Evaluation of anticancer activity of matricaria recutita in eac and dla tumour bearing mice



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EVALUATION OF ANTICANCER ACTIVITY OF

MATRICARIA RECUTITA IN EAC AND DLA TUMOUR BEARING MICE.

Protocol of Dissertation Submitted

By

MISS .VINUTARANI.V.DEVARADDI



To

Rajiv Gandhi University of Health Sciences



Bangalore, Karnataka.

Under the guidance of



Dr. V. M. CHANDRASHEKHAR

Assistant Professor




Department Of Pharmacology,

HANAGAL SHRI KUMARESHWAR COLLEGE OF PHARMACY,

BAGALKOT- 587101, KARNATAKA

(2012-2013)

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA-BANGALORE
ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECT FOR

DISSERTATION


1.

Name of the candidate and address




VINUTARANI.V.DEVARADDI

Department of Pharmacology,

H.S.K. College of Pharmacy,

B.V.V.S campus,

Bagalkot-587101, Karnataka.



2.

Name of institution


H.S.K. College of Pharmacy,

B.V.V.S campus,

Bagalkot-587101, Karnataka.




3.

Course of study and subject



Master of Pharmacy in Pharmacology




4.

Date of admission to course




1 Jan 2012



5.

Title of topic :



Evaluation of Anticancer activity of Matricaria recutita in EAC and DLA tumors bearing Balb/c mice.






6.

7.

8.



BRIEF RESUME OF THE INTENDED WORK

    1. Need for the study:

Cancer is the second leading cause of death in the world1. Throughout history and across the world, the plant kingdom has provided a variety of medicines for cancer treatment, currently over 60٪ of the drugs are derived in one or other way from natural source including plant, marine organism and micro-organism. There are worldwide efforts to discover anticancer agents from plants2.

Cancer is a disease characterized by uncontrolled multiplication and spread of abnormal forms of body’s own cells. It is one of the major causes of death in the developed countries. One in three people will be diagnosed with cancer during their life time and in 2001, 270,000 new cases were reported in the UK with lung and bowel cancer comprising the large category closely followed by breast and prostate cancer1. Heart diseases have declined by 45%, but increasing cancer death still is continued since 1997 according to the data base of the National Cancer Institute Survival Epidemiology and End {NCISEE}3.

Remarked differences can be found in incidence of cancers depending upon genetic and environmental factors. The carcinogenicity by UV rays and chemical agents are the major causes for cancer4. Cigarette smoking is probably the most significant single factor that contributes to the development of cancer5. Cancer can be treated with chemotherapeutic agents along with radiation and surgery. The chemotherapeutic agents provide a temporary improve of symptoms and signs of cancers and most of the chemotherapeutic agents are given in combination with the radiation therapy and surgery, but patient has to consume for the long duration and these drugs are more toxic and economically burden to the patients6.

Several molecules isolated from various marine organisms (algae, fungi, invertebrates and vertebrates) are currently under study at an advanced stage of clinical trials, either directly or in the form of structural modification. Some of them have already been marketed as drugs7. The novel anti-tumor agent trabectedin and aplidine are under phase III clinical trials8,9. With the above the background main goal of the present study is to explore the anticancer activity of Matricari recutita. an in vitro and in vivo evaluation in EAC bearing mice.



6.2 REVIEW OF LITERATURE:
Plant Profile:

Title of the plant : Matricaria recutita Linn.

Family : Asteraceae (Compositae)

Synonyms : Matricaria, German Chamomile, Sweet False Chamomile’s.

Habitat : German Chamomile is annual herb originally from Europe which has to the wild and now naturalized in almost every continent. It can now be found growing along fence rows and in sunny open field from Southern Canada to Northern U.S. West to Minnesota.

Chemical constituents:

The plant contains Terpenoids like α-Bisabolol, α-Bisabolol oxide A and B, Chamazulene, Sesquiterpenes. Flavonoids like Apigenin, Luteolin, Quercetin, Coumarins, Umbelliferone and others are Anthemic acid, Choline, Tannin and Polysaccharides.



Medicinal uses :

Matricaria recutita Linn. has been used medicinally for thousands of years and is widely used in Europe. It is a popular treatment for numerous ailments including sleep disorders, anxiety, digestion, intestinal conditions, skin infections, inflammations (including eczema), wound healing, infantile colic, teething pains and diaper rash. In the USA, Chamomile is the used externally for wounds, ulcers, eczema, gout, neuralgia, rheumatic pain, hemorrhoids and leg ulcers etc 10.

