Document NG4E0EMk3bpDJMEonRJ1L5JQQ

RESEARCH TOXICOLOGY CENTRE - ROMA 7850 CHROMOSOME ABERRATIONS IN CHINESE HAMSTER OVARY CELLS IN VITRO FINAL REPORT RTC Study No.: 52420 Sponsor: SOLVAY SOLEXIS S.p.A. Viale Lombardia, 20 20021 Bollate (MI) Italy Total number of pages: 49 RTC S.p.A. Via Tito Speri, 12 00040 Pomezia (Roma) - ITALY Tel.: + 39.0691095.1 Fax: e-mail: www.rtc.it rtc.it RTC S.p.A. Via Tito Speri, 12 00040 Pomezia (Roma) - ITALY Tel.: + 39.06.91095.1 Fax: +39.06.912.2233 P.O. Box 15301-00143 - Roma Eur Laurentino RTC S.p.A. Capitals sociale Euro 5.164.000 C.C.I.A.A. n' 375376 Reg. Soc. Trib. di Roma n' 2828/72 Cod. Fisc.: 00653120584 Partita IVA: 00920611001 COMPLIANCE STATEMENT We, the undersigned, hereby declare that the following report constitutes a true and faithful account of the procedures adopted, and the results obtained in the performance of the study. The aspects of the study conducted by Research Toxicology Centre S.p.A. were performed in accordance with: A. Good laboratory practice for non clinical laboratory studies, U.S. Food and Drug Administration, Code of Federal Regulations, 21 Part 58, 22 December 1978 and subsequent revisions. B. Decreto Legislativo 27 Gennaio 1992 n. 120, Adoption of88/320/EEC and 90/18/EEC Directives on the inspection and verification ofgood laboratory practice (G.U. 18 Febbraio 1992 n. 40) and subsequent revisions. C. Directive 2004/10/EC of European Parliament and of the Council of 11 February 2004. On the harmonisation of laws, regulations and administrative provisions relating to the application of the principles of good laboratory practice and the verification of their applications for tests on chemical substances. D. ENV/MC/CHEM(98) 17 OECD principles on Good Laboratory Practice (as revised in 1997). Date Date RTC Study No.: 52420 Pagel QUALITY ASSURANCE STATEMENT (Relevant to the aspects of the study conducted by Research Toxicology Centre S.p.A.) Study phases monitored by RTC's QAU according to current relevant Standard Operating Procedures PROTOCOL CHECK Quality Assurance Inspections Da Month Year Inspection Report to Study Director Report to Company Mana ement 13.02.2006 13.02.2006 13.02.2006 PROCESS-BASED INSPECTIONS RELATED TO TIDS TYPE OF STUDY Dose preparation Cell treatment Cell harvesting Slide preparation Slide staining Slide coding Slide scoring 04.05.2006 30.05.2006 05.05.2006 05.05.2006 22.06.2006 19.01.2006 16.03.2006 11.05.2006 31.05.2006 24.05.2006 18.05.2006 26.06.2006 20.01.2006 22.03.2006 Other process-based inspections were carried out on routine activities not directly related to this type of study. The relevant documentation is kept on file although specific inspection dates are not reported here. Associated laboratories and support functions are subject to regular facility inspections. FINAL REPORT Review of this report by RTC's QAU found the reported methods and procedures to describe those used and the results to constitute an accurate re resentation of the recorded raw data. Review completed Date RTC Study No.: 52420 Page3 Contents Page 1. SUMMARY...................................................................................................................... 5 2. INTRODUCTION........................................................................................................... 7 2.1 Purpose........................................................................................................................ 7 2.2 Study organisation....................................................................................................... 7 3. MATERIALS AND METHODS.................................................................................... 9 3.1 Test item...................................................................................................................... 9 3.2 Control substances ...................................................................................................... 9 3.3 S9 tissue homogenate.................................................................................................. 10 3.4 Methods....................................................................................................................... 10 4. RESULTS......................................................................................................................... 11 4.1 Solubility test .............................................................................................................. 11 4.2 Assay for chromosomal aberrations............................................................................ 11 4.3 Selection of dose-levels for scoring............................................................................ 12 4.4 Assay results ............................................................................................................... 13 5. ANALYSIS OF RESULTS............................................................................................. 14 5.1 Statistical analysis....................................................................................................... 14 5.2 Criterion for outcome.................................................................................................. 14 5.3 Evaluation ................................................................................................................... 14 6. CONCLUSIONS .............................................................................................................. 15 7. TABLES 1-11 ................................................................................................................... 16 8. KEY TO TABLES4-6..................................................................................................... 20 9. KEY TO TABLES7-9..................................................................................................... 24 10. APPENDIX I- Historical background incidences of aberrant cells................................... 30 11. APPENDIX II - Study Protocol........................................................................................ 31 12. APPENDIX m - Certificate of analysis........................................................................... 46 13. APPENDIX IV - S9 production of quality control certificate.......................................... 48 RTC Study No.: 52420 Page4 1. SUMMARY 1.1 The test item 7850 was assayed for the ability to cause chromosomal damage in Chinese hamster ovary cells, following in vitro treatment in the absence and presence of S9 metabolic activation. 1.2 Two assays for chromosomal damage were performed. The first assay employed dose-levels of 5000, 2500, 1250, 625, 313, 156, 78.1, 39.1, 19.5, 9.77 and 4.88 g/ml both in the absence and presence of S9 metabolism. For the second assay, dose-levels of 450, 300, 200, 133, 88.9, 59.3, 39.5 and 26.3 g/ml were employed. Solutions of the test item were prepared in ethanol. In the first assay, both in the absence and presence of S9, the cells were treated for 3 hours and the harvest time of 20 hours, corresponding to approximately 1.5 cell cycle, was used. As negative results were obtained, a second assay in the absence of S9 metabolism was performed using a continuous treatment until harvest at 20 hours. Each experiment included appropriate negative and positive controls. Two cell cultures were prepared at each test point. 1.3 Dose-levels were selected for the scoring of chromosomal aberrations on the basis of the cytotoxicity of the test item treatments as determined by the reduction of cell counts at the time of harvesting. The treatment-levels selected for scoring were the following: Experiment S9 1 + 2 - Treatment time (hours) 3 20 Harvest time (hours) 20 20 Dose-level (g/ml) 313, 156 and 78.1 300, 200 and 133 One hundred metaphase spreads were scored for chromosomal aberrations from each culture. 