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FINAL REPORTGLPStudy

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Study initiation date: July 28, 2011

Draft Report date: September O3, 2012

Final Report date September O7 2012

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CONTENT

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%GLP COMPLIANCE STATEMENTSTATEMENT OF THE MANAGEMENTABBREVIATIONSSUMMARY1. INTRODUCTION

. Objective of the study

. Project Staff

. Study personnel

. Regulatory Guidelines 9

. GLP Compliance 10

. Facilities Management and Animal Husbandry 10

. Animal Welfare Act Compliance 10TEST CONDITIONS 10

2.1. Experimental Design V 102.2. Description of the Test and Reference Item 112.3. Test System I 11 .2.4. Allocation 122.5. Justification for Test System 12 L2.6. Housing 122.7. Diet 122.8. Water 122.9. Acclimation 122.10. Animal Identification 133. METHODS 13

Procedure 13Intra-osseous Administration Surgery 13Anaesthesia 13Antibiotic Treatment 14Analgesic/Anti-inflammatory Treatment 14Clinical Obsen/ations and Mortality 14Body Weight 14

3.2. Terminal Obsen/ation 143.3. Tibia Preparation 153.4. Individual Bone Parts Processing 153.5. Histological and Biocompatibility Evaluation 154. RESULTS 164.1. Clinical Signs and Mortality A 164.2. Body Weight 164.3. lntra-osseous Administration 164.4. Gross Pathology Examination 164.5. Histological and Biocompatibility Evaluation 175. CONCLUSION 176. ARCHIVING 18

. AMENDMENTS AND DEVIATIONS 18

. DISTRIBUTION 18APPENDIXES PagesAppendix 1: Photodocumentation of Tibia Preparation 38Appendix 2: Photodocumentation of Histological and Biocompatibility Evaluation 8QAU statement 1Certificate of GLP 2

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Document: | Final report I Identification No.: I17/11/PStudy director: I Lukas Recek I Date: ISeptember 07, 2012

GLP COMPLIANCE STATEMENT

Name of the Study:

Identification No.:

Name of the Test Items:

Name of Reference Item:

Study initiation date:

Date of Draft Report:

Date of Final Report:

Hypro-Oss bone substitution material, PreclinicalImplantation Study in Beagle Dogs

17/11/P

Hypro-Oss

Dexabone®

July 28, 2011

September 03, 2012

September O7, 2012 .

I the undersigned hereby declare that the objectives laid down in the protocol were achievedand as no untoward incidents occurred to adversely affect the quality or integrity of thestudy, I consider the data generated to be valid. This final report fully and accuratelyreflects the procedures used and data generated in the course of this study. This study wasperformed in compliance the OECD Principles of Good Laboratory PracticeC (97)186/Final, agreed study plan and BioTe:st Standard Operating Procedures.

Date: Signature: .. ..."..... ........... .. I

Lukas Recek ‘Study director

I Confidential I Page No.1 I 3/18 I

Bm I Hypro-Oss bone substitution material, Preclinical Implantation Study_ I 'I'BSts.no. in Beagle Dogs

Document: I Final report I Identification No.: 17/11/P IStudy director: I Lukas Recek I Date: September 07, 2012

STATEMENT OF THE MANAGEMENT

According to the conditions of the basic contract SD/013/2011 from April 04,. 2011andamendment 01 from December 27, 2011 between Hypro Otrokovice, sY.~r.o. (as Sponsor)’ andBioTest s.r.o. (as Test Facility) “Hypro-Oss bone substitution material, Preclinical ImplantationStudy in Beagle Dogs” has been performed in compliance with CSN EN ISO 10993-6 Biologicalevaluation of medical devices - Part 6: Tests for local effects after implantation and OECDPrinciples of Good Laboratory Practice.

Date: Signature: ......................... .. I

- Jan ZabskyTest facility man er

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ABBREVIATIONS

H&E Hematoxylin and eosin stainingGLP Good Laboratory PracticeSOP Standard Operation ProcedureQAU Quality Assurance UnitNo. NumberFig. Figurei.m. Intramuscularp.o. OrallyGroup 1 Animal sacrificed 4 weeks after administrationGroup 2 Animal sacrificed 14 weeks after administration

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Docurnent I ifinairepon I ldenfificafion No; I17H1/PStudy director: Lukas Reéek I Date: ISeptember 07, 2012

SUMMARYThe purpose of this study was to evaluate the influence of bone substitution material in twosizes of granules in comparison to reference material on bone tissue of tibia in Beagle dogsafter intra-osseous administration.The study was conducted on 12 dogs (6 males and 6 females) obtained from BioTest s.r.o.,Czech Republic and WOBE Ltd, Hungary breeding facilities.Three or four intra-osseous administration sites (diameter 3 mm) were drilled into both tibias(total number 6 or 8 sites per animal) in each dog and filled with Test Item or Reference Item.The dogs were fasted overnight for min. 12 hours prior to, and 3 hours after the administration.Water was provided ad Iibitum.During the administration the animals were in total anesthesia under supen/ision of theveterinarian. Antibiotic therapy as a post surgery prophylaxis was used during first 14 or 24 daysafter the administration. AnaIgesic/anti-inflammatory treatment was used during first 8 or 18days after the administration to avoid unnecessary pain and inflammation. Antibiotic therapy andanalgesic/anti-inflammatory treatment were under supervision of the veterinarian.

