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BIOEQUIVALENCE STUDY CENTRE , Department of Pharmaceutical Technology, Jadavpur University, Kolkata . 700 032

Tel: (033) 24146967 Fax: (033) 2414618616266, Email: tkpaL12@yahoo.com, Website: www.biostudy.in,

TITLE: S

SPONSOR: 1

'L-H..1 l .LJ! ' INVESTIGATOR:

CLINI CAL PHAILl\'IACOLOG1ST:

ARTICLE: 100mg.

In Collaborationwith TAAB BIOSTIJDY SERVICES, No_67/lB, Ibrahimpur Road, Floor, Flat No_6,Kolkata - 700032_ '

Approved by DCGI vide Letter No. 4-14/97-DC p t (I ) dt. 21'( OCT 2002ISO 9001 :2000 Certified

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BIOEQUIVALENCE STUDY CENTREDepartment0/Pharmaceutical Technology. Jadavpur University. Kolkata - 700 032 . r w ~ el: (033) 24146967Fax: (033) 2414618616266 ISO9001:2000Certified

CONTENTS

4TATEMENTOF COMPLIANCE -1--PERSONNEL INVOLVED 1N TIlE STUDY .5_6 PPEFACE SUMMARY AND CONCLUSION 7

General 77 -Schedule

MATERIALS ANDMEIHODS . 8Test System 8

METHODS 9-9andomization, Numbering & Grouping ofAnimals 9 -Route ofAdministrationf------ JustifIcation for Selection ofWister Strain Rats for the Study 9Route ofAdministration and Reason for Choice 9 Dose P!f'Paration 9

OBSERVATIONS 1010 -- Symptoms

Mortality 10Body Weight 10 .Food Consumption 10

TERM1NAL STUDIES 10,RESULTS 12 IClinical Signs Mortality Body Weight 1 2 1

Food Consumption 13Haematologicai Investigations 13Biochemical Investigations 13

13 -- Organ Weight Necropsy 14Histopathology 14

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BIOEQUIVALENCE STUDY CENTRE... Department of Pharmaceutical Technology, Jadavpur University, Kolkata . 700 032Tel: (033)24146967 Fax: (033) 24146186 J6266, E-mail tkpal 12@yahoo.com, Website: www.biostudy.in,

. . -. .INSTITUTIONAL ANIMALETHlqSCQMMlllEETaabBiostudy -Servicesi No.67J1B. ibrahlmpur Roadf ardFJoort Flat No. 6,BioequivatenceSludyCentret,.Oepartmeo\ofPharmaceuticaITeehriotogy,. ' Jadavpur University, .Kolkata ... 700.,032The members of the Institutional Animal Ethics Committee. (lAEC) navesautiniZed. t h ~ various aspects QftheprotOcols tor carrying out s u b " ' f ~ n i c l a c : ~ tt>xicitystudyofthe following drug on rats andmiee andJlave apptqved- t h e ~ to be pedc;mned. at the TaahBiostudy Services$ No. 6 7 / 1 B , . 1 b r a h i m p u r R o ~ 3Rt FlOOr, Flat No:; .6,a i ~ v a 1 e n c e S t u d y _ C e n t r e : r DePartment of PharmaceuucalTechnology, JaMVpurU n i v e r s i t y ~ Koikata .... 700032, West BengaL

Narneofthe drug: IACECLOFENAC TABLETS loom--;]. . .... . ... ..P ...........w .lA \ ; '

. . . P r o e ( D t ~ ) T . K P a 1 _ .ChairmanllisUtationalAnimalBtbicsCommitte. II ' ... .. '._ .. , . " . . ",'. . pto(f)rgr.K.p., .~ n ; ~ , J t f t ~ . ~ . . T " " t _ ~ o Y . ~ t e E $ 1.... ~ O W ' ~ . , J . v . '. ~ " ' 1 0 0 - D 1 2

Approved by DCGI vide Letter No. 4-14/97-DC Pt (I) dt. 21st'OCT 2002SO 9001 :2000Celtlfled

