Test Facility Certification M.S. 85/2009

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1 Page: 1 of 59 Final Report 2009/717 AMi SET UP AND VALIDATION OF A METHOD FOR THE DETERMINATION OF VIRAL INACTIVATION BY MEANS OF CHEMICAL AND PHYSICAL TREATMENTS ON HUMAN CANCELLOUS BONE TISSUE BLOCKS Study program: 2009/717 AMi Contract n: PARA PARA PARA Sponsor: ARTHRO KINETICS MAGNESITSTRASSE 1 OBJECT KREMS AN DER DONAU Test item: HUMAN CANCELLOUS BONE TISSUE BLOCKS Study Director:... (Dr. L. Brambilla) Released on:... This test report cannot be reproduced partially without a written approval of the Test Facility

2 Page: 2 of 59 INDEX COMPLIANCE WITH GOOD LABORATORY PRACTICE... 4 QUALITY ASSURANCE STATEMENT... 5 SUMMARY... 6 INTRODUCTION... 9 TERMS AND DEFINITIONS BIBLIOGRAPHY FILING PROCEDURES TEST ITEM: Experimental Report 2009/717.A1 - Method set up in order to determinate the virus inoculum volume and the inoculum absorption time of human bone tissue block Experimental conditions Acceptance criteria RESULTS DEVIATIONS observations Experimental Report 2009/717.A2 Method set up in order to validate a recovery method EXPERIMENTAL conditions Efficacy criteria RESULTS DEVIATION OBSERVATIONS Experimental Report 2009/717.A3 - Check of suppression of HYDROGEN PEROXIDE SOLUTION 3% activity and cytotoxicity EXPERIMENTAL conditions Efficacy criteria RESULTS DEVIATION OBSERVATIONS Experimental Report 2009/717.A4 - Viral inactivation kinetics of HYDROGEN PEROXIDE SOLUTION 3%. 27 EXPERIMENTAL conditions Accaptance criteria RESULTS DEVIATION OBSERVATIONS Experimental Report 2009/717.A5 - Viral inactivation effectiveness with HYDROGEN PEROXIDE SOLUTION 3% (Sponsor step 2.1 of Sponsor VA-07 procedure) EXPERIMENTAL conditions RESULTS DEVIATION OBSERVATIONS Experimental Report 2009/717.A6 - Gamma rays treatment viral inactivation effectiveness (step 2.2 as per Sponsor VA-07 procedure)..37 EXPERIMENTAL conditions Efficacy criteria RESULTS DEVIATION OBSERVATIONS Experimental Report 2009/717.A7 - Method set up in order to validate a recovery method (96% EtOH) Experimental conditions Efficacy criteria RESULTS DEVIATION OBSEVATIONS... 44

3 Page: 3 of 59 Experimental Report 2009/717.A8 - Check of suppression of ETHANOL 96% activity and cytotoxicity Experimental conditions Efficacy criteria RESULTS DEVIATION OBSERVATIONS Experimental Report 2009/717.A9 - Viral inactivation kinetics of ETHANOL 96% Experimental conditions Efficacy criteria RESULTS DEVIATION OBSERVATIONS Experimental Report 2009/717.A10 - Viral inactivation effectiveness with ETHANOL 96% (Chemical cleaning step of Sponsor VA-07 procedure) Experimental conditions Efficacy criteria RESULTS DEVIATION OBSERVATIONS CONCLUSIONS 5 9 ATTACHMENTS...5 9

4 Page: 4 of 59 COMPLIANCE WITH GOOD LABORATORY PRACTICE I the undersigned declare that the studies described in this report have been conducted under my supervision and in compliance with the following standards of Good Laboratory Practice: - Organisation for Economic Co-operation and Development, ISBN , Paris United States Food and Drug Administration, Title 21 Code of Federal Regulations Part 58, Federal Register 22 December 1978, and subsequent amendments - Italian Ministerial Decree of June 26th, 1986 "Application of Good Laboratory Practice on chemical substances and criteria for issuing the authorizations foreseen by Presidential decree n. 927/81 art. 6" - Legislative decree of January 27th, 1992, n Enforcement of Community Directives n. 88/320/CEE, n. 90/18/CEE and following decree of 5 August 1999 Enforcement of Community Directives n. 1999/11/CE and 1999/12/CE concerning inspection and verification of Good Laboratory Practice - Directive 2004/10/CE issued by European Parliament and Council dated February 11th concerning the drawing of the legislative, regulatory and administrative dispositions relative to the application of Good Laboratory Practice rules, to the control of their application on the assays performed on the chemical substances - Decree of the Italian Ministry of Health October 12 th, 2009, Certification 85/2009 authorizing Biolab S.p.A. to perform analyses in compliance with the principles of good laboratory practices - Legislative decree n. 50 of March the 2 nd, Enforcement of Community Directives 2004/9/CE e 2004/10/CE, concerning the inspection and verification of Good Laboratory Practice and the drawing of the legislative, regulatory and administrative dispositions relative to the application of Good Laboratory Practice rules, to the control of their application on the assays performed on the chemical substances (GU n.86 of April the 13 th, 2007). STUDY DIRECTOR (Dr. L. Brambilla) DATE

5 Page: 5 of 59 QUALITY ASSURANCE STATEMENT I the undersigned below certify the dates on which the inspections on this study have been done and the relevant observations have been reported to the Director of the Study and to the assay centre Management: PHASE OF STUDY DATE OF INSPECTION / REPORTING Pre-experimental period Experimental period Post-experimental period Documentation: - Study program - Amendment #1 to the Study program - Raw data - Final report GLP QUALITY ASSURANCE (Dr. C. Ronaghi) DATE

6 Page: 6 of 59 SUMMARY The aim of the study was to validate three different viral inactivation steps of the same sterilization process of a medical device, i.e. dynamic immersion in HYDROGEN PEROXIDE SOLUTION 3%, GAMMA RAYS TREATMENT and dynamic immersion in ETHANOL 96%. The test item consisted of human cancellous bone tissue to be used in orthopedic, traumatic and oncologic surgeries (the products are used in order to cover or to fill bone defects). For this purpose the following 6 viral suspensions were used as closely representative viruses which may contaminate the human bone tissue: - Adenovirus type 5, human ATCC VR-5 (as model virus for non enveloped DNA viruses with a medium/high resistance to chemical agents, according to standard EN14476) - Poliovirus type 1 ATCC VR-1562 (chat strain) (as a model virus for non enveloped RNA viruses with a high resistance to chemical agents, according to standard EN14476) - Human herpes virus (Herpes simplex Type1; HSV-1) ATCC VR- 733 (as a model virus for enveloped DNA viruses with medium resistance of chemical agents) - Bovine viral diarrhea virus (BVDV) ATCC VR-534 (as model virus for HCV according to EMEA and German RKI/DVV guidelines and as a model virus for enveloped RNA viruses with low resistance to chemical agents) - Hepatitis A virus Strain HM175/18f [HM175 cytopathic clone B] (HAV) ATCC VR-1402 (as a model virus for non enveloped RNA viruses with a high resistance to chemical agents) - Porcine parvovirus NADL-2 (PPV) ATCC VR-742 (as a model virus for non enveloped DNA viruses with a high resistance to chemical agents). The study was performed in stepwise manner: 1. determination the virus inoculum volume and the inoculum absorption time of human bone tissue block by means of a methylene blue solution. The spongiosa cubes were put into sterile 50ml Falcon tube with a screw-cap together with 15 ml of the virus suspension. The Falcon tubes were placed in a desiccator under negative pressure (0.2 bar, obtained using a vacuum pump). See Experimental Report 2009/717.A1 for more details. 2. validation of virus titre recovery method both from the test solution in which each human bone tissue block was immersed and directly from each human bone tissue block, after different inoculum exposures ( hours; 24 hours after inoculum was tested as well in order to simulate the experimental conditions for gamma rays treatment). See Experimental Report 2009/717.A2 for more details. 3. the check of suppression of HYDROGEN PEROXIDE SOLUTION 3% activity by means of catalase solution and check of test solution cytotoxicity; at the same time the stability of HYDROGEN PEROXIDE SOLUTION 3% was also determined by titration of active O 2. See Experimental Report 2009/717.A3 for more details. 4. determination of viral inactivation kinetics on HYDROGEN PEROXIDE SOLUTION 3% after minutes and hours of contact times, according to standard EN14476 test method (quantitative suspension test). See Experimental Report 2009/717.A4 for more details.

7 Page: 7 of chemical viral inactivation test by means of immersion in HYDROGEN PEROXIDE SOLUTION 3% (immersion procedure under dynamic conditions) against the 6 test virus strains at the contact time validated in the experimentations A2 and A3. See Experimental Report 2009/717.A5 for more details. 6. gamma rays viral inactivation test (gamma irradiation dose: 25.0 kgy) against the 6 test virus strains in validated test conditions defined in the experimentations A2. See Experimental Report 2009/1005.A6 for more details. 7. validation of a recovery method both from the solution in which each human bone tissue block was immersed and directly from human bone tissue block after different inoculum exposures for EtOH 96% (1-3 hours). See Experimental Report 2009/717.A7 for more details. 8. the check of suppression of ETHANOL 96% activity and check of its cytotoxicity See Experimental Report 2009/717.A8 for more details. 9. determination of viral inactivation kinetics on ETHANOL 96% after minutes and 1-3 hours contact times, according to standard EN14476 test method (quantitative suspension test). See Experimental Report 2009/717.A9 for more details. 10. chemical viral inactivation test by means of immersion in ETHANOL 96% (immersion procedure under dynamic conditions) against the 6 test virus strains at the contact time validated in the experimentations A2 and A3. See Experimental Report 2009/717.A10 for more details. On the basis of the results obtained in the sterilization process of human cancellous bone tissue blocks, the viral inactivation step by immersion in HYDROGEN PEROXIDE SOLUTION 3% after 16 hours of contact time has shown good virucidal efficacy against the viruses tested; some residual virus related CPE (cytopatic effect) was observed only for the Porcine Parvovirus (PPV) ATCC VR-742 and Hepatitis A virus (HAV) ATCC VR-1402, but both viral titre reductions resulted 2 Log. The viral inactivation step by immersion in ETHANOL SOLUTION 96% after 3 hours contact time has shown complete efficacy against all viruses tested, i.e. no residual viruses were detected in the assay at the end of the virus incubation period. Due to the relatively low virus recovery from the carriers, a viral titre reduction >4Log, as required by the reference standard EN14476:2005+A1:2006, was not always detectable; however in all validation steps each virus titre was sufficiently high to at least enable a titre reduction >2 Log.

8 Page: 8 of 59 The Gamma rays irradiation step (irradiation dose: 25 kgy), instead, did not shown good efficacy against the viruses tested; in particular this treatment had shown low effectiveness against Poliovirus type 1 ATCC VR-1562, Adenovirus type 5 ATCC VR-5, Porcine Parvovirus (PPV) ATCC VR-742 and Hepatitis A virus (HAV) ATCC VR-1402; a viral titre reduction > 2Log was obtained only against Human herpes virus (HSV-1) ATCC VR-733 and Bovine viral diarrhea virus (BVDV) ATCC VR-534, in compliance with the validity assay criteria. The two chemical viral inactivation steps adopted in the sterilisation process, i.e. immersion in EtOh 96% solution for at least 3 hours and immersion in H2O2 3% solution for at least 16 hours show an overall efficacy in inactivating the 6 test viruses globally >4Logs and therefore should be considered effective in removing possible viral contaminations. In particular, the EtOh 96% viral inactivation step causes a complete inactivation of viral inoculum for all viruses tested.

9 Page: 9 of 59 INTRODUCTION A study was conducted on behalf of ARTHRO KINETICS to demonstrate the virucidal effectiveness of three different viral inactivation steps, in accordance with the Sponsor method and European regulations. The study was performed at the Test Facility Biolab S.p.A. of Vimodrone (MI) via B. Buozzi n. 2 (Italy). EXPERIMENTATION START END RESEARCHERS Method set up in order to determinate the virus inoculum volume and the inoculum absorption time of human bone tissue block (2009/717.A1) Method set up in order to validate a recovery method (2009/717.A2) Check of suppression of HYDROGEN PEROXIDE SOLUTION 3% activity and cytotoxicity (2009/717.A3) Viral inactivation kinetics on HYDROGEN PEROXIDE SOLUTION 3% (2009/717.A4) Viral inactivation effectiveness with HYDROGEN PEROXIDE SOLUTION 3% (2009/717.A5) Oct 13 th 2009 Oct 13 th 2009 L.Brambilla/F.Faccioli Oct 29 th 2009 March 15 th 2010 L.Brambilla/F.Faccioli Nov 13 th 2009 April 09 th 2010 L.Brambilla/F.Faccioli Nov 17 th 2009 March 23 rd 2010 L.Brambilla/F.Faccioli March 31 st 2010 April 20 th 2010 L.Brambilla/F.Faccioli Gamma rays treatment effectiveness (2009/717.A6) April 23 rd May 31 st 2010 L.Brambilla/F.Faccioli Method set up in order to validate a recovery method (96% EtOH) (2009/717.A7) March 31 st 2010 April 20 th 2010 L.Brambilla/F.Faccioli Check of suprression of ETHANOL 96% (2009/717.A8) Feb 17 th 2010 March 29 th 2010 L.Brambilla/F.Faccioli Viral inactivation kinetics on ETHANOL 96% (2009/717.A9) March 31 st 2010 April 20 th 2010 L.Brambilla/F.Faccioli Viral inactivation effectiveness with ETHANOL 96% (2009/717.A10) April 01 st 2010 April 13 th 2010 L.Brambilla/F.Faccioli

10 Page: 10 of 59 According to a Sponsor s request, an amendment to the Study Program 2009/717 AMi was issued on January 28 th 2010, aiming to complete and integrate the method validation of the viral inactivation process described in the study program by the assay of two additional viral strains (PPV and HAV). Study Program 2009/717 AMi was amended in order to integrate the method validation of viral inactivation process by means of an additional step described in the experimentations 2009/717.A7, 2009/717.A8, 2009/717.A9 and 2009/717.A10, i.e. by means of ETHANOL 96% washing. In this report: - doses are expressed as grams of the test substance per 100 millilitres of water (%) - the virus titres are expressed as TCID50: 50% infecting dose of a virus suspension or that dilution of the virus suspension that induce a CPE in 50% of cell culture units. TERMS AND DEFINITIONS Virucidal: Virucidal activity: a chemical agent or a formulation that inactivates viruses under certain conditions. the capability of a product to produce a reduction in the number of viruses under certain conditions.

