Pharmacological studies on matricaria recutita L. In acute and chronic inflammation




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PHARMACOLOGICAL STUDIES ON MATRICARIA RECUTITA L. IN ACUTE AND CHRONIC INFLAMMATION
Protocol of Dissertation Submitted

By

Mr. PARVINDER SINGH

To

Rajiv Gandhi University of Health Sciences

Bangalore, Karnataka.

Under the guidance of

Mr. Chandrashekhar V.M.

Associate Professor




DEPARTMENT OF PHARMACOLOGY

HANAGAL SHRI KUMARESHWAR COLLEGE OF PHARMACY

BAGALKOT-587101, KARNATAKA

(2010-2011)

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA-BANGALORE
ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECT FOR

DISSERTATION


1.

Name of the candidate and address




PARVINDER SINGH

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



22 July 2010





5.

Title of topic:



Pharmacological studies on Matricaria recutita Linn. in Acute and

Chronic inflammation.





6.

7.

8.



BRIEF RESUME OF INTENDED WORK
6.1 NEED FOR THE STUDY
Inflammation is part of the complex biological responses of vascular tissue to harmful stimuli such as pathogens, damaged cells, or irritants. When inflammation occurs, chemical substances from WBCs released into blood and affected tissues in an attempt to rid the body of foreign substances. Inflammation may result in severe inflammatory reactions such as rheumatoid arthritis, atherosclerosis, lung fibrosis and the life threatening hypersensitivity reactions to insect bite, drugs and toxins1. The anti-inflammatory drugs would ideally control the squeal of inflammation. Inflammation is a protective attempt by organism to remove injurious stimuli and to initiate the healing process. Inflammation is one of the responses of the organism to the pathogen which prolongs infections and wound healing will be delayed.

Inflammation can be classified as either acute or chronic inflammation. Acute inflammation is the initial response of the body to harmful stimuli and is achieved by the increased movement of plasma and leukocytes (especially granulocytes) from the blood into the injured tissues. A cascade of biochemical events propagates and matures the inflammatory response, involving the local vascular system or immune system, and various cells within the injured tissue. Prolonged inflammation, known as Chronic inflammation, leads to a progressive shift in the type of cells present at the site of inflammation and is characterized by simultaneous destruction and healing of the tissue from the inflammation process2. Consequently anti-inflammatory drugs are extensively employed in virtually all branches of medicine. Search continues to develop drugs that have potent anti inflammatory activity with minimum side effects, because most of present available anti-inflammatory agents have reported for intestinal bleeding and gastric ulceration3.

There are several plant derived preparations, which showed potential anti-inflammatory activity such as Conyza floribunda, Bonafousia longituba, etc. With continuation of our traditional screening of anti-inflammatory agents from plant origin4, the present study was designed to evaluation of Pharmacological activity of Matricaria recutita in acute and chronic inflammatory conditions, with establishing its possible mechanism of action as an anti-inflammatory agent.

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 etc5.

Pharmacological actions : Matricaria recutita is the potentially used as an antioxidant6, neuroprotective7, diabetic complications8, cardiac effects9, antimicrobial activity10, anti proliferative and apoptic activity11. Cardiovascular effects as increase atrial rate, relaxed thoracic aorta, hemodynamic effects and anti ulcer activity. Chamomile’s main active constituents are apigenin and bisabolol. Apigenin have cardio protective activity12, 13, 14, 15.
6.3 OBJECTIVE OF STUDY

The traditionally well known Matricaria recutita (M.R) will be extracted by using non-polar and polar solvents and extract will be used for the present study with following objectives.



  1. In vitro antioxidant activity of Matricaria recutita

  2. Effect of Matricaria recutita extract on acute inflammation

  3. Effect of Matricaria recutita extract on chronic inflammation

MATERIALS AND METHODS:

7.1 Source of data:

The data will be based on animal experiment as per the parameter studied for the model. Selection of dose will be based on earlier acute toxicity studies7 and animals were divided into following groups:



  1. Acute inflammatory models

Group I : Normal saline 10 mg/kg, p.o.