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BIOEQUIVALENCE STUDY CENTREDepartment ofPharmaceutical Technology,Jadavpur University, Kolmta - 700032 1 1 I i V ~ Tel: (033) 24146967 Fax: (033) 2414618616266 1S09001:2CDIJCertifiedSTATEMENT OF COMPLIANCEProject No. TOXSC (r) ACFT 0610Test Artic::1e ACECLOFENAC TABLETS 10Omg.Project Title : Subchl'Onic Oral Toxicity Study (28 days)Bach no. : KOO4fTI013We hereby attest to the authenticity of the study and guarantee that the data is correct andaccurate to the best of our knowledge and that the study was performed in accordance withthe procedUre and the principles of Good Laboratory Practice for the 1esting of chemicals.We hereby attest that this study was conducted in compliance with Protocol submitted to andapproved by the sponsor.The study also complies with the schedule Y of Drugs and Cosmetic Act (I940} andregulations of the Committee for the Purpose of Control and Supervision of Experiments onAnimals.Prof (Dr.) T. K Pal 0\-1PChief Investigator Signature Date

Dr. S. Agarwal J -1:- Jo' f-Quality Assurance Unit Signature Date

Dr. T. K Chattaraj, M.D. (fi t/(I({ ZtlllO/Clinical Pharmacologist Signature DateACECLOFENAC TABLETS 100mg._KUSUll

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BIOEQUIVALENCE STUDY CENTREDepartmentofPharmaceutical Technology, Jadavpur University, Kolkata - 700 032 IruvSTel: (033) 24146967 Fax: (033) 24146186/6266 ISO 9001:2000CertifiedPERSONNEL 1 i ~ V O L VED IN mE STUDY

Cbief Investigator Prof (Dr.) T. K Pal

Clinical Pbarmacologist Dr. T. K Chanaraj, M.D.

Quality Assurance Unit Prof (Dr.) S. G. DastidarDr. S. AgarwalDr. Anjan Das, M. D.Investigating TeamMr. Anirbandeep BoseMr. Soumendra DamarMr. Nilendra ChatterjeeMr. Bikash RoyMr. Subhro DuttaMr. Arun GhoraiMr. Sanjay GhoraiMr. Ratan Gupta

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BIOEQUIVALENCE STUDY CENTREDepartment ofPharmaceutical Technology,Jadavpur University, Kolkata - 700 032 " .- r d V ~Q " . O ~ ' " Tel: (033) 24146967 Fax: (033) 2414618616266 ISO 9001:2000 Certified

SUMMARY AND CONCLUSIONThe present investigation deals with Sub-chronic Toxicity Test (28 days) of"CF"Ln]fENAC 'T'.tDT Frn;;:: 1 n RMC on'XT;rlar D.,.., ++1-."" .......""0., 0 mO'fKg .., ..,.,.noN,.,..MP.. . . : . t" " , " .U , .ait...U.LI..It:.I..a.lr.::J' ... " " " ~ I"' -J . " .1 . ; )..."" . r ' - U ~ c.:u. LL lv iU i . v J. ' I t : Y ~ , .Ll.lOJ,l.""JII..5,5 mgIKg and 10 mg/kg body weight in accordance with the schedule Y of drugs andCosmetic Act (1940). The animals of both sex from control and different dose groups exhibited normal body weight gain 1:luoughout the dosing period of 28 days. The quantity of food consumption by the animals was f01.md to be comparable with that of the control animal group. "There was no abnormality and lesion found from Histopathological Exarn:ination of the organs viz. Adrenals, Heart, Kidneys, Liver, Lungs, Stomach. " The supplied drug ACECLOFENAC TABLETS lOOMG. did no t show any significant feature ofhaematological toxicity, behavioral toxicity, hepatotoxicity and nepherotoxicity in any of the specimen.

