Document DMy11EdpJy14JZRQJQ8L0D8Bn

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 Sterility 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 1-4 ............................................................................................................................. 16 8. KEY TO TABLES 5-8.............................................................................................................. 21 9. KEY TO TABLES 9-14............................................................................................................ 26 10. APPENDIX I - Study Protocol and First Amendment to Protocol ........................................... 33 11. APPENDIX II - Certificate of analysis ..................................................................................... 52 12. APPENDIX III - Historical control data ................................................................................... 54 13. APPENDIX IV - S9 production and quality control certificates .............................................. 55 RTC Study No.: 76240 Page 4 1. SUMMARY 1.1 The test item C6O4 CYCLIC 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 main experiments for chromosomal damage were performed. In the first experiment, the cells were treated for 3 hours with C6O4 CYCLIC in the presence and absence of S9 metabolism. The cells were then harvested after 20 hours, corresponding to approximately 1.5 cell cycle. Dose levels of 5000, 2500, 1250, 625, 313, 156, 78.1, and 39.1 g/ml were used in the absence and presence of S9 metabolism. Solutions of the test item were prepared in sterile distilled water of injectable grade. Since no induction of chromosomal aberrations were obtained in the first main assay, in agreement with the study protocol, a second main experiment was performed where cells were treated with C6O4 CYCLIC in the absence of S9 metabolism and harvested after 20 hours. A continuous treatment until harvest was used. Besides, in the first main experiment, following treatment with the test item in the presence of S9 metabolism, marked increases in the number of endoreduplicated cells over the concurrent negative control were observed. Since no adequate toxicity was observed at any dose level for the evaluation of genotoxic effects, an additional treatment was performed in order to confirm the observed results following the approval of a protocol amendment. A modified dose range was adopted for this treatment in order to investigate more closely those doses most likely to exhibit endoreduplication. For the second main experiment, the dose levels selected for treatment were 2000, 1330, 889, 593, 395, 263, 176 and 117 g/ml. Both experiments 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 as determined by the reduction of population doubling. On the basis of the above described results, the dose levels referred to active ingredient selected for scoring were the following: Experiment S9 No. 1 + - + 2 - Treatment time (hours) 3 3 20 Harvest time (hours) 20 20 Dose level (g/ml) 1250, 625 and 313 2000, 1330 and 889 889, 593 and 395 One hundred metaphase spreads were scored for chromosomal aberrations from each culture, with the exception of one replicate culture treated with Cyclophosphamide (first main experiment), where 75 metaphases were examined. In addition, for the first main experiment, due to the high frequency of aberrant cells (excluding gaps), found for replicate cultures treated with Mitomycin-C, scoring was terminated at 50 metaphases. RTC Study No.: 76240 Page 5 1.4 For the first experiment, following treatment with the test item C6O4 CYCLIC, no statistically significant increase in the incidence of cells bearing aberrations including or excluding gaps was observed in the absence or presence of S9 metabolism. In the presence of S9 metabolism, marked increases in the number of endoreduplicated cells over the control were observed. An increase in the number of endoreduplicated cells was also observed in the absence of S9 metabolism at the highest dose selected for scoring. For the second experiment, following treatment with the test item in the absence of S9 metabolism, slight increases but not statistically significant in the incidence of cells bearing aberrations, excluding gaps, over the control values were observed at all dose levels selected for scoring. More remarkable increases of aberrant cells were observed when including gaps. In the presence of S9 metabolism, statistically significant increases in the incidence of aberrant cells including and excluding gaps were observed at the highest dose selected for scoring (2000 g/ml). The incidences exceeded the range of our historical values for negative controls when excluding gaps. Marked increases in the number of endoreduplicated cells over the controls were seen at the intermediate and high dose levels selected for scoring. 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. 