Clinical signs and mortality were recorded once a day during the acclimation and healingperiods. During the antibiotic treatment the daily observation also focused on the regenerationcourse of tissue and status of sutures. Body weight was individually recorded in all the animalsat delivery, before surgery procedure and then once in two weeks during the observation period.

At the end of the experimental part (4 and 14 weeks after the administration) the dogs weresacrificed and the tibias were taken out and documented by means of photographs. Theadministration sites and relevant draining lymph nodes were evaluated macroscopically.Abnormalities were recorded with details of location, color, shapeiand size. After that the tibiaswere fixed in 10% formaldehyde solution. IAfter, at the least, 14 days in the formaldehyde solution the tibias were cut up with saw intoindividual parts of bone each one contained a single administration site. If possible, the incisionin bone was led at a distance of min. 5 mm from outside circumference of the administrationsite. The cut parts with administration sites were transported in formaldehyde solution togetherwith appropriate documentation to Prof. Ctibor Povysil (Department of Pathology; The 1stFaculty of Medicine, Charles University) for next preparation and for histological andbiocompatibility evaluation.ResultsClinical Observation IAll the dogs were in good health condition throughout the acclimation and during healing period;no clinical symptoms of toxicity were obsen/ed. IHealing of post-surgery wounds went off without significant complication except only in dogM028 from Group 1 where healing process of the post-surgery woundsby secondary intentionwith granulation was obsen/ed. All the individual body weight values of the dogs were withinnormal range, corresponding to the animals’ age.Gross pathology ExaminationPartially healed surgical wounds with remnants of absorbable sutures were found in the skin ofboth left and right tibial areas in the animals sacrificed 4 weeks after the administration. In somecases the subcutis showed the presence of a mild cavity in the administration site. Thementioned cavity was filled with non purulent, sanguineous, liquid. I I IIn the animals sacrificed 14 weeks after the administration, both skin and subcutis werecompletely healed without any signs of inflammation. i

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Histeolciagicalttande2Bio¢o%mpatibilityEtvalueattientt Evaluation of L bottht tbatchest "Of. Y Test and Reference 1 ilterinsi revealed: neither) signs [of chronicinflammatory reaction not giant cell} reactiori which Ycouldtrelate to response to eforeign body. - 2Four weeks Lafteri thepitntra-eossetous 2 admtinistraetion; ftheflTes2t ; item; was tmostly 2 surrounded bycollagen tissue wathoutpres<-;-nee Orinraammamry elements. Occ2atsi'onea||ey,: an". oceu rrence of tiny.Iameuaor the bonetissueobeen/ed. However itjwa2s}YtimpossibIeaeteetetll withcertaintywhetherit isnewbonetissuetormtationortremnantsoforigma!trabecuIaeofbone. Fourteenweeksafier by t9

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Document: I Final report Identification No.1 I 17/11/P

fest 5 |-0 in Beagle Dogs

Study director: Lukas Reéek Date: ISeptember 07, 2012

Histological and Biocompatibility EvaluationEvaluation of both batches of Test and Reference Items revealed neither signs of chronicinflammatory reaction nor giant cell reaction which could relate to response to foreign body.Four weeks after the intra-osseous administration, the Test item was mostly surrounded bycollagen tissue without presence of inflammatory elements. Occasionally, an occurrence of tinylamella of the bone tissue was observed. However it was impossible to tell with certaintywhether it is new bone tissue formation or remnants of original trabeculae of bone.Fourteen weeks after intra-osseous administration, the Test item was also surrounded bycollagen tissue. Higher occurrence of new bone tissue formation in collagen tissue wasobserved. New bone tissue was originally in close contact with granules of the Test Item. Themost significant evidence of new bone tissue formation on Test Item were findings of new bonetissue surrounding the granules of Test Item in close contact with it. Furthermore, la presence ofnew bone tissue in remnants of Haversian canals of Test Item was observed.No difference between the two batches (batch No. 0210, size of granules: 0.5-1.0 mm and batchNo. 0310, size ofgranules: 1.0-2.0 mm) was found.Four weeks after the intra-osseous administration, the Reference Item was surrounded bycollagen tissue without signs of inflammatory response. IFourteen weeks after the intra-osseous administration, a formation of Iamellae of new bonetissue was found in neighboring areas of the Reference Item, occasionally with osteoblasticexternal edge. The internal edge of new bone tissue was in contact with the surface ofReference Item. lntergrowth of new bone tissue into Reference Item or Test Item was notobsen/ed in any of the cases. No difference between the two batches (batch No. 09FA29110,size of granules: 0.5-1.0 mm and batch No. N035, size of granules: 1.0-2.0 mm) was found.Conclusion IThe intra-osseous administration in all the animals was performed according to the study planwithout any complication, and the planned number of administration sites was drilled into bothtibias of each dog. The healing of post-surgery wounds was without any complication exceptonly in one animal (M028) where healing process by secondary intention with granulationrwasobserved without any connection to administered Items. According to clinical obsen/ation andgross pathology examination, both the Test and Reference Items were well tolerated: no signsof inflammatory reaction or pathological influence of both the Items were observed insurrounding soft tissue or in bone tissue. The described findings were closely connected tohealing process of the post-surgery wounds and corresponded to physiological course of woundhealing.According to histological evaluation, the Test Item caused no inflammatory or other changesresembling a reaction to foreign body, which would indicate unsuitability of the Test Item.Despite particular differences in character of Test and Reference Items both showedgradualformation of new bone tissue around granules of bone substitution material without influence ofdifferent size of granules (0.5-1.0 mm or 1.0-2.0 mm). However, batch No. 0310 containsgranules of smaller size and, consequently, of smaller surface; therefore, a smaller amount ofnew bone tissue could be expected in comparison with the granules of batch No. 0210. TheTest Item without reference to used batch corresponded to Reference Item in histology findingsand new bone tissue formation. The observed findings can be considered as an appropriateresponse to tested bone substitution material. I

