CYTOTOXICITY TEST: MEM ELUTION. Test Substance: LEGA PER CERAMICA CERAM 450. Test Report N 0103/04. Study performed for

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1 CYTOTOXICITY TEST: MEM ELUTION Test Substance: LEGA PER CERAMICA CERAM 450 Test Report N 0103/04 Study performed for NUOVA FRANCO SUISSE ITALIA S.r.l. Via Dell Artigianato, 26 PERAGA DI VIGONZA PD by BIOCHEM S.a.s. Via Marzocchi 2/A ZOLA PREDOSA BO Test Report 0103/04 Page 1/5

2 TEST FOR CYTOTOXICITY: MEM ELUTION MANAGEMENT OF STUDY Head of Biology substitute: Laura Sperli QUALITY ASSURANCE: Quality Assurance Manager: Cinzia Bernardi, dr. HEAD OF LABORATORY: Giovanni Bassini, Ch.Eng. TIME SCHEDULE OF STUDY The study was started the 27 th of January 2004 and was completed the 30 th of January Test Report 0103/04 Page 2/5

3 TEST FOR CYTOTOXICITY: MEM ELUTION ANALISI CHIMICO-FISICHE Ref. Your Order of January, 2004 Test Substance Description: LEGA PER CERAMICA Code: Ceram 450 Sterilisation: not sterile Receiving number: Receiving date: January 23 rd, 2004 Sampling carried out by: NUOVA FRANCO SUISSE Test Method ISO Part 5th: Tests for in vitro Cytotoxicity Ed USP XXVI Ed.2003 <87> Elution Test Summary of practice Cell cultures are grown to a near-confluent monolayer in cultures dishes. The medium is aspirated and replaced with undiluted liquid extract. Cell cultures are observed after 24 and 48h-contact for the assessment of the presence or absence of a cytotoxic effect from the test extract. Target cells: BSCL 56 /L 929 (Mouse connective tissue) Extraction conditions: 5,4 g of the specimen was extracted in 27 ml of Cell Culture Medium MEM (4 g / 20 ml) at 37 C for 24 hours.(iso Part 12 and <88> USP XXVI Ed.) Reagents: MEM with Earle's salts added with foetal bovine serum, L- glutamine and some antibiotics. Positive control: Phenol 0.5 % solution Negative control: 4 g of polycarbonate extracted as the sample. Incubation: Test extract, positive and negative controls are incubated 48 h at 37 C in a 5% CO 2 atmosphere and inspected for the result evaluation. Test Report 0103/04 Page 3/5

4 Apparatus ANALISI CHIMICO-FISICHE Incubator, which maintains the cultures at 37 C, 5% CO 2 ; Microscope, with inverted phase contrast optics; Water Bath; Laminar Flow Cabinet; Sterile Disposable; Tissue Culture Dishes. Interpretation of Results: The determination of the cytotoxicity of liquid extracts is performed examining the cells under the microscope to assess general morphology, vacuolation, detachment, cell lysis, membrane integrity. The change from normal morphology is rated on a reactivity grade from 0 to 4 (see Grading system).the responses obtained from the Negative Control and the Positive Control are measured. Grading Reactivity Reactivity description 0 None 1 Slight 2 Mild 3 Moderate 4 Strong No cells showing damage, intracellular granulation, no cell lysis. <20% damaged cells (these cells are round, without intracellular granulation,) occasionally cell lysis 20-50% damaged cells (these cells are round, without intracellular granulation,) cell lysis 50-70% damaged cells (these cells are round, without intracellular granulation,) wide cell lysis) % damaged cells (these cells are round, without intracellular granulation,) nearly complete destruction of the cell layers Test Report 0103/04 Page 4/5

5 RESULTS AFTER 48H INCUBATION SCORE Positive control % damaged cells 4 Positive control % damaged cells 4 Positive control % damaged cells 4 Negative control 1 No cells showing damage 0 Negative control 2 No cells showing damage 0 Negative control 3 No cells showing damage 0 MEM control 1 No cells showing damage 0 MEM control 2 No cells showing damage 0 MEM control 3 No cells showing damage 0 Extract of the test material 1 No cells showing damage 0 Extract of the test material 2 No cells showing damage 0 Extract of the test material 3 No cells showing damage 0 Extract of the test material 4 No cells showing damage 0 Extract of the test material 5 No cells showing damage 0 The present Test Report is exclusively referred to the tested sample. The present Test report mustn t be partially reproduced without Biochem and SINAL authorisation. Test carried out by: Laura Sperli Issue authorised by: Head of laboratory Giovanni Bassini, Ch.Eng. Zola Predosa, February, 02 nd 2004 Test Report 0103/04 Page 5/5