1.5 On the basis of these results it is concluded that C6O4 CYCLIC induces chromosomal aberrations in Chinese hamster ovary cells after in vitro treatment under the reported experimental conditions. It is also concluded that the test item inhibits cell cycle progression and chromosome segregation under the reported experimental conditions. RTC Study No.: 76240 Page 6 2. INTRODUCTION 2.1 Purpose This report describes the experiment performed to assess the clastogenic activity of C6O4 CYCLIC 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: - Test method B.10 in vitro mammalian chromosome aberration test described in Council Regulation (EC) No. 440/2008. - 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 Toxicology, in vitro Toxicology and Immunology Department Research Toxicology Centre S.p.A. (RTC) Via Tito Speri, 12/14 00040 Pomezia (Roma) Italy Principal dates Study protocol approved by Study Director: 09-Mar-2009 Study commenced: 17-Mar-2009 (Main assay I treatment) Study completed: 08-Jul-2009 (Main Assay II completion of scoring) Personnel involved in the study Study Monitor: Dr I. Colombo Study Director: P. Ciliutti, Biol. D. Scorers of slides L. Caserta, Lab. Tech. A. Dello Russo, Lab. Tech. S. Duca, Lab. Tech. C. Marabottini, Biol.D. Archiving RTC Study No.: 76240 Page 7 The original data arising from this study, microscope slides, the original final protocol and a copy of the final report consigned will be stored in the archives of Research Toxicology Centre S.p.A. for a period of 3 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. An aliquot of the test item will be retained within the archives of the testing facility for a period of 10 years after which it will be destroyed. RTC Study No.: 76240 Page 8 3. MATERIALS AND METHODS 3.1 Test item Details of the test item received at RTC were as follows: Name : Label name : Batch : Expiry date : Received from : Date received : Amount received : Description from the Sponsor : Description at first use : Container : Storage at RTC : RTC reference number : C6O4 CYCLIC cC6O4 (solution 16%) 150/28 31-Dec-2020 Solvay Solexis S.p.A. 05-Mar-2009 337.94 ml yellowish solution yellowish liquid colourless glass bottle room temperature 11663 On 16 March 2009 and 27 March 2009 two sub-samples of 7 ml and 5ml, respectively, of the test item were transferred from the Formulation Unit to the Department of Genetic Toxicology, in vitro Toxicology and Immunology and stored under the same conditions. Solutions of the test item, as received, were prepared immediately before use in sterile distilled water. Solutions were prepared on a weight/volume basis. Concentrations were expressed in terms of active ingredient. All test item solutions were used within 20 minutes of the preparation. 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. A copy of the certificate of analysis of the test item can be found in Appendix II of this report. 3.2 Control substances The solvent used in this study was sterile distilled water of injectable grade, batch no. 08G2803, obtained from Bieffe, Italy. Solutions of Mitomycin-C, batch 117K1683, and Cyclophosphamide, batch 097K1311, both obtained from Sigma were prepared in sterile distilled water immediately prior to use and served as positive controls. Untreated cultures were included in the experimental scheme and acted as reference control for cultures treated with the positive controls. RTC Study No.: 76240 Page 9 3.3 S9 tissue homogenate Two rat S9 liver tissue fractions: lot numbers 2273 and 2378 were used for main experiments 1 and 2 respectively. The S9 liver tissue fractions had the following characteristics: Lot Number Inducing Agents Preparation date Expiry date Received from Date received Storage at RTC Strain Sex of donors Protein content : 2273 : Phenobarbital 5,6-Benzoflavone : 01-May-2008 : 01-May-2010 : : 29-Jul-2008 : Approximately -80C : Sprague Dawley : Male : 37.6 mg/ml Lot Number Inducing Agents Preparation date Expiry date Received from Date received Storage at RTC Strain Sex of donors Protein content : 2378 : Phenobarbital 5,6-Benzoflavone : 18-Feb-2009 : 18-Feb-2011 : . : 24-Mar-2009 : Approximately -80C : Sprague Dawley : Male : 37.8 mg/ml Production and quality control certificates can be found in Appendix IV of this report. 3.4 Methods The methods used were in compliance with the attached Study Protocol and First amendment to the Protocol (Appendix I), with the exception that, due to a technical oversight, 75 metaphases and not 100 were scored for culture no. 21 treated with Cyclophosphamide. This deviation from the protocol was not considered to have affected the integrity of the study since the expected response of the test system to the positive control was demonstrated. RTC Study No.: 76240 Page 10 4. RESULTS 4.1 Sterility test Since the test item is an aqueous sample, a preliminary sterility test was performed. An aliquot of 500 l of the test item, as supplied, was added to 4.5 ml of nutrient broth and incubated at 37C for about 24 hours before evaluating the outcome of the test. Based on the results obtained it was not considered necessary to filter the test item before use in any experimental procedure. 4.2 Assay for chromosomal aberrations For the first main assay, dose levels of 5000, 2500, 1250, 625, 313, 156, 78.1 and 39.1 g/ml were employed both in the absence and presence of S9 metabolism. 