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Bi@ Hypro-Oss bone substitution material, Preclinical Implantation Study:1 ‘I Test 5.-ta». in Beagle Dogs

Document: I Final report I Identification No.: I17/11/PStudy director: Lukas Recek I Date: ISeptember 07, 2012

1. INTRODUCTION

1.1. Objective of the studyThe purpose of this study was to evaluate the influence of bone substitution material containingtwo different sizes of granules in comparison to reference material on bone tissue of tibia inBeagle dogs after intra-osseous administration.

1.2. Project StaffSponsor: Hypro Otrokovice, s.r.o.

Pflstavni 568, Otrokovice765 02, Czech RepublicTel.:+420 577 159 724e-mail: hypro@,h\Lpro.cz

Sponsors Representative: Antonin Galatik,Hypro Otrokovice, s.r.o. ,Pfistavni 568, Otrokovice765 02, Czech RepublicTel.: +420 605 147 956e-mail: hypro@hypro.cz

Test Facility: BioTest s.r.o., Pod Zamkem 279, Konarovice281 25, Czech Republic

Test Facility Manager: Jan Zabsky, IBioTest s.r.o., Pod Zamkem 279, Konarovice281 25, Czech RepublicTel.: +420 313129 374e-mail: zabsky@biotest.cz

Project Manager: Jana Prochazkova,BioTest s.r.o., Pod Zamkem 279, Konarovice281 25, Czech RepublicTel.: +420 313129 374e-mail: prochazkova@.biotest.cz

Study Director: Lukas Recek,BioTest s.r.o., Pod Zamkem 279, Konarovice281 25, Czech RepublicTel.: +420 313129 374e-mail: recek@biotest.cz

Quality Assurance Manager: Vladimlra Maminakova,BioTest s.r.o., Pod Zamkem 279,281 25 Konarovice, Czech RepublicTel.: +420 313129 374e-mail: maminak0va@biotest.czJana Novakova,BioTest s.r.o., Pod Zamkem 279,281 25 Konarovice, Czech RepublicTel.: +420 313 129 374e-mail: jnovakova@bi0test.cz

IConfidential IPaQe N0-I I 8/18 I

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Document: I Final report I Identification No.1 I17/11/PStudy director: I Lukas Recek I Date: ISeptember 07, 2012

Histological and Biocompatibility Ctibor Povysil,Evaluation Non-GLP activity: Institute of Pathology 1th Faculty of Medicine

Charles UniversityStudniskova 2, Praha 2128 00, Czech RepublikTel.: +420 224 968 660e-mail: ctibor.povvsil@lf1.cuni.cz

1.3. Study personnel IThe following individuals were the primary contributors and supen/isory personnel participatingin this study.Study Director: Lukas Reéek, I

BioTest s.r.o., Pod Zamkem 279, Konarovice281 25, Czech RepublicTel.: +420 313 129 374e-mail: recek@biotest.cz

Responsible Person for Experimental Lukas Reéek,Conducting: BioTest s.r.o., Czech RepublicClinical obsen/ation: Lukas Resek

BioTest s.r.o., Czech RepublicSupervisor, Animal Care: Zuzana Gilarova,

BioTest s.r.o., Czech RepublicTel.: +420 313 129 374e-mail: gilarova@biotest.cz

Veterinarian: Lukas Panek,BioTest s.r.o., Pod Zamkem 279, Konarovice281 25, Czech RepublicTel.: +420 313129 374 Ie-mail: panek@biotest.cz

Gross Pathology: Ales Brejcha,BioTest s.r.o., Pod Zamkem 279, Konarovice281 25, Czech RepublicTel.: +420 313 129 374e-mail: breicha@biotest.cz

Histological and Biocompatibility Ctibor Povysil,Evaluation Non-GLP activity: Institute of Pathology 1th Faculty of Medicine

Charles UniversityStudniskova 2, Praha 2

I 128 00, Czech.RepublikTel.: +420 224 968 660

‘e-mail: ctibor.povysil@lf1.cuni.cz

1.4. ReguIatoryGuideIinesThe study was carried out on basis:CSN EN ISO 10993-6 Biological evaluation of medical devices - Part 6: Tests for local effectsafter implantation.