1.4 Following treatment with 7850, for the first experiment, increases in the incidences of cells bearing gaps over the control were observed at all dose-levels selected for scoring in the presence of S9 metabolism. An increase in the incidence of cells bearing aberrations excluding gaps, reaching statistical significance (p<0.05) was observed at the highest dose selected for scoring. The incidence was within the range of historical values observed in our laboratory for negative controls, therefore was not considered biologically meaningful. For both experiments, no statistically significant increase in the incidence of cells bearing aberrations including or excluding gaps was observed following treatment with the test item in the absence of S9 metabolism. Statistically significant increases in the number of cells bearing aberrations (including and excluding gaps) were observed following treatments with the positive controls Cyclophosphamide and Mitomycin-C, indicating the correct functioning of the test system. RTC Study No.: 52420 Page5 1.5 It is concluded that 7850 does not induce chromosomal aberrations in Chinese hamster ovary cells after in vitro treatment in the presence or absence of metabolic activation, under the reported experimental conditions. RTC Study No.: 52420 Page6 2. INTRODUCTION 2.1 Purpose describes the experiment performed to assess the clastogenic activity of 7850 in Chinese hamster ovary cells following in vitro treatment in the absence or presence of S9 metabolic activation. The study was designed to comply with the experimental methods indicated in: - EEC Council Directive 2000/32, Annex 4A. - OECD Guideline No. 473 (Adopted: 21st July 1997). 2.2 Study organisation Sponsor SOLVAY SOLEXIS S.p.A. Viale Lombardia, 20 20021 Bollate (MI) Italy Location of study Genetic and Cellular Toxicology Department Research Toxicology Centre S.p.A. (RTC) Via Tito Speri, 12 00040 Pomezia (Roma) Italy Principal dates Study protocol approved by Study Director: 18-Jan-2006 Study commenced: 07-Mar-2006 (Main assay I treatment) Study completed: 19-Jun-2006 (Main Assay II completion of scoring) Personnel involved in the study Study Monitor: Study Director: Scorers of slides RTC Study No.: 52420 Page 7 Archiving: The original data arising from this study, prepared microscope slides, and a copy of the consigned final report will be stored in the archives of Research Toxicology Centre S.p.A. for a period of three years from the date of consignment of the report. At the completion of this period the Sponsor will be contacted for despatch or disposal of the material, or further archiving. An aliquot of the test item will be retained within the archives of the testing facility for a period of ten years after which it will be destroyed. RTC Study No.: 52420 Page8 3. MATERIALS AND METHODS 3.1 Test item Details of the test item received at RTC were as follows: Name Batch C.A.S. No. Received from Date received Amount received Description Expiry date Container Storage at RTC RTC reference number 7850 3223 ON 329238-24-6 SOLVAY SOLEXIS 20-Feb-2006 100 g Colourless liquid 3 1-Dec-20 12 Opaque plastic bottle Room temperature 10022 On 27-Feb-2006 a 12g sub-sample of the test item was transferred from the Formulation Unit to the Department of Genetic and Cellular Toxicology and stored under the same conditions. Solutions of the test item, as received, were prepared immediately before use in ethanol. Solutions were prepared on a weight/volume basis without correction for the displacement due to the volume of the test item. Concentrations were expressed in terms of material as received. All test item solutions were used within 25 minutes of the initial formulation. No assay of test item stability, nor its concentration and homogeneity in solvent were undertaken. All dose-levels in this report are expressed to three significant figures. 3.2 Control substances The solvent used in this study was ethanol, batch no. 4G297304I obtained from Carlo Erba, Italy. Solutions of Mitomycin-C (batch 104K033 1 obtained from Sigma and batch 1 149026 10605077 obtained from Fluka) and Cyclophosphamide (batch 075K 166 1 obtained from Sigma) were prepared in sterile distilled water immediately prior to use and served as positive controls. Injectable grade distilled water (batch 03H28-02) was obtained from Bieffe, Trieste, Italy. Untreated cultures were included in the experimental scheme and acted as reference control for the positive control cultures. RTC Study No.: 52420 Page9 3.3 S9 tissue homogenate The rat S9 liver tissue fraction used in this study had the following characteristics: Lot Number Inducing Agents Preparation date Expiry date Received from Date received Storage at RTC S train Sex of donors Protein content 1 876 Phenobarbital - 5,6-Benzoflavone 09-Jun-2005 09-Jun-2007 MOLTOX, Molecular Toxicology, Inc. 1 4-Jul-2005 Approximately -80C S prague Dawley Male 37.2 mg/ml A production and quality control certificate can be found in Addendum N of this report. 3.4 Methods The methods used were in compliance with the attached S tudy Protocol with the exception that an additional experiment was performed with the continuous treatment in the absence of S9 metabolism where no dose-level showed an adequate cytotoxicity for the scoring of chromosomal aberrations. Data concerning this experiment are not presented in the report and will be archived as indicated in the Study Protocol. RTC Study No. : 52420 Page 10 4. RESULTS 4.1 Solubility test The test item was found to be miscible with ethanol at 500 mg/ml. Aliquots of stock solutions exceeding 62.5 mg/ml added to supplemented Ham' s Fl0 medium gave opacity or few particles in suspension in the medium. On the basis of the above mentioned result, the cytogeneti c experiments were performed using a maximum dose-level of 5000 g/ml. During the first main assay, particles in suspension were observed at the beginning of treatment at the dose levels of 5000 and 2500 g/ml both in the absence and presence of S9 metabolism. Slight opacity was observed at the dose of 1250 glml both in the absence and presence of S9 metabolism At the end of treatment, dose-related opacity and precipitation were observed at the dose levels of 5000, 2500, 1250 and 625 glml both in the absence and presence of S9. During the second assay slight opacity of the medium was observed at the beginning of treatment at the dose-level of 450 g/ml. At the end of treatment slight precipitation was also observed. 4.2 Assay for chromosomal aberrations The first assay employed dose-levels of 5000, 2500, 1250, 625, 313, 156, 78.1, 39.1, 19.5, 9.77 and 4.88 g/ml both in the absence and presence of S9 metabolism. For the second assay, dose-levels of 450, 300, 200, 133 , 88.9, 59.3 , 39.5 and 26.3 g/ml were employed. In the first experiment, both in the absence and presence of S9 metabolic activation, the treatment time was 3 hours after which the cells were allowed to recover prior to harvesting. The harvest time of 20 hours, corresponding to approximately 1.5 cell cycle, was used. As negative results were obtained, a second experiment without S9 metabolic activation, was performed with continuous treatment until sampling at 20 hours. Appropriate negative and positive control cultures were included in each experiment. Positive control treated cultures received Mitomycin-C 0.30 and 0.45 g/ml for 3 hours treatment and 0.15 and 0.10 g/ml for the continuous treatment in the absence of S9 metaboli sm or Cyclophosphamide 15.0 and 23.0 g/ml i n the presence of S9. Two cultures were prepared at each test point. Air-dried slides were prepared from each culture and stained with 3% Giemsa. Following treatment, the pH and osmolality of the treatment media at the higher dose-levels were determined for both experiments. Slight dose-related reductions of these parameters over the control values were observed in the first main assay at the higher dose-levels of 5000 and 2500 g/ml both in the absence and presence of S9 metabolism. These results are not presented in this report but, are retained in the study file and archived as indicated in the study protocol. RTC Study No. : 52420 Page 11 4.3 Selection of dose-levels for scoring Cell counts were performed at harvesting time for eac h culture of each treatment series, and the results are presented in Tables 1 to 3. In the first main assay, following treatment in the absence of S9 metabolism, severe toxicity was observed at the dose-levels of 5000, 2500, 1250 and 625 g/ml, where no cells or few cells were recovered. Mild toxicity was observed at the dose-level of 3 13 g/ml reducing the cell count to 75% of the control. Slight toxicity was observed at the dose-levels of 156 and 78.1 g/ml (cell counts approximately 80% o f the control). No remarkable toxicity was observed over the remaining dose-range. Following treatment in the presence of S9 metabolism, severe toxicity was observed at the higher dose-levels of 5000, 2500 and 1 250 g/ml, where no cells were recovered. Marked toxicity was observed at the dose-level of 625 g/ml reducing the cell count to 34% of the control. Mild and slight toxicity was observed at the dose-levels of 3 1 3 and 1 56 g/ml (cell counts 77% and 85% of the control respectively). No toxicity was observed over the remaining dose-range. In the second main assay, after the continuous treatment until sampling at 20 hours, severe toxicity was observed at the highest dose-level of 450 g/ml, where few cells were recovered. Moderate toxicity was observed at the dose-level of 300 g/ml where cell count was reduced to 58% of the control. Slight toxicity was observed at the dose-level of 200 g/ml (cell count 82% of the control). No remarkable toxicity was observed over the remaining dose-range. The highest dose-level selected for the scoring of aberrations should be a concentration causing moderate toxicity (ideally the reduction of cell count should be approximately 50%) and treatments reducing the cell count value to below 20% of the relevant control should not be scored. If no toxicity is observed then the highest practicable dose-level should be selected. On the basis of the above results the dose-levels selected for scoring were: Experiment S9 1 -/+ 2 - Treatment time (hours) 3 20 Harvest time (hours) 20 20 Dose-level (g/ml) 3 1 3, 156 and 78. 