11 Page: 11 of 59 BIBLIOGRAPHY 1. EN Chemical disinfectants and antiseptics Quantitative test in virucidal suspension for chemical disinfectants and antiseptics used in human medicine Test method and requirements (phase2, step1) April Addendum A1 October VA-07 section 1, amount of annexes: 0 - Virus inactivation study - Details regarding samples, process steps (Sponsor procedure). 3. ISO , Medical devices utilizing animal tissues and their derivatives Part 3: Validation of the elimination and/or inactivation of viruses and transmissible spongiform encephalopathy (TSE) agents EMEA - the European Agency for the Evaluation of Medicinal Products CPMP/BWP/268/95 14 February, Annals of transplantation vol. 8, No 2, 2003 Peracetic Acid-Ethanol treatment of Allogeneic avital Bone Tissue Transplants a Reliable Sterilization Method. 6. VA-07 section 1, amount of annexes: 0 - Virus inactivation study - Details regarding samples, process steps. 7. E.P. 6th edition 2009 (6.4) Hydrogen peroxide solution (3 per cent) 8. Cell and Tissue Banking 2: , kluver Academic Publisher. Printed in the Netherlands. Comparison of the efficacy of virus inactivation methods in allogenic avital bone tissue transplants 29 märz VA-07 section 1, amount of annexes: 0 - Virus inactivation study - Details regarding samples, process steps version 03, November 18 th 2009 (Sponsor procedure). FILING The study program, amendments, all raw data and a copy of the final report are filed in the archives of the Test facility for ten years after the issuing of the final report. No retained sample will be kept. At the end of the conservation period, the Sponsor may request an extension of the conservation of all or part of the products for a further period, or their restitution. A suitable agreement shall be drafted in this case. PROCEDURES All procedures used during this study are recorded in the Procedures Manual of the Test facility.

12 Page: 12 of 59 TEST ITEM: IMMERSION IN HYDROGEN PEROXIDE SOLUTION 3% The sample representative of the disinfectant product used in the treatment consists of transparent colourless liquid contained into a dark jar (1l). Product HYDROGEN PEROXIDE SOLUTION 30% Manufacturer MERCK Composition HYDROGEN PEROXIDE SOLUTION 30% Stability Batch not provided K CoA See Attachment N.1 Manufacturing date Not provided Expiry date May 31 st 2011 Receiving EUITVI-4528 Date September 22 nd, 2009 #ID S GAMMA RAYS STERILISATION Irradiation facility Irradiation dose Irradiation data Receiving Date #ID spongiosa blocks Gammatom S.p.A. Via XXIV Maggio, 14 Guanzate (CO) 25.0 kgy April 22 nd 2010 May 19 th 2010 (carrier inoculated with HAV) April 23 rd 2010 May 20 th 2010 (carrier inoculated with HAV) S TEST CARRIERS (PROVIDED BY THE SPONSOR) Raw material Blocks #ID spongiosa blocks Batch Cancellous bone (defatted, cleaned, lyophilized): this status of bone material is comparable with the status before cleaning with 3% H 2 O 2 15 x 15 x 30 mm S (11 x blocks for pre testing; packaged in units to 5&6) S (30 x blocks for pre testing; packaged in units to 6) S (spongiosa blocks) BXL ( S, S) BXL ( S)

13 Page: 13 of 59 IMMERSION IN WITH ETHANOL 96% The sample representative of the disinfectant product used in the treatment consists of transparent colourless liquid contained into a plastic jar (2.5 l). Product ETHANOL 96% Manufacturer MERCK Composition ETHANOL 96% Stability Batch not provided 09H CoA See Attachment N.1 Manufacturing date Not provided Expiry date August 2014 Receiving EUITVI-5882 Date November 27 th, 2009 #ID S TEST CARRIERS (PROVIDED BY THE SPONSOR) Raw material Blocks #ID Batch Cancellous bone (defatted, cleaned, lyophilized) treated only with ether: this status of bone material is comparable with the status before cleaning with 96% EtOH 15 x 15 x 30 mm S (80 x blocks; packaged in units 2 blocs in 50 ml centrifuge tube) SE A Manufacturing date Jan 22 nd 2010 Expiry date Receiving Not provided EUITVI-6863 Date January 28 th 2010 #ID spongiosa blocks S

14 Page: 14 of 59 Experimental Report 2009/717.A1 - Method set up in order to determinate the virus inoculum volume and the inoculum absorption time of human bone tissue block EXPERIMENTAL CONDITIONS 1. MEDIA AND REAGENTS Reagents Methylene blue solution. 2. EQUIPMENT AND MATERIALS Vortex Chronometer Micropipettes Sterile Falcon 50 ml Dessicator Vacuum pump VELP ARBORE GILSON GHIARONI 3. EXPERIMENTAL DESIGN Test temperature The test was conducted at 20 C ±1 C Test Carrier The test carriers, provided by the Sponsor, consist of human cancellous bone tissue (defatted, cleaned, lyophilized) which are comparable with the status before cleaning with 3% H 2 O 2; they are blocks with the following dimensions:15 x 15 x 30 mm (equivalent volume 6.75 cm 3 ). 4. ASSAY EXECUTION Assay The method set up was performed in order to determinate the virus inoculum volume and the inoculum absorption time of human bone tissue block by means of a methylene blue solution (colorant solution) pouring the spongiosa cubes into sterile 50ml Falcon with a screw-cap together with 15 ml of the virus suspension that were placed in a desiccator under negative pressure (0.2 bar, obtained using a vacuum pump). ACCEPTANCE CRITERIA As per ISO the inoculum shall not be more than 6.75 ml +10%.

15 Page: 15 of 59 RESULTS The volume of colorant solution necessary to completely cover the carrier resulted equivalent to 15 ml; following are shown the absorption results: INITIAL INOCULUM VOLUME PRESSURE CONDITIONS ABSORPTION TIME 15 g 0.2 bar (equivalent to 15 cmhg) 15 minutes Replica 1 Replica 2 Replica 3 RESIDUAL VOLUME 12.1 g (or ml) 11.3 g (or ml) 11.7 g (or ml) ABSORPTION VOLUME 2.9 g (or ml) 3.7 g (or ml) 3.3 g (or ml) MEAN OF THE ABSORPTION VOLUME 3.3 g (or ml) DEVIATIONS The study did not undergo deviations compared to the study program. OBSERVATIONS On the basis of the results obtained in compliance with the assay validity criteria, the mean of the absorption volume results CONFORM after 15 minutes of immersion time in the presence of a negative pressure.

16 Page: 16 of 59 Experimental Report 2009/717.A2 Method set up in order to validate a recovery method The recovery method was validated in order to define the procedure to use in the experimentation A5 and A6. EXPERIMENTAL CONDITIONS 1. ASSAY SYSTEM Virus strains Adenovirus type 5, human ATCC VR-5 Poliovirus type 1 (chat strain) ATCC VR-1562 Human herpes virus (Herpes simplex Type1; HSV-1) ATCC VR- 733 Bovine viral diarrhea virus (BVDV) ATCC VR-534 Hepatitis A virus Strain HM175/18f [HM175 cytopathic clone B] (HAV) ATCC VR-1402 Porcine parvovirus NADL-2 (PPV) ATCC VR-742 Test virus suspension The viral suspensions were kept frozen at 70 C; before their use they were multiplied in the following appropriate cellular lines: HOST CELLS INCUBATIONS VIRUS STRAINS LINE TIMES (Days) Adenovirus type 5, human Hela 7 Poliovirus type 1 (chat strain) Hela 7 Human herpesvirus (Herpes simplex Type1; HSV-1) Vero 7 Bovine viral diarrea virus (BVDV) MDBK 7 Hepatitis A virus Strain HM175/18f [HM175 cytopathic clone B] (HAV) FRhK-4 12 Porcine parvovirus NADL-2 (PPV) ST 7 Cellular debris was removed by means of double centrifugation at low speed, and the supernatant containing the virus was used for the test. 2. CELL CULTURES, MEDIA AND REAGENTS Cell cultures MDBK (NBL-1 Kidney) HeLa (cells of human uterine carcinoma) Vero FRhK-4 ST ATCC CCL-22 ATCC CCL-2 ATCC CCL-81 ATCC CRL-1688 ATCC CRL-1746 The cellular culture was kept frozen at < 70 C; before viral inoculum, it was showed itself as confluent monolayer.

17 Page: 17 of 59 Culture Medium EMEM: Eagle s minimal essential medium BIOWHITTAKER - LONZA FBS (2%) Foetal Bovine Serum BIOWHITTAKER LONZA NEAA (1%) Non-Essential Amino Acid BIOWHITTAKER LONZA PEN-STREP (1%) antibiotics BIOWHITTAKER LONZA PBS Phosphate Buffer Saline BIOWHITTAKER - LONZA Reagents WFI Water for injection EUROSPITAL BSA Buffer bovine serum albumin (3% in distilled water) SIGMA 3. EQUIPMENT AND MATERIALS Incubator Centrifuge Deepfreeze CO 2 incubator Vortex Chronometer Micropipettes Microdishes 96 wells Inverted microscope phmeter Vessel containing water and ice Water bath Dessicator Vacuum pump ARBORE HERAEUS Angelantoni Scientifica PBI VELP ARBORE GILSON CAMBREX LEITZ BECKMAN ARBORE 4. EXPERIMENTAL DESIGN Test temperature The test was conducted at 20 C ±1 C Interfering substance Clean conditions: The used interfering substance was prepared with a concentration 10 times higher than the final concentration of 0.03%. Bovine albumin 0.3 g Diluent q.s. to 100 ml Test carriers The test carriers, provided by the Sponsor, consisted of human bone tissue (defatted, cleaned, lyophilized) which were comparable with their manufacturing process status before cleaning with 3% H 2 O 2 ; they consisted of blocks with the following size:15 x 15 x 30 mm (equivalent volume 6.75 cm 3 ). Inoculum preparation The inoculum was preparated by mixing each virus suspension with interfering substance in ratio 9:1. Carrier Inoculum Test carriers were inoculated with each inoculum preparation by separately using the inoculum volume validated in the experimentation A1.

18 Page: 18 of 59 Contact times The following exposure times were used: hours (immersion in sterile water) 24 hours (in vial) 5. ASSAY EXECUTION Assay of viral activity (virus titration) Serial dilutions 1:10 were prepared with culture Medium, starting from virus stock solution of viral suspension. 0.1 ml were transferred in dishes with 96 wells containing the cellular confluent monolayer (>90%) without culture Medium. Each dilution of viral suspension was placed onto dishes sixfold. The 12 wells of the upper part and the 12 wells of the lower part of the dish did not receive the viral inoculum and were used as control of cellular line. After 1 hour of incubation at 37 C ±1 C 0.1ml of culture Medium were added to viral inoculum. After the appropriate incubation period, the cellular culture was observed with inverted microscope to detect any cytopatic effect (CPE) due to viral suspension. After this detection the infecting activity (TCID 50 evaluation) was calculated by means of Spearman Karber method. Assay control For each contact time, 2 carriers test were inoculated with the 6 viral suspension test separately. The carrier test was immersed into a ml (ratio 1:5 with carrier volume as indicated by the Sponsor procedure VA-07) of sterile water. At the end of contact time a viral recovery was determined respectively on the water solution and on the carrier itself in order to determine the best immersion time which was to be used in the efficacy test by means of disinfection with HYDROGEN PEROXIDE SOLUTION 3%. The recovery validation on the carrier at 24 hours was validated for the viral inactivation test by means of gamma rays treatment. Validation of viral recovery in the immersion system (water) At the and of each contact time a viral titration was performed in order to determine the best immersion time which was used in the efficacy test (by means of disinfection with HYDROGEN PEROXIDE SOLUTION 3%). For this purpose, serial dilutions 1:10 were prepare with culture Medium, starting from the water containing the inoculated carrier. 0.1 ml were transferred in dishes with 96 wells containing the cellular confluent monolayer (>90%) without culture Medium. Each dilution of viral suspension was placed onto dishes sixfold. The 12 wells of the upper part and the 12 wells of the lower part of the dish will not receive the viral inoculum and were used as control of cellular line. After 1 hour of incubation at 37 C ±1 C 0.1ml of culture Medium were added to viral inoculum. The test was performed in duplicate. After the appropriate incubation period, the cellular culture was observed with inverted microscope to detect any cytopatic effect (CPE) due to viral suspension. After this detection the infecting activity (TCID 50 evaluation) was calculated by means of Spearman Karber method.