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1.5. e_iGLeP?Compliancie This nion”-clinicealiIaiboratorye %si%’cu'd%y]required;Strictiieciifnipleianceiwith tihee%OECD Principles of Good.Laborat0ry iPr%actic1e C ‘(9_7')_'|__"_8"6/_Fii"iQ|_'€1'_l1"d_"'_2_0O.4/'1‘O/EC;_-Ith€_:_CZ6Ch'_5|El'W'_ No; 378/2007; and Decreeof eMinisit|3/70f Health and 1Mine%is’triy of ricuiwre oftheczech Repiuiblic No.86/2008 iC0||ectioYnes oflaws about GoodIab0rat0|*yeipracticefortestiengfofdrugs; ii e i i e -I iQuality Assuranicei Review: e The study idesciiribed in ithis;|niterimeireportwasi subjected to QualityAssurance j evaluations Qriaboraiory processes daita _ii %iTheeei%%%peir3Qnne|effrom the;Quae;;tyAssuranew <QAu>inspe@ied westudy QAF’r@9ramrelevant$OP$ ofBioTees_r.@.

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Document: Final report I Identification No.: I17/11/PStudy director: Lukas Reéek I Date: ISeptember 07, 2012

1.5. GLP ComplianceThis non-clinical laboratory study required strict compliance with the OECD Principles of GoodLaboratory Practice C (97)186/Final and 2004/10/EC, the Czech law No. 378/2007 and Decreeof Ministry of Health and Ministry of Agriculture of the Czech Republic No.86/2008 Collections oflaws about Good laboratory practice for testing of drugs.Quality Assurance Review: The study described in this Interim report was subjected to QualityAssurance evaluations of laboratory processes and data. The personnel from the QualityAssurance Unit (QAU) inspected the study in accordance with the QA Programme, relevantSOPs of BioTest s.r.o. and the OECD GLP Principles. The QAU performed the study-basedinspections min. three times, and the facility-based or process-based inspections according tothe year plan. The Statement of QAU is included in the Final report.

1.6. Facilities Management and Animal HusbandryAnimal care was in compliance with the SOPs of BioTest s.r.o., the European convention for theprotection of vertebrate animals used for experimental and other scientific purposes (ETS 123),the Czech Collection of laws No. 246/1992, inclusive of the amendments, on the Protection ofanimals against cruelty, and Public Notice of the Ministry of Agriculture of the Czech Republic,Collection of laws No. 207/2004 as amended, on keeping and exploitation of experimentalanimals. BioTest s.r.o. is a holder of the Accreditation Certificate for users issued by CentralCommittee for Animal Protection of the Czech Republic.

1.7. Animal Welfare Act ComplianceThe study was prepared for this type of experiment and approved by the Institutional AnimalCare and Use Committee (IACUC) and the Committee for Animal Protection of the Ministry ofIndustry and Trade of the Czech Republic (17/2011). Procedures used in this plan wasdesigned to conform to accepted practices and to minimize or avoid causing pain, distress, ordiscomfort to the animals. In those circumstances in which required study procedures werelikely to cause more than momentary or slight pain or distress, the animals received appropriateanalgesics and anesthetics.The number of animals selected for use in this study is considered to be the minimum numbernecessary to meet scientific and regulatory guidelines for this type of study.

2. TEST CONDITIONS

2.1. Experimental DesignProcedure: Date:Study Initiation Date: July 28, 2011Animal Receipt: July 29, 2011, August 09, 2011Acclimation: 5-13 DaysInitiation of Experimental Part: July 29, 2011, August 09, 2011intra-osseous administration: Group 1: Animal No. M042, F019 August 9, 2011

M026, F022 August 10, 2011M028, F045 August 11, 2011

Group 2: Animal No. M027, F024 August 3, 2011M020 August 4, 2011M021, F029 August 5, 2011F9776 August 18, 2011

Antibiotic Treatment: During first 14- 24 days after administrationAnalgesic/Anti-inflammatoiy During first 8 - 18 days administrationTreatment:

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Procedure:End of the Experimental Part:

Date:August 31, 2012

Clinical observation: DailyDetailed clinical obsen/ation: WeeklyBody weight: At delivery, before surgery and than once in two weeksHealing period: Groups 1:

4 weeks after intra-osseous administrationGroups 2:

14 weeks after intra-osseous administrationTibia preparation: Group 1: Animal No. M042, F019 September 6, 2011

M026, F022 September 7, 2011M028, F045 September 8, 2011

Group 2: Animal No. M027, F024 November 9, 2011M020 November 10, 2011M021, F029 November 11, 2011F9776 November 24, 2,011

Taking of tibia (documentation of tibia by photographs)Draft report date: September 03, 2012Final report date: September 07, 2012

2.2. Description of the Test and Reference ItemName of the Test Item Hypro-OssDescription: Bone substitution material .Summary formula: Ca5HO13P3+atelocollagen type ITest Item stability: . 5 yearsStorage condition: -25°to +50°C, avoid direct sun lightBatch No.: 0210 (Size of granules: 0.5-1.0 mm)Batch No.: 0310 (Size of granules: 1.0-2.0 mm)Sterility: Yes

Name of the Reference Item: Dexabone®Description: Bone substitution materialStorage condition: 5° to 30°C, avoid direct sun light, heatBatch No.: 09FA29110 (Size of granules: 0.5-1.0 mm)Batch No.: N035 (Size of granules: 1.0-2.0 mm)Sterility: Yes

2.3. Test SystemSpecies & Strain: Beagle dogsSupplier: BioTest s.r.o., Konarovice, Pod Zamkem 279,

Czech RepublicWOBE Ltd, Budapest, 1164 Garmada u. 10, Hungaiy

Age on delivery: 6-22.5 monthsExpected weight at delivery: 9-12.2 kgNumber of animals per group: Group 1: 6 (3 males and 3 females) I

Group 2: 6 (3 males and 3 females)Animal identification: Individual identification by ear-tattooThe dogs were vaccinated against: parvovirosis, canine distemper, leptospirosis, parainfluenza,canine hepatitis and rabies.