1 300, 200 and 133 For the first main assay, at the dose-level of 625 (g/ml) cell count was reduced to 34% of the control, however, due to the toxicity of treatment, an insufficient number of eligible metaphases were recovered for the scoring of aberrations. RTC Study No.: 52420 Page 12 4.4 Assay results One hundred metaphase spreads were scored for chromosomal aberrations from each culture. The results are presented in Tables 4, 5 and 6. In these tables the numbers and types of aberrations are presented, together with the total number of aberrations (chromatid type and chromosome type) including and excluding gaps. The total number of aberrant metaphases including and excluding gaps are also shown. For the first experiment, following treatment with the test item in the presence of S9 metabolism, increases in the incidences of cells bearing gaps over the controls were observed at all dose-levels selected for scoring The incidences reached statistical significance for one replicate culture at the lowest dose. A slight increase in the incidence of cells bearing aberrations excluding gaps, reaching statistical significance (p<0.05) for one replicate culture and for the pooled cultures, was observed at the highest dose selected for scoring. The incidence was within the range of historical values observed in our laboratory when considering the pooled cultures, therefore was not considered biologically meaningful. An increase in the number of endoreduplicated cells over the control was mainly observed at the highest dose selected for scoring. Following treatment in the absence of S9 metabolism, slight increases in the incidence of cells bearing gaps over the control values were observed at the highest and intermediate dose-levels selected for scoring after the short treatment. A slight increase in the incidences of cells bearing gaps was also observed at the highest dose selected for scoring after the continuous treatment. No relevant increase in the incidence of cells bearing aberrations excluding gaps was observed at any dose-level selected for scoring. A few endoreduplicated cells and polyploid cells were noted for the short and continuous treatment respectively. Marked increases in the frequency of cells bearing aberrations (including and excluding gaps) were seen in the cultures treated with the positive control substances, indicating the correct functioning of the assay system. The modal number of chromosomes observed in the cells of untreated cultures (without S9 metabolism) was 21 (72.5% in the first experiment and 89.0% in the second experiment). The frequency of cells containing 20 and 22 chromosomes was 1.0% and 26.5% in the first experiment, 0.0%, and 11.0% in the second experiment. For both experiments no metaphases were found with 19 or 23 chromosomes. RTC Study No. : 52420 Page 13 5. ANALYSIS OF RESULTS 5.1 Statistical analysis For the statistical analysis, Fisher's Exact Test is used to compare the number of cells bearing aberrations (assumed to be Poisson distributed) in control and treated cultures. The analysis is performed using sets of data either including or excluding gaps. The results of the statistical analysis are presented in Tables 7, 8 and 9. For the first experiment, following treatment with the test item in the presence of S9 metabolism, a statistically significant increase in the incidence of cells bearing gaps was observed for one replicate culture at the lowest dose selected for scoring. A slight but significant increase (p<0.05) in the incidence of cells bearing aberrations excluding gaps was observed for one replicate at the highest dose selected for scoring. A statistically significant increase in the incidence of cells bearing aberrations excluding gaps was also observed for the pooled cultures from the highest dose selected for scoring. The incidence of aberrant cells, however, was within the range ofhistorical values observed in our laboratory for negative controls, therefore was not considered biologically meaningful. For both experiments, no statistically significant increase in the incidence of cells bearing aberrations including or excluding gaps was observed following treatment with the test item in the absence of S9 metabolism. 5.2 Criterion for outcome In this assay, the test item is considered to have clastogenic properties if the following criteria are all fulfilled: (i) Statistically significant increases in the incidence of cells bearing aberrations are observed at any dose-level over the concurrent control. (ii) The increases must exceed the historical control values. (iii) The increases are reproduced in both replicate cultures. The evaluation is based on the set of results, which excludes gaps. A more detailed explanation of the criteria for evaluation ofthe results is given in the Study Protocol. 5.3 Evaluation On the basis of the above mentioned results and in accordance with the criteria for the outcome of the study the test item was not considered to induce chromosomal aberrations in Chinese hamster ovary cells in vitro. Statistically significant increases in aberrant cells compared with the relevant control values were seen in cultures treated with the positive controls Mitomycin-C and Cyclophosphamide, indicating the correct functioning of the assay system. RTC Study No. : 52420 Page 14 6. CONCLUSIONS A summary of the results is presented in Tables 1 0 and 1 1 giving the incidence of cells bearing aberrations (excluding gaps) and the relative cell count for each test point. The statistical significance of the recorded numbers of cells bearing aberrations is also shown. On the basis of these results it is concluded that FLUOROLINK 7850 does not induce chromosomal aberrations in Chinese hamster ovary cells after in vitro treatment in the absence or presence of S9 metabolic activation, under the reported experimental conditions. RTC Study No. : 52420 Page 15 7. TABLES 1-11 RTC Study No. : S24:JO Page 16 7 8 5 0 : CHROMOSOME ABERRATIONS TABLE 1 - C e l l growth resul t s - Wi thout metabol i c act iva t i on STUDY NO . : 5 2 4 2 0 MAIN ASSAY : 1 SOLVENT : ETHANOL TREATMENT TIME : 3 hours SAMPLING TIME : 2 0 hours T r e a tme n t Dos e - l eve l Cul ture N viable ( g/ml ) No . cel l sx1 0 6 /ml Mean Re lat ive c e l l growth ( % ) Un t r e a t e d 1 0 . 63 0 . 63 107 2 0 . 63 Solvent 1% 3 0 . 59 0 . 59 100 4 0 . 59 Tes t Item 4 . 88 25 0 . 62 0 . 61 103 26 0 . 60 Tes t I tem 9 . 77 23 0 . 61 0 . 60 102 24 0 . 59 Tes t Item 19 . 5 21 0 . 55 0 . 56 94 22 0 . 56 Test I t em 39 .1 19 0 . 51 0 . 56 95 20 0 . 61 Tes t I tem 78 . 1 17 0 . 47 0 . 49 83 18 0 . 51 Tes t Item 156 15 0 . 48 0 .48 81 16 0 . 47 Tes t I tem 313 13 0 . 43 0 . 44 75 14 0 . 45 Te s t I tem 625 11 0 . 02 0 . 02 3 12 0 . 02 Tes t Item 1250 9 0 . 00 0 . 00 0 10 0 . 00 Test I tem 2500 7 0 . 00 0 . 00 0 8 0 . 00 Te st I t em 5000 5 0 . 00 0 . 00 0 6 0 . 00 M i t omyc in-C 0 . 30 27 0 . 47 0 . 48 75 28 0 . 48 Mitomyc in-C 0 . 45 29 0 . 41 0 . 42 67 30 0 . 43 RTC Study No. : 52420 Page 1 7 7 8 5 0 : CHROMOSOME ABERRATI ONS TABLE 2 - C e l l growth resu l t s - With metabol i c act iva t i on STUDY NO . : 5 2 4 2 0 MAIN ASSAY : 1 SOLVENT : ETHANOL TREATMENT TIME : 3 hours SAMPLING TIME : 2 0 hours Treatment Do s e - l evel Cul ture N viable Mean ( g/ml ) No . cel l s * 1 0 * * 6 /ml Unt reated 31 0 . 62 0 . 62 32 0 . 62 Solvent 1% 33 0 . 61 0 . 59 34 0 . 56 Te s t I t em 4 . 