19 Page: 19 of 59 Validation of viral recovery on test carriers At the end of contact time, a centrifugation at G during 5 minutes was performed on each carrier for three times consecutively, in order to recover the residual adsorbed virus. Then a titration was performed by means of serial decimal dilutions with culture Medium, starting from the carrier. 0.1 ml were transferred in dishes with 96 wells containing the cellular confluent monolayer (>90%) without culture Medium. Each dilution of viral suspension was placed onto dishes sixfold. The 12 wells of the upper part and the 12 wells of the lower part of the dish did not receive the viral inoculum and were used as control of cellular line. After 1 hour of incubation at 37 C ±1 C 0.1ml of culture Medium were added to viral inoculum. The test was performed in duplicate. After the appropriate incubation period, the cellular culture was observed with inverted microscope to detect any cytopatic effect (CPE) due to viral suspension. After this detection the infecting activity (TCID 50 evaluation) was calculated by means of Spearman Karber method. 6. CALCULATIONS AND EXPRESSION OF THE RESULTS Determination of TCID 50 The infecting activity was determined by means of Spaerman Karber method that uses the following formula to calculate the value of TCID 50 : -Log TCID 50 = - (-x 0 ) - {[R/100]- 0.5} x Log dilution factor where: x 0 = log 10 of the lowest dilution with 100% of positive reaction (CPE) R = sum (%) of positive cultures Log TCID 50 values were rounded to two significant ciphers as shown in Annex D table D.1 of EN Rounding is automatically performed by excel datasheet. EFFICACY CRITERIA The minimum titre of the virus suspensions in the virus control shall be sufficiently high to enable a titre reduction of at least 2 Logs to verify the method.

20 Page: 20 of 59 RESULTS Validation of the recovery method For each virus strains, the Log TCID 50 obtained in the presence of a bovine albumin solution with a final concentration of 0.03% (simulating clean conditions) at the different contact times, are shown in the table here below: Contact times Poliovirus Type 1 ATCC VR Adenovirus Type 5 ATCC VR - 5 Human herpesvirus (HSV-1) ATCC VR-733 Bovine viral diarrea virus (BVDV) ATCC VR-534 Porcine Parvovirus (PPV) ATCC VR-742 Hepatitis A virus (HAV) ATCC VR CARRIER h h h h h h WATER h h h h h h CARRIER h h h h h h WATER h h h h h h

21 Page: 21 of 59 DEVIATION No deviation has been recorded from study program. OBSERVATIONS On the basis of the results obtained in compliance with the assay validity criteria, the recovery method results VALIDATED; each residual virus titre was expressed as Log TCID 50 and was sufficiently high to enable a titre reduction of at least 2 Logs to verify the method.

22 Page: 22 of 59 Experimental Report 2009/717.A3 - Check of suppression of HYDROGEN PEROXIDE SOLUTION 3% activity and cytotoxicity The hydrogen peroxide 3% neutralization method was validated in order to define the procedure to be used in the experimentation A4, A5 and A6. EXPERIMENTAL CONDITIONS 1. ASSAY SYSTEM Virus strains Adenovirus type 5, human ATCC VR-5 Poliovirus type 1 (chat strain) ATCC VR-1562 Human herpesvirus (Herpes simplex Type1; HSV-1) ATCC VR- 733 Bovine viral diarrea virus (BVDV) ATCC VR-534 Hepatitis A virus Strain HM175/18f [HM175 cytopathic clone B] (HAV) ATCC VR-1402 Porcine parvovirus NADL-2 (PPV) ATCC VR-742 Test virus suspension The viral suspensions were kept frozen at 70 C; before their use they were multiplied in the following appropriate cellular line: HOST CELLS INCUBATIONS VIRUS STRAINS LINE TIMES (Days) Adenovirus type 5, human Hela 7 Poliovirus type 1 (chat strain) Hela 7 Human herpes virus (Herpes simplex Type1; HSV-1) Vero 7 Bovine viral diarrhea virus (BVDV) MDBK 7 Hepatitis A virus Strain HM175/18f [HM175 cytopathic clone B] (HAV) FRhK-4 12 Porcine parvovirus NADL-2 (PPV) ST 7 Cellular debris were removed by means of double centrifugation at low speed, and the supernatant containing the virus was used for the test. 2. CELL CULTURES, MEDIA AND REAGENTS Cell cultures MDBK (NBL-1 Kidney) HeLa (cells of human uterine carcinoma) Vero FRhK-4 ST ATCC CCL-22 ATCC CCL-2 ATCC CCL-81 ATCC CRL-1688 ATCC CRL-1746 The cellular culture was kept frozen at < 70 C; before viral inoculum, it was showed itself as confluent monolayer.

23 Page: 23 of 59 Culture Medium EMEM: Eagle s minimal essential medium BIOWHITTAKER - LONZA FBS (2%) Foetal Bovine Serum BIOWHITTAKER LONZA NEAA (1%) Non-Essential Amino Acid BIOWHITTAKER LONZA PEN-STREP (1%) antibiotics BIOWHITTAKER LONZA PBS Phosphate Buffer Saline BIOWHITTAKER - LONZA Reagents WFI Water for injection EUROSPITAL BSA Buffer bovine serum albumin (0.3% w/v in distilled water) SIGMA Catalase solution (0.025% final concentration) SIGMA Sulphuric acid SIGMA Potassium permanganate standard solution 0.02 M in H 2 O SIGMA 3. EQUIPMENT AND MATERIALS Incubator Centrifuge Deepfreeze CO 2 incubator Vortex Chronometer Micropipettes Microdishes 96 wells Inverted microscope phmeter Vessel containing water and ice Water bath ARBORE HERAEUS Angelantoni Scientifica PBI VELP ARBORE GILSON CAMBREX LEITZ BECKMAN ARBORE 4. EXPERIMENTAL DESIGN Test temperature The test was conducted at 20 C ±1 C Concentration The test substance was prepared at concentrations 1.25 times higher than the concentrations requested by the test. The test substance was tested at the final concentration of 3% (diluted into sterile water). Contact time The test was performed at the time zero of contact. Interfering substance Clean conditions: The used interfering substance was prepared with a concentration 10 times higher than the final concentration of 0.03%. Bovine albumin 0.3 g Diluent q.s. to 100 ml

24 Page: 24 of ASSAY EXECUTION Assay of viral activity (virus titration) Serial dilutions 1:10 were prepared with culture Medium, starting from virus stock solution of viral suspension. 0.1 ml were transferred in dishes with 96 wells containing the cellular confluent monolayer (>90%) without culture Medium. Each dilution of viral suspension was placed onto dishes sixfold. The 12 wells of the upper part and the 12 wells of the lower part of the dish did not receive the viral inoculum and were used as control of cellular line. After 1 hour of incubation at 37 C ±1 C 0.1ml of culture Medium were added to viral inoculum. After the appropriate incubation period, the cellular culture was observed with inverted microscope to detect any cytopatic effect (CPE) due to viral suspension. After this detection the infecting activity (TCID 50 evaluation) was calculated by means of Spearman Karber method. Check of suppression of disinfectant activity 1 ml of interfering substance was added to 1 ml of viral suspension and to 8 ml of the HYDROGEN PEROXIDE SOLUTION 3% inactivated with 0.025% catalase solution (in order to obtain a final in test concentration of the test sample equal to 3%). At time zero, 0.5 ml of the afore described solution was diluted into 4.5 ml of culture Medium + 2% FBS in iced bath. Then the solution was left in iced bath for 30 minutes ±10 seconds. After this period serial dilutions 1:10 with culture Medium were performed. The solutions were incubated at 37 C ±1 C in water-bath for 5 minutes. 0.1 ml of the solutions were transferred sixfold onto microdishes, incubated at 37 C ±1 C for 1 hour, then 0.1 ml of culture Medium were added to the viral inoculum. After the appropriate incubation period, the cellular culture was observed with inverted microscope to detect any cytopatic effect (CPE) due to viral suspension. After this detection the infecting activity (TCID 50 ) was calculated by means of Spearman Karber method. Check of cytotoxicity of the test substance To evaluate any cytopatic effect of the test substance, a cytotoxicity test was conducted by mixing 8 ml of the HYDROGEN PEROXIDE SOLUTION 3% inactivated with 0.025% catalase solution with 2 ml of sterile water. Then serial dilutions 1:10 were performed with culture Medium. 0.1 ml of each dilution of the test substance were put on dishes sixfold. The 12 wells of the upper part and the 12 wells of the lower part of the dish will not receive the viral inoculum and were used as control of cellular line. After 1 hour of incubation at 37 C ±1 C 0.1ml of culture Medium were added to each well. Immediately after the addition of the test substance and daily for the appropriated days, the cellular culture was observed with inverted microscope to detect any cytopatic effect (CPE) due to test substance. Titration of active O2 The titration of active O 2 was evaluated at the same time of neutralization check and was performed according to E.P. 6 th edition 2009 (6.4) Hydrogen peroxide solution (3 per cent), diluting 10.0 g to ml with sterile water. To 10.0 ml of this solution were added 20 ml of dilute sulphuric acid and titrated with 0.02 M potassium permanganate until a pink colour was obtained.

25 Page: 25 of CALCULATIONS AND EXPRESSION OF THE RESULTS Determination of TCID 50 The infecting activity was determined by means of Spearman Karber method that uses the following formula to calculate the value of TCID 50 : -Log 10 TCID 50 = - (-x 0 ) - {[R/100]- 0.5} x log 10 dilution factor where: x 0 = log 10 of the lowest dilution with 100% of positive reaction (CPE) R = sum (%) of positive cultures Log TCID 50 values were rounded to two significant ciphers as shown in Annex D table D.1 of EN Rounding is automatically performed by excel datasheet. Titration of active O2 1 ml of 0.02 M potassium permanganate is equivalent to mg of H 2 O 2 or 0.56 ml of oxygen. EFFICACY CRITERIA 1. Check of suppression of disinfectant activity: the difference of the value of TCID 50 among the cellular cultures treated with the inactivated test substance and the ones treated only with the viral inoculum must be 0.5 log The test substance shall not be cytotoxic at the concentrations tested, i.e. its contribution in terms of CPE shouldn t be visible in the test; in any case, the cytotoxicity shall be low enough to at least enable a titre reduction of 4 Log to verify the method. Titration of active O2 The specification for Active O 2 is ± 10% from the title of nominal active O 2. RESULTS Check of suppression of disinfectant activity: HYDROGEN PEROXIDE SOLUTION 3% Interfering substance: PARAMETERS: Virus titre (Log TCID 50 ) virus control BSA 0.03% final in test concentration (Log TCID 50 ) H2O2 3% VIRUS STRAINS 0 min 60 min 0 min Poliovirus Type 1 ATCC VR-1562 Adenovirus Type 5 ATCC VR-5 Human herpesvirus (HSV-1) ATCC VR-733 Bovine viral diarrea virus (BVDV) ATCC VR-534 Porcine Parvovirus (PPV) ATCC VR-742 Hepatitis A virus (HAV) ATCC VR-1402 Virus titre Log TCID 50 Reduction

26 Page: 26 of 59 Cytotoxicity of HYDROGEN PEROXIDE SOLUTION 3% TEST SUBSTANCE HOST CELL LINE CPE HYDROGEN PEROXIDE SOLUTION 3% Hela Vero MDBK FRhK-4 ST NOT CYTOTOXIC NOT CYTOTOXIC NOT CYTOTOXIC NOT CYTOTOXIC NOT CYTOTOXIC Titration of active O2 TEST SUBSTANCE HYDROGEN PEROXIDE SOLUTION 3% CONTACT TIMES ACTIVE Cl 2 ACTIVE Cl 2 THEORETICAL (g) % VS THEORETICAL T min min min min hours hours hours hours DEVIATION No deviation has been recorded from study program. OBSERVATIONS On the basis of the results obtained in compliance with the assay validity criteria, the suppression of disinfectant activity results VALIDATED; the difference of the value of TCID 50 among the cellular cultures treated with the inactivated test substance and the ones exposed only with the viral inoculum was resulted <0.5 log. The test substance resulted NOT CYTOTOXIC at the concentrations tested against each cell line used, i.e. its contribution in terms of CPE was not visible in the test; so, this low level of cytotoxicity was enough to at least enable a titre reduction of 4 Log to verify the method. The specification for Active O 2 was resulted ± 10% from the title of nominal active O 2, so it was possible defined the test substance HYDROGEN PEROXIDE SOLUTION 3% stable during the contact time used in the test (16 hours).