Pharmacological actions :

Matricaria recutita is the potentially used as an antioxidant 11, neuroprotective12, diabetic complications13, cardiac effects 14, antimicrobial activity 15, anti proliferative and apoptic activity16. Cardiovascular effects as increase atrial rate, relaxed thoracic aorta17, hemodynamic effects and anti ulcer activity18. Chamomile’s main active constituents are apigenin and bisabolol. Apigenin have cardio protective activity 19, 20.

6.3 Objectives of the study:

The main goal of the present study is to evaluate the anticancer activity of Matricaria recutita. species with the following objectives:



  1. In vitro evaluation of anticancer activity of the extract of Matricaria recutita. species.

  2. In vivo evaluation of anti cancer activity of Matricaria recutita extracts in EAC and DAL Tumour cells bearing mice.

  3. Effect of the Matricaria recutita. extracts on physiological and haematological parameters in EAC EAC and DAL Tumour cells bearing mice.

  4. The effect of the Matricaria recutita. extracts on biochemical and histopathological parameter changes in EAC and DAL Tumour cells bearing mice.


METHODS AND MATERIALS:

7.1. Source of data: All the data will be collected from the experimental animal model and the animals will be subdivided into following groups and the dose of extract of Matricaria recutita.. was selected on the basis of acute toxicity test as per OECD guidelines.

I Anticancer activity of Matricaria recutita. against Ehrlich Ascites Carcinoma bearing Balb/c mice

a) Group I : Normal/positive control (n=8)

b) Group II : Tumor control (n=8)

c) Group III : 5-Flurouracil (n=8)

d) Group IV : Low dose of methanol extract (n=8)

e) Group V : Moderate dose of methanol extract (n=8)

f) Group VI : High dose of methanol extract (n=8)


II Anticancer activity of Matricaria recutita. against Daltons lymphoma Ascites (DLA) bearing Balb/c mice

a) Group I : Normal/positive control (n=8)

b) Group II : Tumor control (n=8)

c) Group III : 5-Flurouracil (n=8)

d) Group IV : Low dose of methanol extract (n=8)

e) Group V : Moderate dose of methanol extract (n=8)

f) Group VI : High dose of methanol extract (n=8)

7.2 Materials :

Animals : Female Balb/c mice

Chemicals : All the chemicals of AR grade

Instruments : Haemocytometer, Binocular microscope, Centrifuge machine etc.



7.3 Methods:

Preparation of Plant extract:-

The plant will be authenticated and collected from NBRI, Lucknow in ideal conditions will be air dried under the shade and powdered to a fine texture of uniform size by passing through the sieve # 44. The powder collected will be extracted with polar and non polar solvent and the extract obtained will be used for the present study.



In vitro studies: Tryphan Blue Exclusion Method (Cell viability test)21:

This is one of the methods to assess cytotoxicity of anticancer compounds. It comes under preliminary screening of anticancer compounds. This test is based on the principle that the living cell membrane has the ability to prevent the entry of dye. Hence, they remain unstained and can be easily distinguished from dead cells, which take the dye.



Method: For EAC

The Ascitic fluid was withdrawn from animals which were induced with EAC cell lines. Ascitic fluid of 0.1 ml was aspirated and it was diluted with 2ml of phosphate buffered saline (PBS). To this equal volume of different extracts were mixed and this mixture was incubated for 3hrs at 370C. Then the mixture was added to 0.5ml of tryphan blue and mixed thoroughly. The diluted suspension was charged into haemocytometer. The viable cells (unstained) were counted in a WBC chamber under a microscope and the mean numbers of cells in four chambers were calculated as follows

Total number of cells = Mean number of cells Dilution factor (2) ×10 4

Method: For DLA22

Cell lines:

Dalton s Lymphoma Ascites (DLA) tumour cells were obtained through the courtesy of Amala Cancer Research Centre, Thrissur, Kerala, India.