1Phief Investigator : Prof (Dr.) T. K Pal ri4linical Phannacologist : Dr. T. K Chattaraj, M.D. 1:t-In Charge of.Quality Assurance Unit : Dr. S. Agarwal

ACECLOFENAC TABLETS lOOmgo_KUSUM6

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BIOEQUIVALENCE STUDY CENTREDepartmentofPharmaceutical Technology,Jadavpur University, Kolkata - 700 032 ' ;- :::, -

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BIOEQUIVALENCESTuDy CENTREDepartment ofPharmaceutical Technology, Jadavpur University, Kolkala - 700031 . rw[alTel: (033) 24146967Fax: (033) 1414618616166 ISO 9001:2000Certified

MATERIALS AND METHODTEST SYSTEMSpecies RatStrain Wister Strain RatsSource Animal house, IleB, KolkataSex Male and femaleAge 6to8weeksNo. Ofanimals per dose level 6 per sex per doseAcclimation Seven days prior to dosingIdentification ofanimals By cage number and individual marking onfurDiet PelIeted feed supplied by

Mis Ghosh Enterprise, KolkataWater Aquaguard pure water in glass bottles

ad libitumHousing The rats were housed 6 each; of the same sex

in polycarbonate cages provided withbedding of husk The temperature wasmaintained in between 20 to 24C andrelative humidity between 30 to 70%; 12hours each of dark and light cycle wasmaintained.

Dose Male: 0 mgIKg , 2 mgIKg, 5 mgIKg and10 mglkg body weightFemale: 0 mgIKg , 2 mgIKg, 5 mglKgand 10 mglkg body weight.

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BIOEQUIVALENCE STUDY CENTREDepartment ofPharmaceutical Technology,Jadavpur UniverSity, Kolkata - 700 032 r d V ~MsootTel: (033) 24146967Fax: (033) 2414618616266 ISO 9001:2000 CertifiedMETHODSRandomization, Numbering and Grouping of AnimalsForty eight rats i.e. 24 male and 24 female healthy rats were divided into four groups of6 rats~ " , p ~ ~ " ; .. r ; t ~ < " ' ' ' ' .. r ~ " ' ' ' ' ' ; v ' m ' g +l.e ; t ~ s e ~ 4 : ' {\ ........fKg 2 ..."gIK.... ;;:: mgIK"'" ~ ~ A 1 { \ V""1. " ~ A 1 . . ~ . J .VW UV.:>V ~ v u V " 1 . ~ \ A . < I V u. UV VJ . v UJ . 5 'L , lU 1......50 j . .1.....5 (Ulu J .Vrnglkg Animals were allowed acclimation period of 7 days to laboratory conditions prior tothe initiation ofdosing Rats were assigned to six per cage sex wise and the individual animalwas fur marked with picric acid. The females were nulliparous and not pregnant.Route ofAdministration: OmI (gavages). Justification for selection ofWister Strain Rats for the Study

1) One of the rodent species recommended as test system for the use in toxicity studies, 2) This test system has been demonstrated to be sensitive to toxins, 3) Widely used throughout indilstryfor the evaluation oftoxicity ofvarious products, 4) Historical data and evidence at the facility.

Route ofAdministration and Reason for ChoiceOral, fOT 28 days. The oral route was selected for use because,

I) The dosage can be accurately administered,2) _One of the proposed routes for toxicity testing,

Oral route is considered to be a potential route ofhuman exposure.DOSE PREPARATIONACECLOFENAC TABLETS lOOmg. suspended in water was administered to rats at thedose levels of 2 rngIKg, 5 rngIKg and 10 rnglkg in the dose volume of Im1J 100g body

. weight. The test article suspensions were freshly prepared every day for 28 days. The controlanimals were administered vehicle only.

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BIOEQUIVALENCE STUDY CENTRE Department ofPharmaceutical Technology, Jadavpur University, Kolkata - 700 032 . I J i i V ~ Tel: (033) 24146967 Fax: (033) 2414618616206 ISO 9001:2000 CertifiedOBSERVATIONS Symptoms IAll animals were obsefved daily for clinical signs.- The time of onset,1 intensity and duration

Iof these symptoms, i f any, were recorded. Mortality I All animals were observed twice daily for mortality during the period ~ the study.