27 Page: 27 of 59 Experimental Report 2009/717.A4 - Viral inactivation kinetics of HYDROGEN PEROXIDE SOLUTION 3% EXPERIMENTAL CONDITIONS 1. ASSAY SYSTEM Virus strains Adenovirus type 5, human ATCC VR-5 Poliovirus type 1 (chat strain) ATCC VR-1562 Human herpes virus (Herpes simplex Type1; HSV-1) ATCC VR- 733 Bovine viral diarrhea virus (BVDV) ATCC VR-534 Hepatitis A virus Strain HM175/18f [HM175 cytopathic clone B] (HAV) ATCC VR-1402 Porcine parvovirus NADL-2 (PPV) ATCC VR-742 Test virus suspension The viral suspensions were kept frozen at 70 C; before their use they were multiplied in the following appropriate cellular line: HOST CELLS INCUBATIONS VIRUS STRAINS LINE TIMES (Days) Adenovirus type 5, human Hela 7 Poliovirus type 1 (chat strain) Hela 7 Human herpes virus (Herpes simplex Type1; HSV-1) Vero 7 Bovine viral diarrhea virus (BVDV) MDBK 7 Hepatitis A virus Strain HM175/18f [HM175 cytopathic clone B] (HAV) FRhK-4 12 Porcine parvovirus NADL-2 (PPV) ST 7 Cellular debris were removed by means of double centrifugation at low speed, and the supernatant containing the virus was used for the test. 2. CELL CULTURES, MEDIA AND REAGENTS Cell cultures MDBK (NBL-1 Kidney) HeLa (cells of human uterine carcinoma) Vero FRhK-4 ST ATCC CCL-22 ATCC CCL-2 ATCC CCL-81 ATCC CRL-1688 ATCC CRL-1746 The cellular culture was kept frozen at < 70 C; before viral inoculum, it was showed itself as confluent monolayer. Culture Medium EMEM: Eagle s minimal essential medium BIOWHITTAKER - LONZA FBS (2%) Foetal Bovine Serum BIOWHITTAKER LONZA NEAA (1%) Non-Essential Amino Acid BIOWHITTAKER LONZA PEN-STREP (1%) antibiotics BIOWHITTAKER LONZA PBS Phosphate Buffer Saline BIOWHITTAKER - LONZA Catalase solution (0.025% final concentration) SIGMA

28 Page: 28 of 59 Reagents WFI Water for injection EUROSPITAL BSA Buffer bovine serum albumin (0.3% in distilled water) SIGMA 3. EQUIPMENT AND MATERIALS Incubator Centrifuge Deepfreeze CO 2 incubator Vortex Chronometer Micropipettes Microdishes 96 wells Inverted microscope phmeter Vessel containing water and ice Water bath ARBORE HERAEUS Angelantoni Scientifica PBI VELP ARBORE GILSON CAMBREX LEITZ BECKMAN ARBORE 4. EXPERIMENTAL DESIGN Test temperature The test was conducted at 20 C ±1 C Disinfectant concentration The test substance was prepared at concentrations 1.25 times higher than the concentrations requested by the test. The test substance was tested at the final concentration of 3% (diluted into sterile water). Contact times The following contact times were used: minutes, hours Water for injection (WFI) Interfering substance Clean conditions: The used interfering substance was prepared with a concentration 10 times higher than the final concentration of 0.03%. Bovine albumin 0.3 g Diluent q.s. to 100 ml 5. ASSAY EXECUTION Assay of viral activity (virus titration) Serial dilutions 1:10 were prepared with culture Medium, starting from virus stock solution of viral suspension. 0.1 ml were transferred in dishes with 96 wells containing the cellular confluent monolayer (>90%) without culture Medium. Each dilution of viral suspension was placed onto dishes sixfold. The 12 wells of the upper part and the 12 wells of the lower part of the dish will not receive the viral inoculum and were used as control of cellular line. After 1 hour of incubation at 37 C ±1 C 0.1ml of culture Medium were added to viral inoculum. After the appropriate incubation period, the cellular culture was observed with inverted microscope to detect any cytopatic effect (CPE) due to viral suspension. After this detection the infecting activity (TCID 50 evaluation) was calculated by means of Spearman Karber method.

29 Page: 29 of 59 Check of cellular sensitivity to virus To verify if the HYDROGEN PEROXIDE SOLUTION 3% inactivated with catalase modifies the cellular sensitivity to viral infection, this procedure was followed: 0.1 ml of the HYDROGEN PEROXIDE SOLUTION 3% inactivate with 0.025% catalase solution were put on the microdish containing the cellular confluent monolayer; the other microdish was treated with 0.1 ml of PBS. After 1 hour of incubation at 37 C ±1 C, the test substance and PBS were removed and the viral inoculum in the volume of 0.1ml were performed with culture Medium. After 1 hour of incubation at 37 C ±1 C 0.1 ml of culture Medium were added to the viral inoculum. After the appropriate incubation period, the cellular culture was observed with inverted microscope to detect any cytopatic effect (CPE) due to viral suspension. After this detection the infecting activity (TCID 50 ) was calculated by means of Spearman Karber method both in the cellular culture treated with the test substance and in that treated with PBS. Check of viral inactivation (on Poliovirus type 1) 2 ml of viral suspension were mixed to 8 ml of PBS and to 10 ml of formaldehyde at 0.7% final. Before and after a contact of 30, 60 minutes, 0.2 ml of this solution were mixed with 1.8 ml of culture Medium + 2% FBS in an iced bath; then serial dilutions 1:10 were performed with PBS + 2% FBS always in iced bath. The solutions were incubated at 37 C ±1 C at bain-marie for 5 minutes and were transferred (0.1 ml for each well in sixfold) onto microdishes, were incubated at 37 C ±1 C for 1 hour. Then 0.1 ml of culture Medium were added to the viral inoculum. After the appropriate incubation period, the cellular culture was observed with inverted microscope to detect any cytopatic effect (CPE) due to viral suspension. After this detection the infecting activity (TCID 50 ) was calculated by means of Spearman Karber method. Assay 1 ml of interfering substance was mixed with 1 ml of viral suspension, then vortexed. 8 ml of the assay sample were added to this solution and left in contact for the test time. After the contact time, 0.5 ml of the solution (after neutralization) were mixed with 4.5 ml of culture Medium in iced bath, then serial dilutions 1:10 with culture Medium were performed. 0.1 ml of this solution was put onto dishes in sixfold in the microdish containing the cellular confluent culture (>90%). The 12 wells of the upper part and the 12 wells of the lower part of the dish will not receive the viral inoculum and were used as control of cellular line. After 1 hour of incubation at 37 C ±1 C 0.1 ml of culture Medium were added to the viral inoculum. After the appropriate incubation period, the cellular culture was observed with inverted microscope to detect any cytopatic effect (CPE) due to viral suspension. In a second time the same test described above, was performed with water instead of the test substance, after 0 and 60 minutes (virus control). After this detection the infecting activity (TCID 50 ) was calculated by means of Spearman Karber method both in the cellular culture treated with the test substance and in that treated with PBS.

30 Page: 30 of CALCULATIONS AND EXPRESSION OF THE RESULTS Determination of TCID 50 The infecting activity was determined by means of Spearman Karber method that uses the following formula to calculate the value of TCID 50 : -Log TCID 50 = - (-x 0 ) - {[R/100]- 0.5} x Log dilution factor where: x 0 = Log of the lowest dilution with 100% of positive reaction (CPE) R = sum (%) of positive cultures Log TCID 50 values were rounded to two significant figures as shown in Annex D table D.1 of EN Rounding is automatically performed by excel datasheet. Evaluation of the virucidal activity The virucidal activity of the product test solution was performed for each exposure time. The Logreduction willbe performed by subtracting the logarithmic titre TCID 50 at any test point from the logarithmic titre TCID 50 of the virus control. ACCAPTANCE CRITERIA 1. A contact time is considered effective when at least 4 Log reduction in viral titre is obtained. 2. Check of cellular sensitivity to virus: the difference of the value of TCID 50 among the cellular cultures treated with the test substance and the ones not treated with the test substance (i.e. PBS only) must be < 1 log Check of viral inactivation (Poliovirus): the difference of the value of TCID 50 among the cells treated with inactivated viral suspensions and the ones treated with active viral suspensions must be, after 60 minutes of incubation between 2logs and 4.5logs and between 0.5logs and 2.5logs after 30 minutes of incubation for Poliovirus type 1.

31 Page: 31 of 59 RESULTS The results of Log reduction at the different contact times are shown in the table here below. TEST SUBSTANCE: HYDROGEN PEROXIDE SOLUTION CONCENTRATION: 3.0% VIRUS Log REDUCTIO N Bovine viral Human Poliovirus diarrea Porcine Hepatitis A Adenovirus herpesvirus type 1 virus Parvovirus virus (HAV) Type 5 (HSV-1) ATCC (BVDV) (PPV) ATCC ATCC ATCC VR-5 ATCC VR-1562 ATCC VR-742 VR-1402 VR-733 VR min min min h h h h h REDUCTION >4 Log AFTER TIMES 2 HOURS 2 HOURS 30 MIN 1 HOURS 8 HOURS 16 HOURS DEVIATION No deviation has been recorded from study program. OBSERVATIONS On the basis of the results obtained, in compliance with the assay validity criteria, the test substance HYDROGEN PEROXIDE SOLUTION 3.0% provided the following results: - causes a reduction > 4Log against Poliovirus type 1 ATCC VR-1562 after 2 hours of contact time, using a 0.03% final concentration of bovine albumin, in compliance with EN 14476:2005+A1: causes a reduction > 4Log against Adenovirus type 5 ATCC VR-5 after 2 hours of contact time, using a 0.03% final concentration of bovine albumin, in compliance with EN 14476:2005+A1: causes a reduction > 4Log against Human herpes virus (HSV-1) ATCC VR-733 after 30 minutes of contact time, using a 0.03% final concentration of bovine albumin, in compliance with EN 14476:2005+A1: causes a reduction > 4Log against Bovine viral diarrhea virus (BVDV) ATCC VR-534 after 60 minutes of contact time, using a 0.03% final concentration of bovine albumin, in compliance with EN 14476:2005+A1: causes a reduction > 4Log against Porcine Parvovirus (PPV) ATCC VR-742 after 8 hours of contact time, using a 0.03% final concentration of bovine albumin, in compliance with EN 14476:2005+A1: causes a reduction > 4Log against Hepatitis A virus (HAV) ATCC VR-1402 after 16 hours of contact time, using a 0.03% final concentration of bovine albumin, in compliance with EN 14476:2005+A1:2006.

32 Page: 32 of 59 Experimental Report 2009/717.A5 - Viral inactivation effectiveness with HYDROGEN PEROXIDE SOLUTION 3% (Sponsor step 2.1 of Sponsor VA-07 procedure) EXPERIMENTAL CONDITIONS 1. ASSAY SYSTEM Virus strains Adenovirus type 5, human ATCC VR-5 Poliovirus type 1 (chat strain) ATCC VR-1562 Human herpes virus (Herpes simplex Type1; HSV-1) ATCC VR- 733 Bovine viral diarrhea virus (BVDV) ATCC VR-534 Hepatitis A virus Strain HM175/18f [HM175 cytopathic clone B] (HAV) ATCC VR-1402 Porcine parvovirus NADL-2 (PPV) ATCC VR-742 Test virus suspension The viral suspensions were kept frozen at 70 C; before their use they were multiplied in the following appropriate cellular line: HOST CELLS INCUBATIONS VIRUS STRAINS LINE TIMES (Days) Adenovirus type 5, human Hela 7 Poliovirus type 1 (chat strain) Hela 7 Human herpes virus (Herpes simplex Type1; HSV-1) Vero 7 Bovine viral diarrhea virus (BVDV) MDBK 7 Hepatitis A virus Strain HM175/18f [HM175 cytopathic clone B] (HAV) FRhK-4 12 Porcine parvovirus NADL-2 (PPV) ST 7 Cellular debris were removed by means of double centrifugation at low speed, and the supernatant containing the virus was used for the test. 7. CELL CULTURES, MEDIA AND REAGENTS Cell cultures MDBK (NBL-1 Kidney) HeLa (cells of human uterine carcinoma) Vero FRhK-4 ST ATCC CCL-22 ATCC CCL-2 ATCC CCL-81 ATCC CRL-1688 ATCC CRL-1746 The cellular culture was kept frozen at < 70 C; before viral inoculum, it was showed itself as confluent monolayer. Culture Medium EMEM: Eagle s minimal essential medium BIOWHITTAKER - LONZA FBS (2%) Foetal Bovine Serum BIOWHITTAKER LONZA NEAA (1%) Non-Essential Amino Acid BIOWHITTAKER LONZA PEN-STREP (1%) antibiotics BIOWHITTAKER LONZA PBS Phosphate Buffer Saline BIOWHITTAKER - LONZA Catalase solution (0.025% final concentration) SIGMA

33 Page: 33 of 59 Reagents WFI Water for injection EUROSPITAL BSA Buffer bovine serum albumin (0.3% in distilled water) SIGMA 2. EQUIPMENT AND MATERIALS Incubator Centrifuge Deepfreeze CO 2 incubator Vortex Chronometer Micropipettes Microdishes 96 wells Inverted microscope phmeter Vessel containing water and ice Water bath Dessicator Vacuum pump ARBORE HERAEUS Angelantoni Scientifica PBI VELP ARBORE GILSON CAMBREX LEITZ BECKMAN ARBORE GHIARONI 3. EXPERIMENTAL DESIGN Test temperature The test was conducted at C (according to Sponsor procedure). Disinfectant concentration The test substance was tested at the final concentration of 3% (diluted into sterile water). Contact times The following contact time was used: 16 hours (according to Sponsor procedure). Interfering substance Clean conditions: The used interfering substance was prepared with a concentration 10 times higher than the final concentration of 0.03%. Bovine albumin 0.3 g Diluent q.s. to 100 ml Test Carriers The test carriers, provided by the Sponsor, consist of human cancellous bone tissue (defatted, cleaned, lyophilized) which are comparable with the status before cleaning with 3% H 2 O 2 ; they are blocks with the following dimensions:15 x 15 x 30 mm (equivalent volume 6.75 cm 3 ). Inoculum preparation The inoculum was preparated mixing each virus suspension with interfering substance in ratio 9:1. Carrier Inoculum Test carriers were inoculated with each inoculum preparation separately using the inoculum volume validated in the experimentation A1.