In vitro cytotoxic assay

DLA was maintained by serial transplantation from mice to mice .The ascetic fluid of the DLA was drawn out from the donor mice carrying tumour for 7-9 days.the freashly drawn ascetic fluid from the peritoneal cavity was washed thrice with phosphate buffer saline (PBS,pH7.4) and diluted in PBS to a concentration of 106 cells/ml.One million cells were incubated with various concentrations of the herbal extract() in a total volume of 1ml for 3 hours at 370C. 0.1% carboxy methyl cellulose (CMC) was used as control.After incubation the viability of the cells was determined by the tryphan blue exclusion method23



Effect of ethanolic herbal extract residues on ascites tumour bearing animals

BALB/c mice (20-25 g) were obtained from the Breeding section, Amala Cancer Research

Centre, Thrissur. They were kept in groups of six in well ventilated cages in air controlled room, fed with normal mice chow (Sai Feeds, Bangalore, India) and water adlibitum. All animal experiments were conducted after permission from the Institutional Ethical Committee. DLA cells were aspirated from the peritoneal cavity and washed three times with PBS. Ascites tumour cells were then inoculated to three groups (6 animals / groups) of BALB / c mice by injecting one million cells in to their peritoneal cavity. After 24 h of tumour inoculation two different dosages of herbal extract residues were given and continued for ten consecutive days.

Group I : Untreated control for DLA

Group II : DLA cells + 10 mg/kg b.wt of Matricaria recutita herbal extract residue in 0.1% CMC (ip) – low dosage

Group III : DLA cells +50 mg/kg b.wt of Matricaria recutita herbal extract residue in 0.1% CMC (ip) – high

The death pattern of animals due to tumour burden was noted every day and the percentage of increase in life span was calculated using the formula, T-C/C X 100 where ‘T’ and ‘C’ are the number of days the treatedand control animals survived respectively24 .

Haematological parameters

On the 15th day post tumour inoculation, blood was collected in heparinzed tubes from the respective animals by tail vein and the haematological parameters such as white blood cell count, red blood cell count and percentage of haemoglobin were determined by following the standard procedures .24



Tumour cell count

0.1 ml of the ascitic fluid was aseptically withdrawn on the 15th day post tumour inoculation using a 1 ml syringe and diluted with 0.9 ml of PBS (pH 7.4) to adjust the cell count to 1x106 cells. From the stock 0.1 ml was taken and mixed with 0.8 ml of PBS and then incubated for 3 hours at 37 0C. 0.1 ml of tryphan blue was then added to this Then a drop of the resulting solution was loaded on the heamocytometer and the number of tumour cells randomly in every 100 cells was counted 24.



Estimation of SGPT and SGOT

On 15th day post tumour inoculation, blood from the tail vein of treated and control mice was withdrawn individually into test tubes which were then centrifuged at low speed for about 10-15 min to collect the serum for enzyme analysis following the method 25.



In vivo Anticancer activity of extracts:

Induction of Ehrlich Ascites Carcinoma:

The Ascitic carcinoma bearing mice (donor) was taken 15 days after tumor transplantation. The Ascitic fluid was drawn using an 18-gauge needle into sterile syringe. A small amount was tested for microbial contamination. Tumor viability was determined by Trypan blue exclusion test and cells were counted using haemocytometer. The Ascitic fluid was suitably diluted in normal saline to get a concentration of 106 cells/ml of the tumor cell suspension. This was injected by intraperitonial route to obtain ascitic tumor. The mice were weighed on the day of tumor inoculation and then for three each days. Treatment was started 24 hours after tumor inoculation. 5-Flurouracil (Standard) and extracts were administered till the 9th day by intraperitonial route26.



Induction of DLA27:

DLA was maintained by serial transplantation from mice to mice .The ascetic fluid of the DLA was drawn out from the donor mice carrying tumour for 7-9 days.the freashly drawn ascetic fluid from the peritoneal cavity was washed thrice with phosphate buffer saline (PBS,pH7.4) and diluted in PBS to a concentration of 106 cells/ml. Ascites tumour cells were then inoculated to three groups (6 animals / groups) of BALB / c mice by injecting one million cells in to their peritoneal cavity. After 24 h of tumour inoculation two different dosages of herbal extract residues were given and continued for ten consecutive days.


A]. Physical parameters :

  1. Percentage increase in body weight as compared to day-0.

  2. Median survival time (MST).

  3. Percentage increase in life span (%ILS) 28.

  4. Mean survival time (MEST).

  5. Cell viability test (% survivors of malignant cells in ascitic fluid) 29.

B]. Haematological parameters30.

Total WBC, DLC, RBC and Hemoglobin content.



