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le pena Gy 084 Signatur C604 CYCLIC (S. typhimurium and E. coli) FINALREPORT Viale Lombardia, 20 20021Bollate (MI) aa ttt Tnape, 214 ComteSoc Ero 8164000 BECER TcroRrevIeuEcin sar JE A 1 hende ws sense ore preprtisopnfr a ---- oD sy va 750 roe harmonisation of laws, regulations and administrative provisions relating io the application of (Relevant toQtUhAosLeIaTspYecAtsSoSfUtRheANstCudEyScToAndTuEctMedEbNyTRTC $.p.A) Study phases monitored by RTC's QAU `According to curent relevant Standard Operating Procedures PROTOCOL CHECK PRREOLCAETSESD-TBAOSTEHDISINTSYPPEECTOIFOSNTSUDY TDeossteepxreecputairoant,ion Qualit(yDAasysuMroanncteh IYnesapre)ctions Inspection | Reportio | Report to Study Company Director | Management 9.032000 | 19.03.2000 | 19.03.2000 1131.00212.2000099 -- 1116002122000009 tOytpheerofprstoucdeys.s-Tbhaeserdelienvsapnetctdioocnusmewnetraeticoanrriisedkeoputtoonnfrloeutailntehoaucgthivistpieecsifnioctidnisrpeeccttliyonredlaatteesd atroe (nhoits rAespsoorctieadtehedrel.aboratories and support functions are subject to regular facility inspections. RFeIvNiAewL RoEfPtOhiRs Treport by RTCs QAU found the reported Review completed methods and procedure o describe those used an the rests 0 || constitute an accurate representationofthe recorded raw data. Date (Head of Quality Assurance) RTC Study No.: 76250 Page 4 Contents Page 1. SUMMARY ................................................................................................................................ 7 2. INTRODUCTION ...................................................................................................................... 8 2.1 Purpose ............................................................................................................................. 8 2.2 Principles of the method ................................................................................................... 8 2.3 Study organisation ............................................................................................................ 8 3. MATERIALS AND METHODS.............................................................................................. 10 3.1 Test item ......................................................................................................................... 10 3.2 Control items .................................................................................................................. 10 3.3 Media .............................................................................................................................. 11 3.4 S9 tissue homogenate...................................................................................................... 12 3.5 Bacterial strains .............................................................................................................. 13 3.6 Methods .......................................................................................................................... 13 3.6.1 Preliminary toxicity test.................................................................................................. 13 3.6.2 Main experiments ........................................................................................................... 13 3.6.3 Incubation and scoring.................................................................................................... 14 4. RESULTS ................................................................................................................................. 15 4.1 Sterility test..................................................................................................................... 15 4.2 Toxicity test .................................................................................................................... 15 4.3 Assay for reverse mutation ............................................................................................. 15 5. ANALYSIS OF RESULTS ...................................................................................................... 16 5.1 Criteria for outcome of the assays .................................................................................. 16 5.2 Evaluation ....................................................................................................................... 16 6. CONCLUSION......................................................................................................................... 17 7. KEY TO TABLES 1-12............................................................................................................ 18 7.1 Structure of Tables 3-12 ................................................................................................. 18 7.2 Regression line................................................................................................................ 18 Tables TABLE 1 - Toxicity test without metabolic activation ....................................................................... 19 TABLE 2 - Toxicity test with metabolic activation ............................................................................ 20 TABLE 3 - Experiment 1 - Plate incorporation method - TA1535..................................................... 21 TABLE 4 - Experiment 1 - Plate incorporation method - TA1537..................................................... 22 TABLE 5 - Experiment 1 - Plate incorporation method - WP2uvrA.................................................. 23 TABLE 6 - Experiment 1 - Plate incorporation method - TA98......................................................... 24 TABLE 7 - Experiment 1 - Plate incorporation method - TA100....................................................... 25 TABLE 8 - Experiment 2 - Preincubation method - TA1535 ............................................................. 26 TABLE 9 - Experiment 2 - Preincubation method - TA1537 ............................................................. 