88 55 0 . 59 0 . 60 56 0 . 61 Te s t I t em 9 . 77 53 0 . 59 0 . 61 54 0 . 62 Te st I t em 19 . S 51 0 . 62 0 . 61 52 0 . 59 Te s t I tem 39 . 1 4s 9o 0 . 60 0 . 61 0 . 61 Test I t em 78 . 1 47 0 . 61 0 . 59 48 0 . 57 Tes t Item 156 45 0 . 51 0 . 50 46 0 . 48 Tes t Item 313 43 0 . 44 0 . 45 44 0 . 46 Test Item 625 41 0 . 17 0 . 20 42 0 . 23 Test I t em 1250 39 0 . 00 0 . 00 40 0 . 00 Te s t I t em 2500 37 0 . 00 0 . 00 38 0 . 00 Tes t I t em 5000 35 0 . 00 0 . 00 36 0 . 00 Cyc l ophosphamide 1 5 . 0 57 0 . 44 0 . 43 58 0 . 42 Cyc l ophosphamide 2 3 . 0 59 0 . 35 0 . 37 60 0 . 38 Re lat ive c e l l growth ( % ) 106 100 103 103 103 103 101 85 77 34 0 0 0 69 59 R TC Study No. : 52420 Page 18 7 8 5 0 : CHROMOSOME ABERRATIONS TABLE 3 - Ce l l growth resul t s - Wi thout metabo l i c act iva t i on STUDY NO . : 5 2 4 2 0 MAIN ASSAY : 2 SOLVENT : ETHANOL TREATMENT TIME : 2 0 hours SAMPLING TIME : 2 0 hours T r e a tme n t Dos e - l evel Cul ture N viable Mean ( 1,tg / ml ) No . cells * l0 * * 6 /ml Relative cell growth ( % ) Un t r e a t ed 85 0 . 79 0 . 78 142 86 0 . 76 Solvent 1% 87 0 . 50 0 . 55 100 88 0 . 59 Tes t I t em 26 .3 103 0 . 60 0 . 60 110 104 0 . 60 Tes t Item 39 . 5 101 0 . 58 0 . 59 107 102 0 . 59 Te s t I t em 59 . 3 99 0 . 57 0 . 56 102 100 0 . 54 Test Item 88 . 9 97 0 . 52 0 . 54 99 98 0 . 56 Test I tem 133 95 0 . 53 0 . 51 93 96 0 . 48 Tes t Item 200 93 0 . 47 0 . 45 82 94 0 . 42 Tes t I t em 300 91 0 . 31 0 . 32 58 92 0 . 32 Tes t Item 450 89 0 . 02 0 . 02 90 0 . 01 M i t omyc in-C 0 . 10 105 106 0 . 40 0 . 38 0 . 39 50 Mitomyc in-C 0 . 15 107 108 0 . 37 0 . 41 0 . 39 50 R TC Study No. : 52420 Page 19 8. KEY TO TABLES 4-6 This table shows, for each test culture used in the main assay and by treatment group totals, the types and numbers of aberrations, identified as follows: Gaps Del Exch - This refers to either chromatid or chromosome gaps. Interstitial or terminal deletions. Exchanges: a) Chromatid exchanges Includes symmetrical and asymmetrical exchanges; intra- and inter chromosome exchanges. b) Chromosome exchanges Includes both dicentric and ring types. Other H: ER: PP: These include Heavily damaged cells (more than 5 aberrations/cell) Endoreduplicated cells Polyploid cells Isolocus - Includes isochromatid and isolocus breaks when these cannot be distinguished. Tot.abs - Total number of aberrations observed. Cells with abs. - Cells with aberrations. RTC Study No.: 52420 Page20 7 8 5 0 : CHROMOSOME ABERRAT IONS TABLE 4 - Scor ing o f Aberrations - Wi thout me tabo l ic act ivat i on STUDY NO . : 5 2 4 2 0 MAIN ASSAY : 1 SOLVENT : ETHANOL TREATMENT TIME : 3 hours SAMPLING TIME : 2 0 hours Treatment Un t rea t e d Solvent Test Item Test I t em Tes t I t em Mitomyc in-C Dose-level Cul ture Cel l s Chroma t i d Chromosome I s o l ocus ( g/ml ) No . Scored Gaps Del Exch Del Exch Other - 1% 78 .1 156 313 0.30 1 100 6 1 0 0 0 0 0 2 100 2 0 0 0 0 0 0 200 8 1 0 0 0 0 0 3 100 3 0 0 0 1 0 0 4 100 3 0 0 0 0 0 0 200 6 0 0 0 1 0 0 17 100 6 0 0 0 0 0 0 18 100 1 0 0 0 0 0 0 200 7 0 0 0 0 0 0 15 100 5 1 0 0 0 0 0 16 100 6 1 0 0 0 0 0 200 11 2 0 0 0 0 0 13 100 6 0 0 0 1 0 0 14 100 3 1 0 0 0 0 2 ER 200 9 1 0 0 1 0 2 ER 27 100 8 29 30 0 0 4 0 28 100 11 26 25 4 1 1 0 200 19 55 55 4 1 5 0 Tot . abs ( +gaps ) Tot . abs ( -gaps ) Cells Cells wi th abs wi th abs ( +gaps ) ( -gaps ) 7 1 2 0 9 1 4 1 3 0 7 1 6 0 1 0 7 0 6 1 7 1 13 2 7 1 4 1 11 2 71 63 68 57 139 12 0 7 1 1 0 8 1 4 1 3 0 7 1 4 0 1 0 5 0 6 1 6 12 2 5 1 4 1 9 2 47 45 40 38 87 83 RTC Study No.: 52420 Page 21 7 8 5 0 : CHROMOSOME ABERRATI ONS TABLE 5 - Scoring of Aberrations - With me tabo l i c activa t i on STUDY NO . : 5 2 4 2 0 MAIN ASSAY : 1 SOLVENT : ETHANOL TREATMENT TIME : 3 hours SAMPLING TIME : 2 0 hours Treatment Dose-level Cul ture C el l s Chroma t i d Chromosome Isolocus ( g/ml ) No . Scored Gaps Del Exch Del Exch Othe r Tot . abs ( +gaps ) Tot . abs ( -gaps ) Cells Cells with abs wi th abs ( +gaps ) ( -gap s ) Un t r e a t e d - So lvent 1% Test Item 78 . 1 Test I tem 156 Test Item 313 Cyclophospharnide 1 5 . 0 31 100 5 0 0 0 0 0 0 5 0 32 100 4 0 0 0 0 0 lER 4 0 200 9 0 0 0 0 0 l ER 9 0 33 100 10 0 0 0 0 0 0 10 0 34 100 11 0 0 0 0 0 0 11 0 200 21 0 0 0 0 0 0 21 0 47 100 9 0 0 0 0 0 0 9 0 48 100 28 3 0 0 0 0 2 ER 31 3 200 37 3 0 0 0 0 2 ER 40 3 45 100 9 2 0 0 0 0 0 11 2 46 100 18 0 1 0 0 0 3ER 19 1 200 27 2 1 0 0 0 3 ER 30 3 43 100 12 0 0 0 1 0 SER 13 1 44 100 13 5 0 0 0 0 6ER 18 5 200 25 5 0 0 1 0 llER 31 6 57 100 15 25 38 0 0 8 0 58 100 19 23 26 0 0 11 0 200 34 48 64 0 0 19 0 86 71 79 60 165 131 5 0 3 0 8 0 9 0 8 0 17 0 9 0 19 3 28 3 10 2 15 1 25 3 10 1 16 5 26 6 46 42 43 40 89 82 RTC Study No.: 52420 Page 22 7 8 5 0 : CHROMOSOME ABERRATIONS TABLE 6 - Scor ing o f Aberra t i ons - Wi thout metabol ic act iva t i on STUDY NO . : 5 2 4 2 0 MAIN AS SAY : 2 SOLVENT : ETHANOL TREATMENT TIME : 2 0 hours SAMPLING TIME : 2 0 hours Treatment Untreated S o lven t Tes t I tem Tes t Item Tes t Item Mitomyc in-C Dose -level Cu lture Cells Chroma t i d Chromosome I s o locus ( g/ml ) No . Scored Gaps Del Exch Del Exch O t he r - 85 100 2 0 0 0 0 0 0 86 100 0 0 0 0 0 0 0 200 2 0 0 0 0 0 0 1% 87 100 2 0 0 0 0 0 0 88 100 3 0 0 0 0 0 0 200 5 0 0 0 0 0 0 133 95 100 2 0 0 0 0 0 0 96 100 0 0 0 0 0 0 0 200 2 0 0 0 0 0 0 200 93 100 2 0 0 0 0 0 lPP 94 100 4 0 0 0 0 0 l PP 200 6 0 0 0 0 0 2PP 300 91 100 1 0 0 0 0 0 lPP 92 100 7 0 0 0 0 0 0 200 8 0 0 0 0 0 l PP 0 . 10 105 100 9 1 15 0 0 0 0 106 100 2 1 16 0 0 0 1H 200 11 2 31 0 0 0 lH Tot . abs ( +gaps ) Tot . abs ( -gaps ) Cells Cells with abs wi th abs ( +gaps ) ( -gap s ) 2 0 0 0 2 0 2 0 3 0 5 0 2 0 0 0 2 0 2 0 4 0 6 0 1 0 7 0 8 0 25 16 19 17 44 33 2 0 0 0 2 0 2 0 3 0 5 0 2 0 0 0 2 0 2 0 4 0 6 0 1 0 7 0 8 0 17 13 19 18 36 31 RTC Study No.: 52420 Page 23 9. KEY TO TABLES 7-9 This table summarises the statistical analyses for the individual treated cultures and for the treatment groups using the pooled data. The analyses are presented both including and excluding gaps from the data-sets. The following abbreviations are used: P. Value The calculated probability value obtained from the comparison of the treated with the controls, using Fisher's Exact Test. The test item treatments are compared with the relevant solvent or untreated controls as appropriate (according to the vehicle used for the test material). Positive control treatments are compared with the untreated controls. Sig. Significance level. The significance level ofthe achieved P-value. For the test item treatments, correction is made for multiple comparisons (as indicated at the foot of each table). For positive control treatments and for all dose-levels combined significance is indicated as: * Statistically significant at P<0.05 ** Statistically significant at P<0.0 1 *** Statistically significant at P<0.00 1 # No statistic was calculated since the proportion of cells bearing aberrations was identical for the treated and relevant negative control cultures. NS Not significant. RTC Study No.: 52420 Page 24 7 8 5 0 : CHROMOSOME ABERRATIONS TABLE 7 - Stat i s t ical Analys i s - Wi thout metabo l i c ac t ivat i on STUDY NO . : 5 2 4 2 0 MAIN ASSAY : 1 SOLVENT : ETHANOL TREATMENT TIME : 3 hours SAMPLING TIME : 2 0 hours Comparison with Negat ive control Cel l s with aberrations ( + gaps ) ( - gaps ) Dose .g/ml CULT . P . values Sig . P . values Sig . Te s t I t em Test I tem Test Item Al l dose leve l s M i t omyc in-C 78 . 1 17 18 Pooled 156 15 16 Pooled 313 13 14 Pooled 0 . 30 27 28 Pooled 0 . 5292 0 . 1914 0 . 3856 0 .2371 0 . 2371 0 . 1738 0 . 3669 0 . 5292 0 . 3998 0 .3909 0 .0000 0 . 0000 0 . 0000 N.S. N.S. N.S. N.S. N. S . N. S . N. S . N.S. N.S. N.S. *** *** *** 0 . 6668 0 . 6668 0 . 5000 0 . 5564 0 . 5564 0 . 5000 0 . 5564 0 . 5564 0 . 5000 0 . 6328 0 .0000 0 . 0000 0 . 0000 N.S. N.S. N.S. N.S. N. S . N.S. N.S. N.S. N.S. N.S. *** *** *** In thi s table correc t ion i s made for the mul t iple compari s ons o f the test i t em treatment s with the negat ive contro l s . For individual cul tures , s ince s ix compari sons are made , the required " p " values for signi f icance are 0 . 0 0 9 ( * ) , 0 . 0 0 2 ( * * ) and 0 . 0 0 0 2 ( * * * ) . For treatment l eve l s , s ince three compari s ons are made the required " p " values for sign i f i cance are 0 . 0 17 ( * ) , 0 . 0 0 3 ( * * ) and 0 . 