IBody ~ e i g h t . . . . ' . IThe weIght of each rat was recorded on day 0 and at weeldy mterval, throughout the courseof the study. The groups: mean body weights were calculated. IFood consumption IIThe quantity of food consumed by groups consisting of six rats eac* ,vas recorded weeklyand the food consumption per rat was calculated for comrol and dose droops.TERMINAL STUDIES ILaboratory Investigations IIFollowing laboratory investigations were carried out prior to sacrifice on completion ofdosing period of 28 days in animals fasted over-night Blood samPlf were collected fromorbItal smus followmg mommg usmg hepann as antIcoagulant. I 'Haematological Investigations' . IFollowing haematological parameters were studied using MEDONIC 620 Cell Counter.Hb : Hemoglobin (g%) RBC : Red Blood Corpuscles (x106 / cmm)Rt. : Reticulocyte (%) MCV : Mean Corpuscu1!ar Volume ( 11m3) HCT : Hematocrit (%) MCH : Mean Corpusculal Hemoglobin (pg) Platelets: (x 105 /cmm) MCHC : Mean Corpuscn1ar Hemoglobin Concentration (010) N : Neutrophils (%) WBC : White Blood Cocpuscles (x 1i f cmm)L : Lymphocytes (%) E : Eosinopmls (%)M : Monocytes (%)

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BIOEQUIVALENCE STUDY CENTREDepartmentofPharmaceutical Technology,Jadavpur University, Kolkata - 700 032 . : :r d V ~I I I . . n 2Tel: (033) 24146967 Fax: (033) 2414618616266 1809001:2000 CrifiedBiochemical investigationsFollowing biochemical parameters were studied using MlCROLAB-300. TOTAL SERUM PROTEIN (g01o) BUN: Blood Urea Nitrogen (mg'lo) SGPT: SerumGlutamic Pyruvic Transaminase (lUlL) SGOT: Serum Glutamic Oxaloacetic Transaminase (lUlL) SA P : SerumAlkaline Phosphatase (lUlL) BLOOD SUGAR (mg'lo) NecropsyAll animals were sacrificed on day 29, using cervical dislocation technique. Necropsy of all animals was carried out and the weights of the following organs were recorded: Liver, kidneys and heart. The organ weights were recorded as absolute values and their relative values (i.e. per cent of the body weight) were calculated. HistopathologyFollowing tissue samples of organs from control and animals treated at the highest dose level of 10 mg/kg were preserved in 10% fonnalin for histopathological examination. Adrena1s; Heart; Kidneys; Liver; Lungs; Stomach; Adrenals, Heart;. Kidneys, Liver, Lungs and Stomach oflow and intermediate dose group animals were preserved for possible histopathological examination, in case the' histopathological examination ofhigh dose group animals is indicative of abnormalities associated with the treatment.

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BIOEQUIVALENCE STUDY CENTREDepartmentofPharmaceutical Technology. ITriVSadavpur University, Kolkata - 700 032 . a. . 00'"Tel: (033) 24146967Fax: (033) 24146186/6266 1509001:2000CertlfedRESULTS Clinical signs (Table B) Male-Group I (0 mglkg) : Animals were free of intoxicating signs throughout the dosing period of 28 days (animal nos. 1 to 6). Group n (2 mglkg) : Animals were free of intoxicating signs throughout the dosing period of 28 days (animal nos. 13 to 18). Group III (5 mglkg) : Animals were free of intoxicating signs throughout the dosing period of28 days (animal nos.25 to 30). . Group IV (10 mglkg) : Animals were free of intoxicating signs throughout the dosing period of 28 days (animal nos.37 to 42).

FemaleGroup I (0 mglkg) : Animals were free of intoxicating signs throughout the dosing period of 28 days (animal nos.7 to 12). Group n (2 mglkg) : Animals were free of intoxicating signs throughout the dosing 1 period of28 days (animal nos. 19 to 24). Group III (5 mglkg) : Animals were free of intoxicating signs throughout the dosing period . of28 days (animal llos.31 to 36). Group IV (lO mglkg) : Animals were free of intoxicating signs throughout the dosing period of28 day'S (animal nos.43 to 48).

Mortality (Table C). Male and Female All animals from control and different dose groups survived through thedosing period -- 1 of28 days.

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BIOEQUIVALENCE STUDY CENTREDepartmentofPharmaceutical Technology. . .Jadavpur University, Kolkata - 700 032 . , OIMSDt'"el: (033) 24146967Fax: (033) 24146186/6266 ISO9001:2000Certified

BODY WEIGHT (TABLE D; APP.I; FIG.I & mMaIe-Animals from control and the different dose groups exhibited' nonna!. body weight gainthroughout the dosing period of28 days.FemaleAnimals from control and the different dose groups exhibited normal body weight gainthroughout the dosing period of28 days.