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. Since no induction of chromosomal aberrations was obtained in the first main assay, in agreement with the study protocol, a second main experiment was performed where cells were treated with C6O4 CYCLIC in the absence of S9 metabolism and harvested after 20 hours. A continuous treatment until harvest was used. For the first main experiment, following treatment with the test item in the presence of S9 metabolism, marked increases in the number of endoreduplicated cells over the control were observed. Since no adequate toxicity was observed at any dose level for the evaluation of genotoxic effects, following the approval of a protocol amendment, for the second main experiment an additional treatment in the presence of S9 metabolism was performed in order to confirm the observed results. For this experiment a modified dose range was adopted in order to investigate more closely those doses most likely to exhibit endoreduplication. For the second main experiment, the dose levels selected for treatment were 2000, 1330, 889, 593, 395, 263, 176 and 117 g/ml. Appropriate negative and positive control cultures were included in the experiment. Positive control treated cultures received Mitomycin-C 0.30 and 0.45 g/ml for 3 hours treatment or 0.10 and 0.15 g/ml for the continuous treatment in the absence of S9 metabolism or Cyclophosphamide 15.0 and 23.0 g/ml in 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. No relevant effect on pH of the treatment media was observed at any dose level tested. Due to the presence of water in the solvent control cultures, a slight increase in osmolality over the control was observed for cultures treated at the highest dose level selected for treatment. The values however were comparable to those observed in untreated control cultures. 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.: 76240 Page 11 During the main experiments, no precipitation or opacity of the media was observed at the beginning or end of treatment. 4.3 Selection of dose levels for scoring At the time of harvesting, cell counts were performed for each culture and cytotoxicity was evaluated as determined by the reduction of population doubling (PD) over the relevant control. Population doubling is the log of the ratio of the final cell count at harvesting (N) to the starting (baseline) count (X0) divided by the log of 2: PD = log (N/ X0)]/log 2 Results are presented in Tables 1 to 4. In the first main experiment, following treatment in the absence or presence of S9 metabolism, a severe toxicity was observed at the higher dose levels of 5000 and 2500 g/ml, where no cells or few cells where recovered at harvesting. In the absence of S9, moderate toxicity was observed at the next lower dose level of 1250 g/ml where PD was 75% of the control. No cytotoxicity was observed over the remaining dose range. In the presence of S9 metabolism, no remarkable toxicity was observed over the remaining dose range. In the second main experiment following treatment in the absence of S9, a severe toxicity was observed at the higher dose levels of 2000 and 1330 g/ml, where few cells were recovered at harvesting. Marked toxicity was also observed at the dose levels of 889 and 593 g/ml where the PD was 31% of the control. No remarkable toxicity was observed over the remaining dose range. In the presence of S9 metabolism distinct toxicity was observed at the highest dose level of 2000 g/ml where the PD was 51% of the control. Slight toxicity reducing the PD at approximately 80% of the control 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 PD should be approximately 50%). If no toxicity is observed then the highest practicable dose level should be selected. On the basis of the above described results the dose levels selected for scoring were the following: Experiment S9 No. 1 + - + 2 - Treatment time (hours) 3 3 20 Harvest time (hours) 20 20 Dose level (g/ml) 1250, 625 and 313 2000, 1330 and 889 889, 593 and 395 The dose levels of 0.30 and 0.10 g/ml were selected for the scoring of cultures treated with Mitomycin-C (first and second main experiment respectively) and the dose level of 15.0 g/ml was selected for the scoring of cultures treated with Cyclophosphamide. RTC Study No.: 76240 Page 12 4.4 Assay results One hundred metaphase spreads were scored for chromosomal aberrations from each culture, with the exception of one replicate culture treated with Cyclophsphamide (first main experiment), where 75 metaphases were examined. In addition, for the first main experiment, due to the high frequency of aberrant cells (excluding gaps), found for replicate cultures treated with Mitomycin-C, scoring was terminated at 50 metaphases. The results are presented in Tables 5 to 8. 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 is also shown. For the first experiment, following treatment with the test item C6O4 CYCLIC in the absence or presence of S9 metabolism, no remarkable increase over the control values in the number of chromosome aberrations including or excluding gaps was observed. Following treatment in the presence of S9 metabolism, marked increases in the number of endoreduplicated cells over the control were observe. An increase in the number of endoreduplicated cells was also observed in the absence of S9 metabolism at the highest dose selected for scoring. For the second experiment, following treatment with the test item in the absence of S9 metabolism, slight increases in the incidence of cells bearing aberrations excluding gaps, over the control values were observed at all dose levels selected for scoring. More remarkable increases of aberrant cells were observed when including gaps. In the presence of S9 metabolism, increases in the incidence of aberrant cells including and excluding gaps, were observed at the highest dose selected for scoring (2000 g/ml). The incidences exceeded the range of our historical values for negative controls when excluding gaps. Additionally the presence of a heavily damaged cell, bearing more than five aberrations was also noted. In this experiment the induction of endoreduplicated cells was confirmed since marked increases were seen at the intermediate and high dose levels selected for scoring. 