0 0 0 3 ( * * * ) . RTC Study No. : 52420 Page 25 7 8 5 0 : CHROMOSOME ABERRATIONS TABLE 8 - Stat i s tical Analys i s - Wi th metabo l i c ac t iva t i on STUDY NO . : 5 2 4 2 0 MAIN ASSAY : 1 SOLVENT : ETHANOL TREATMENT TIME : 3 hours SAMPLING TIME : 2 0 hours Compari son wi th Negat ive control Cel l s wi th aberrations ( + gap s ) ( - gap s ) Dose g/ml CULT . P . values Sig . P . values Sig . Tes t I t em Te s t I t em Tes t I tem Al l dose level s Cyc l opho sphamide 78 . 1 47 48 Pooled 156 45 46 Pooled 313 43 44 Pooled 15 . 0 57 58 Pooled 0 . 5208 0 . 0083 0 . 0564 0 . 4082 0 . 0663 0 . 1267 0 . 4082 0 . 0413 0 . 0981 0 . 0480 0 . 0000 0. 0000 0. 0000 N. S . * N.S. N.S. N. S . N. S . N. S . N. S . N. S . * *** *** *** # 0 . 0363 0 . 1241 0 . 1104 0 .3334 0 . 1241 0 . 3334 0 . 0038 0 . 0150 0 . 0308 0 . 0000 0 .0000 0 . 0000 N.S. N.S. N. S . N. S . N. S . N.S. N.S. * * * *** *** *** I n this table c orrec t i on i s made for the mul t iple c ompari sons o f the test i t em treatment s wi th the negat ive contro l s . For individual cul tures , s ince six comparisons are made , the requi red " p " values for s igni f icance are 0 . 0 0 9 ( * ) , 0 . 0 02 ( * * ) and 0 . 0 0 0 2 ( * * * ) . For treatment leve l s , s ince three compari sons are made the required " p " va lues for s i gni f icance are 0 . 0 1 7 ( * ) , 0 . 0 0 3 ( * * ) and 0 . 0 0 0 3 ( * * * ) . RTC Study No. : 52420 Page 26 7 8 5 0 : CHROMOSOME ABERRATI ONS TABLE 9 - Stat i s t ical Analys i s - Wi thout metabo l i c ac t iva t i on STUDY NO . : 5 2 4 2 0 MAIN AS SAY : 2 SOLVENT : ETHANOL TREATMENT TIME : 2 0 hours SAMPLING T IME : 2 0 hours Comparison wi th Negat ive control Cel l s wi th aberrations ( + gaps ) ( - gaps ) Dose g/ml CULT . P . values Sig . P . values Sig . Tes t I tem Test Item Test Item Al l dose level s Mitomyc in-C 133 95 96 Pooled 200 93 94 Pooled 300 91 92 Pooled 0 . 10 105 106 Pooled 0 . 5707 0 . 1295 0 . 2245 0 . 5707 0 . 3484 0 . 5000 0 . 3490 0 . 0625 0 .2873 0 . 5670 0. 0000 0. 0000 0 . 0000 N.S. N.S. N.S. N. S . N. S . N. S . N. S . N.S. N. S . N.S. *** *** *** # # # # # # # # # # 0 . 0000 0 .0000 0 . 0000 N.S. N.S. N. S . N.S. N.S. N.S. N.S. N.S. N.S. N. S . *** *** *** In thi s tabl e correc t i on i s made for the mul t iple c ompari sons o f the test i tem treatments with the negat ive contro l s . For individua l cul tures , s ince s ix compar i s ons are made , the required " p " values for s igni ficance are 0 . 0 0 9 ( * ) , 0 . 0 02 ( * * ) and 0 . 0 0 0 2 ( * * * ) . For treatment l eve l s , s ince three compari s ons are made the required " p " va lues for signi f icance are 0 . 0 17 ( * ) , 0 . 0 0 3 ( * * ) and 0 . 0 0 0 3 ( * * * ) . RTC Study No. : 52420 Page2 7 7 8 5 0 : CHROMOSOME ABERRATIONS TABLE 10 - Summary Tabl e STUDY NO . : 5 2 4 2 0 MAIN ASSAY : 1 SOLVENT : ETHANOL TREATMENT TIME : 3 hours SAMPLING TIME : 20 hours Presence of S9 metabo l i sm Treatment Dose .g/ml %CA ( Re l . ce l l growth ) Absence o f S 9 metabol i sm %CA ( Rel . cell growth ) Un t re a t e d S o lvent Tes t I tem Te s t I t em Tes t I t em Mitomyc in-C Cyc l ophosphamide 1% 78 . 1 156 313 0.30 15 . 0 0.0 106) 0.0 100) 1 .5 N.S. ( 101) 1 . 5 N. S . ( 85) 3.0 * 77 ) 41 . 0 *** ( 69) 0.5 107 ) 0.5 100) 0 . 0 N. S. ( 83) 1 . 0 N. S . ( 81) 1 . 0 N .S . ( 75) 41 . 5 *** ( 75) Key : % CA Rel . cell * ** *** Percentage o f cel l s bearing aberrat i ons ( exc luding gaps ) growth : Relative to solvent control s ( percent ) Not tested or not selected for the scoring of aberrations Stat i s t ica l ly s ign i f icant at P< 0 . 0 5 Stat i s t ica l ly s i gni f icant at P< 0 . 0 1 Stat i s t i ca l ly s igni f icant at P< 0 . 0 0 1 RTC Study No. : 52420 Page 28 7 8 5 0 : CHROMOSOME ABERRATIONS TABLE 1 1 - Summary Table STUDY NO . : 5 2 4 2 0 MAIN AS SAY : 2 SOLVENT : ETHANOL TREATMENT TIME : 2 0 hours SAMPLING TIME : 2 0 hours Presence of S9 metabo l i sm T r e a tme n t Dose .g /ml %CA ( Re l . ce l l growt h ) Abs ence o f S 9 metabo l i sm %CA ( Rel . cell growth ) Unt reated So lven t Tes t I tem Test I tem Tes t I t em Mitomyc in-C 1% 133 200 300 0 . 10 0.0 142 ) 0.0 100) 0 . 0 N. S . ( 93 ) 0 . 0 N. S. ( 82) 0 . 0 N. S . ( 58) 15 . 5 *** ( 50) Key : % CA Rel . cell * ** *** Percentage o f cel l s bearing aberrat i ons ( excluding gap s ) growth : Relat ive to s olvent contro l s ( percent ) Not tes ted or not selected for the scoring o f aberrat i ons Stat i s t ically s i gn i f i cant at P< 0 . 0 5 Stat i s t i c a l ly s i gni f i cant at P< 0 . 0 1 Stat i s t ical ly s i gni f i cant at P< 0 . 0 0 1 RTC Study No. : 52420 Page 29 10. APPENDIX I - Historical background incidences of aberrant cells CHROMOSOME ABERRATIONS IN CHINESE HAMSTER OVARY CELLS Background incidences (%) of aberrant cells (Years 1990-2005) Mean SD (crn-1) n Minimum Maximum Mean SD (crn-1) n Minimum Maximum Mean SD (crn-1) n Minimum Maximum I Absence of S9 Presence of S9 metabolism 20 hours sampling time 20 hour sampling time + 2aps I - gaps + gaps - gaps UNTREATED 1 .6 0.5 2.0 0.5 1.5 0.7 1 .7 0.6 68 68 47 47 0.0 0.0 0.0 0.0 6.0 3.0 7.0 2.5 SOLVENT 1 .8 0.6 1 .9 0.7 1 .6 0.8 1 .7 0.9 51 51 35 35 0.0 0.0 0.0 0.0 8.0 4.0 8.0 4.0 POSITIVE 41.1 39.1 37.6 35.4 1 5 .2 1 4 .4 14.8 14.7 71 71 49 49 15.0 14.5 15.5 15.5 77.0 76.0 67.0 66.0 SD = standard deviation n = number of experiments RTC Study No. : 52420 Page30 11. APPENDIX Il - Study Protocol RTC Study No. : 52420 Page 31 '111Y NINIAL\NN rol 11 RESEARCH TOXICOLOGY CENTRE ROMA Version 05/1 7850 CHROMOSOME ABERRATIONS IN CHINESE HAMSTER OVARY CELLS IN VITRO Final Protocol prepared for SOLVAY SOLEXIS S.p.A. Viale Lombardia, 20 20021 Bollate (MI) Italy by RESEARCH TOXICOLOGY CENTRE, S.p.A. Via Tito Speri, 12 00040 Pomezia (Roma) Italy RTC Enquiry No. 52420 Conmestelal *env. RTC S.p.A. Via Tito Spen. 12 00040 Poraatia (Roma) - ITALY Tel.: .39.06.91095.1 Pax: 39.06.910.5737 RTC Study No.: 52420 - 1 of 14 - Hood Oft mod Aelkielodsbstion RTC RDA. Via Tito SP110. 12 00040 Pornazia (Roma) - ITALY TaL 39.06 91095.1 Faun 39 08.912.2233 P.O.Box 16301-00143 - Roma Elarlsurenlino January 2006 RTC 9.9A. Capita* socials Euro 5.164.000 C.CJAA_ n' 375378 Rap. Soc. Trib. dl Roma 2828/72 Cod. Fla..: 00653120554 Panda NA: 00920611001 Page 32 Version 05/1 CHROMOSOME ABERRATIONS IN CHINESE HAMSTER OVARY CELLS IN VITRO MANAGEMENT OF STUDY Scientific Director Head of Genetic and Cellular Toxicology Study Director Sponsor SOLVAY SOLEXIS S.p.A. Viale Lombardia, 20 2002 1 Bollate (Ml) Italy Monitor QUALITY ASSURANCE Quality Assurance Manager LOCATION OF STUDY The study will be performed at Research Toxicology Centre, S.p.A. Via Tito Sperl, 1 2 00040 Pomezia, ROMA. Italy The laboratory facilities, archives and administration are located at this site. TIME SCHEDULE OF STUDY The study will be conducted with a time schedule agreed between the Sponsor and RTC. TEST ITEM IDENTITY The test itern will be 850 RTC Enquiry No. 52420 RTC Study No. : 52420 - 2 of 14 January 2006 Page 33 Version 05/1 CHROMOSOME ABERRATIONS IN CIDNESE HAMSTER OVARY CELLS IN VITRO 1. INTRODUCTION 1.1 Objective To assay the test item for the ability to induce chromosome aberrations in Chinese hamster ovary (CHO) cells, after treatment in the presence and absence of S9 metabolism. 1 .2 Regulatory requirements This study will be conducted in compliance with the GLP regulations of: US FDA [21 CFR part 58, 22 December 1 978] and subsequent revi;ions; Directive 2004/10/EC of the European Parliament and of the Council of 1 1 February 2004; ENV/MC/CHEM(98) 1 7 "OECD principles on Good Laboratory Practice - as revised in 1 997"; Decreto Legislativo no. 120 of 27 January 1 992 and subsequent revisions. In addition, the study is designed following the experimental methods indicated in the guidelines of: EEC Council Directive 2000/32, Annex 4A. OECD Guideline No. 473 (adopted July 1 997). 