34 Page: 34 of ASSAY EXECUTION Viral activity (virus titration) Serial dilutions 1:10 were prepared with culture Medium, starting from virus stock solution of viral suspension. 0.1 ml were transferred in dishes with 96 wells containing the cellular confluent monolayer (>90%) without culture Medium. Each dilution of viral suspension was placed onto dishes sixfold. The 12 wells of the upper part and the 12 wells of the lower part of the dish will not receive the viral inoculum and were used as control of cellular line. After 1 hour of incubation at 37 C ±1 C 0.1ml of culture Medium were added to viral inoculum. After the appropriate incubation period, the cellular culture was observed with inverted microscope to detect any cytopatic effect (CPE) due to viral suspension. After this detection the infecting activity (TCID 50 evaluation) was calculated by means of Spearman Karber method. Assay For each contact time, 1 test carrier was inoculated with the 6 viral suspension test separately. The test carrier was immersed into a vessel containing ml (ratio 1:5 with carrier volume as indicated by the Sponsor procedure) of HYDROGEN PEROXIDE SOLUTION 3% protected from light and inside an orbital shalker (Dessicator with only shaker active). At the end of contact time a viral recovery was determined respectively on the HYDROGEN PEROXIDE SOLUTION 3% and on the carrier in order to determine residual virus. At the same time the test described above, was performed with water instead of the disinfectant product, after 0 and 16 hours or another validated contact time (virus control). Viral recovery in the immersion system At the and of each contact time a viral titration was performed on disinfectant test solution in order to determinate the residual virus title. At this purpose, serial dilutions 1:10 were prepare with culture Medium, starting from the HYDROGEN PEROXIDE SOLUTION 3% containing the inoculated carrier. 0.1 ml were transferred in dishes with 96 wells containing the cellular confluent monolayer (>90%) without culture Medium. Each dilution of viral suspension was placed onto dishes sixfold. The 12 wells of the upper part and the 12 wells of the lower part of the dish will not receive the viral inoculum and were used as control of cellular line. After 1 hour of incubation at 37 C ±1 C 0.1ml of culture Medium were added to viral inoculum. The test was performed in duplicate. After the appropriate incubation period, the cellular culture was observed with inverted microscope to detect any cytopatic effect (CPE) due to viral suspension. After this detection the infecting activity (TCID 50 evaluation) was calculated by means of Spearman Karber method. Viral recovery on carrier test As per Experimentation 2009/717.A2.

35 Page: 35 of CALCULATIONS AND EXPRESSION OF THE RESULTS Determination of TCID 50 The infecting activity was determined by means of Spearman Karber method that uses the following formula to calculate the value of TCID 50 : -Log TCID 50 = - (-x 0 ) - {[R/100]- 0.5} x Log dilution factor where: x 0 = Log of the lowest dilution with 100% of positive reaction (CPE) R = sum (%) of positive cultures Log TCID 50 values were rounded to two significant figures as shown in Annex D table D.1 of EN Rounding is automatically performed by excel datasheet. Evaluation of the virucidal activity The virucidal activity of the product test solution was performed for each exposure time. The logreduction willbe performed by subtracting the logarithmic titre TCID50 at any test point from the logarithmic titre TCID 50 of the virus control. 6. EFFICACY CRITERIA At least 2 Log reduction in viral titre. RESULTS The results of Log reduction on the test carriers and on the residual immersion system are shown following. TEST SUBSTANCE: HYDROGEN PEROXIDE SOLUTION 3.0% INTERFERING SUBSTANCE: BSA 0.03% final in test concentration CONTACT TIME: VIRUS STRAINS CARRIER 1 CARRIER 2 Poliovirus Type 1 ATCC VR-1562 Adenovirus Type 5 ATCC VR-5 Human herpes virus (HSV-1) ATCC VR-733 Bovine viral diarrhea virus (BVDV) ATCC VR-534 Porcine Parvovirus (PPV) ATCC VR-742 Hepatitis A virus (HAV) ATCC VR HOURS RESIDUAL IMMERSION SYSTEM 1 RESIDUAL IMMERSION SYSTEM 2 >2.50 >2.50 >3.33 >3.33 >4.50 >4.50 >4.83 >4.83 >3.67 >3.67 >4.50 >4.50 >3.50 >3.50 >4.00 >

36 Page: 36 of 59 DEVIATION No deviation has been recorded from study program. OBSERVATIONS On the basis of the results obtained in compliance with the assay validity criteria, the test substance HYDROGEN PEROXIDE SOLUTION 3.0% provided the following results: - causes a reduction > 2Log from cuboids and causes a reduction > 3Log from supernatant against Poliovirus type 1 ATCC VR-1562 after 16 hours of contact time using a 0.03% final concentration of bovine albumin in compliance with the Sponsor procedure requirements; no test virus was recovered after treatment. - causes a reduction > 4Log against Adenovirus type 5 ATCC VR-5 after 16 hours of contact time from both supernatant and cuboids using a 0.03% final concentration of bovine albumin in compliance with the Sponsor procedure requirements; no test virus was recovered after treatment. - causes a reduction > 3Log from cuboids and causes a reduction > 4Log from supernatant against Human herpes virus (HSV-1) ATCC VR-733 after 16 hours of contact time using a 0.03% final concentration of bovine albumin in compliance with the Sponsor procedure requirements; no test virus was recovered after treatment. - causes a reduction > 3Log against Bovine viral diarrhea virus (BVDV) ATCC VR-534 after 16 hours of contact time from both supernatant and cuboid using a 0.03% final concentration of bovine albumin in compliance with the Sponsor procedure requirements; no test virus was recovered after treatment. - causes a reduction > 2Log from cuboids and causes a reduction > 3Log from supernatant against Porcine Parvovirus (PPV) ATCC VR-742 after 16 hours of contact time using a 0.03% final concentration of bovine albumin in compliance with the Sponsor procedure requirements. - causes a reduction > 2Log from cuboids and causes a reduction > 3Log from supernatant against Hepatitis A virus (HAV) ATCC VR-1402 after 16 hours of contact time from both supernatant and cuboid using a 0.03% final concentration of bovine albumin in compliance with the Sponsor procedure requirements; no test virus was recovered after treatment.

37 Page: 37 of 59 Experimental Report 2009/717.A6 - Gamma rays treatment viral inactivation effectiveness (step 2.2 as per Sponsor VA-07 procedure) EXPERIMENTAL CONDITIONS 1. ASSAY SYSTEM Virus strains Adenovirus type 5, human ATCC VR-5 Poliovirus type 1 (chat strain) ATCC VR-1562 Human herpes virus (Herpes simplex Type1; HSV-1) ATCC VR- 733 Bovine viral diarrhea virus (BVDV) ATCC VR-534 Hepatitis A virus Strain HM175/18f [HM175 cytopathic clone B] (HAV) ATCC VR-1402 Porcine parvovirus NADL-2 (PPV) ATCC VR-742 Test virus suspension The viral suspensions were kept frozen at 70 C; before their use they were multiplied in the following appropriate cellular line: HOST CELLS INCUBATIONS VIRUS STRAINS LINE TIMES (Days) Adenovirus type 5, human Hela 7 Poliovirus type 1 (chat strain) Hela 7 Human herpes virus (Herpes simplex Type1; HSV-1) Vero 7 Bovine viral diarrhea virus (BVDV) MDBK 7 Hepatitis A virus Strain HM175/18f [HM175 cytopathic clone B] (HAV) FRhK-4 12 Porcine parvovirus NADL-2 (PPV) ST 7 Cellular debris were removed by means of double centrifugation at low speed, and the supernatant containing the virus was used for the test. 8. CELL CULTURES, MEDIA AND REAGENTS Cell cultures MDBK (NBL-1 Kidney) HeLa (cells of human uterine carcinoma) Vero FRhK-4 ST ATCC CCL-22 ATCC CCL-2 ATCC CCL-81 ATCC CRL-1688 ATCC CRL-1746 The cellular culture was kept frozen at < 70 C; before viral inoculum, it was showed itself as confluent monolayer. Culture Medium EMEM: Eagle s minimal essential medium BIOWHITTAKER - LONZA FBS (2%) Foetal Bovine Serum BIOWHITTAKER LONZA NEAA (1%) Non-Essential Amino Acid BIOWHITTAKER LONZA PEN-STREP (1%) antibiotics BIOWHITTAKER LONZA PBS Phosphate Buffer Saline BIOWHITTAKER - LONZA

38 Page: 38 of 59 Reagents WFI Water for injection EUROSPITAL BSA Buffer bovine serum albumin (3% in distilled water) SIGMA 2. EQUIPMENT AND MATERIALS Incubator Centrifuge Deepfreeze CO 2 incubator Vortex Chronometer Micropipettes Microdishes 96 wells Inverted microscope phmeter Vessel containing water and ice Water bath Dessicator Vacuum pump ARBORE HERAEUS Angelantoni Scientifica PBI VELP ARBORE GILSON CAMBREX LEITZ BECKMAN ARBORE GHIARONI 3. EXPERIMENTAL DESIGN Test temperature The test was conducted at C (according to Sponsor VA-07 procedure). Interfering substance Clean conditions: The used interfering substance was prepared with a concentration 10 times higher than the final concentration of 0.03%. Bovine albumin 0.3 g Diluent q.s. to 100 ml Gamma irradiation dose 25.0 kgy according to Sponsor procedure VA-07. Test Carriers The test carriers, provided by the Sponsor, consist of human cancellous bone tissue (defatted, cleaned, lyophilized) which are comparable with the process status before cleaning with 3% H 2 O 2; they were blocks with the following size:15 x 15 x 30 mm (equivalent volume 6.75 cm 3 ). Inoculum preparation The inoculum was preparated mixing each virus suspension with interfering substance in ratio 9:1. Carrier Inoculum Test carriers were inoculated with each inoculum preparation separately using the inoculum volume validated in the experimentation A1.

39 Page: 39 of ASSAY EXECUTION Viral activity (virus titration) Serial dilutions 1:10 were prepared with culture Medium, starting from virus stock solution of viral suspension. 0.1 ml were transferred in dishes with 96 wells containing the cellular confluent monolayer (>90%) without culture Medium. Each dilution of viral suspension was placed onto dishes sixfold. The 12 wells of the upper part and the 12 wells of the lower part of the dish did not receive the viral inoculum and were used as control of cellular line. After 1 hour of incubation at 37 C ±1 C 0.1ml of culture Medium were added to viral inoculum. After the appropriate incubation period, the cellular culture was observed with inverted microscope to detect any cytopatic effect (CPE) due to viral suspension. After this detection the infecting activity (TCID 50 evaluation) was calculated by means of Spearman Karber method. Assay After inoculum each carrier was sent to gamma irradiation treatment according to Sponsor requirements. The test was performed in Gammatom S.p.A. irradiation facility located in Via XXIV Maggio, 14 Guanzate (CO) Italy. At the end of gamma rays treatment the carrier was sent to Biolab S.p.A in order to determinate a residual virus. At the same time the test described above, was performed without gamma ray treatment (virus control). Viral recovery on carrier test As per Experimentation 2009/717.A2. 5. CALCULATIONS AND EXPRESSION OF THE RESULTS Determination of TCID 50 The infecting activity was determined by means of Spearman Karber method that uses the following formula to calculate the value of TCID 50 : -Log 10 TCID 50 = - (-x 0 ) - {[R/100]- 0.5} x log 10 dilution factor where: x 0 = log 10 of the lowest dilution with 100% of positive reaction (CPE) R = sum (%) of positive cultures Log TCID 50 values were rounded to two significant ciphers as shown in Annex D table D.1 of EN Rounding is automatically performed by excel datasheet. Evaluation of the virucidal activity The virucidal activity of the product test solution was performed for each exposure time. The Log reduction was performed by subtracting the logarithmic titre TCID 50 at any test point from the logarithmic titre TCID 50 of the virus control. EFFICACY CRITERIA At least 2 Log reduction in viral titre.

40 Page: 40 of 59 RESULTS The results of Log reduction on the test carriers after Gamma rays treatment are shown following. TREATMENT: INTERFERING SUBSTANCE: Gamma Ray 25 kgy BSA 0.03% final concentration VIRUS CARRIER 1 CARRIER 2 Poliovirus Type 1 ATCC VR-1562 Adenovirus Type 5 ATCC VR-5 Human herpes virus (HSV-1) ATCC VR-733 Bovine viral diarrhea virus (BVDV) ATCC VR-534 Porcine Parvovirus (PPV) ATCC VR-742 Hepatitis A virus (HAV) ATCC VR >2.33 >2.33 >2.67 > DEVIATION No deviation has been recorded from study program. OBSERVATIONS On the basis of the results obtained, in compliance with the assay validity criteria, the GAMMA RAYS treatment provided the following results: - causes a reduction of about 1Log against Poliovirus type 1 ATCC VR-1562 using a 0.03% final concentration of bovine albumin in compliance with the Sponsor procedure requirements. - causes a reduction of about 2Log against Adenovirus type 5 ATCC VR-5 using a 0.03% final concentration of bovine albumin in compliance with the Sponsor procedure requirements. - causes a reduction > 2.5Log against Human herpes virus (HSV-1) ATCC VR-733 using a 0.03% final concentration of bovine albumin in compliance with the Sponsor procedure requirements; no test virus was recovered after treatment. - causes a reduction > 2Log against Bovine viral diarrhea virus (BVDV) ATCC VR-534 using a 0.03% final concentration of bovine albumin in compliance with the Sponsor procedure requirements; no test virus was recovered after treatment. - causes a reduction <1Log against Porcine Parvovirus (PPV) ATCC VR-742 using a 0.03% final concentration of bovine albumin in compliance with the Sponsor procedure requirements. - causes a reduction <1Log against Hepatitis A virus (HAV) ATCC VR-1402 using a 0.03% final concentration of bovine albumin in compliance with the Sponsor procedure requirements.