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 (64.25%). The frequency of cells containing 20, 22 and 23 chromosomes was 0.75%, 34.75% and 0.25%. No metaphases were found with 19 chromosomes. RTC Study No.: 76240 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 9 to 12. Following treatment with the test item in the absence of S9 metabolism, no statistically significant increase in the incidence of cells bearing aberrations including or excluding gaps was observed. In the presence of S9 metabolism, statistically significant increases in the incidence of cells bearing aberrations, including and excluding gaps, exceeding the range of our historical controls, were observed at the highest dose level selected for scoring. 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 of the results is given in the Study Protocol. 5.3 Evaluation Based on the positive results obtained in the presence of S9 metabolism, together with increases in the numbers of endoreduplicated cells and in accordance with the criteria for the outcome of the study, the test item was considered to induce chromosomal aberrations and inhibition of cell cycle progression 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.: 76240 Page 14 6. CONCLUSIONS A summary of the results are presented in Tables 13 and 14 giving the incidence of cells bearing aberrations (excluding gaps), the incidence of endoreduplicated cells and the relative population doubling 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 C6O4 CYCLIC induces chromosomal aberrations in Chinese hamster ovary cells after in vitro treatment under the reported experimental conditions. It is also concluded that the test item inhibits cell cycle progression and chromosome segregation under the reported experimental conditions. RTC Study No.: 76240 Page 15 7. TABLES 1-4 RTC Study No.: 76240 Page 16 C6O4 CYCLIC: CHROMOSOME ABERRATIONS TABLE 1 Population doubling results - Without metabolic activation STUDY NO.: 76240 MAIN ASSAY: 1 SOLVENT: STERILE DISTILLED WATER OF INJECTABLE GRADE TREATMENT TIME: 3 hours SAMPLING TIME: 20 hours ___________________________________________________________________________ Treatment Dose level Culture No.viable Mean Relative PD (g/ml) No. cellsx106/ml (%) ___________________________________________________________________________ Untreated - 1 0.76 0.73 106 2 0.70 ___________________________________________________________________________ Solvent 10% 3 0.68 0.70 100 4 0.71 ___________________________________________________________________________ Test Item 39.1 19 0.71 0.71 102 20 0.71 ___________________________________________________________________________ Test Item 78.1 17 0.71 0.70 100 18 0.69 ___________________________________________________________________________ Test Item 156 15 0.71 0.71 102 16 0.70 ___________________________________________________________________________ Test Item 313 13 0.71 0.70 100 14 0.69 ___________________________________________________________________________ Test Item 625 11 0.73 0.72 104 12 0.71 ___________________________________________________________________________ Test Item 1250 9 0.56 0.58 75 10 0.59 ___________________________________________________________________________ Test Item 2500 7 0.33 0.33 0 8 0.33 ___________________________________________________________________________ Test Item 5000 5 0.00 0.00 0 6 0.00 ___________________________________________________________________________ Mitomycin-C 0.30 21 0.48 0.46 42 22 0.43 ___________________________________________________________________________ Mitomycin-C 0.45 23 0.56 0.52 57 24 0.48 ___________________________________________________________________________ Note: Baseline cell count: 0.33 cells x 106/ml RTC Study No.: 76240 Page 17 C6O4 CYCLIC: CHROMOSOME ABERRATIONS TABLE 2 - Population doubling results - With metabolic activation STUDY NO.: 76240 MAIN ASSAY: 1 SOLVENT: STERILE DISTILLED WATER OF INJECTABLE GRADE TREATMENT TIME: 3 hours SAMPLING TIME: 20 hours ___________________________________________________________________________ Treatment Dose level Culture No.viable Mean Relative PD (g/ml) No. cellsx106/ml (%) ___________________________________________________________________________ Untreated - 25 0.66 0.66 116 26 0.66 ___________________________________________________________________________ Solvent 10% 27 0.62 0.60 100 28 0.58 ___________________________________________________________________________ Test Item 39.1 43 0.64 0.62 106 44 0.60 ___________________________________________________________________________ Test Item 78.1 41 0.65 0.62 106 42 0.59 ___________________________________________________________________________ Test Item 156 39 0.66 0.64 111 40 0.62 ___________________________________________________________________________ Test Item 313 37 0.63 0.62 106 38 0.61 ___________________________________________________________________________ Test Item 625 35 0.61 0.62 106 36 0.63 ___________________________________________________________________________ Test Item 1250 33 0.61 0.59 97 34 0.56 ___________________________________________________________________________ Test Item 2500 31 0.25 0.26 0 32 0.26 ___________________________________________________________________________ Test Item 5000 29 0.00 0.00 0 30 0.00 ___________________________________________________________________________ Cyclophosphamide 15.0 45 0.44 0.46 48 46 0.48 ___________________________________________________________________________ Cyclophosphamide 23.0 47 0.38 0.38 20 48 0.37 ___________________________________________________________________________ Note: Baseline cell count: 0.33 cells x 106/ml RTC Study No.: 76240 Page 18 C6O4 CYCLIC: CHROMOSOME ABERRATIONS TABLE 3 - Population doubling results - Without metabolic activation STUDY NO.: 76240 MAIN ASSAY: 2 SOLVENT: STERILE DISTILLED WATER OF INJECTABLE GRADE TREATMENT TIME: 20 hours SAMPLING TIME: 20 hours ___________________________________________________________________________ Treatment Dose level Culture No.viable Mean Relative PD (g/ml) No. cellsx106/ml (%) ___________________________________________________________________________ Untreated - 49 0.57 0.55 102 50 0.53 ___________________________________________________________________________ Solvent 10% 51 0.54 0.54 100 52 0.54 ___________________________________________________________________________ Test Item 117 67 0.47 0.49 89 68 0.51 ___________________________________________________________________________ Test Item 176 65 0.47 0.50 91 66 0.52 ___________________________________________________________________________ Test Item 263 63 0.50 0.50 91 64 0.49 ___________________________________________________________________________ Test Item 395 61 0.51 0.50 91 62 0.49 ___________________________________________________________________________ Test Item 593 59 0.24 0.30 31 60 0.36 ___________________________________________________________________________ Test Item 889 57 0.29 0.30 31 58 0.31 ___________________________________________________________________________ Test Item 1330 55 0.14 0.15 0 56 0.16 ___________________________________________________________________________ Test Item 2000 53 0.04 0.05 0 54 0.06 ___________________________________________________________________________ Mitomycin-C 0.10 69 0.34 0.34 45 70 0.33 ___________________________________________________________________________ Mitomycin-C 0.15 71 0.29 0.28 23 72 0.27 ___________________________________________________________________________ Note: Baseline cell count: 0.23 cells x 106/ml RTC Study No.: 76240 Page 19 C6O4 CYCLIC: CHROMOSOME ABERRATIONS TABLE 4 - Population doubling results - With metabolic activation STUDY NO.: 76240 MAIN ASSAY: 2 SOLVENT: STERILE DISTILLED WATER OF INJECTABLE GRADE TREATMENT TIME: 3 hours SAMPLING TIME: 20 hours ___________________________________________________________________________ Treatment Dose level Culture No.viable Mean Relative PD (g/ml) No. cellsx106/ml (%) ___________________________________________________________________________ Untreated - 73 0.64 0.61 94 74 0.57 ___________________________________________________________________________ Solvent 10% 75 0.67 0.65 100 76 0.63 ___________________________________________________________________________ Test Item 117 91 0.55 0.55 84 92 0.54 ___________________________________________________________________________ Test Item 176 89 0.53 0.54 82 90 0.54 ___________________________________________________________________________ Test Item 263 87 0.52 0.52 79 88 0.52 ___________________________________________________________________________ Test Item 395 85 0.52 0.53 80 86 0.54 ___________________________________________________________________________ Test Item 593 83 0.55 0.55 84 84 0.55 ___________________________________________________________________________ Test Item 889 81 0.54 0.55 84 82 0.55 ___________________________________________________________________________ Test Item 1330 79 0.53 0.54 82 80 0.54 ___________________________________________________________________________ Test Item 2000 77 0.46 0.39 51 78 0.31 ___________________________________________________________________________ Cyclophosphamide 15.0 93 0.43 0.44 67 94 0.45 ___________________________________________________________________________ Cyclophosphamide 23.0 95 0.44 0.43 64 96 0.42 ___________________________________________________________________________ Note: Baseline cell count: 0.23 cells x 106/ml RTC Study No.: 76240 Page 20 8. KEY TO TABLES 5-8 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 interchromosome 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.: 76240 Page 21 C6O4 CYCLIC: CHROMOSOME ABERRATIONS TABLE 5 - Scoring of Aberrations - Without metabolic activation STUDY NO.: 76240 MAIN ASSAY: 1 SOLVENT: STERILE DISTILLED WATER OF INJECTABLE GRADE TREATMENT TIME: 3 hours SAMPLING TIME: 20 hours _______________________________________________________________________________________________________________________________ Cells Cells Treatment Dose level Culture Cells Chromatid Chromosome Isolocus Tot.abs Tot.abs with abs with abs (g/ml) No. Scored Gaps Del Exch Del Exch Other (+gaps) (-gaps) (+gaps) (-gaps) _______________________________________________________________________________________________________________________________ Untreated - 1 100 0 0 0 0 0 0 0 0 0 0 0 2 100 0 0 0 0 0 0 0 0 0 0 0 200 0 0 0 0 0 0 0 0 0 0 0 Solvent 10% 3 100 3 0 0 0 0 0 0 3 0 3 0 4 100 1 0 0 0 0 0 0 1 0 1 0 200 4 0 0 0 0 0 0 4 0 4 0 Test Item 313 13 100 2 0 0 0 0 0 0 2 0 2 0 14 100 2 2 0 1 0 0 0 5 3 3 1 200 4 2 0 1 