C] Biochemical parameters :

After the collection of the blood samples the mice were sacrificed and their liver was excised, rinsed in ice-cold normal saline followed by cold 0.15 mol/lit Tris-Hcl buffer (pH 7.4), blotted dry and weighed. The homogenate 10% w/v was prepared in 0.15 mol/lit Tris-Hcl buffer and a portion of it was utilized for the measurement of LPO31 and other portion of the same after precipitating with TCA was used for the estimation of glutathione. The remaining homogenate was centrifuged at 1500 rpm for the estimation of SOD, catalase and protein content32.



Statistical analysis:

FOR EAC:


All the data were expressed in mean + SEM. The significance of differences in mean between control and treated animals for different parameters was determined by using one way ANOVA followed by Dunnett’s multiple comparison (Post-hoc) test. Percentage tumor inhibition can be calculated by Chi-Square test.

FOR DLA:

Results were expressed as mean + standard deviation and Student’s ʻt’ test was used.



7.4 Does the study require any investigation or to be conducted on patients or other humans/animal? If so please describe briefly

Yes, the above study requires to be carried out in mice. The effects of preparation on cancer will be studied by considering physiological, pathological and biochemical parameters using animal models.



7.5 Has ethical clearance been obtained from your institution for performing various tests on animals?

Yes, the study is cleared from institutional animal ethics committee. copy is enclosed with protocol.



References:

  1. Madhusudhan S, Middleton MR. The immerging role of DNA repair proteins as predictive, prognostic, and therapeutics in cancer. Cancer Treat.2005;31:603-617.

  2. Newman DJ, Cragg, Snader KM. The natural product as a source of new drugs over the period 1981-2001. J Nat Prod.2003;66:1022-1037.

  3. Rang HP, Dale MM, Ritter JM, Flower RJ. Rang and Dale’s Pharmacology, Churchil Livingstone Elsevier.2007;6:718-719.

  4. Brunton LP, Goodman and Gilman’s. The Pharmacological Basis of therapeutics. 2006;11:1315-1316.

  5. Aaithiresain K, Boopathy NS, Kavitha S. Costal vegetation underexplored source of anticancer drugs. Nat Prod Rad.2006;5:115-119.

  6. Mary AK, Brian AK, Robin L, Joseph BG, Wayne AK, Williams BR. Applied Therapeutics. American Cancer Society, Cancer facts and figures 2007. Atlanta, GA: American cancer society.2007;9:881-882.

  7. Prajapati ND, Kumar U. Agro Dictionary of Medicinal plants. Jodhpur. India.2003: 218.

  8. Kodrechi MB, Kornprobst JM. Marine pharmacology: Potentialities in the treatment of Infectious diseases, Osteoporosis and Alzheimer’s disease. Adv Biochem Eng /Biotechnol. 2005; 97: 105-131.

  9. Vankesteren C, Devought MM, Lopez-Lozarol L, Mahot PA, Schellens JH, Jimeno JM, Beijin J, Yondelis. Ctrabectrdin ET-743. The development of an anticancer agent of marine origin. Anticancer Drugs.2003;14:481-50.

  10. Newel CA, Anderson LA, Phillipson JD, Herbal medicines. A guide for health care professionals. London, Pharmaceutical press.1996;9:296.

  11. Pereira RP, Faachinetto R, de Souza Prestes A, Puntel RL, Santos da Silva GN, Heinzmann BM, Burger ME, Morel AF, Morsch VM, Rocha JB. Antioxidants effects of different extracts from Melissa officinalis, Matricariarecutita and Cymbopogoncitratus. Neurochem Res.2009; 34(5):973-983.

  12. Chandrashekhar VM, Ranpariya VL, Ganapathy S, Parashar A, Muchandi AA. Neuroprotective activity of Matricaria recutita Linn against global model of ischemia in rats. J Ethanopharmacol.2010;127:645-651.

  13. Kato A, Minoshima Y, Yamamoto J, Adachi I, Watson AA, Nash RJ. Protective effects of dietry Chamomile tea on diabetic complication. J Agric Food Chem.2008;56(17):8206-8211.

  14. Lawrence G, Raman Reddy CV, Robert FG. Cardial effects of Chammomile tea. J Clin Pharmacol.1973;13:475-479.

  15. Nogueira JC, DinizMde F, Lima EO. Anti microbial activity of plants in acute otitisexterna Braz J Otorhinolarygol.2008;74(1):118-24.

  16. Srivastava JK, Gupta S. Antiproliferative and apoptic effects of chamomile extract in various human cancer cells. J Agric Food Chem.2007;55(23):9470-9478.