41 Page: 41 of 59 Experimental Report 2009/717.A7 - Method set up in order to validate a recovery method (96% EtOH) EXPERIMENTAL CONDITIONS 1. ASSAY SYSTEM Virus strains Adenovirus type 5, human ATCC VR-5 Poliovirus type 1 (chat strain) ATCC VR-1562 Human herpes virus (Herpes simplex Type1; HSV-1) ATCC VR- 733 Bovine viral diarrhea virus (BVDV) ATCC VR-534 Hepatitis A virus Strain HM175/18f [HM175 cytopathic clone B] (HAV) ATCC VR-1402 Porcine parvovirus NADL-2 (PPV) ATCC VR-742 Test virus suspension The viral suspensions were kept frozen at 70 C; before their use they were multiplied in the following appropriate cellular line: HOST CELLS INCUBATIONS VIRUS STRAINS LINE TIMES (Days) Adenovirus type 5, human Hela 7 Poliovirus type 1 (chat strain) Hela 7 Human herpes virus (Herpes simplex Type1; HSV-1) Vero 7 Bovine viral diarrhea virus (BVDV) MDBK 7 Hepatitis A virus Strain HM175/18f [HM175 cytopathic clone B] (HAV) FRhK-4 12 Porcine parvovirus NADL-2 (PPV) ST 7 Cellular debris were removed by means of double centrifugation at low speed, and the supernatant containing the virus was used for the test. 2. CELL CULTURES, MEDIA AND REAGENTS Cell cultures MDBK (NBL-1 Kidney) HeLa (cells of human uterine carcinoma) Vero FRhK-4 ST ATCC CCL-22 ATCC CCL-2 ATCC CCL-81 ATCC CRL-1688 ATCC CRL-1746 The cellular culture was kept frozen at < 70 C; before viral inoculum, it was showed itself as confluent monolayer. Culture Medium and Reagents EMEM: Eagle s minimal essential medium BIOWHITTAKER - LONZA FBS (2%) Foetal Bovine Serum BIOWHITTAKER LONZA NEAA (1%) Non-Essential Amino Acid BIOWHITTAKER LONZA PEN-STREP (1%) antibiotics BIOWHITTAKER LONZA PBS Phosphate Buffer Saline BIOWHITTAKER - LONZA WFI Water for injection EUROSPITAL

42 Page: 42 of EQUIPMENT AND MATERIALS Incubator Centrifuge Centrifuge MicroSpin Deepfreeze CO 2 incubator Vortex Chronometer Micropipettes Microdishes 96 wells Inverted microscope phmeter Water bath Columns MicroSpin TM S400 HR Dessicator Vacuum pump ARBORE THERMO EPPENDORF Angelantoni Scientifica PBI VELP ARBORE GILSON CAMBREX LEITZ BECKMAN ARBORE GE HEALTHCARE GHIARONI 4. EXPERIMENTAL DESIGN Test temperature The test was conducted at 20 C ±1 C Test carriers The test carriers provided by the Sponsor consist of human bone treated with a routine cleaning process including ether step, which are comparable with the status before cleaning with 96% EtOH; they are blocks with the following dimensions:15 x 15 x 30 mm (equivalent volume 6.75 cm 3 ). Inoculum preparation The inoculum was preparated mixing each virus suspension with water in ratio 9:1. Carrier Inoculum Test carriers were inoculated with each inoculum preparation separately using the inoculum volume validated in the experimentation A1. Contact times The following exposure times to viral inoculum were used: 1 3 hours (immersion in sterile water) 5. ASSAY EXECUTION Assay of viral activity (virus titration) Two virus titration were performed, one for the virus suspension as such and one on the virus suspension after filtration with S400 HR columns MicroSpin. Of each one, serial dilutions 1:10 were prepared with culture Medium, starting from virus stock solution of viral suspension. 0.1 ml were transferred in dishes with 96 wells containing the cellular confluent monolayer (>90%) without culture Medium. Each dilution of viral suspension was placed onto dishes sixfold. The 12 wells of the upper part and the 12 wells of the lower part of the dish will not receive the viral inoculum and were used as control of cellular line. After 1 hour of incubation at 37 C ±1 C 0.1ml of culture Medium were added to viral inoculum. After the appropriate incubation period, the cellular culture was observed with inverted microscope to detect any cytopatic effect (CPE) due to viral suspension. After this detection the infecting activity (TCID 50 evaluation) was calculated by means of Spearman Karber method.

43 Page: 43 of 59 Assay control For each contact time, 2 carriers test were inoculated with the 6 viral suspension test separately. The carrier test was immersed into a ml (ratio 1:5 with carrier volume as indicated by the Sponsor procedure VA-07) of sterile water. At the end of contact time a viral recovery was determined respectively on the water and on the carrier in order to determine the best immersion time which was used in the efficacy test by means of disinfection with ETHANOL 96%. Validation of viral recovery in the immersion system (water) At the and of each contact time a viral titration was performed in order to validate the best immersion time which was used in the efficacy test (by means of disinfection with ETHANOL 96%). At this purpose, serial dilutions 1:10 were prepare with culture Medium, starting from the residual water containing the inoculated carrier after filtration with S400 HR columns MicroSpin. 0.1 ml were transferred in dishes with 96 wells containing the cellular confluent monolayer (>90%) without culture Medium. Each dilution of viral suspension was placed onto dishes sixfold. The 12 wells of the upper part and the 12 wells of the lower part of the dish will not receive the viral inoculum and was used as control of cellular line. After 1 hour of incubation at 37 C ±1 C 0.1ml of culture Medium were added to viral inoculum. The test was performed in duplicate. After the appropriate incubation period, the cellular culture was observed with inverted microscope to detect any cytopatic effect (CPE) due to viral suspension. After this detection the infecting activity (TCID 50 evaluation) was calculated by means of Spearman Karber method. Validation of viral recovery on carrier test At the end of contact time, a centrifugation at G during 5 minutes was performed on each carrier for three times consecutively, in order to recover the residual adsorbed virus. Then a titration was performed by means of serial decimal dilution with culture Medium, starting from the centrifuged carrier after filtration with S400 HR columns MicroSpin. 0.1 ml were transferred in dishes with 96 wells containing the cellular confluent monolayer (>90%) without culture Medium. Each dilution of viral suspension was placed onto dishes sixfold. The 12 wells of the upper part and the 12 wells of the lower part of the dish will not receive the viral inoculum and were used as control of cellular line. After 1 hour of incubation at 37 C ±1 C 0.1ml of culture Medium were added to viral inoculum. The test was performed twice. After the appropriate incubation period, the cellular culture was observed with inverted microscope to detect any cytopatic effect (CPE) due to viral suspension. After this detection the infecting activity (TCID 50 evaluation) was calculated by means of Spearman Karber method. 6. CALCULATIONS AND EXPRESSION OF THE RESULTS Determination of TCID 50 The infecting activity was determined by means of Spearman Karber method that uses the following formula to calculate the value of TCID 50 : -Log TCID 50 = - (-x 0 ) - {[R/100]- 0.5} x Log dilution factor where: x 0 = Log of the lowest dilution with 100% of positive reaction (CPE) R = sum (%) of positive cultures Log TCID 50 values were rounded to two significant ciphers as shown in Annex D table D.1 of EN Rounding is automatically performed by excel datasheet.

44 Page: 44 of 59 EFFICACY CRITERIA The minimum titre of the virus suspensions in the virus control shall be sufficiently high to enable a titre reduction of at least 2 Logs to verify the method. RESULTS PRODUCT: Ethanol 96% vol CONCENTRATION: 80.00% VIRUS Log TCID 50 Contact time Poliovirus Type 1 ATCC VR-1562 Adenovirus Type 5 ATCC VR - 5 Human herpes virus (HSV- 1) ATCC VR-733 Bovine viral diarrhea virus (BVDV) ATCC VR-534 Porcine Parvovirus (PPV) ATCC VR-742 Hepatitis A virus (HAV) ATCC VR-1402 CARRIER h h RESIDUAL WATER h h CARRIER h h RESIDUAL WATER h h DEVIATION No deviation has been recorded from study program. OBSEVATIONS On the basis of the results obtained, in compliance with the assay validity criteria, the recovery method results VALIDATED; each residual virus titre was expressed as Log TCID 50 and was sufficiently high to enable a titre reduction of at least 2 Logs to verify the method.

45 Page: 45 of 59 Experimental Report 2009/717.A8 - Check of suppression of ETHANOL 96% activity and cytotoxicity The Ethanol 96% neutralization method was validated in order to define the procedure to use in the experimentation A9 and A10. EXPERIMENTAL CONDITIONS 1. ASSAY SYSTEM Virus strains Adenovirus type 5, human ATCC VR-5 Poliovirus type 1 (chat strain) ATCC VR-1562 Human herpes virus (Herpes simplex Type1; HSV-1) ATCC VR- 733 Bovine viral diarrhea virus (BVDV) ATCC VR-534 Hepatitis A virus Strain HM175/18f [HM175 cytopathic clone B] (HAV) ATCC VR-1402 Porcine parvovirus NADL-2 (PPV) ATCC VR-742 Test virus suspension The viral suspensions were kept frozen at 70 C; before their use they were multiplied in the following appropriate cellular line: HOST CELLS INCUBATIONS VIRUS STRAINS LINE TIMES (Days) Adenovirus type 5, human Hela 7 Poliovirus type 1 (chat strain) Hela 7 Human herpes virus (Herpes simplex Type1; HSV-1) Vero 7 Bovine viral diarrhea virus (BVDV) MDBK 7 Hepatitis A virus Strain HM175/18f [HM175 cytopathic clone B] (HAV) FRhK-4 12 Porcine parvovirus NADL-2 (PPV) ST 7 Cellular debris were removed by means of double centrifugation at low speed, and the supernatant containing the virus was used for the test. 9. CELL CULTURES, MEDIA AND REAGENTS Cell cultures MDBK (NBL-1 Kidney) HeLa (cells of human uterine carcinoma) Vero FRhK-4 ST ATCC CCL-22 ATCC CCL-2 ATCC CCL-81 ATCC CRL-1688 ATCC CRL-1746 The cellular culture was kept frozen at < 70 C; before viral inoculum, it was showed itself as confluent monolayer. Reagents WFI Water for injection EUROSPITAL BSA Buffer bovine serum albumin + sheep erythrocytes (3% in distilled water) SIGMA

46 Page: 46 of 59 Culture Medium EMEM: Eagle s minimal essential medium BIOWHITTAKER - LONZA FBS (2%) Foetal Bovine Serum BIOWHITTAKER LONZA NEAA (1%) Non-Essential Amino Acid BIOWHITTAKER LONZA PEN-STREP (1%) antibiotics BIOWHITTAKER LONZA PBS Phosphate Buffer Saline BIOWHITTAKER - LONZA 2. EQUIPMENT AND MATERIALS Incubator Centrifuge Centrifuge MicroSpin Deepfreeze CO 2 incubator Vortex Chronometer Micropipettes Microdishes 96 wells Inverted microscope phmeter Water bath Columns MicroSpin TM S400 HR ARBORE THERMO EPPENDORF Angelantoni Scientifica PBI VELP ARBORE GILSON CAMBREX LEITZ BECKMAN ARBORE GE HEALTHCARE 3. EXPERIMENTAL DESIGN Test temperature The test was conducted at 20 C ±1 C Interfering substance Dirty conditions: The used interfering substance was prepared with a concentration 10 times higher than the final concentration of 0.3%: bovine albumin 3 g diluent q.s. to 100 ml sheep erythrocytes 3 ml 8 ml of defibrinated sheep blood were centrifuged at 800 rpm; the supernatant was eliminated and the erythrocytes were suspended in diluent ; the procedure was repeated till the supernatant was clarified. 3 ml of erythrocytes were suspended in 97 ml of bovine albumin solution. Control (without sheep erythrocytes) A bovine albumin solution at a concentration 10 times greater than the final test concentration (0.3%) was used: Bovine albumin 3 g Diluent q. s. to 100 ml Concentration The test substance was used neat therefore the final in test concentration was 80% (maximum concentration testable), corresponding to 76.8% of active matter.