Group II : Control receives 0.1ml of 0.5% Arachidonic acid in 0.2 M carbonate buffer.

Group III : Standard drug Diclofenac (10 mg/kg, i.p).

Group IV : Matricaria recutita extract (100mg/kg, p.o) treatment.

Group V : Matricaria recutita extract (200mg/kg, p.o) treatment.

Group VI : Matricaria recutita extract (300mg/kg, p.o) treatment.



  1. Chronic inflammatory models

Group I : Normal saline 10ml/kg, p.o.

Group II : Control receives 0.1ml of Freund’s adjuvant in normal saline.

Group III : Standard drug Diclofenac (10 mg/kg, i.p).

Group IV : Matricaria recutita extract (100mg/kg, p.o) treatment.

Group V : Matricaria recutita extract (200mg/kg, p.o) treatment.

Group VI : Matricaria recutita extract (300mg/kg, p.o) treatment.


7.2 Materials:

Plant : Matricaria recutita Linn.

Chemicals : Arachidonic acid, Acetic acid, Methanol.

Animals : Male Albino Rats, Swiss Albino Mice.

Instruments : Research microscope, Centrifuge, Digital Plethysmograph.

7.3 Methods:

7.3.1 Preparation of plant extract:

The plant 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

7.3.2 DPPH radical scavenging assay.

The free radical scavenging activity was measured in terms of hydrogen donating or radical scavenging ability, using the stable radical, DDPH. A 0.1 mM solution of DDPH in methanol was prepared and 0.1 ml of this solution was added to 3.0 ml of Control (without the test compound, but an equivalent amount of methanol) and test compound solution prepared at different concentrations (6.25-500 μg/ml) in different test tubes. Thirty minutes later, the absorbance was measured at 517 nm. The concentration of extract at which maximum percentage inhibition of DPPH radical and IC50 values were determined with the help of standard graph16. The percentage hydroxyl radical scavenging is calculated by the formula:

DPPH radical scavenging activity = 1 – (Absorbance of sample / Absorbance of blank) x 100.

7.3.3 In vitro peroxidation activity.

The degree lipid peroxidation was evaluated by estimating the thiobarbituric acid reactive substances (TBARS). Briefly, different concentrations of the extract (6.25-500 μg/ml) were added to the liver homogenate. Lipid peroxidation was initiated by adding 100 μl of 15 mmol FeSO4 solution to 3 ml of liver homogenate. After 30 minutes, 100μl of this reaction mixture was placed in test tube containing 1.5ml of 10% trichloro acetic acid (TCA) and centrifuged after 10 minutes. The supernatant was separated and mixed with 1.5ml of 0.67% thiobarbituric acid (TBA) in 50% acetic acid. The mixture was heated in water bath at 85oC for 30 minutes to complete the reaction. The intensity of the pink coloured complex was measured at 553nm with UV Spectrophotometer17, 18.



7.3.4 In vitro hydroxyl radical scavenging activity.

Various concentrations (6.25-500 μg/ml) of extracts were taken in different test tubes and evaporated to dryness. 1ml of Iron EDTA solution (0.13% ferrous ammonium sulfate and 0.26% EDTA), 0.5% of EDTA (0.018%) and 1ml of DMSO (0.85% v/v in 0.1 M phosphate buffer, pH 7.4) were added to these tubes and the reaction was initiated by adding 0.5 ml 0.22% ascorbic acid. Test tubes were capped tightly and heated on water bath at 80-90oC for 15mins. The reaction was terminated by the addition of 1ml ice cold TCA (17.5% w/v), 3 ml of Nash reagent (75.0 g ammonium acetate, 3 ml glacial acetic acid and 2 ml acetyl acetone were mixed and raised to one liter with distilled water) was added to all the test tubes and left at room temperature for 15 minutes for colour development. Intensity of yellow colour formed was measured spectrophotometrically at 412 nm against reagent blank19, 20. Maximum percentage inhibition of hydroxyl radical scavenging is calculated by the formula:

% Hydroxyl radical scavenging activity = 1-(Absorbance of sample / Absorbance ok blank) x 100.

