AOT) Body weight (grams ) Calculated dose (ml) 1 Head 175mg/kg ml 2 Neck 550mg/kg ml 3 Back 2000mg/kg ml 4 Base of the tail 2000mg/kg ml 5 No mark 2000mg/kg ml Husbandry condition: 1. Housing : Rats were housed in each cage of poly propylene with stainless steel top grill. The dry husk was used as bedding material and was changed every morning. 2. Environment : The animal were exposed to 12 hours light and 12 hours dark cycle with The relative humidity 50 to 70 % and the ambient temperature was 22 ± 03 c. 3. Diet : Amruth brand rat pellete feed supplied by Pranav Agro Ltd, was provided throughout the study period except on previous night of dosing i.e (overnight) fasting before dosing. The drinking water was given ad libitum Propylene bottles with stainless steel sipper tube. 8

9 2.4 Preparation of Test formulation for administration: 1. Test drug : Soberex Capsule 2. Vehicle : Gum acacia 3. Dose preparation : The test formulation supplied by sponsor was made in to Fine suspension in vehicle with suitable concentration All the animal were dosed constant dose volume. (1 ml/ 100g body weight) 175 mg/kg, 550mg/kg, 2000mg/kg 4. Schedule : single dose per animal a) Administration : The test formulation was administered through oral route at different dose levels to respective animal through oral feeding needle on to disposable syringe b) Dose fixation : According to the AOT Software. C) Route : oral d) Dose : 175mg/kg, 550mg/kg, 2000mg/kg test substance e) Dose volume : 1ml/100g animal 9

10 3. Observation: 3.1 Examination of Physical and Behavioural changes : The animal was observed continuously for 4 hours after the dosing. The careful cage side observation was done without disturbing the animal attention and at the end of the every hour the animal were individually exposed to open arena for recording the behavioural changes like increased or decreased motor activity, convulsions, straub s reaction, muscle spasm, catatonia, spasticity, ophisthotonus, hyperesthesia, muscle relaxation, anaesthesia, arching and rolling, lacrimation, salivation, diarrhoea, writhing, mode of respiration, changes in skin colour etc. exitus, CNS depression hypo activity, passivity, relaxation, ataxia, narcosis, etc. 3.2 Mortality: All the animals were observed at ½, 1, 2, 3, 4, 24 h, 48 h after dosing and there daily once for mortality during the entire period of the study (i.e.14 days). after 10

11 4. RESULT: 4.1 Physical and behavioural examination: There were no physical and behavioural changes-(except mild increase in motor activity and irritability seen in animal dosed 550mg/kg and 2000mg/kg) in all the treated animals on day one at ½, 1,2,3,4 hours intervals after dosing and there after once daily for 14 consecutive days. Thus the data obtained from the study on single dose administration of Soberex Capsule oral administration up to 14 days of observation period does not result in any physical and behavioural changes. 4.2 Mortality: All the animals belonging to the treated group were survived throughout the 14 days observation period after dosing. 11

12 Conclusion: The test drug did not produce any mortality up to the dose of 2000mg/kg per oral which is equivalent to 22.4g total dose for a human being weighing 70 kg man. At the dose level studied the drug also did not produce any observable toxic effect except for mild irritation in animal in dose550mg/kg and 2000mg/kg and thus it could be concluded that the test drug is without any toxic potential even at the dose of 2000mg/kg in animals equivalent to 22.4g for human being. Dr. B. Ravishankar 12

13 Pharmacology Laboratory SDM Centre for research in Ayurveda & allied sciences, Kuthpadi, Udupi PRO FORMA FOR RECORDING SIGNS AND SYMPTOMS DURING ACUTE TOXICITY STUDIES Group: 175mg/kg Date: 13/01/12 Dose: 1.13ml Animal: Rat-1(220g) Route: Oral Drug: Soberex Capsule SIGNS & SYMPTOMS Basal 30 min 1h 2h 3h 4h 24h 48h General impression N N N N N N N N Increased motor activity Convulsion: Tonic Clonic Straubs reaction Muscle spasm Catatonia Opisthotonus Hyperaesthesia Decreased motor activity Muscle relaxation Anaesthesia Arching and rolling Lacrimation Diarrhoea Writhing Salivation Viscid Watery Respiration Stimulation Depression Failure Skin colour Blanching Cyanosis Vasodilatation Grip strength(kg)-mean N N N N N N N N Visual placing response N N N N N N N N Tail pinch response N N N N N N N N Auditory response N N N N N N N N mucus membrane N N N N N N N N Piloerection Experiment conducted by: Head of the lab (Director): N: Normal 13