FOOD CONSuMPTION (TABLEE; FIG.ill & IV)Male and FeinaleDuring the dosing period and at termination the quantity of food consumed by animals fromdifferent dose groups was found to be comparable with that by control ammals. HAEMATOLOGICAL INVESTIGATIONS (TABLE F; APP.II) Male and female - at termination of dosing on day 29, no significant changes were observed in the values of different parameters studied when compared with controls and values obtained were within normal biological and laboratory limits. BIOCHEMICAL INVESTIGATIONS (TABLE G; APP. llI) Male and Female...;. At termination-on day 29, allbiocbemica1 parameters studied ie . Total scnnn protein, SGPT, SOOT, SAP, BUN and BLOOD SUGAR were found to be comparable with controls and were within the normal biological and laboratory limits. ORGAN WEIGHTS (TABLE H & 1; APP. IV, V) Male and FemaleAnimals from control and the different dose groups exhibited nonna! body weight gainthroughout the dosing period of28 days.

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BIOEQUIVALENCE STUDY CENTREDepartment ofPharmaceutical Technology,Jadavpur University, Kolkata - 700 032 . : :uv[tlJ

M_DnTel.' (033) 24146967 Fax: (033) 2414618616266 ISO 9001:2000 Certllied

NECROPSY (TABLE J; APP.VI) Summary ofnecropsy findings in male and female animals is described in Table 1. The gross pathological examination revealed no abnormality attributable to the trea1nlent.

mSTOPAmOLOGY (TABLE K; APP.VI) Summary of histopathological observations is given in Table no.K. Th e histopathological observations on individual. animal given in App.Vl. Histopathological examination ofanimals from high dose group revealed no abnormality attributable to ,the treatment.

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BIOEQUIVALENCE STUDY CENTRE Department o fPharmaceutical Technology, Jadavpur University, Kolkata - 700032 ruv!i!Tel: (033) 24146967 Fax: (033) 2414618616266 ISO9001:2000CerUfied

SUB CHRONIC ORAL TOXICITY STUDY OF ACECLOFENAC TABLETS lOOMG .

IN RATS

FIG NO. I: MALE (RAt) GROUP MEAN BO DY l132 WF...JGBT (g) I

I

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BIOEQUIVALENCE STUDY CENTREDepartment ofPharmaceutical Technology,Jadavpur University, Kolkala - 700032 . rw[t1Tel: (033) 24146967 Fax: (033) 2414618616266 /SO 9001:2000 Ce:tifiedSUB ,CHRONIC ORAL TOXICITY STUDY OF

ACECLOFENAC TABLETS lOOMG. ' IN RATS

FIG NO. D: FEMALE (RAT) GROUP MEAN BODYWEIGIIT(g)

132 . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

130UJJf:-

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BIOEQUIVALENCE STUDY CENTREDepartment ofPharmaceutical Technology,Jadavpur University, Kolkata - 700 032 .: :a DD2Ewrael: (033) 24146967 Fax: (033) 2414618616266 ISO9001:2000Certified\

SUB CHRONIC ORAL TOXICITY STUDY OFACECLOFENAC TABLETS lOOMG.

JNRATS

FIG NO. m: MALE (RAT) GROUP MEAN FOOD

7/'eli!.5zS 600ZouCl 5oo

4

.

.,------'l

CONSUMPI10N (gIanimal).

/I__ - . - . - " r - . - - ~ - - l

-_.- ~ - - - - - - - = ~ ~ --"~ - ' .,---

0 7 14 21 28DAYS

- -Orng/kg ~ ~ 2 m g / k g - 5m g / k g ~ 1 0 m g / k g

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BIOEQUIVALENCE STUDY CENTREDepartment ofPharmaceutical Technology,Jadavpur University, Kolkata - 700032Tel.' (033) 24146967Fax.' (033) 2414618616266 ISO 9001:2000Certified

SUBCBRONIC ORAL TOXICITY STIJDY OFACECLOFENAC TABLETS l 0 0 M G ~ .

lNRATS

FIG NO. IV: FEMALE (RAT) GROUP MEAN FOODCONSUMPTION (gIanimal)

7

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