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2.4. Allocation

Group Type ofmaterial Batch No. position

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F022Hypro-Oss 0310 left tibia 10Dexabone N035 right tibia 10

M026,M028 FO45*

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M027,M020 FO24*

Hypro-Oss 0310 left tibia 10Dexabone N035 MO21* F9776,

F029right tibia 10*four administration sites were drilled into both tibias (total number 8 sites per animal), unlike

other animal where three administration sites were drilled.

2.5. Justification for Test SystemBeagle dog is a common animal model for conducting pre-clinical studies. The dimension oftibia allows using this animal model to biological evaluation study. This study should provideddata of bone tissues reaction to the administered intra-osseous bone substitution material.

2.6. HousingThe study animals were individually housed in pens, environmentally monitored and ventilatedrooms maintained at a temperature of 15-21°C and a relative humidity of 30-70 %. Fluorescentlighting provided illumination approximately 12 hours per day. Cleaning of pens and surroundingarea was performed on daily bases. Food and water containers were changed and sanitizedtwice a week.

2.7. DietDuring the acclimation and study periods the dogs were fed with standard pellet diet ofmonitored quality. At a minimum of 2 times per year it is analyzed for possible toxic ormicrobiological contamination. There were no contaminants in the diet at levels that couldreasonably be expected to affect the purpose or integrity of the study. The certificates areavailable at BioTest archive. Amount of 30g per kg of body weight of feeding mixture DELICANEXTRA (Delican, Czech Republic) was provided per animal per day. .

2.8. WaterWater of monitored quality, analyzed twice per year (the certificates are available at BioTestarchive) was supplied ad /ibitum during the acclimation and study period. There were nocontaminants in the water at levels that could reasonably be expected to affect the purpose orintegrity of the study.

2.9. AcclimationThe dogs were acclimated for 5-13 days. No prophylactic or therapeutic treatment wasadministered during the acclimation or study periods without approval of the study director. Onlyanimals in good health conditions were used for the study.

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2.10. Animal IdentificationEach dog was individually identified by ear-tattoo. Pen cards were labelled with the studynumber and animal number.

3. .METHODS

3.1. Procedure

3.1.1. intra-osseous Administration SurgeryThe surgery, preparation of the administration sites, and the administration of Test andReference Items was carried out in Test Facility by Test Facility’s veterinarian.

Three or four intra-osseous administration sites (diameter 3 mm) were drilled into both tibias(total number 6 or 8 sites per animal) in each dog (see section 2.4.). The administration siteswere filled with Test Item or Reference Item. '

The dogs were fasted overnight for min. 12 hours prior to, and 3 hours after the administration.Water was provided ad /ibitum.

Course of the intra-osseous administration was provided in follow steps: .Step 1: Pre-medication

- by Medetomidine and Midazolam (see section 3.1.2.).Step 2: Anaesthesia .

- initiated by Ketamine (see section (3.1.2.).Step 3: Inhalation Anaesthesia

- the dogs were intubated by tracheal catheter to lead anaesthesia by inhalationprovided by a mixture of oxygen and lsoflurane (see section 3.1.2.).

Step 4: Preparation of surgical place on left tibia IStep 5: intra-osseous administration of the Test ItemStep 6: Preparation of surgical place on right tibiaStep 7: intra-osseous administration of the Reference Item iStep 6: Post surgery treatment I

- after the administration the dogs went through an antibiotic and analgesictreatment (see sections 3.1.3. and 3.1.4.).

3.1.2. AnaesthesiaDuring the administration the animals were in total anesthesia under supervision of theveterinarian.Pre-medication by Medetomidine and Midazolam was performed to prepare the dogs foranaesthesia and to help provide the optimum conditions for the surgery. The anaesthesia wasinitiated by Ketamine and then a tracheal intubation was performed to lead anaesthesia byinhalation. The inhalation anaesthesia was carried out using a mixture of oxygen gas andlsofluran.Type: I Dose: I Administration: Purpose:Medetomidine '. . i P d‘ t'(Domnon Pfizer) I 0.35 pg/kg I m I reme ica ion

gdazglam Roche S r 0 ) I 1 mg/kg i.m. I Premedicationormicu , .. .