14 Pharmacology Laboratory SDM Centre for research in Ayurveda & allied sciences, Kuthpadi, Udupi PRO FORMA FOR RECORDING SIGNS AND SYMPTOMS DURING ACUTE TOXICITY STUDIES Group: 550mg/kg Date: 16/01/12 Dose: 0.7ml Animal: Rat-2(130g) Route: Oral Drug: Soberex Capsule SIGNS & SYMPTOMS Basal 30 min 1h 2h 3h 4h 24h 48h General impression N N N N N N N N Increased motor activity Convulsion: Tonic Clonic Straubs reaction Muscle spasm Catatonia Opisthotonus Hyperaesthesia Decreased motor activity Muscle relaxation Anaesthesia Arching and rolling Lacrimation Diarrhoea Writhing Salivation Viscid Watery Respiration Stimulation Depression Failure Skin colour Blanching Cyanosis Vasodilatation Grip strength(kg)-mean N N N N N N N N Visual placing response N N N N N N N N Tail pinch response N N N N N N N N Auditory response N N N N N N N N mucus membrane N N N N N N N N Piloerection Experiment conducted by: Head of the lab (Director): N: Normal 14

15 Pharmacology Laboratory SDM Centre for research in Ayurveda & allied sciences, Kuthpadi, Udupi PRO FORMA FOR RECORDING SIGNS AND SYMPTOMS DURING ACUTE TOXICITY STUDIES Group: 2000mg/kg Date: 19/01/12 Dose: 3.4ml Animal: Rat-3(175g) Route: Oral Drug: Soberex Capsule SIGNS & SYMPTOMS Basal 30 min 1h 2h 3h 4h 24h 48h General impression N active active active active active N N Increased motor activity Convulsion: Tonic Clonic Straubs reaction Muscle spasm Catatonia Opisthotonus Hyperaesthesia Decreased motor activity Muscle relaxation Anaesthesia Arching and rolling Lacrimation Diarrhoea Writhing Salivation Viscid Watery Respiration Stimulation Depression Failure Skin colour Blanching Cyanosis Vasodilatation Grip strength(kg)-mean N N N N N N N N Visual placing response N N N N N N N N Tail pinch response N N N N N N N N Auditory response N N N N N N N N mucus membrane N N N N N N N N Piloerection Experiment conducted by: Head of the lab (Director): N:Normal 15

16 Pharmacology Laboratory SDM Centre for research in Ayurveda & allied sciences, Kuthpadi, Udupi PRO FORMA FOR RECORDING SIGNS AND SYMPTOMS DURING ACUTE TOXICITY STUDIES Group: 2000mg/kg Date: 21/01/12 Dose: 3.7ml Animal: Rat-4(190g) Route: Oral Drug: Soberex Capsule SIGNS & SYMPTOMS Basal 30 min 1h 2h 3h 4h 24h 48h General impression N N active N N N N N Increased motor activity Convulsion: Tonic Clonic Straubs reaction Muscle spasm Catatonia Opisthotonus Hyperaesthesia Decreased motor activity Muscle relaxation Anaesthesia Arching and rolling Lacrimation Diarrhoea Writhing Salivation Viscid Watery Respiration Stimulation Depression Failure Skin colour Blanching Cyanosis Vasodilatation Grip strength(kg)-mean N N N N N N N N Visual placing response N N N N N N N N Tail pinch response N N N N N N N N Auditory response N N N N N N N N mucus membrane N N N N N N N N Piloerection Experiment conducted by: Head of the lab (Director): N: Normal 16