7.3.5 Superoxide anion scavenging activity.

The reaction mixture containing nitroblue tetrazolium solution (156 μM NBT in 100 mM phosphate buffer, pH 7.4), NADH solution (465 μM in 100 mM phosphate buffer, pH 7.4) were mixed with different concentrations of the test compounds and the reaction was started by the addition of Phenazine methosulphate solution (60 μM PMS in 100 mM phosphate buffer, pH 7.4). The final reaction mixture was incubated at 25oC for 5 minutes, and then absorbance was measured at 560 nm in a UV spectrophotometer. A decrease in absorbance of the reaction mixture is indicative of an increasing superoxide anion scavenging activity21.



In vivo studies

7.3.6 Acute inflammatory models

7.3.6.1 Arachidonic acid induced rat paw oedema.

In this experiment different groups (as given above) of rats were pretreated with Matricaria recutita given (100mg/kg, 200mg/kg and 300mg/kg) p.o and the standard drugs [Cyproheptadine and Nimesulide (the dual blockers)]. Paw oedema was induced by single injection of 0.1ml of 0.5% Arachidonic acid in 0.2M carbonate buffer (pH 8.4) into the right hind paw (subplantar) of rats 30 mins after drug treatment. Hind paw volume was measured 1 hour after arachidonic acid injection by using digital Plethysmograph22.



7.3.6.2 Acetic acid induced peritoneal inflammation.

Acetic acid (0.05N; 0.4mL) in normal saline was administered to each animal (i.p.) 1 h after the administration of the test substances. Three hours later, the animals were killed under anaesthesia, and the protein content in the peritoneal exudates was determined and writhing response was observed by the method of Turner (1965). The time of writhing and the number of writhings in 15 mins were noted23.



7.3.6.3 Carrageenin-induced rat paw oedema (pre and post treatment).

The control vehicle, test and standard drugs were administered 30 minutes prior to the administration (pre) of Carrageenin (1%). The paw volumes were measured immediately and thereafter at hourly intervals, for 6 hours, following the administration of Carrageenin24. The same experiment was repeated with the test extract of M.R., administered after 2 hours following Carrageenin administration (post)25.


7.3.7 Chronic inflammatory models

7.3.7.1 Induction of arthritis.

Following anesthesia (45mg/kg of Ketamine chloride, i.p.), 0.1 ml of Freund’s adjuvant was injected in the sub-plantar tissue of right posterior paw of rat. Every day animals were carefully and thoroughly inspected, by examining the affected paw and the animals general status. Evaluation of anti-inflammatory effects of Matricaria recutita extract was performed by monitoring the oedema in the right paw. In control animals, sub-plantar injection of Freund’s adjuvant produced a pronounced local oedema after few hours with progressive increase, reaches to maximum the 8th day after inoculation or in the 30th day after induction of arthritis26.



7.3.7.2 Chronic treatment:

On day 0, the animals were subjected to behavioral test and assessment of body weight and right paws measurement, followed by the injection of Freund’s adjuvant in the right paw (Test I). On the 10th day after adjuvant administration, all tests are repeated (Test-II). On 15th day, animals were treated with distilled water (1ml/kg of body weight), and drug (lower & higher dose) and again subjected to the tests the next day (Test-III). The test solution was administered daily and testing application was done on 30th day after administration of Freund’s adjuvant tests IV, V, VI and VII respectively27.



7.3.8 Statistical Evaluation: The collected data will be evaluated by ANOVA method by Dunnett’s multiple comparison test method and will be confirmed by the Student’s t-test for intergroup differences.

    1. Does the study require any investigations or to be conducted on patients or other animals? If so please describe briefly.

Yes, for this study rats and mice will be used, before sacrificing the animals for FCA parameters, rats will be sacrificed by deep anesthesia using anesthetic agents.