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Type: I Dose: Administration: I Purpose:K t ' . . . . .(ijaikrglgin Bio‘/eta a S ) 5 mg/kg i.m. Initiation of anaesthesialsofluran C t f(lsofluran Nicholas Piramal, Torrex 0 5-2% inhalation any Ou OChiesi CZ) ' anaesthesia

3.1.3. Antibiotic TreatmentThe antibiotic therapy as a post surgery prophylaxis was used during first 14 days after theadministration. This therapy was under supen/ision of the veterinarian who prolonged thescheduled treatment period in animal No. M028 by 10 days.Type: I Dose: (mg/dog) I Frequency: Administration:Cl" d ' DI ' .CIlrI'Dfg';3;C'n( aacm I 150 I lwiceaday p.o. I

3.1.4. Analgesic/Anti-inflammatory TreatmentThe analgesic/anti-inflammatory treatment was used during first 8 days after the administrationto avoid unnecessary pain and inflammation. This therapy was under supervision of theveterinarian who prolonged the scheduled treatment period in animal No. M028 by 10 days.Type: I Dose: (mg/kg) I Frequency: I Administration:Acidum tolfenamicum(Tolfedine Cs inj.,Vétoquinol s.r.o.) I 4 every other day I i'm' I

3.1.5. Clinical Observations and MortalityDaily Observations: All the dogs were observed for clinical signs, normality of foodconsumption, morbidity or mortality once a day during the acclimation and healing periods.During the antibiotic treatment the daily observation also focused on the regeneration course oftissue and status of sutures.Detailed Clinical Observations: All the dogs were observed for detail clinical signs weeklyafter intra-osseous administration. The detailed clinical observation was mainly focused on theregeneration course of tissue and status of sutures.The clinical obsen/ations also included: Signs of toxicity, changes in the skin and fur, eyes andmucous membranes, respiratory, circulatory, autonomic and central nen/ous system,somatomotor activity and behaviour pattern, changes in gait, posture and response to handling,the presence of clonic or tonic movements and stereotypes (SOP SN-TOX-00).

3.1.6. Body WeightAll dogs were individually weighed at delivery, before surgery procedure and then once in twoweeks during observation period.

3.2. Terminal ObservationAt the end of the experimental part (4 and 14 weeks after the administration) the dogs weresacrificed (preparation T61, lnter\/et International V.T.) and the tibias were taken out.Administration sites and relevant draining lymph nodes were evaluated macroscopically.Abnormalities were recorded with details of location, colour, shape and size.

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3.3. Tibia PreparationFour weeks in Group 1 and fourteen weeks in Group 2 after the intra-osseous administration thedogs were sacrificed and the tibias were taken out and documented by photographs. After thatthe tibias were fixed in 10% formaldehyde solution.After, at least, 14 days in the formaldehyde solution the tibias were cut up with saw intoindividual parts of bone each one contained a single administration site. If possible, the incisionin bone was led at a distance of min. 5 mm from outside circumference of the administrationsite. The cut parts with administration sites were transported in formaldehyde solution togetherwith appropriate documentation to Prof. Ctibor Povysil (Department of Pathology 15‘ Faculty ofMedicine Charles University) for next preparation and for histological and biocompatibilityevaluation.Test Facility was responsible for the isolation of the tibias, cutting of the isolated bone intoindividual parts each containing single administration site and transport the presen/ed individualparts to Prof. Ctibor Povysil (Department of Pathology 1th Faculty of Medicine CharlesUniversity).

The following procedures were under Non-GLP conditions:stProf. Ctibor Povysil (Department of Pathology 1 Faculty of Medicine Charles University) was

responsible for the individual bone parts processing, preparation of thin sections, histologicaland biocompatibility evaluation. I

3.4. Individual Bone Parts ProcessingNon-GLP conditions FAfter failure of obtain histological section appropriate for evaluation from Animal No. F019(Group 1, Hypro-Oss, batch No. 0210, position 1) method of individual parts of the boneprocessing for histological evaluation was changed. Instead of embedding in methylmethacrylate filler the individual parts of the bone with administration sites were decalcified andthen histologically evaluated.

Technological procedure of decalcifying of individual bone parts:Step 1: Washing of the samples - running waterStep 2: Decalcification — for 4 daysStep 4: Neutralisation ~ for 2-3 hours IStep 3: Washing of the samples ~ running water

3.5. Histological and Biocompatibility EvaluationNon-GLP conditionsThe thin section (5-8 um) of administered site were prepared and stained with hematoxylin andeosin. Histological evaluation of inflammation signs in attached tissue (Granulocytes,Leukocytes, Plasma cells, T-cells etc) and biocompatibility evaluation of each administration sitewas performed using an optical microscope (Olympus BX-51, Tokyo, Japan).Sections analysis and results evaluation were performed by Prof. Povysil (Department ofPathology 15‘ Faculty of Medicine Charles University).

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4. RESULTS I

4.1. Clinical Signs and MortalityAll the dogs were in good health condition throughout the acclimation and during healing period,no clinical symptoms of toxicity were obsen/ed.Healing of post-surgery wounds after intra-osseous administration went off without significantcomplication except only in dog M028 from Group 1 where healing process of the post-surgerywounds by secondary intention with granulation was obsen/ed. Due to the healing process theantibiotic and analgesic/anti-inflammatory treatment was prolonged by 10 days.

4.2. Body WeightThe following body weights were recorded:

Table 1: Body weight (kg) — Group 1 — Individual animal dataWeek IatdeliveryI 1 3 4

Animal No. I I

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All the individual body weights of the dogs were within normal range, corresponding to the ageof the animals.

4.3. intra-osseous AdministrationThe administration was performed without any complication according to procedure in the Studyplan. The planned number of administration sites was drilled into both tibias of each dog (totalnumber 6 or 8 sites per animal) and filled with Test Item or Reference Item.