1 .3 Principles of the assay This assay detects structural changes in the chromosomes of cultured mammalian cells induced by test agents. The Chinese hamster ovary cell line is useful for this work because of its stable aneuploid karyotype (modal number is 2 1 chromosomes), short cell cycle (1214 hours) and its high plating efficiency. The cell line is derived from an explant of the ovary of the Chinese hamster (Cricetulus griseus, 2n = 22). In the assay method, exponentially growing cultures of these cells are exposed to the test substance. Since the cell line has lost much of its ability to metabolise indirect mutagens to reactive forms, the test is performed both in the presence and in the absence ofan S9 metabolising system. The cells are exposed to the test item both in the absence and presence of metabolic activation for a short treatment time (3 hours) and post treatment mitosis are harvested for analysis at a time corresponding to approximately 1 .5 cell cycle lengths (20 hours) to allow detection of chemicals which may cause cell cycle delays. The spindle poison colcemid is used to arrest the cells in metaphase. The cells are subsequently brought into suspension and air-dried slide preparations are made and stained. The chromosomes in the metaphase spreads are examined by microscopy for evidence of chromosomal damage. RTC Enquiry No. 52420 RTC Study No. : 52420 - 3 of 14 - January 2006 Page 34 Version 05/1 Clear positive results with and/or without S9 metabolism will not be confirmed in further experiments. If negative or equivocal results both with and/or without S9 metabolism are obtainep, an additional experiment will be perfonned with continuous treatment in the absence of metabolic activation and harvest time at approximately 1 .5 cell cycle lengths. Any additional experiment will be performed following discussion with the Sponsor. 2. TEST ITEM 2.1 It is the responsibility of the Sponsor to supply the test item, accompanied by analytical data confirming the identity, purity, stability, strength and composition of the item, the solubility and stability in the proposed vehicle and details of any known hazards to laboratory staff. The test item should be accompanied by a certificate of analysis. 2.2 Approximately one year after the submission of the final report, remaining amounts of the test item will be destroyed by incineration. An aliquot of the item will be retained within the archives of the testing facility for a period of ten years after which it will be destroyed. 2.3 The test item identity is indicated on previous pages of this protocol. 2.4 Unless otherwise indicated by the Sponsor, the storage conditions for the test item will be room temperature. 2.S The precautions necessary when handling either the test item or prepared formulations of the test item are based on information supplied by the Sponsor. The minimum safety precautions necessary are detailed under the RTC Hazard Classification System, according to RTC standard procedures. 2.6 The amount of test item received and used will be recorded according to standard procedures. 2.7 Fresh solutions of the test item will be prepared for each day's work; solutions will be prepared on a weight/volume basis without correction for the displacement due to the volume occupied by the test item. Unless specified by the Sponsor, concentrations of solutions will be expressed in terms of material as received, and not of active constituents. Preferred solvents will be sterile distilled water, culture medium, DMSO, ethanol, acetone. Other solvents may be used as necessary. 2.8 No assay of the test item stability, nor its concentration and homogeneity in the solvent/vehicle will be undertaken, nor samples of formulated test item consigned to the Sponsor, without express instructions from the Sponsor. No determination of the absorption of the test item in the test system will be made without express instructions from the Sponsor. RTC Enquiry No. 52420 RTC Study No. : 52420 - 4 of 14 January 2006 Page 35 Version 05/1 3. MATERIALS AND METHODS 3.1 Chinese hamster ovary (CHO) cells Chinese hamster ovary cells were obtained from Dr. A.T. Natarajan (State University of Leiden). This cell line derives from the CHO isolate originally described by Kao and Puck (1 968). The karyotype, generation time and plating efficiency have been checked in this laboratory. The cells are checked at regular intervals for the absence ofmycoplasmal contamination. Permanent stocks of CHO cells are stored in l iquid nitrogen, and subcultures are prepared from these stocks for experimental use. Cultures are grown in Ham's Fl0 medium supplemented with 15% Newborn Calf serum. All incubations are at 37C in a 5% carbon dioxide atmosphere (100% humidity nominal). Culture flasks are labelled during subculturing and experimental use with their identity and appropriate codices. 3.2 Culture medium The medium used for the growth ofthe cells has the following composition: Ham's F.10 Streptomycin sulphate 50 mg/ml Penicillin G 50,000 ID/ml Newborn Calf Serum 499.0 ml 1 .0 ml 88.2 ml 3.3 S9 mix The S9 l iver tissue fraction will be prepared according to RTC standard procedures or will be obtained from an appropriate supplier (MOLTOX, Molecular Toxicology, Inc., USA). Induction of drug metabolising enzyme-levels is routinely performed using phenobarbitone and betanaphtoflavone (mixed induction). Induction with Aroclor 1 254 will be performed if specifically requested by the Sponsor. Records pertaining to the preparation ofthe S9 tissue fraction are kept on file at RTC. The mixture of S9 tissue fraction and cofactors (S9 mix) will be prepared in the following proportions: S9 tissue fraction NADP (0.l M) G-6-P (0.lM) KCl (0.33M) MgC12 (0. lM) Hepes (0.2M) H .B .S.S. 3.0 ml 0.4 ml 0.5 ml 1 .0 ml 0.4 ml 1 .0 ml 3 .7 ml 1 0.0 ml RTC Enquiry No. 52420 RTC Study No. : 52420 - 5 of 14 - January 2006 Page 36 Version 05/1 3.4 Control items Positive control treatments are included in every experiment. The positive control items, Mitomycin-C and Cyclophosphamide, are obtained commercially and characterised by their labelling. Fresh solutions in sterile distilled water will be prepared for each day's work. Determination of the stability and concentration of solutions of these agents will not be undertaken since it is sufficient to provide evidence for the correct expected response of the test system to them. 4. SELECTION OF DOSE LEVELS 4.1 Selection of dose levels for treatment The highest dose-level to be used is determined according to the solubility of the test item in the culture medium and solvent vehicle, but will not exceed a maximum concentration of 5 mg/ml. Up to two dose-levels at which precipitation is observed may be included in the treatment series. Seven lower dose-levels will be used, spaced at approximately equal intervals. If an additional experiment is performed, dose-levels will be selected on the basis of the results obtained in the first experiment. Space intervals will be selected according to the results obtained (e.g. toxicity) in the first experiment. Positive control treatments will be Mitomycin-C in the absence of S9 metabolism and Cyclophosphamide in the presence of S9 metabolism, both at the appropriate concentrations to give a clear positive response. Each experiment will include also solvent vehicle and untreated control cultures. Duplicate cultures are prepared for each experimental test point. When it seems advisable, further testpoints or controls may be included in the experiment. 5. ASSAY PROCEDURE 5.1 Preparation of the test cultures Approximately 20 hours before treatment an appropriate number of flasks for the experiment are prepared from a single pool of cells. Each 25 sq.cm. flask is seeded with 300,000 cells in supplemented Ham's Fl0. These cells should be in exponential growth phase at the time of treatment. The following section describes treatment procedures which will be followed in the first experiment. RTC Enquiry No. 52420 RTC Study No.: 52420 - 6 of 14 - January 2006 Page 37 Version 05/1 5.2 Treatment ofthe cultures Treatment medium without metabolic activation is prepared for each test point in the following proportions: Test item or control solution Supplemented Ham's Fl0 medium 0.05 ml 4.95 ml Treatment medium with metabolic activation is prepared containing the test item and S9 mix in the following proportions: Test item or control solution S9 mix Supplemented Ham's FI O medium 0.05 ml 0.50 ml 4.45 ml Both in the absence and presence of S9 metabolism, the cultures are incubated in this treatment media for three hours. The medium is then removed and the flasks are washed twice with Ca/Mg-free Phosphate Buffered Saline (PBS). Fresh medium is added and the cultures are incubated for a further 17 hours (Recovery Period). Colcemid (0.2 g/ml final concentration) is added for the last three hours of the recovery period, leading up to harvesting. In this way cultures are prepared for harvesting at 20 hours after treatment commenced. Where it is necessary to adjust the composition of the treatment medium (for example, where culture medium is used as the solvent), the final composition of the treatment medium will be indicated in the Final Report. 