  17. Lorenzo PS, Rubio MC, Medina JH. Involvement of monoamine oxidase and noradrenaline uptake in the positive chronotropic effects of apigenin in rat atria. Euro J Pharmacol.1996; 312:203-207.

  18. Ko FN, Huang TF, Teng CM. Vasodilatory action mechanism of apigenin isolated from apium graveollens in rat thoracic aorta. Biochem Biophys Acta.1991;14:1115(1):69-74.

  19. Gould L, Reddy CVR, Gomprecht RF. Cardiac effects of chamomile tea. J Clin Pharmacol New Drugs.1973;13:475-479.

  20. Tamasdon S, Cristea E, Mihele D. Action upon gastric secretion of Robiniae Flores, chamomillae Flores and strobulilupuli extracts. Faracia.1981;29:71-75.

  21. Lowry OH, Rosebrough NJ, Farr AL, Randall R. Protein measurement with the Folinphenol reagent. Biol Chem.1951:193:265-275.

  22. Ramasamy Ganesan,Papanasam Kamalraj, Krishnasamy Muthuchelian.Protective effects of ethanolic extract residue isolated from the bark of Terminalia Arjuna against DLATumour Cells.Asian Pacific J Cancer Prev,11,803-808.

  23. Talwar GP(1974).Hand book of practical immunology.In Talwar GP (Eds)National Book Trust, New Delhi,336-9.

  24. Ramnath V, Kuttan G,Kuttan R (2002) Antitumor effect of abrin on transplanted tumours in mice .Indian J Physiolpharmacol,4,69-77.

  25. Reitman S,Frankel S(1957).A calorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases.Am J Clin Pathol,28,56.

  26. Naigonkar AV, Burande MD. A manual of medical laboratory technology. Nirali’s Prakashan.1996;2:284..

  27. Ramasamy Ganesan,Papanasam Kamalraj, Krishnasamy Muthuchelian.Protective effects of ethanolic extract residue isolated from the bark of Terminalia Arjuna against DLATumour Cells.Asian Pacific J Cancer Prev,11,803-808.

  28. Umadevi P, Emerson SF, Sharada AC. In vivo tumor inhibitory and radiosensitising effects of an Indian Medicinal Plant, Plumago rosea , on experimental mouse tumors. Indian J Exp Biol. 1994;32:523-528.

  29. Mazumdar UK, Gupta M, Suryyendubkas M, Dilip M. Anticancer activity of Hygrophylia Spinosa on EAC and sarcoma-180 induced mice. Ind J Exp Biol.1997;35:473-477.

  30. Ellman GL. Tissue sulphydryl groups. Arch Biochem Biophys.1979;82:70-77.

  31. Onkava H, Onishi N, Yagi K. Assay for lipid peroxidation in animal tissue by thiobarbituric acid reaction. Anal Biochem.1979:95;351-358.

  32. Kakkar P, Dos B, Vishwanathan PN. A modified spectrophotometric assay of Superoxide Dismutase, Ind J Biochem Biophys.1984;21:130-132.







9


SIGNATURE OF THE CANDIDATE




Vinutarani .V. D


10


REMARKS OF THE GUIDE

The plant is reported for treating very potential for various diseases. With this, our present study involves the search of new agent and validation of extract of Matricaria recutita for its anticancer activity.





11




NAME AND DESIGNATION OF THE GUIDE



Dr. V.M.CHANDRASHEKHAR

Associate Professor





12


SIGNATURE







13


CO-GUIDE





Dr. Nanu R. Rathod

Assistant Professor





14


SIGNATURE







15


HEAD OF THE DEPARTMENT



Dr. I. S. MUCHCHANDI

H.O.D. ,Department of Pharmacology

H.S.K.College of Pharmacy,

B.V.V.S.Campus,Bagalkot-587101




16


SIGNATURE







17


REMARKS OF THE PRINCIPAL




18


NAME OF THE PRINCIPAL



Dr. I.S. MUCHCHANDI

H.O.D. ,Department of Pharmacology

H.S.K. College of Pharmacy,

B.V.V.S.Campus,Bagalkot-587101




19


SIGNATURE







OFFICE OF THE INSTITUTIONAL ANIMAL ETHICS COMMITTEE (IAEC)

HANGAL SHRI KUMARESHWAR COLLEGE OF PHARMACY,

BAGALKOT-587101, KARNATAKA.