4.4. Gross Pathology ExaminationGroup 1 IThree males (M042, M026, M028) and three females (F019, F022, F045) were sacrificed 4weeks after the intra-osseous administration. Partiallyhealed surgical wounds with remnants ofabsorbable sutures were found in the skin of both left and right tibial areas of all the dogs. In

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some cases, the subcutis exhibited the presence of a mild cavity in the administration site. Thementioned cavity was filled with non purulent, sanguineous liquid, see Appendix 1.In all the cases, the tibial bones including implantation sites were removed and fixed in 10%neutral formaldehyde. I

Group 2Three males (M027, M020, M021) and three females (F024, F9776, F029) were sacrificed 14weeks after the intra-osseous administration. In all the cases, both skin and subcutis werecompletely healed without any signs of inflammation, see Appendix 1.In all the cases, the tibial bones including implantation sites were removed and fixed in 10%neutral formaldehyde.

4.5. Histological and Biocompatibility EvaluationEvaluation of both batches of Test Item revealed neither signs of chronic inflammatory reactionnor giant cell reaction which could be related to a response to foreign body.Four weeks after the intra-osseous administration, the Test item was mostly surrounded bycollagen tissue without the presence of inflammatory elements. Occasionally, an occurrence oftiny lamella of the bone tissue was obsen/ed. However, it was impossible to tell with certaintywhether it is new bone tissue formation or remnants of original trabeculae of bone, see Figs 1and 2 in Appendix 2. .Fourteen weeks after intra-osseous administration, the Test item was also surrounded bycollagen tissue. A higher occurrence of new bone tissue formation in collagen tissue wasobsen/ed. New bone tissue was originally in close contact with the Test Item granules, see Figs3-7 in Appendix 2. The most significant evidence of new bone tissue formation on Test Item wasthe findings of new bone tissue surrounding the granules of Test Item in close contact with it,see Figs 5-7 in Appendix 2. Furthermore, the presence of new bone tissue in remnants ofHaversian canals of Test Item was observed, see Fig. 4 in Appendix 2.No difference between the two batches (batch No. 0210, size of granules: 0.5-1.0 mm and batchNo. 0310, size of granules: 1.0-2.0 mm) was found.

Similarly, the Test Item evaluation of both batches of Reference Item revealed neither signs ofchronic inflammatory reaction nor giant cell reaction which could be related to a response toforeign body.Four weeks after the intra-osseous administration, the Reference Item was surrounded bycollagen tissue without signs of inflammatory response, see Figs 8 and 9 in Appendix 2.Fourteen weeks after the intra-osseous administration, a formation of Iamellae of new bonetissue was found in neighbouring areas of the Reference Item, occasionally with osteoblasticexternal edge. Internal edge of new bone tissue was in contact with the surface of ReferenceItem. The lntergrowth of new bone tissue into Reference Item or Test Item was not observed inany of the cases. No difference between the two batches (batch No. 09FA29110, size ofgranules: 0.5-1.0 mm and batch No. N035, size of granules: 1.0-2.0 mm)was found. I

5. CONCLUSION .

The intra-osseous administration in all the animals was performed according to the study planwithout any complication, and the planned number of administration sites was drilled into bothtibias of each dog. The healing of post-surgery wounds was without any complication exceptonly in one animal (M028) where healing process by secondary intention with granulation wasobsen/ed without any connection to administered Items. According to clinical observation andgross pathology examination, both the Test and Reference Items were well tolerated: no signs

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of inflammatory reaction or pathological influence of both the Items were obsen/ed insurrounding soft tissue or in bone tissue. The described findings were closely connected tohealing process of the post-surgery wounds and corresponded to physiological course of woundhealing.According to histological evaluation, the Test Item caused no inflammatory or other changesresembling a reaction to foreign body, which would indicate unsuitability of the Test Item.Despite particular differences in character of Test and Reference Items both showed gradualformation of new bone tissue around granules of bone substitution material without influence ofdifferent size of granules (0.5-1.0 mm or 1.0-2.0 mm). However, batch No. 0310 containsgranules of smaller size and, consequently, of smaller surface; therefore, a smaller amount ofnew bone tissue could be expected in comparison with the granules of batch No. 0210. TheTest Item without reference to used batch corresponded to Reference Item in histology findingsand new bone tissue formation. The obsen/ed findings can be considered as an appropriateresponse to tested bone substitution material.

6. ARCHIVING

All data pertaining to the conduct of this study will be stored in the Archives of BioTest s.r.o.,Konarovice for a minimum period of 10 years. After 10 years, the Sponsor’s consent will besought and the archived material will either be destroyed or will be transferred to the Sponsor.Material to be archived: Study plan with amendments, correspondence, all the documentationand raw data related to the Test Item, test system and test conditions, draft report and finalreport with statement of QAU, records of QAU inspections. I

7. AMENDMENTS AND DEVIATIONS

The study plan was amended by:Amendment No. 01 of August 9, 2011: Exclusion of animal F043 from the study, followingevaluation and from report of the study due to health complication after intra-osseousadministration and inclusion animal F9776 into study.Amendment No. 02 of July 30, 2012: Change of head of QAU.Amendment No. 03 of August 31, 2012: Specification of Draft report date.Amendment No. 04 of September 05, 2012: Specification of Final report date.Deviations:Fourth administration sites were added on left tibia of animal F043 due to unsuitable thirdadministration site.Deviation from determined range of the room temperature (15-21°C) and relative air humidity(30-70%) were recorded during the study. I

The deviations that occurred were announced to, and approved by Study Director, and therecords of the deviations are enclosed in the raw data file of the study. The integrity of the studywas not affected in any case.