0 0 0 7 3 5 1 Test Item 625 11 100 0 0 0 0 0 0 0 0 0 0 0 12 100 4 0 0 0 0 0 0 4 0 4 0 200 4 0 0 0 0 0 0 4 0 4 0 Test Item 1250 9 100 0 0 0 0 0 0 3ER 0 0 10 100 1 0 0 0 0 0 5ER 1 0 200 1 0 0 0 0 0 8ER 1 0 0 0 1 0 1 0 Mitomycin-C 0.30 21 75 1 4 51 3 1 10 0 70 69 41 40 22 100 0 0 40 8 2 2 2H 52 52 33 33 175 1 4 91 11 3 12 2H 122 121 74 73 _______________________________________________________________________________________________________________________________ RTC Study No.: 76240 Page 22 C6O4 CYCLIC: CHROMOSOME ABERRATIONS TABLE 6 - Scoring of Aberrations - With metabolic activation STUDY NO.: 76240 MAIN ASSAY: 1 SOLVENT: STERILE DISTILLED WATER OF INJECTABLE GRADE TREATMENT TIME: 3 hours SAMPLING TIME: 20 hours _______________________________________________________________________________________________________________________________ Cells Cells Treatment Dose level Culture Cells Chromatid Chromosome Isolocus Tot.abs Tot.abs with abs with abs (g/ml) No. Scored Gaps Del Exch Del Exch Other (+gaps) (-gaps) (+gaps) (-gaps) _______________________________________________________________________________________________________________________________ Untreated - 25 100 4 0 0 0 0 0 1ER,1PP 4 0 26 100 4 2 0 0 0 0 0 6 2 200 8 2 0 0 0 0 1ER,1PP 10 2 4 0 5 2 9 2 Solvent 10% 27 100 1 0 0 0 0 0 0 1 0 1 0 28 100 2 0 0 0 0 0 0 2 0 2 0 200 3 0 0 0 0 0 0 3 0 3 0 Test Item 313 37 100 2 1 0 0 0 0 4ER 3 1 3 1 38 100 2 0 0 0 1 0 7ER 3 1 3 1 200 4 1 0 0 1 0 11ER 6 2 6 2 Test Item 625 35 100 4 0 0 0 0 0 18ER 4 0 4 0 36 100 0 0 0 0 0 0 10ER 0 0 0 0 200 4 0 0 0 0 0 28ER 4 0 4 0 Test Item 1250 33 100 4 0 0 0 0 0 14ER 4 0 34 100 0 0 0 0 0 0 12ER 0 0 200 4 0 0 0 0 0 26ER 4 0 4 0 0 0 4 0 Cyclophosphamide 15.0 45 50 5 13 31 0 1 3 1H 53 48 35 35 46 50 4 20 25 0 0 1 1H 50 46 34 33 100 9 33 56 0 1 4 2H 103 94 69 68 _______________________________________________________________________________________________________________________________ RTC Study No.: 76240 Page 23 C6O4 CYCLIC: CHROMOSOME ABERRATIONS TABLE 7 - Scoring of Aberrations - Without metabolic activation STUDY NO.: 76240 MAIN ASSAY: 2 SOLVENT: STERILE DISTILLED WATER OF INJECTABLE GRADE TREATMENT TIME: 20 hours SAMPLING TIME: 20 hours _______________________________________________________________________________________________________________________________ Cells Cells Treatment Dose level Culture Cells Chromatid Chromosome Isolocus Tot.abs Tot.abs with abs with abs (g/ml) No. Scored Gaps Del Exch Del Exch Other (+gaps) (-gaps) (+gaps) (-gaps) _______________________________________________________________________________________________________________________________ Untreated - 49 100 1 0 0 0 0 0 0 50 100 2 0 0 0 0 0 0 200 3 0 0 0 0 0 0 1 0 2 0 3 0 1 0 2 0 3 0 Solvent 10% 51 100 0 1 0 0 0 0 1PP 1 1 1 1 52 100 1 0 0 0 0 0 0 1 0 1 0 200 1 1 0 0 0 0 1PP 2 1 2 1 Test Item 395 61 100 0 0 0 0 4 0 0 4 4 4 4 62 100 2 0 0 0 1 1 1ER 4 2 4 2 200 2 0 0 0 5 1 1ER 8 6 8 6 Test Item 593 59 100 3 1 0 0 1 0 0 5 2 4 1 60 100 3 4 0 0 0 0 2PP,1ER 7 4 6 3 200 6 5 0 0 1 0 2PP,1ER 12 6 10 4 Test Item 889 57 100 0 2 0 0 0 1 0 3 3 3 3 58 100 2 1 0 0 0 0 0 3 1 3 1 200 2 3 0 0 0 1 0 6 4 6 4 Mitomycin-C 0.10 69 100 3 6 19 0 0 9 1PP 37 34 24 23 70 100 2 5 18 0 2 2 0 29 27 19 18 200 5 11 37 0 2 11 1PP 66 61 43 41 _______________________________________________________________________________________________________________________________ RTC Study No.: 76240 Page 24 C6O4 CYCLIC: CHROMOSOME ABERRATIONS TABLE 8 - Scoring of Aberrations - With metabolic activation STUDY NO.: 76240 MAIN ASSAY: 2 SOLVENT: STERILE DISTILLED WATER OF INJECTABLE GRADE TREATMENT TIME: 3 hours SAMPLING TIME: 20 hours _______________________________________________________________________________________________________________________________ Cells Cells Treatment Dose level Culture Cells Chromatid Chromosome Isolocus Tot.abs Tot.abs with abs with abs (g/ml) No. Scored Gaps Del Exch Del Exch Other (+gaps) (-gaps) (+gaps) (-gaps) _______________________________________________________________________________________________________________________________ Untreated - 73 100 5 1 0 0 0 0 4ER 6 1 5 1 74 100 5 1 0 0 0 0 2ER 6 1 5 1 200 10 2 0 0 0 0 6ER 12 2 10 2 Solvent 10% 75 100 2 0 0 0 0 0 1ER 2 0 2 0 76 100 3 1 0 0 0 0 1ER 4 1 4 1 200 5 1 0 0 0 0 2ER 6 1 6 1 Test Item 889 81 100 2 0 0 0 0 0 3ER 2 0 2 0 82 100 7 0 0 0 0 0 2ER 7 0 7 0 200 9 0 0 0 0 0 5ER 9 0 9 0 Test Item 1330 79 100 2 0 0 0 0 0 11ER 2 0 80 100 1 0 0 0 0 0 11ER 1 0 200 3 0 0 0 0 0 22ER 3 0 2 0 1 0 3 0 Test Item 2000 77 100 4 0 1 0 1 2 19ER 8 4 7 3 78 100 6 2 1 0 1 3 11ER,1H 13 7 10 6 200 10 2 2 0 2 5 30ER,1H 21 11 17 9 Cyclophosphamide 15.0 93 100 2 13 32 0 1 5 1H 53 51 36 36 94 100 5 23 42 0 1 13 1H 84 79 45 45 200 7 36 74 0 2 18 2H 137 130 81 81 _______________________________________________________________________________________________________________________________ RTC Study No.: 76240 Page 25 9. KEY TO TABLES 9-14 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 between the treated and 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 of the 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<0.01 *** Statistically significant at P<0.001 # 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.: 76240 Page 26 C6O4 CYCLIC: CHROMOSOME ABERRATIONS TABLE 9 - Statistical Analysis - Without metabolic activation STUDY NO.: 76240 MAIN ASSAY: 1 SOLVENT: STERILE DISTILLED WATER OF INJECTABLE GRADE