17 Pharmacology Laboratory SDM Centre for research in Ayurveda & allied sciences, Kuthpadi, Udupi PRO FORMA FOR RECORDING SIGNS AND SYMPTOMS DURING ACUTE TOXICITY STUDIES Group: 2000mg/kg Date: 23/01/12 Dose: 3.92ml Animal: Rat-5(200g) Route: Oral Drug: Soberex Capsule SIGNS & SYMPTOMS Basal 30 min 1h 2h 3h 4h 24h 48h General impression N N active Active N N N N Increased motor activity Convulsion: Tonic Clonic Straubs reaction Muscle spasm Catatonia Opisthotonus Hyperaesthesia Decreased motor activity Muscle relaxation Anaesthesia Arching and rolling Lacrimation Diarrhoea Writhing Salivation Viscid Watery Respiration Stimulation Depression Failure Skin colour Blanching Cyanosis Vasodilatation Grip strength(kg)-mean N N N N N N N N Visual placing response N N N N N N N N Tail pinch response N N N N N N N N Auditory response N N N N N N N N mucus membrane N N N N N N N N Piloerection Experiment conducted by: Head of the lab (Director): N: Normal 17

18 Rat No.1 Time of drug administration Time interval (min) Exitus Hypo activity Passivity Relaxation Ataxia Narcosis Ptosis Exophthalmo ses Hyperactivit y Irritability Stereotypy Tremors Convulsion Straub tail Analgesia Others Pharmacology Laboratory SDM Centre for research in Ayurveda & allied sciences, Kuthpadi, Udupi Drug : Soberex Capsule Date : 13/01/12 Group : 175mg/kg Route: Oral Animal: Rat-1(220g) Study: Acute oral toxicity CNS Depression ANS CNS Stimulation Basa l h 48 h Experiment conducted by: Head of the lab (Director): 18

19 Rat No.2 Time of drug administration Time interval (min) Exitus Hypo activity Passivity Relaxation Ataxia Narcosis Ptosis Exophthalmo ses Hyperactivit y Irritability Stereotypy Tremors Convulsion Straub tail Analgesia Others Pharmacology Laboratory SDM Centre for research in Ayurveda & allied sciences, Kuthpadi, Udupi Pro forma for assessing Gross behaviour Drug : Soberex Capsule Date : 16/01/12 Group : 550mg/kg Animal: Rat-2(130g) Route : Oral Study: Acute oral toxicity CNS Depression ANS CNS Stimulation Basa l h 48 h Experiment conducted by: Head of the lab (Director): 19

20 Rat No3 Time of drug administration Time interval (min) Exitus Hypo activity Passivity Relaxation Ataxia Narcosis Ptosis Exophthalmo ses Hyperactivit y Irritability Stereotypy Tremors Convulsion Straub tail Analgesia Others Pharmacology Laboratory SDM Centre for research in Ayurveda & allied sciences, Kuthpadi, Udupi Pro forma for assessing Gross behaviour Drug : Soberex Capsule Date : 19/01/12 Group : 2000mg/kg Animal: Rat-3(175g) Route: Oral Study: Acute oral toxicity CNS Depression ANS CNS Stimulation Basa l h h Experiment conducted by: Director: 20

21 Rat No4 Time of drug administration Time interval (min) Exitus Hypo activity Passivity Relaxation Ataxia Narcosis Ptosis Exophthalmo ses Hyperactivit y Irritability Stereotypy Tremors Convulsion Straub tail Analgesia Others Pharmacology Laboratory SDM Centre for research in Ayurveda & allied sciences, Kuthpadi, Udupi Proforma for assessing Gross behaviour Drug : Soberex Capsule Date : 21/01/12 Group : 2000mg/kg Animal: Rat-4(190g) Route: Oral Study: Acute oral toxicity CNS Depression ANS CNS Stimulation Basa l h h Experiment conducted by: Head of the lab (Director): 21

22 Rat No.5 Time of drug administration Time interval (min) Exitus Hypo activity Passivity Relaxation Ataxia Narcosis Ptosis Exophthalmo ses Hyperactivit y Irritability Stereotypy Tremors Convulsion Straub tail Analgesia Others Pharmacology Laboratory SDM Centre for research in Ayurveda & allied sciences, Kuthpadi, Udupi Pro forma for assessing Gross behaviour Drug : Soberex Capsule Date :23/01/12 Group : 2000mg/kg Animal: Rat-5(200g) Route: Oral Study: Acute oral toxicity CNS Depression ANS CNS Stimulation Basa l h h Experiment conducted by: Head of the lab (Director): 22