5.3 Additional experiment If negative or equivocal results both with and/or without S9 metabolism are obtained, an additional experiment is performed with continuous treatment and a harvest time at approximately 1 .5 cell cycle lengths. This experiment is conducted only in the absence of S9 and treatment medium is prepared for each test point as described in section 5.2. The treatment media are added to the tubes and the cultures are incubated for twenty hours. Colcemid is added (0.2 g/ml final concentration) for the last three hours of the treatment period, leading up to harvesting. Cells are harvested at twenty hours after beginning oftreatment. Negative results with metabolic activation need to be confirmed on a case-by-case basis. In those cases where confirmation of negative results is considered necessary, an additional experiment will be carried out following approval of an amendment to the protocol. If there is evidence of a cell cycle delay, the clastogenic potential may be readily detected by treatment/sampling times longer than 1 .5 cell cycle lengths. In this case an additional experiment may be performed after discussion with the Sponsor. The performance of this experiment will be carried out following approval of an amendment to the protocol. RTC Enquiry No. 52420 RTC Study No. : 52420 - 7 of 14 January 2006 Page 38 Version 05/1 5.4 Osmolality and pH measurement For each experiment, the osmolality and pH of treatment media (at the higher dose-levels of treatment), with and without S9 metabolic activation will be checked. If considered appropriate, data will be presented in the Final Report. 5.5 Harvesting and slide preparation The medium is removed from the flasks, and the cells are brought into suspension by trypsinization. The cell suspension is centrifuged and the cell pellet is resuspended in hypotonic solution. The cells are then fixed in freshly prepared methanol:acetic acid fixative and washed two times with fixative. A few drops of the cell suspension obtained in this way are dropped onto clean, wet, grease-free, glass slides and air-dried to produce metaphase chromosome spreads. At least three slides will usually be prepared, each labelled with the identity of the culture. The slides are stained in 3% Giemsa in tap water and rinsed in tap and distilled water. The slides are then made permanent with Eukitt. 5.6 Selection of dose-levels for scoring of chromosomal aberrations At the time ofharvesting, cell counts will be performed for each culture. The highest dose-level selected for scoring should show a significant reduction in cell count. Ideally the reduction should be approximately 50% of the control and treatments reducing cell count values to below 20% of the control should not be used. If the test item does not induce toxicity at any dose-level, then the highest treatment level will be selected as the highest dose-level for scoring. Two lower dose-levels will be also selected for the scoring of chromosomal aberrations. The lowest dose-level for scoring should be on the borderline of cytotoxicity. The intermediate dose-level will be evenly spaced between the two. Where it seems advisable, the mitotic index (number of metaphases per 1000 cells) will also be determined in order to have additional information on the cytoto,licity of treatment. 5.7 Metapbase analysis The slides are randomly assigned code numbers by a person not subsequently involved in slide evaluation, and any other identification marks are concealed. Metaphase spreads judged to be of sufficient quality to permit scoring are examined at high magnification. Metaphases that differ from the modal chromosomal complement by more than two centromeres are not scored. Polyploid and endoreduplicated cells encountered will be recorded, but not included in the count of eligible metaphases. From 1 00 eligible metaphases per test culture, the number of chromosomes, the specific types and numbers of aberrations are recorded. The Vernier readings of aberrant or equivocal metaphases are recorded. RTC Enquiry No. 52420 - 8 of 1 4 - January 2006 RTC Study No. : 52420 Page 39 Version 05/1 If more than 50% of the cells are found to contain aberrations (other than gaps), then at the discretion of the Study Director, scoring for that culture may be terminated at 50 metaphase spreads only. Where it seems advisable, additional metaphases may be scored using microscope slides that have not previously been examined. These slides will be re-coded prior to scoring. 6. REPORTING 6.1 Presentation of data The data is presented in the form of tables giving frequencies of chromosome exchanges and deletions, chromatid exchanges and deletions, gaps, and other anomalies (polyploid cells, endoreduplication, seriously damaged cells etc.). The total number of aberrant metaphases (including and excluding gaps), the relative cell counts, and the number of metaphases analysed for each test culture are also shown. These are all tabulated by individual treated cultures, and by dose-level groups. The modal number of chromosomes in the cell line will be recorded together with the distribution ofchromosome numbers. 6.2 Statistical evaluation of the data The numbers of cells bearing aberrations in the control and treated cultures are compared using Fisher's exact test. The comparison is performed both including and excluding gaps from the aberration counts. The values obtained at all the treatment dose-levels combined are compared with the control values. Since multiple dose-levels are compared with the negative controls, the problem of Type I error (chance 'positive' results) arises. Accordingly, significance levels for each treatment-level will also be presented after application of Bonferroni's correction. The solvent/vehicle controls will be used as the reference point for comparison in the statistical evaluation and the evaluation of the results. 6.3 Evaluation ofthe results For a test item to be considered clastogenic, four criteria must be met: (i) Increases over the concurrent controls If any dose-level shows a statistically significant increase in aberration-bearing cells, this will be considered as evidence of a clastogenic effect. (ii) Increase over historical controls If the increases fall within the range of values normally observed in the negative control cultures, the test item cannot be judged clastogenic. Any significant increases over the concurrent solvent/vehicle controls will therefore be compared with historical control values derived from recent studies. The historical data-base will be included as an appendix in the Study Report. RTC Enquiry No. 52420 RTC Study No. : 52420 - 9 of 1 4 - January 2006 Page 40 Version 05/1 (iii) Reproducibility Any increases observed must be present in both replicate cultures. (iv) Biological sgnificance The conclusions must be consistent with the underlying biology of the assay. Specifically: - It is not required that an increased response is observed at increasing dose-levels, but dose-related activity will be taken as further evidence of a clastogenic effect. - The types of aberrations observed will be taken into consideration. - An increased incidence of an aberration type with an exceedingly low background frequency will be considered as evidence of a clastogenic effect. The variability inherent in biological material may inevitably lead to the generation of equivocal data sets [eg. (i) dose-related increases over concurrent controls that do not exceed historical values or (ii) increases over historical values which are not significantly greater than concurrent controls]. In such cases it is strongly recommended that further evaluation should be undertaken in the form ofa further experimental treatment of the relevant treatment series and scoring. 6.4 Reporting procedure A Draft Report will be supplied, and a Final Report issued subsequently to include any . agreed changes or amendments. If any corrections or additions are required to the Final Report, these will be in the form of an amendment by the Study Director. The amendment will clearly identify that part of the Final report that is being added to or corrected, and the reasons for the changes, and will be signed and dated by the person responsible. 