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

Yes, the study is cleared from Institutional Animal Ethics Committee (IAEC). The copy is enclosed with this protocol.
References:


  1. Robbins S. L. and Ramzi S. C. Pathologic basis of diseases. 7th ed. Elsevier. 2005:48.




  1. Ferrero-Miliani L., Nielsen O.H., Andersen P.S., Girardin S.E. Chronic inflammation: importance of NOD2 and NALP3 in interleukin-1beta generation. Clin Exp Immunol.2007; 147 (2): 227–35.




  1. Rang H. P., Dale M. M., Ritter J. M., Flower R. J. Rang and Dale’s Pharmacology, 6th ed. Churchil Livingstone Elsevier 2007:226-247.




  1. Heras B. de les, Slowing K., Benedi J., Carretero E., Ortega T., Toledo C., Bermejo P. Anti-inflammatory and anti-oxidant activity of plants used in traditional medicine in Ecuador. J Ethanopharmacol 1998; 61(2):161-166.




  1. Newel C.A., Anderson L.A., Phillipson J.D., Herbal medicines. A guide for health care professionals. London, Pharmaceutical press 1996; 9: 296.




  1. Pereira R.P., Faachinetto R., de Souza Prestes A., Puntel R.L., Santos da Silva G.N., Heinzmann B.M., Burger M.E., Morel A.f., MorschV.M., Rocha J.B. Antioxidants effects of different extracts from Melissa officinalis, Matricaria recutita and Cymbopogoncitratus. Neurochem Res 2009; 34(5):973-83.




  1. Chandrashekhar V.M., Ranpariya V.L., Ganapathy S., Parashar A., Muchandi A.A. Neuroprotective activity of Matricaria recutita Linn against global model of ischemia in rats. J Ethanopharmacol. 2010; 127:645-651.




  1. Kato A., Minoshima Y., Yamamoto J., Adachi I., Watson A.A., Nash R.J. Protective effects of dietry Chamomile tea on diabetic complication. J Agric Food Chem. 2008; 56(17): 8206-11.




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




  1. Nogueira J.C., Diniz Mde F., Lima E.O. Anti microbial activity of plants in acute otitis externa Braz J Otorhinolarygol 2008; 74(1):118-24.




  1. Srivastava J.K., Gupta S. Antiproliferative and apoptic effects of chamomile extract in various human cancer cells. J Agric Food Chem 2007; 55 (23):9470-8.




  1. Lorenzo P.S., Rubio M.C., Medina J.H. Involvement of monoamine oxidase and noradrenaline uptake in the positive chronotropic effects of apigenin in rat atria. Euro J Pharmacol 1996; 312:203-7.




  1. Ko F.N., Huang T.F., Teng C.M. Vasodilatory action mechanism of apigenin isolated from apiumgraveollens in rat thoracic aorta. Biochem Biophys Acta 1991; 1115:69-74.




  1. Gould L., Reddy C.V.R., Gomprecht R.F. Cardiac effects of chamomile tea. J Clin Pharmacol New Drugs 1973; 13:475-479.




  1. Tamasdon S., Cristea E., Mihele D. Action upon gastric secretion of Robiniae Flores, Chamomillae Flores and strobuli lupuli extracts. Faracia 1981; 29:71-75.




  1. Wanger H., Bladt S. Plant drug analysis. Springer Verlag Publishers, Berlin, 1984:125




  1. Ganapathy S., Chandrashekhar V.M., Chitme H.R., Lakshmi Narsu M. Free radical scavenging activity of gosssypin and nevedensin. An in-vitro evaluation. Indian J Pharmacol 2007; 39(6):281-283.




  1. Madan Mohan P., Raghavan G., Ajay Kumar Singh R., Palpu P. Free radical scavenging potential of Saussareacostus. Acta Pharma. 2005; 55: 297-304.




  1. Singh R.P., Chidambra Murthy K.N., Jayaprakash G.K. Studies on the antioxidant activity of Pomegranate (Punicagranatum) peel and seed extracts using in-vitro models. J Agric Food Chem. 2002; 50: 81-86.