REG NO.821/01/a/CPCSEA,Dated:6th AUG 2004 UNDER THE RULES 5(a) OF THE

“BREEDING OF AND EXPERIMENTS ON ANIMAL (control and supervision)

RULES 1998”

Ref: HSK CP/IAEC, Clear/1-8/2012-13

CERTIFICATE

This is to certify that Miss. Vinutarani.V.Devaraddi a student of FIRST M.Pharm is permitted to carry out experiments on animals for the dissertation / thesis work entitled “Evaluation of Anticancer activity of Matricaria recutita in EAC and DLA tumour bearing mice” as per details mentioned and after observing the usual formalities laid down by IAEC as per provision made by CPCSEA.



Animal house in charge Chairman

Form B

See rule [6 (a) and 8 (a)]

PART A

1]

Name and address of the establishment:


H.S.K. COLLEGE OF PHARMACY

BAGALKOT, KARNATAKA.

2]

Date and registration Number of the

Establishment:



821 /01/a CPCSEA.


3]

Name, address and registration NO. of the

Breeder from whom acquired and the date of

Acquisition:

OFFICE OF CPSEA,

MINISTRY OF ENVIRONMENT AND

FOREST, 3RD SEAWARD ROAD,

VALMIKINAGAR, THRIRUVANMIYUR,

CHENNAI-600041.


4]

Place where the animals are presently kept:


ANIMAL HOUSE,

H.S.K. COLLEGE OF PHARMACY,

BAGALKOT, KARNATAKA.


5]

Place where experiment is to be performed:


DEPARTMENT OF PHARMACOLOGY,

H.S.K. COLLEGE OF PHARMACY,

BAGALKOT, KARNATAKA.


6]

The date on which the experiment is to be commence and the duration of the experiment :


1 Dec 2012 ,6 months



The protocol form for the research proposal - PART B in the case of experiments using other than non-human primate animals for a few projects, PART C for use of non-human primate for new projects and PART D for use of non-human primate for extension of ongoing projects-should be duly filled, signed and annexed with this form.

Dated: Signature

Place: (NAME AND DESIGNATION)


PART –B
Protocol form for research proposal to be submitted to the committee on use of small animals / animals other than non-human Primate in biomedical research for ONGOING / NEW PROJECTS.

1.

Project title :

EVALUATION OF ANTICANCER ACTIVITY OF MATRICARIA RECUTITA IN EAC DLA TUMOUR BEARING MICE.


2.

Investigation(s) :

Designation





Dr. V.M.CHANDRASHEKHAR

Assistant professor



3.

Department(s) :


Department of Pharmacology,

H.S.K. College of Pharmacy,

Bagalkot, Karnataka.

4.

(a) Funding source(s) : if any

----


(b) Are sufficient funds available for purchase and maintenance of the animals



Yes



(c)

Duration of present project:




(1) Number of months:

6 months



(2) Date of start of the project:

( Experiment)



1 Dec 2012



(3) Date of termination of the project:


1 May 2012



5.

Date by which approval is needed in case the project is to be funded by outside agency (If less than six weeks from the date of admission, please justify below).

----




6.

Summary of project briefly summarize in laymen’s term the background, the objective and the experimental approach

(a)Background:

Enclosed



(b)Objectives:

Enclosed



(c)Experimental procedure:

Enclosed



7.

(a)

Name of species

Balb/c mice



Age

Sex

Weight

4-8 weeks


Female

20-30 g/20-25


(b)

Rationale for selection

Approximate number of animals required during the first 12 months.

96


Justification of number (define treatment group and number per group)

12 groups,

Each group containing eight animals.



Number of animals housed per week

30


8.

List all invasive Non-Surgical Animals Procedures and Potentially stressful Non-invasive procedures to be used (example: IM injection, foot pad injection, venapunctures).

Invasive Non-surgical animal procedures.



Procedure and approximate frequency:

6 months Enclosed



Anaesthetic and /or Analgesic and dosage:

Not applicable



Test substance injected and /or applied:

Post Orally Administered





9.

Does the protocol prohibit the use of anaesthetic and analgesic for the conduct of painful procedures?

No


10.

With surgical procedure/Experimental procedure be performed?
No



(a)Will the animal be sacrificed after surgery?
No


(b)Give anticipated post-operative survival time:
No

11.

Will hazardous agent such as radio isotopes, carcinogens, radiation exposure, microbial and parasitic agent be administered to animals?
No.

INVESTIGATOR SIGNATURE



DATE: _________________



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