6.5 Final Report The following information and data will be included in the final report: - name and address of the facility performing the study and the dates on which the study was initiated and completed; - objective and procedures stated in the approved protocol, including approved changes to the original protocol; - the test item, identified by name, chemical name or chemical number; - method used; - data generated while conducting the study; - statistical methods employed for analysing the data; RTC Enquiry No. 52420 - 1 0 of 14 - January 2006 RTC Study No. : 52420 Page 41 Version 05/1 - a summary of the data, an analysis of the data and a statement ofthe conclusions drawn from the analysis; - any unforeseen circumstances that may have affected the quality or integrity of the study; - historical solvent/vehicle control data; - the name and signature ofthe Study Director; - the location where all raw data, specimens and final report are to be stored; - Quality assurance statement. One original unbound, one copy bound and a PDF version will be supplied. 6.6 Records kept Full records will be maintained of all aspects of study conduct, along with the results of all measurements and observations. Prior to final archiving of the study data a full list will be prepared of all records associated with the study. 6.7 Archiving All raw data, records and documentation arising from this study and a copy of the final report consigned, generated during the course of this study will be retained at RTC. Archiving will be provided for a period of 3 years after which the Sponsor will be contacted for instructions regarding despatch or disposal of the material. As a further option, archiving space can be rented for an additional time. The signed Final Protocol and the top copy ofthe Final Report will be despatched to and archived by the Sponsor. 7. STUDY CONDUCT 7.1 Language English language and Italian language version of the study protocol, Standard Operating Procedures and other study documents may be used interchangeably. Similarly, English and Italian renderings of chemical names, including that of the test item will be considered to be equivalent. 7 .2 Scientific decisions The procedures described in this protocol may not comprehensively cover all the circumstances that can arise in the assay of test item. When the study director considers it advisable to modify the procedures described for the selection of a solvent, selection of dose-levels, interpretation ofthe outcome ofthe study or other aspects of the study conduct, he/she will record carefully the decision he/she has reached and the reasoning which led to it. RTC Enquiry No. 52420 RTC Study No.: 52420 - 1 1 of 14 - January 2006 Page 42 Version 05/1 7.3 Quality assurance The study is subjected to the procedure for quality assurance as defined by the relevant GLP regulations. Specifically: - the protocol is inspected for compliance; - procedures ofthe laboratories concerned will be inspected at intervals adequate to assure the integrity of the study; - the final report is reviewed to ensure that it accurately describes the methods and relevant Standard Operating Procedures and that the results are in agreement with the raw data; - periodic reports on these activities are made to management and the Study Director. All raw data pertaining to the study are available for inspection by the Study Monitor (for scientific monitoring) or the Quality Assurance Unit of the Sponsor (compliance monitoring). 8. DEPARTURES FROM REGULATORY REQUIREMENTS Items which are the responsibility of the Sponsor are indicated in sections 2. 1 , 2.4, 2.7, 2.8 and 3.3 ofthis protocol. Since full compliance with regulatory requirements may depend on the performance of some of these items, the Sponsor should ensure that appropriate actions are initiated or undertaken. RTC Enquiry No. 52420 RTC Study No. : 52420 - 12 of 14 - January 2006 Page 43 Version 05/1 9. REFERENCES Galloway S.M. et al. (1 994) Report from working group on in vitro tests for chromosomal aberrations Mutation Research, 3 12 241-261 Hsu T.C. et al. (1 977) Cytogenetic assays of chemical clastogens using mammalian cells in culture. Mutation Research 45 233-247 Kao F.T. and Puck T.T. (1 968) Genetics ofSomatic mammalian cells VII. Proceedings ofthe National Academy of Science (USA) 60 1275-128 1 Preston R.J. et al. (1981). Mammalian in vivo and in vitro cytogenetic assays: A report ofthe US EPA's Gene-tox program Mutation Research 87 143-1 88 Scott D. et al. (1 990) Metaphase chromosome aberration assay in vitro in: Basic mutagenicity tests: UKEMS recommended procedures D.J. Kirkland (editor), Cambridge University Press, 62-86 Seeberg A.H. and R. Forster (1988). Testing above the limit of solubility. Environmental Mutagenesis 11 (S1 1) 92 RTC Enquiry No. 52420 RTC Study No. : 52420 - 1 3 of l4 - January 2006 Page 44 STUDY TITLE TEST FACILITY RTC ENQUIRY NO. TEST ITEM Version 05/1 PROTOCOL APPROVAL PAGE CHROMOSOME ABERRATIONS IN CHINESE HAMSTER OVARY CELLS IN VITRO RESEARCH TOXICOLOGY CENTRE S.p.A. Via Tito Sperl, 1 2 00040 Pomezia (RM) Italy 52420 7850 APPROVED BY Study Director RELEASED BY SPONSOR AUTIIORISED BY SPONSOR icology SOLVAY SOLEXIS S.p.A. Viale Lombardia, 20 2002 1 Bollate (MI) Italy Name and Title Please print or type your name and company status below your signature RTC Enquiry No. 52420 RTCStudy No. : 52420 - 14 of 14 - January 2006 Page 45 12. APPENDIX m - Certificate of analysis RTC Study No. : 52420 Page46  COLOHBO_I Page FLK785J_32230N_ PFPE e-.p2 s2pul SAMPLE d&t.e NOY 30 2000 aolv.nt Acetone file /apaee/MYFIDS- /COLQIBOJ/i'LK78SO- _32230N_001.fid ACQUISITION sfrq 282. 116 tn Fl 9 at 0 . 500 np 75170 75188.0 fb 41400 .. 10 tpwx 61 pw 41 . 5 dl 2 . 000 tof 2400.0 nt 1000 et 1000 .J.oc.C n gain not 1.1aed FUGS il "'dp DISPtAY -41SU6.6 .. 39502.5 .. 0 2,0 lum 158.01 io 03.57 rn 22965.4 rfp -39578 .8 th 0 in 140.HS i ph -......DEC. , VT dfrq 299.917 dn Hl dpwr 30 dof oia mllll Wlf 200 PROCESSING lb 2.00 lsfid -1 wt.file proc Lp fn 131072 .1:eact procplot _, IS=484 FLK785 0_3223ON_00 1 . jpeg MOL . WEIGHT 551 EQU I V . WEIGHT 551 Cl-1 . 0 "I. C3 C2=9 .1 1. CJ " -OCF2O-" "-OCF (CF3) o- -OCF3 = -OCFCF3COOH -OCF2CL = - -OCF (CF)) CF2CL = -OCF2CF (CF3) CL = -OCF2COOH = 3.3 0.8 1.1 15 . 8 29. 7 49. 2 [!] IBOLLATE jSOLEXIS COLOMBO_! Page FLK7850_3223ON_ PFPE Pulse Sequence : s2pul I I I I I I I I I I '-o '---r--130 -80 -130 -140 - 1 5 0 ppm 0 . 32 1. 74 120 . 00 18 . 50 -t... -o . oo -0. 19 0 . 51 '-l 13. APPENDIX IV - S9 production of quality control certificate RTC Study No. : 52420 Page 48 MOLTOXTM POST MITOCHONDRIAL SUPERNATANT (S-9) PRODUCTION & QUALITY CONTROL CERTIFICATE LOT NO.: 187 SPECIES: Rat PREPARATION DATE:,,Iont 09, 2005 PART NO.: 11-105 STRAIN: Sorazue Dawlev EXPIRATION DATE: lot 09` 2007 VOLUME: 5 ml TISSUE: Liver REFERENCE: Matsushima, et, al., In: In Vitro MetaboliQ BUFFER: 0,154MKOI INDUCING AGENT(s): phengtgrtile - 1.7.12enzsfilys2rie Activation in Mutagennii Malin fEJ, de Scats, Ed.). Elnyier, 1976, o, 85, STORAGE: At or below-70C BIOCHEMISTRY: PROTEIN . 37.2mg/ml Assayed according to the method of Lowry et al., JBC 193:265, 1951 using bovine serum albumin as the standard. ALKOXYRESORUFIN-0-DEALKYLASE ACTIVITIES Fold Activity P450 EROD IA I , IA2 indmstisn 101.3 Assays for ethoxyresorufin-0-deethylase (EROD), pentoxy-, benzyl- and methoxyresorufin-0-dealkylases (PROD, BROD, & PROD 2B1 21.0 MROD) were conducted using a modification of the methods of Burke, et al., Biochem Pharm 34:3337, 1985. Fold- BROD 2B1 35.6 inductions were calculated as the ratio of the sample vs. uninduced specific activities (SA's). Control SA's (pmoles/min/ MROD 1A2 20.3 mg protein) were 43.8, 20.6, 64.4, & 15.5 for EROD, PROD, BROD and MROD, respectively. BIOASSAY: TEST FOR THE PRESENCE OF ADVENTITIOUS AGENTS Samples of S-9 were assayed for the presence of contaminating microflora by plating I.0 ml volumes on Nutrient Agar and Minimal Glucose (Vogel-Bonner E, supplemented with 0.05 mM L-histidine and D biotin) media. Triplicate plates were read after 24 - 48 h incubation at 35C. The tested samples met acceptance criteria. - PROMUTAGEN ACTIVATION No. his+ Revertants EtBr/ CPA/ TA98 TA1535 302.4 2222 The ability of the sample to activate ethidium bromide (EtBr) and cyclophosphamide (CPA) to intermediates mutagenic to TA98 and TA1535, respectively, was determined according to Lesca, et al,, Mutation Res 129:299, 1984. Data were expressed as revertants per g EtBr or per mg CPA. Dilutions of the sample S9, ranging from 0.2 - 1.0% in S9 mix, were tested for their ability to activate benzo(a)pyrene (BP) and 2-aminoanthracene (2-AA) to intermediates mutagenic to T A100. Assays were conducted using duplicate plates as described by Maron & Ames (Mutat. Res.113:173, 1983). l S9 oer olate/naliber his' revertants oer plate Promutazep 0 14 2.4 54 BP (5 g) 116 158 279 406 527 608 2-AA (2.5 g) 128 200 426 1621 1613 864. MOLECULAR TOXICOLOGY, INC. 157 Industrial Park Dr, Boone, NC28604 RTC Study No.: 52420 Page 49