  1. Klein S.M., Cohen G., Cederbaum A.J. Production of formaldehyde during metabolism of dimethyl sulphoxide by hydroxyl radical generating system. Biochemistry 1991; 20: 6006-6012.




  1. Nishimiki M., Rao N.A. The occurrence of superoxide anion in the reduced Phenazine methosulphate and molecular oxygen. Biochem Biophys Res Commun.1972; 46: 849-853.




  1. DiMartino M.J., Griswold D.E., Berkowitz B.A., Poste G., Lewis A.J. Pharmacological characterization of the anti-inflammatory properties of a new dual inhibitor of lipoxygenase and cyclooxygenase. Agents Actions.1987; 20: 113-123.




  1. Sen T., Nasralla Hussam S.H., Nag Chaudhuri A.K. Studies on the anti inflammatory and related pharmacological activities of Psidium guavaja: a preliminary report. Phytother Res. 1995b; 9: 118-122.




  1. Winter C.A., Risley E.A., Nuss G.W. Carrageenin induced oedema in hind paw of the rat as an assay for anti inflammatory drugs. Proc Soc Exp Biol Med. 1962; 111: 544-547




  1. Boughton-Smith N.K., Deakin A.M, Follenfant R.L., Whittle B.J., Garland L.G. Role of oxygen radicles and arachidonic metabolites in the reverse passive Arthus reaction and Carrageenin paw oedema in rats. Br J Pharmacol. 1993; 110: 896-902.




  1. Suthakaran R., Kavimani S., Venkappayya D., Farhat S., Suganthi K. Inflammation: Synthesis and pharmacological investigation of some new 4(3H)- Quinazolinone analogs as antioxidant, antihistaminic, anti-inflammatory and antitumor agents. Rasayan J Chem 2008; 1:2:263-275.




  1. Monica L.A., Eduardo H., Santos R., Ana A.B. de silva., Seirgo T., Maria de L., Sara V. Evaluation of acute and chronic treatments with Harpagophytum procumbens on Freund’s adjuvant induced arthritis in rats. J Ethnopharmacol 2004; 91: 325-330.







9


SIGNATURE OF THECANDIDATE




PARVINDER SINGH


10


REMARKS OF THE GUIDE



Matricaria recutita Linn has been mentioned as very good medicinal plant in various literatures. The present study was evaluated scientifically for its anti-inflammatory activity


11




NAME AND DESIGNATION OF THE GUIDE

Mr. V.M. Chandrashekhar

Associate Professor



12


SIGNATURE







13


CO-GUIDE







14


SIGNATURE







15


HEAD OF THE DEPARTMENT



Dr. I.S. MUCHANDI


16


SIGNATURE







17


REMARKS OF THE PRINCIPAL





18


NAME OF THE PRINCIPAL



Dr. I.S. MUCHANDI


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/2001-11/1-12

CERTIFICATE

This is to certify that Mr. Parvinder Singh, A student of first M.Pharm is permitted to carry out experiments on animal for the dissertation / thesis work entitled as “Pharmacological studies on Matricaria recutita L. on acute and chronic inflammation” as per details mentioned and after observing the usual formalities lay 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,3RDSEAWARD 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 :






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:

Pharmacological studies on Matricaria recutita L. in Acute and Chronic inflammation.




2.

Investigation (S):

Designation



Mr. Chandrashekhar V.M.

Associate 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:

6 months


(1) Number of months:





(2) Date of start of the project:

( Experiment)







(3) Date of termination of the project:





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

Male Albino rats



Age

Sex

Weight

4-8 weeks


Male

200-250 g


(b)

Rationale for selection

Approximate number of animals required during the first 12 months.

150 rats


40 mice

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

Twenty four groups, each group six 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, vena punctures).

Invasive surgical animal procedures.



Procedure and approximate frequency:

Enclosed



Anaesthetic and /or Analgesic and dosage:

Ketamine hydrochloride 45 mg/kg, i.p.



Test substance injected and /or applied:

Test substance will administer orally.





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:
----

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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