8. DISTRIBUTION

Final Report will be distributed as follows:2 copies Sponsor1 copy Test facility Manager1 copy GLP Archive BioTest s.r.oIn addition to the paper copy, the sponsor will receive the final version in pdf (acrobat) format.

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Study: Hypro-Oss bone substitution material,Preclinical Implantation Study in Beagle Dogs

Date ofthe Final Report: September 07, 2012

QA inspector: V1adimira.Mamifiakova, Jana Novakova

. ‘Dates (day/month/year), ; jI11Sp6C1i0n f. t. of reporting to management

O lmspec Ion and study director

Study plan 28.07.2011 28.07.2011In-process phase ‘ I 03.08.2011 05.08.2011Reporting phase (Final report) 07.09.2012 07.09.2012

The Quality Assurance Unit (QAU) of BioTest s.r.o. has inspected the study and reviewed the

Final report. This report fully and accurately reflected the procedures and raw data generated

during conduct of this study. I ‘ _ I _

QAU certifies that the study presented has been carriediout in accordance with approved Study

Plan, valid Standard Operating Procedures, and in compliance with OECD Principles of Good

Laboratory Practice.

Date: September 07, 2012 Jana Novakova NW

/‘ asses;Headof! Unit

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3,» CERTIFICATI: or2‘L.

ceitifiltat sp.zn./ certificate Ref.l\lo: sul<1s43420/2010

Vydany v souladu s §13 odst. 2 pism. a bod 4 zakona c;378/2007 st», 0 lecivech a o zmenach nekteryclisouvisejicich zakonfi (zakon 0 létiivech), ve znenipozdejsich pfedpisfi

Pfisltisny organ Ceske reptibliky potvrzuje nasledujici:

Testovaci zafizeni:BioTest s.r.o.Sidlo:Pod Zamkem 279, 281 25 KonaroviceAdresa testovaciho zafizeni:Pod Zamkem 279, 281 25 Konarovice

bylo inspektovano v souladu se Elankem 3 smernice2004/9/ES a § IOI, odst. 3 Zakona c. 378/2007 Sb, oleéivech a o zmenach nekterych souvisejicich zakonii(zakon 0 lecivech), ve znenl pozdejsich piedpisfi a priteto inspekci bylo provedeno posouzeni souladu sespravnou Iaboratorni praxi podle smérnice 2004/9/ES.

Na zaklade znalosti ziskanych behem posledni inspekcetohototestovaciho zafizeni, ktera byla provedena dne13.10.2010, je toto testovaci zafizeni povaiovano zasubjekt splfiujici principy spravné laboratorni praxe dane0 Zasadaini spravne Iaboratorni Ipraxe podle OECD

(C(97) 186 v konesnem zneni)o sn1ernici2004/I0/ESa ~o vyhlaskou c. 86/2008 Sb., 0 stanoveni zasad SLP

Tento certifikat odrail stav testovaciho zai“"izeni v casevyse Zminene inspekce a nemelo by se spolehat na to, Zebude odraiet stay shody po uplynuti vice nez tri let oddata inspekce. Po teto dobe-by mél cyt konzultovanvydavajici organ.Pravost tohoto ceitifikatu miiie byt oveiena uvydavajiciho orgéinu.

Rozsah einnostiToxikologicke studie

lakékoli omezeni nebo vysvetleni vztahujici se krozsahu certi tikatu:

Datum: 30.12.2010

GOOD LABORATORY PRACTICE

Issued following in accordance with Section 13.paragraph 2, letter a, point 4 of the Act No 378/200?Coll., on Pharmaceuticals and on Amendments to SomeRelated Acts (Act on Pharmaceuticals), as amended

The competent authority of the Czech Republic confirmsthe following:

Test facility:BioTest s.r.o.Head office:Pod Zamkem 279, 281 25 KonaroviceSite address: I _ IPod Zamkem 279, 281 25 Konarovice

has been inspected in accordance with Art. 3 ofDirective 2004/9/EC and Section I01 paragrapli 3 oftheAct No 378/2007 Coll, on Pharmaceuticals and onAmendments to Some Related Acts (Act onPharmaceuticals), as amended and during this inspectionassessment of conformity with GLP according toDirective 200,4/9/EC has been performed.

From the knowledge gained during inspection ofthis testfacility, the latest of which was conducted on13/10/2010, it is considered that it complies withthe principles of good l8l30l‘21'[Ol’y practice given by0 OECD Principles of Good Laboratory Practice

(C(97) 186 final)o Directive 2004/I 0/EC ando Decree No 86/2008 Coll. on GLP principles I

This certificate reflects the status of the test facility atthe time ofthe inspection noted above and should not berelied upon to reflect the compliance status if more thenthree years have elapsed since the date of inspection.after which time the issuing, authority should beconsulted. aThe authenticity ofthis certificate may be verified withthe issuing authority.

Scope of activitiesToxicological studies ,

Any restrictions or c-1arit‘ying_ remarks related to thescope ofthis certificate:

Date: 30.12.2010

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