47 Page: 47 of ASSAY EXECUTION Assay of viral activity (virus titration) Two virus titration were performed, one for the virus suspension as such and one on the virus suspension after filtration with S400 HR columns MicroSpin. Of each one, serial dilutions 1:10 were prepared with culture Medium, starting from virus stock solution of viral suspension, as per the following scheme: 0.5 ml of viral suspension ml culture Medium. 0.1 ml were transferred in dishes with 96 wells containing the cellular confluent monolayer (>90%) without culture Medium. Each dilution of viral suspension was placed onto dishes sixfold. The 12 wells of the upper part and the 12 wells of the lower part of the dish did not receive the viral inoculum and were used as control of cellular line. After 1 hour of incubation at 37 C ±1 C 0.1ml of culture Medium were added to viral inoculum. After the appropriate period of incubation, the cellular culture was observed with inverted microscope to detect any cytopatic effect (CPE) due to viral suspension. After this detection the infecting activity (TCID 50 evaluation) was calculated by means of Spearman Karber method. Check of suppression of disinfectant activity 1 ml of water was added to 1 ml of viral suspension and to 8 ml of the 96% EtOH. At time zero, the afore described solution was filtrated with S400 HR columns MicroSpin. Then this solution was left in iced bath for 30 minutes ±10 seconds. After this period serial dilutions 1:10 with culture Medium were performed. The solutions were incubated at 37 C ±1 C in water-bath for 5 minutes. 0.1 ml of the solutions were transferred six fold onto microdishes, incubated at 37 C ±1 C for 1 hour, then 0.1 ml of culture Medium were added to the viral inoculum. After the appropriate period of incubation, the cellular culture was observed with inverted microscope to detect any cytopatic effect (CPE) due to viral suspension. After this detection the infecting activity (TCID 50 ) was calculated by means of Spearman Karber method. Check of cytotoxicity of the test substance In order to evaluate the level of cytotoxicity of the test substance, two cytotoxicity tests were conducted: one for the test substance to the concentration of the test itself, and another on the test substance at the test concentration after filtration with S400 HR columns MicroSpin. This test was conducted by mixing 8 ml of the 96% EtOH with 2 ml of sterile water. Then serial dilutions 1:10 were performed with culture Medium. 0.1 ml of each dilution of the test substance were put on dishes sixfold. The 12 wells of the upper part and the 12 wells of the lower part of the dish did not receive the viral inoculum and were used as control of cellular line. After 1 hour of incubation at 37 C ±1 C 0.1ml of culture Medium were added to each well. Immediately after the addition of the test substance and daily until the end of incubation periods, the cellular culture was observed with inverted microscope to detect any cytopatic effect (CPE) due to test substance.

48 Page: 48 of CALCULATIONS AND EXPRESSION OF THE RESULTS Determination of TCID 50 The infecting activity was determined by means of Spearman Karber method that uses the following formula to calculate the value of TCID 50 : -Log 10 TCID 50 = - (-x 0 ) - {[R/100]- 0.5} x log 10 dilution factor where: x 0 = log 10 of the lowest dilution with 100% of positive reaction (CPE) R = sum (%) of positive cultures Log TCID 50 values were rounded to two significant ciphers as shown in Annex D table D.1 of EN Rounding is automatically performed by excel datasheet. EFFICACY CRITERIA 1. Check of suppression of disinfectant activity: the difference of the value of TCID 50 among the cellular cultures treated with the inactivated test substance and the ones treated only with the viral inoculum must be 0.5 Log. 2. The test substance shall not be cytotoxic at the concentrations tested, i.e. its contribution in terms of CPE shouldn t be visible in the test; in any case, the cytotoxicity shall be low enough to at least enable a titre reduction of 4 Log to verify the method. RESULTS Check of suppression of disinfectant activity: ETHANOL 96% vol INTERFERING SUBSTANCE: BSA + sheep erythrocytes 0.3% final concentration PARAMETERS: Virus titre (Log Virus titre Log Virus titre (Log TCID 50 ) virus Reduction (Log TCID control 50 ) EtOh 96% TCID 50 ) VIRUS STRAINS 0 min 60 min 0 min Poliovirus Type 1 ATCC VR-1562 Adenovirus Type 5 ATCC VR-5 Human herpes virus (HSV-1) ATCC VR-733 Bovine viral diarrhea virus (BVDV) ATCC VR-534 Porcine Parvovirus (PPV) ATCC VR-742 Hepatitis A virus (HAV) ATCC VR

49 Page: 49 of 59 Cytotoxicity of ETHANOL 96% vol TEST SUBSTANCE HOST CELLS LINE CPE (after filtration) ETHANOL 96% vol Hela Vero MDBK FRhK-4 ST NOT CYTOTOXIC NOT CYTOTOXIC NOT CYTOTOXIC NOT CYTOTOXIC NOT CYTOTOXIC DEVIATION No deviation has been recorded from study program. OBSERVATIONS On the basis of the results obtained, in compliance with the assay validity criteria, the suppression of disinfectant activity results VALIDATED; the difference of the value of TCID 50 among the cellular cultures treated with the inactivated test substance and the ones treated only with the viral inoculum was resulted <0.5 Log. The test substance ETHANOL 96% was resulted NOT CYTOTOXIC at the concentrations tested against each cell lines used after filtration with S400 HR columns MicroSpin, i.e. its contribution in terms of CPE was corresponded at a low level of cytotoxicity that was enough to at least enable a titre reduction of 4 Log to verify the method.

50 Page: 50 of 59 Experimental Report 2009/717.A9 - Viral inactivation kinetics of ETHANOL 96% EXPERIMENTAL CONDITIONS 1. ASSAY SYSTEM Virus strains Adenovirus type 5, human ATCC VR-5 Poliovirus type 1 (chat strain) ATCC VR-1562 Human herpes virus (Herpes simplex Type1; HSV-1) ATCC VR- 733 Bovine viral diarrhea virus (BVDV) ATCC VR-534 Hepatitis A virus Strain HM175/18f [HM175 cytopathic clone B] (HAV) ATCC VR-1402 Porcine parvovirus NADL-2 (PPV) ATCC VR-742 Test virus suspension The viral suspensions were kept frozen at 70 C; before their use they were multiplied in the following appropriate cellular line: HOST CELLS INCUBATIONS VIRUS STRAINS LINE TIMES (Days) Adenovirus type 5, human Hela 7 Poliovirus type 1 (chat strain) Hela 7 Human herpes virus (Herpes simplex Type1; HSV-1) Vero 7 Bovine viral diarrhea virus (BVDV) MDBK 7 Hepatitis A virus Strain HM175/18f [HM175 cytopathic clone B] (HAV) FRhK-4 12 Porcine parvovirus NADL-2 (PPV) ST 7 Cellular debris were removed by means of double centrifugation at low speed, and the supernatant containing the virus was used for the test. 2. CELL CULTURES, MEDIA AND REAGENTS Cell cultures MDBK (NBL-1 Kidney) HeLa (cells of human uterine carcinoma) Vero FRhK-4 ST ATCC CCL-22 ATCC CCL-2 ATCC CCL-81 ATCC CRL-1688 ATCC CRL-1746 The cellular culture was kept frozen at < 70 C; before viral inoculum, it was showed itself as confluent monolayer. Culture Medium EMEM: Eagle s minimal essential medium BIOWHITTAKER - LONZA FBS (2%) Foetal Bovine Serum BIOWHITTAKER LONZA NEAA (1%) Non-Essential Amino Acid BIOWHITTAKER LONZA PEN-STREP (1%) antibiotics BIOWHITTAKER LONZA PBS Phosphate Buffer Saline BIOWHITTAKER - LONZA

51 Page: 51 of 59 Reagents WFI Water for injection EUROSPITAL BSA Buffer bovine serum albumin + sheep erythrocytes (3% in distilled water) SIGMA 3. EQUIPMENT AND MATERIALS Incubator Centrifuge Centrifuge MicroSpin Deepfreeze CO 2 incubator Vortex Chronometer Micropipettes Microdishes 96 wells Inverted microscope phmeter Water bath Columns MicroSpin TM S400 HR ARBORE THERMO EPPENDORF Angelantoni Scientifica PBI VELP ARBORE GILSON LONZA LEITZ BECKMAN ARBORE GE HEALTHCARE 4. EXPERIMENTAL DESIGN Test temperature The test was conducted at 20 C ±1 C Disinfectant concentration The test substance was used neat therefore the final in test concentration was 80% (maximum concentration testable), corresponding to 76.8% of active matter. Contact times The following contact time was used: minutes, 1 3 hours Interfering substance Dirty conditions: The used interfering substance was prepared with a concentration 10 times higher than the final concentration of 0.3%: bovine albumin 3 g diluent q.s. to 100 ml sheep erythrocytes 3 ml 8 ml of defibrinated sheep blood were centrifuged at 800 rpm; the supernatant was eliminated and the erythrocytes were suspended in diluent ; the procedure was repeated till the supernatant was clarified. 3 ml of erythrocytes were suspended in 97 ml of bovin albumin solution. Control (without sheep erythrocytes) A bovine albumin solution at a concentration 10 times greater than the final test concentration (0.3%) was used: Bovine albumin 3 g Diluent q. s. to 100 ml

52 Page: 52 of ASSAY EXECUTION Assay of viral activity (virus titration) Two virus titration were performed, one for the virus suspension as such and one on the virus suspension after filtration with S400 HR columns MicroSpin. Of each one, serial dilutions 1:10 were prepared with culture Medium, starting from virus stock solution of viral suspension. 0.1 ml were transferred in dishes with 96 wells containing the cellular confluent monolayer (>90%) without culture Medium. Each dilution of viral suspension was placed onto dishes sixfold. The 12 wells of the upper part and the 12 wells of the lower part of the dish will not receive the viral inoculum and were used as control of cellular line. After 1 hour of incubation at 37 C ±1 C 0.1ml of culture Medium were added to viral inoculum. After the appropriate period of incubation, the cellular culture was observed with inverted microscope to detect any cytopatic effect (CPE) due to viral suspension. After this detection the infecting activity (TCID 50 evaluation) was calculated by means of Spearman Karber method. Check of cellular sensitivity to virus To verify if the 96% EtOH modifies the cellular sensitivity to viral infection, this procedure was followed: 0.1 ml of the 96% EtOH after filtration with S400 HR columns MicroSpin were put on dishes in the wells of the microdishes containing the cellular confluent monolayer; the other wells were treated with 0.1 ml of PBS. After 1 hour of incubation at 37 C ±1 C, the test substance and PBS were removed and the viral inoculum in the volume of 0.1ml were performed with the following scheme: 0.5 ml of viral suspension ml culture Medium. After 1 hour of incubation at 37 C ±1 C 0.1 ml of culture Medium were added to the viral inoculum. After the appropriate period of incubation, the cellular culture was observed with inverted microscope to detect any cytopatic effect (CPE) due to viral suspension. After this detection the infecting activity (TCID 50 ) was calculated by means of Spearman Karber method both in the cellular culture treated with the test substance and in that treated with PBS. Check of viral inactivation (on Poliovirus) 2 ml of viral suspension were mixed to 8 ml of PBS and to 10 ml of formaldehyde at 0.7% final. Before and after a contact of 30, 60 minutes, 0.2 ml of this solution were mixed with 1.8 ml of culture Medium + 2% FBS in an iced bath; then serial dilutions 1:10 were performed with PBS + 2% FBS always in iced bath. The solutions were incubated at 37 C ±1 C at bain-marie for 5 minutes and were transferred (0.1 ml for each well in sixfold) onto microdishes, were incubated at 37 C ±1 C for 1 hour. Then 0.1 ml of culture Medium were added to the viral inoculum. After the appropriate period of incubation, the cellular culture was observed with inverted microscope to detect any cytopatic effect (CPE) due to viral suspension. After this detection the infecting activity (TCID 50 ) was calculated by means of Spearman Karber method.

53 Page: 53 of 59 Assay 1 ml of water was mixed with 1 ml of viral suspension, then vortexed. 8 ml of the assay sample were added to this solution and left in contact for the test time. After the contact time, the solution was mixed and filtrated with S400 HR columns MicroSpin TM, then serial dilutions 1:10 with culture Medium were performed and 0.1 ml of this solution were put onto dishes in sixfold in the microdish containing the cellular confluent culture (>90%). The 12 wells of the upper part and the 12 wells of the lower part of the dish will not receive the viral inoculum and were used as control of cellular line. After 1 hour of incubation at 37 C ±1 C 0.1 ml of culture Medium were added to the viral inoculum. After the appropriate period of incubation, the cellular culture was observed with inverted microscope to detect any cytopatic effect (CPE) due to viral suspension. In a second time the same test described above, was performed with water instead of the test substance, after 0 and 60 minutes (virus control). After this detection the infecting activity (TCID 50 ) was calculated by means of Spearman Karber method both in the cellular culture treated with the test substance and in that treated with PBS. 6. CALCULATIONS AND EXPRESSION OF THE RESULTS Determination of TCID 50 The infecting activity was determined by means of Spearman Karber method that uses the following formula to calculate the value of TCID 50 : -Log TCID 50 = - (-x 0 ) - {[R/100]- 0.5} x Log dilution factor where: x 0 = Log of the lowest dilution with 100% of positive reaction (CPE) R = sum (%) of positive cultures Log TCID 50 values were rounded to two significant figures as shown in Annex D table D.1 of EN Rounding is automatically performed by excel datasheet. Evaluation of the virucidal activity The virucidal activity of the product test solution was performed for each exposure time. The logreduction willbe performed by subtracting the logarithmic titre TCID50 at any test point from the logarithmic titre TCID 50 of the virus control. EFFICACY CRITERIA 1. A contact time is considered effective when at least 4 Log reduction in viral titre is obtained. 2. Check of cellular sensitivity to virus: the difference of the value of TCID 50 among the cellular cultures treated with the test substance and the ones not treated with the test substance (i.e. PBS only) must be < 1 Log. 3. Check of viral inactivation (Poliovirus): the difference of the value of TCID 50 among the cells treated with inactivated viral suspensions and the ones treated with active viral suspensions must be, after 60 minutes of incubation between 2 Log and 4.5 Log and between 0.5 Log and 2.5 Log after 30 minutes of incubation for Poliovirus.