TREATMENT TIME: 3 hours SAMPLING TIME: 20 hours ______________________________________________________________________ Comparison with Cells with aberrations Negative control __________________________________ (+ gaps) (- gaps) __________________________________ Dose g/ml CULT. P. values Sig. P. values Sig. ______________________________________________________________________ Test Item 313 13 # N.S. # N.S. 14 0.4294 N.S. 0.3334 N.S. Pooled 0.5002 N.S. 0.5000 N.S. Test Item 625 11 0.1956 N.S. # N.S. 12 0.2564 N.S. # N.S. Pooled # N.S. # N.S. Test Item 1250 9 0.1956 N.S. # N.S. 10 0.4599 N.S. # N.S. Pooled 0.1860 N.S. # N.S. All dose levels 0.4792 N.S. 0.7501 N.S. Mitomycin-C 0.30 21 0.0000 *** 0.0000 *** 22 0.0000 *** 0.0000 *** Pooled 0.0000 *** 0.0000 *** ______________________________________________________________________ In this table correction is made for the multiple comparisons of the test item treatments with the negative controls. For individual cultures, since six comparisons are made, the required "p" values for significance are 0.009(*), 0.002(**) and 0.0002(***). For treatment levels, since three comparisons are made the required "p" values for significance are 0.017(*), 0.003(**) and 0.0003(***). RTC Study No.: 76240 Page 27 C6O4 CYCLIC: CHROMOSOME ABERRATIONS TABLE 10 - Statistical Analysis - With metabolic activation STUDY NO.: 76240 MAIN ASSAY: 1 SOLVENT: STERILE DISTILLED WATER OF INJECTABLE GRADE TREATMENT TIME: 3 hours SAMPLING TIME: 20 hours ______________________________________________________________________ Comparison with Cells with aberrations Negative control __________________________________ (+ gaps) (- gaps) __________________________________ Dose g/ml CULT. P. values Sig. P. values Sig. ______________________________________________________________________ Test Item 313 37 0.3186 N.S. 0.3334 N.S. 38 0.3186 N.S. 0.3334 N.S. Pooled 0.2515 N.S. 0.2493 N.S. Test Item 625 35 0.1707 N.S. # N.S. 36 0.2948 N.S. # N.S. Pooled 0.5000 N.S. # N.S. Test Item 1250 33 0.1707 N.S. # N.S. 34 0.2948 N.S. # N.S. Pooled 0.5000 N.S. # N.S. All dose levels 0.3506 N.S. 0.5623 N.S. Cyclophosphamide 15.0 45 0.0000 *** 0.0000 *** 46 0.0000 *** 0.0000 *** Pooled 0.0000 *** 0.0000 *** ______________________________________________________________________ In this table correction is made for the multiple comparisons of the test item treatments with the negative controls. For individual cultures, since six comparisons are made, the required "p" values for significance are 0.009(*), 0.002(**) and 0.0002(***). For treatment levels, since three comparisons are made the required "p" values for significance are 0.017(*), 0.003(**) and 0.0003(***). RTC Study No.: 76240 Page 28 C6O4 CYCLIC: CHROMOSOME ABERRATIONS TABLE 11 - Statistical Analysis - Without metabolic activation STUDY NO.: 76240 MAIN ASSAY: 2 SOLVENT: STERILE DISTILLED WATER OF INJECTABLE GRADE TREATMENT TIME: 20 hours SAMPLING TIME: 20 hours ______________________________________________________________________ Comparison with Cells with aberrations Negative control __________________________________ (+ gaps) (- gaps) __________________________________ Dose g/ml CULT. P. values Sig. P. values Sig. ______________________________________________________________________ Test Item 395 61 0.0979 N.S. 0.0439 N.S. 62 0.0979 N.S. 0.2585 N.S. Pooled 0.0525 N.S. 0.0609 N.S. Test Item 593 59 0.0979 N.S. 0.5564 N.S. 60 0.0183 N.S. 0.1096 N.S. Pooled 0.0179 N.S. 0.1859 N.S. Test Item 889 57 0.2082 N.S. 0.1096 N.S. 58 0.2082 N.S. 0.5564 N.S. Pooled 0.1421 N.S. 0.1859 N.S. All dose levels 0.0241 * 0.0782 N.S. Mitomycin-C 0.10 69 0.0000 *** 0.0000 *** 70 0.0000 *** 0.0000 *** Pooled 0.0000 *** 0.0000 *** ______________________________________________________________________ In this table correction is made for the multiple comparisons of the test item treatments with the negative controls. For individual cultures, since six comparisons are made, the required "p" values for significance are 0.009(*), 0.002(**) and 0.0002(***). For treatment levels, since three comparisons are made the required "p" values for significance are 0.017(*), 0.003(**) and 0.0003(***). RTC Study No.: 76240 Page 29 C6O4 CYCLIC: CHROMOSOME ABERRATIONS TABLE 12 - Statistical Analysis - With metabolic activation STUDY NO.: 76240 MAIN ASSAY: 2 SOLVENT: STERILE DISTILLED WATER OF INJECTABLE GRADE TREATMENT TIME: 20 hours SAMPLING TIME: 20 hours ______________________________________________________________________ Comparison with Cells with aberrations Negative control __________________________________ (+ gaps) (- gaps) __________________________________ Dose g/ml CULT. P. values Sig. P. values Sig. ______________________________________________________________________ Test Item 889 81 0.4668 N.S. 0.6668 N.S. 82 0.0986 N.S. 0.6668 N.S. Pooled 0.3001 N.S. 0.5000 N.S. Test Item 1330 79 0.4668 N.S. 0.6668 N.S. 80 0.2603 N.S. 0.6668 N.S. Pooled 0.2514 N.S. 0.5000 N.S. Test Item 2000 77 0.0986 N.S. 0.1096 N.S. 78 0.0136 N.S. 0.0063 * Pooled 0.0148 * 0.0100 * All dose levels 0.1863 N.S. 0.2422 N.S. Cyclophosphamide 15.0 93 0.0000 *** 0.0000 *** 94 0.0000 *** 0.0000 *** Pooled 0.0000 *** 0.0000 *** ______________________________________________________________________ In this table correction is made for the multiple comparisons of the test item treatments with the negative controls. For individual cultures, since six comparisons are made, the required "p" values for significance are 0.009(*), 0.002(**) and 0.0002(***). For treatment levels, since three comparisons are made the required "p" values for significance are 0.017(*), 0.003(**) and 0.0003(***). RTC Study No.: 76240 Page 30 C6O4 CYCLIC: CHROMOSOME ABERRATIONS TABLE 13 - Summary Table STUDY NO.: 76240 MAIN ASSAY: 1 SOLVENT: STERILE DISTILLED WATER OF INJECTABLE GRADE TREATMENT TIME: 3 hours SAMPLING TIME: 20 hours ___________________________________________________________________________ Presence of S9 Absence of S9 metabolism metabolism _______________ ________________ Treatment Dose g/ml %CA (Rel.PD) %CA (Rel.PD) __________________________________________________________________________ Untreated - 1.0 ( 116) 0.0 ( 106) Solvent 10% 0.0 ( 100) 0.0 ( 100) Test Item 313 1.0 N.S.( 106) 0.5 N.S.( 100) Test Item 625 0.0 N.S.( 106) 0.0 N.S.( 104) Test Item 1250 0.0 N.S.( 97) 0.0 N.S.( 75) Mitomycin-C 0.30 - 41.7 *** ( 42) Cyclophosphamide 15.0 68.0 *** ( 48) - __________________________________________________________________________ Key: % CA : Percentage of cells bearing aberrations (excluding gaps) Rel.PD : Population doubling relative to solvent controls (percent) - : Not tested or not selected for the scoring of aberrations * : Statistically significant at P<0.05 ** : Statistically significant at P<0.01 *** : Statistically significant at P<0.001 RTC Study No.: 76240 Page 31 C6O4 CYCLIC: CHROMOSOME ABERRATIONS TABLE 14 - Summary Table STUDY NO.: 76240 MAIN ASSAY: 2 SOLVENT: STERILE DISTILLED WATER OF INJECTABLE GRADE TREATMENT TIME: 3 hours (+S9)/20 hours (-S9) SAMPLING TIME: 20 hours ___________________________________________________________________________ Presence of S9 Absence of S9 metabolism metabolism _______________ ________________ Treatment Dose g/ml %CA (Rel.cell %CA (Rel.cell growth) growth) __________________________________________________________________________ Untreated - 1.0 ( 94) 0.0 ( 102) Solvent 10% 0.5 ( 100) 0.5 ( 100) Test Item 395 - 3.0 N.S.( 91) Test Item 593 - 2.0 N.S.( 31) Test Item 889 0.0 N.S.( 84) 2.0 N.S.( 31) Test Item 1330 0.0 N.S.( 82) - Test Item 2000 4.5 * ( 51) - Mitomycin-C 0.10 - 20.5 N.S.( 45) Cyclophosphamide 15.0 40.5 *** ( 67) - __________________________________________________________________________ Key: % CA : Percentage of cells bearing aberrations (excluding gaps) Rel.PD : Population doubling relative to solvent controls (percent) - : Not tested or not selected for the scoring of aberrations * : Statistically significant at P<0.05 ** : Statistically significant at P<0.01 *** : Statistically significant at P<0.001 RTC Study No.: 76240 Page 32 10. APPENDIX I - Study Protocol and First Amendment to Protocol RTC Study No.: 76240 Page 33 RTC Study No.: 76240 Page 34 ---------------------------------------------------------------- ---------------------------------- RTC Study No.: 76240 Page 35 RTC Study No.: 76240 Page 36 RTC Study No.: 76240 Page 37 RTC Study No.: 76240 Page 38 RTC Study No.: 76240 Page 39 RTC Study No.: 76240 Page 40 RTC Study No.: 76240 Page 41 RTC Study No.: 76240 Page 42 RTC Study No.: 76240 Page 43 RTC Study No.: 76240 Page 44 RTC Study No.: 76240 Page 45 RTC Study No.: 76240 Page 46 ---------------- ------------------------------- RTC Study No.: 76240 Page 47 ------------------------- ------------------ -------------------------------------------------------- ------------------ RTC Study No.: 76240 Page 48 - IFjrri'0\ RESEARCH TOXICOLOGY CENTRE - ROMA PROTOCOL AMENDMENT (1) STUDY TITLE : C604 CYCLIC CHROMOSOME ABERRATIONS IN CHINESE HAMSTER OVARY CELLS IN VITRO STUDY NO. : 76240 DATE OF PROTOCOL APPROVAL : 09 March 2009 AMENDMENT: In order to further evaluate results obtained in Main assay 1 in the presence of S9 metabolism, treatment was repeated in Main assay 2. Data will be presented and evaluated following section 6 of the Study Protocol. Reason Effective date : Agreed with the Sponsor : Date of study note 1 (24 Apr 2009). RTC Enquiry Number: 75841 RTC Study Number: 76240 Can m e loall0Ifte RTC 55 A. TItoSperl.12 013340 P en (Ran 4- ITALY Te LA.3911691095.1 Fax : .09n69105737 em all4DJsIELUI - 1 of 3 - Heal Olice aid AM IL istalkoe RTC SPA. VW Tit SpeII.12 LIII40 POM (RCITI4 - rrALY P.O. ta -Rama Eer Lalre leo RTC Study No.: 76240 Page 49 --------------------------------- --------------------------------------- RTC Study No.: 76240 Page 50 --------------- ------------------- RTC Study No.: 76240 Page 51 11. APPENDIX II - Certificate of analysis RTC Study No.: 76240 Page 52 RTC Study No.: 76240 Page 53 12. APPENDIX III - Historical control data CHROMOSOME ABERRATIONS IN CHINESE HAMSTER OVARY CELLS Background incidences (%) of aberrant cells (Years 1990-2008) Absence of S9 20 h sampling time + gaps - gaps Presence of S9 20 h sampling time + gaps - gaps Mean SD (n-1) n Minimum Maximum UNTREATED CONTROLS 1.8 0.5 2.3 0.6 1.5 0.7 1.7 0.7 121 121 92 92 0.0 0.0 0.0 0.0 7.0 3.0 8.0 3.0 Mean SD (n-1) n Minimum Maximum SOLVENT CONTROLS 2.0 0.4 2.4 0.5 1.8 0.7 1.9 0.7 93 93 72 72 0.0 0.0 0.0 0.0 8.0 4.0 8.5 4.0 Mean SD (n-1) n Minimum Maximum POSITIVE CONTROLS 37.4 35.4 38.3 36.0 14.8 14.6 13.8 13.8 124 124 93 93 14.0 12.0 15.5 15.5 77.0 76.0 84.0 82.0 SD = standard deviation n = number of experiments RTC Study No.: 76240 Page 54 13. APPENDIX IV - S9 production and quality control certificates RTC Study No.: 76240 Page 55 RTC Study No.: 76240 Page 56 --------------- RTC Study No.: 76240 Page 57