54 Page: 54 of 59 RESULTS Evaluation of virucidal activity on EtOH 96% The results of Log reduction at the different contact times are shown following. TEST SUBSTANCE Ethanol 96% vol CONCENTRATION 80.00% INTERFERING SUBSTANCE VIRUS CONTACT TIMES Poliovirus Type 1 ATCC VR-1562 Adenovirus Type 5 ATCC VR - 5 BSA + erythrocytes 0.3% Human herpesviru s (HSV-1) ATCC VR-733 Bovine viral diarrea virus (BVDV) ATCC VR- 534 Porcine Parvovirus (PPV) ATCC VR-742 Hepatitis A virus (HAV) ATCC VR min Log Reduction 15 min min hour hours Reduction >4 Log after minutes 5 min 5 min 5 min 5 min 5 min 30 min DEVIATION No deviation has been recorded from study program. OBSERVATIONS On the basis of the results obtained, in compliance with the assay validity criteria, the test substance ETHANOL 96% provided the following results: - causes a reduction > 4Log against Poliovirus type 1 ATCC VR-1562, Adenovirus type 5 ATCC VR- 5, Human herpesvirus (HSV-1) ATCC VR-733, Bovine viral diarrea virus (BVDV) ATCC VR-534 and Porcine Parvovirus (PPV) ATCC VR-742 after 5 minutes of contact time, using a 0.3% final concentration of bovine albumin and erythrocytes, in compliance with EN14476:2005+A1: causes a reduction > 4Log against Hepatitis A virus (HAV) ATCC VR-1402 after 30 minutes of contact time, using a 0.3% final concentration of bovine albumin and erythrocytes, in compliance with EN14476:2005+A1:2006.

55 Page: 55 of 59 Experimental Report 2009/717.A10 - Viral inactivation effectiveness with ETHANOL 96% (Chemical cleaning step of Sponsor VA-07 procedure) EXPERIMENTAL CONDITIONS 1. ASSAY SYSTEM Virus strains Adenovirus type 5, human ATCC VR-5 Poliovirus type 1 (chat strain) ATCC VR-1562 Human herpes virus (Herpes simplex Type1; HSV-1) ATCC VR- 733 Bovine viral diarrhea virus (BVDV) ATCC VR-534 Hepatitis A virus Strain HM175/18f [HM175 cytopathic clone B] (HAV) ATCC VR-1402 Porcine parvovirus NADL-2 (PPV) ATCC VR-742 Test virus suspension The viral suspensions were kept frozen at 70 C; before their use they were multiplied in the following appropriate cellular line: HOST CELLS INCUBATIONS VIRUS STRAINS LINE TIMES (Days) Adenovirus type 5, human Hela 7 Poliovirus type 1 (chat strain) Hela 7 Human herpes virus (Herpes simplex Type1; HSV-1) Vero 7 Bovine viral diarrhea virus (BVDV) MDBK 7 Hepatitis A virus Strain HM175/18f [HM175 cytopathic clone B] (HAV) FRhK-4 12 Porcine parvovirus NADL-2 (PPV) ST 7 Cellular debris were removed by means of double centrifugation at low speed, and the supernatant containing the virus was used for the test. 2. CELL CULTURES, MEDIA AND REAGENTS Cell cultures MDBK (NBL-1 Kidney) HeLa (cells of human uterine carcinoma) Vero FRhK-4 ST ATCC CCL-22 ATCC CCL-2 ATCC CCL-81 ATCC CRL-1688 ATCC CRL-1746 The cellular culture was kept frozen at < 70 C; before viral inoculum, it was showed itself as confluent monolayer. Culture Medium and Reagents EMEM: Eagle s minimal essential medium BIOWHITTAKER - LONZA FBS (2%) Foetal Bovine Serum BIOWHITTAKER LONZA NEAA (1%) Non-Essential Amino Acid BIOWHITTAKER LONZA PEN-STREP (1%) antibiotics BIOWHITTAKER LONZA PBS Phosphate Buffer Saline BIOWHITTAKER - LONZA WFI Water for injection EUROSPITAL

56 Page: 56 of EQUIPMENT AND MATERIALS Incubator Centrifuge Centrifuge MicroSpin Deepfreeze CO 2 incubator Vortex Chronometer Micropipettes Microdishes 96 wells Inverted microscope phmeter Water bath Columns MicroSpin TM S400 HR Dessicator Vacuum pump ARBORE THERMO EPPENDORF Angelantoni Scientifica PBI VELP ARBORE GILSON LONZA LEITZ BECKMAN ARBORE GE HEALTHCARE GHIARONI 4. EXPERIMENTAL DESIGN Test temperature The test was conducted at C (according to Sponsor procedure). Disinfectant concentration The test substance was used neat therefore the final in test concentration was 80% (maximum concentration testable), corresponding to 76.8% of active matter. Contact times The following contact time was used: 3 hours (according to Sponsor procedure). Test Carriers The test carriers provided by the sponsor consist of human bone treated in routine cleaning process including ether step, which are comparable with the status before cleaning with 96% EtOH; they were blocks with the following dimensions:15 x 15 x 30 mm (equivalent volume: 6.75 cm 3 ). Inoculum preparation The inoculum was preparated mixing each virus suspension with water in ratio 9:1. Carrier Inoculum Test carriers were inoculated with each inoculum preparation separately using the inoculum volume validated in the experimentation A1. 5. ASSAY EXECUTION Viral activity (virus titration) Two virus titration were performed, one for the virus suspension as such and one on the virus suspension after filtration with S400 HR columns MicroSpin. Of each one, serial dilutions 1:10 were prepared with culture Medium, starting from virus stock solution of viral suspension. 0.1 ml were transferred in dishes with 96 wells containing the cellular confluent monolayer (>90%) without culture Medium. Each dilution of viral suspension was placed onto dishes sixfold. The 12 wells of the upper part and the 12 wells of the lower part of the dish did not receive the viral inoculum and were used as control of cellular line. After 1 hour of incubation at 37 C ±1 C 0.1ml of culture Medium were added to viral inoculum.

57 Page: 57 of 59 After the appropriate period of incubation, the cellular culture was observed with inverted microscope to detect any cytopatic effect (CPE) due to viral suspension. After this detection the infecting activity (TCID 50 evaluation) was calculated by means of Spearman Karber method. Assay For each contact time, 2 carrier test was inoculated with the 6 viral suspension test separately. The carrier test was immersed into a ml (ratio 1:5 with carrier volume as indicated by the Sponsor procedure) of ETHANOL 96% protect from light under agitation with a frequenz of 45-50/min. At the end of contact time a viral recovery was determined respectively on the 96% EtOH and on the carrier in order to determine residual virus. At the same time the test described above, was performed with water instead of the disinfectant product, after 0 and 3 hours of contact time (virus control). Viral recovery in the immersion system At the and of each contact time a viral titration was performed on disinfectant test solution in order to determinate the residual virus titre. The titration was performed by means of serial decimal dilution with culture Medium, starting from the residual disinfectant test solution (96% EtOH containing the inoculated carrier) after filtration with S400 HR columns MicroSpin. 0.1 ml were transferred in dishes with 96 wells containing the cellular confluent monolayer (>90%) without culture Medium. Each dilution of viral suspension was placed onto dishes sixfold. The 12 wells of the upper part and the 12 wells of the lower part of the dish did not receive the viral inoculum and were used as control of cellular line. After 1 hour of incubation at 37 C ±1 C 0.1ml of culture Medium were added to viral inoculum. The test was performed in duplicate. After the appropriate period of incubation, the cellular culture was observed with inverted microscope to detect any cytopatic effect (CPE) due to viral suspension. After this detection the infecting activity (TCID 50 evaluation) was calculated by means of Spearman Karber method. Viral recovery on carrier test As per Experimentation 2009/717.A2. 6. CALCULATIONS AND EXPRESSION OF THE RESULTS Determination of TCID 50 The infecting activity was determined by means of Spearman Karber method that uses the following formula to calculate the value of TCID 50 : -Log TCID 50 = - (-x 0 ) - {[R/100]- 0.5} x Log dilution factor where: x 0 = Log of the lowest dilution with 100% of positive reaction (CPE) R = sum (%) of positive cultures Log TCID 50 values were rounded to two significant figures as shown in Annex D table D.1 of EN Rounding is automatically performed by excel datasheet. Evaluation of the virucidal activity The virucidal activity of the product test solution will be performed for each exposure time. The logreduction will be performed by subtracting the logarithmic titre TCID 50 at any test point from the logarithmic titre TCID 50 of the virus control.

58 Page: 58 of 59 EFFICACY CRITERIA At least 2 Log reduction in viral titre. RESULTS The results of Log reduction on the test carriers and on the residual immersion system are shown following. TEST SUBSTANCE: ETHANOL 96% CONTACT TIME: VIRUS STRAINS CARRIER 1 CARRIER 2 Poliovirus Type 1 ATCC VR-1562 Adenovirus Type 5 ATCC VR-5 Human herpes virus (HSV-1) ATCC VR-733 Bovine viral diarrhea virus (BVDV) ATCC VR-534 Porcine Parvovirus (PPV) ATCC VR-742 Hepatitis A virus (HAV) ATCC VR-1402 DEVIATION No deviation has been recorded from study program. OBSERVATIONS 3 HOURS RESIDUAL IMMERSION SYSTEM 1 RESIDUAL IMMERSION SYSTEM 2 >2.83 >2.83 >3.50 >3.50 >4.33 >4.33 >4.67 >4.67 >3.50 >3.50 >4.33 >4.33 >3.50 >3.50 >3.67 >3.67 >3.50 >3.50 >3.67 >3.67 >3.17 >3.17 >3.33 >3.33 On the basis of the results obtained, in compliance with the assay validity criteria, the test substance ETHANOL 96% provided the following results: - causes a reduction > 2Log from cuboids and causes a reduction > 3Log from supernatant against Poliovirus type 1 ATCC VR-1562 after 3 hours of contact time in compliance with the Sponsor procedure requirements; no test virus was recovered after treatment. - causes a reduction > 4Log against Adenovirus type 5 ATCC VR-5 after 3 hours of contact time from both supernatant and cuboids in compliance with the Sponsor procedure requirements; no test virus was recovered after treatment. - causes a reduction > 3Log from cuboids and causes a reduction > 4Log from supernatant against Human herpes virus (HSV-1) ATCC VR-733 after 3 hours of contact time in compliance with the Sponsor procedure requirements; no test virus was recovered after treatment. - causes a reduction > 3Log against Bovine viral diarrhea virus (BVDV) ATCC VR-534 after 3 hours of contact time from both supernatant and cuboids in compliance with the Sponsor procedure requirements; no test virus was recovered after treatment. - causes a reduction > 3Log against Porcine Parvovirus (PPV) ATCC VR-742 after 3 hours of contact time from both supernatant and cuboids in compliance with the Sponsor procedure requirements; no test virus was recovered after treatment. - causes a reduction > 3Log against Hepatitis A virus (HAV) ATCC VR-1402 after 3 hours of contact time from both supernatant and cuboids in compliance with the Sponsor procedure requirements; no test virus was recovered after treatment.

59 Page: 59 of 59 CONCLUSIONS On the basis of the results obtained on the sterilization process of human cancellous bone tissue blocks, the inactivation step by immersion in HYDROGEN PEROXIDE SOLUTION 3% after 16 hours of contact time had shown good efficacy against the viruses tested; some residual virus CPE (cytopatic effect) were observed only for the Porcine Parvovirus (PPV) ATCC VR-742 and Hepatitis A virus (HAV) ATCC VR-1402, but both viral titre reductions resulted >2 Log. The inactivation step by immersion in ETHANOL SOLUTION 96% after 3 hours of contact time has shown complete efficacy against all viruses tested, i.e. no residual viruses were detected in the assay at the end of the incubation period. Due to the relatively low virus recovery from the carriers, a viral titre reduction >4Log, as required by the reference standard EN14476:2005+A1:2006, was not always detectable; however in all validation steps each virus titre was sufficiently high to enable a titre reduction of at least 2 Logs. The Gamma rays irradiation step (irradiation dose: 25 kgy), instead, did not shown good efficacy against the viruses tested; in particular this treatment had shown low effectiveness against Poliovirus type 1 ATCC VR-1562, Adenovirus type 5 ATCC VR-5, Porcine Parvovirus (PPV) ATCC VR-742 and Hepatitis A virus (HAV) ATCC VR-1402; a viral titre reduction > 2Logs was obtained only against Human herpes virus (HSV-1) ATCC VR-733 and Bovine viral diarrhea virus (BVDV) ATCC VR-534, in compliance with the validity assay criteria. The two chemical viral inactivation steps adopted in the sterilisation process, i.e. immersion in EtOh 96% solution for at least 3 hours and immersion in H2O2 3% solution for at least 16 hours show an overall efficacy in inactivating the 6 test viruses globally >4Logs and therefore should be considered effective in removing possible viral contaminations. In particular, the EtOh 96% viral inactivation step causes a complete inactivation of viral inoculum for all viruses tested. ATTACHMENTS ATTACHMENT TITLE NUMBER OF PAGES N.1 COA OF H 2 O 2 PROVIDED BY THE SPONSOR 1 N.2 COA OF ETOH 96% PROVIDED BY THE SPONSOR 1 N.3 N.4 N.5 N.6 N.7 N.8 N.9 N.10 N.11 RAW DATA: EXPERIMENTATION 2009/717.A2 RAW DATA: EXPERIMENTATION 2009/717.A3 RAW DATA: EXPERIMENTATION 2009/717.A4 RAW DATA: EXPERIMENTATION 2009/717.A5 RAW DATA: EXPERIMENTATION 2009/717.A6 RAW DATA: EXPERIMENTATION 2009/717.A7 RAW DATA: EXPERIMENTATION 2009/717.A8 RAW DATA: EXPERIMENTATION 2009/717.A9 RAW DATA: EXPERIMENTATION 2009/717.A10