27 TABLE 10 - Experiment 2 - Preincubation method - WP2uvrA ........................................................ 28 TABLE 11 - Experiment 2 - Preincubation method - TA98 ............................................................... 29 TABLE 12 - Experiment 2 - Preincubation method - TA100 ............................................................. 30 RTC Study No.: 76250 Page 5 Contents Page Appendices APPENDIX 1 - Historical control data without metabolic activation (2000-2008)............................ 31 APPENDIX 2 - Historical control data with metabolic activation (2000-2008)................................. 32 Addenda ADDENDUM I - Study Protocol ........................................................................................................ 33 ADDENDUM II - Certificate of analysis............................................................................................ 48 ADDENDUM III - S9 production and quality control certificates ..................................................... 50 RTC Study No.: 76250 Page 6 1. SUMMARY 1.1 The test item C6O4 CYCLIC was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. 1.2 The test item was used as a solution in sterile distilled water. 1.3 The test item C6O4 CYCLIC was assayed in the toxicity test at a maximum dose level of 5000 Pg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 Pg/plate. Concentrations are expressed in terms of active ingredient. No precipitation of the test item was observed at the end of the incubation period at any concentration. No toxicity was observed at any dose level with any tester strain. 1.4 In Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels expressed in terms of active ingredient: 5000, 2500, 1250, 625 and 313 Pg/plate. No toxicity was observed at any dose level with any tester strain both in the absence and presence of S9 metabolic activation. As no relevant increases in revertant numbers were observed at any concentration tested, a pre-incubation step was included for all treatments of Main Assay II. The test item was assayed at the same concentrations employed in Main Assay I. Toxicity, as indicated by thinning of the background lawn and/or reduction in revertant numbers, was observed at higher dose levels with all tester strains, both in the absence and presence of S9 metabolic activation. No precipitation of the test item was noted at the end of the incubation period at any concentration tested, in any experiment. 1.5 The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism. 1.6 It is concluded that the test item C6O4 CYCLIC does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions. RTC Study No.: 76250 Page 7 2. INTRODUCTION 2.1 Purpose This report describes experiments performed to assess the mutagenic activity of the test item to Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and to Escherichia coli strain WP2 uvrA using the procedures developed by Ames et al., 1975 and revised by Maron and Ames, 1983. The study was designed to comply with the experimental methods indicated in: - Test method B.13/14 `Reverse mutation test using bacteria' described in Council Regulation (EC) No. 440/2008. - OECD Guideline for the testing of chemicals No. 471 (Adopted July 1997). - ICH S2A Genotoxicity: Specific Aspects of Regulatory Tests, Step 5. 2.2 Principles of the method Reverse mutation assays employ bacterial strains which are already mutant at a locus whose phenotypic effects are easily detected. The Salmonella tester strains have mutations causing dependence on a particular amino acid (histidine) for growth. The ability of test items to cause reverse mutations (reversions) to histidine-independence can easily be measured. The E. coli tester strains of the WP2 series are similarly mutant at the tryptophan locus. Since many chemicals only demonstrate mutagenic activity after metabolism to reactive forms, in order to detect these "indirect mutagens" the test is performed in the presence and absence of a rat liver metabolising system. 2.3 Study organisation Sponsor: SOLVAY SOLEXIS S.p.A. Viale Lombardia, 20 20021 Bollate (MI) Italy Location of Study: Research Toxicology Centre S.p.A. Genetic Toxicology, in vitro Toxicology and Immunology Department Via Tito Speri, 12/14 00040 Pomezia (Rome) Italy RTC Study No.: 76250 Page 8 Principal dates: Study protocol approved by Study Director: 09-Mar-2009 Study commenced: 20-Mar-2009 (Toxicity assay treatment) Study completed: 30-Mar-2009 (Completion of scoring Main Assay II) Study Director: S. Cinelli, Biol. D. Archiving: The original data arising from this study, 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.: 76250 Page 9 3. MATERIALS AND METHODS The methods used were in compliance with the attached Study Protocol (Addendum I). 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. A certificate of analysis, supplied by the Sponsor, can be found in Addendum II of this report. 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 2 hours and 21 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 items The solvents used in this study were: Sterile distilled water (Bieffe Medital, batch 08G2803). Dimethylsulfoxide (DMSO) (Fluka AG, batch 1331319 30907P11). Positive control treatments used solutions prepared as follows: Sodium azide (Moltox, Inc., batch 6289SA) in distilled water. 9-Aminoacridine (9-AA.HCl, Moltox, Inc., batch 1004AAHC) in DMSO. 2-Nitrofluorene (Moltox, Inc., batch 2210NF) in DMSO. 2-Aminoanthracene (Moltox, Inc., batch 6306AA and Sigma, batch 58F-3462) in DMSO. Methylmethanesulphonate (MMS) (Sigma, batch 018K3765) in distilled water. RTC Study No.: 76250 Page 10 Positive control treatments are indicated in the following table: Tester strain Absence of S9 Presence of S9 TA1535 sodium azide 1 Pg/plate 2-aminoanthracene 1 Pg/plate TA100 sodium azide 1 Pg/plate 2-aminoanthracene 1 Pg/plate (2 Pg/plate) TA1537 9-amino-acridine 50 Pg/plate 2-aminoanthracene 1 Pg/plate TA98 2-nitrofluorene 2 Pg/plate 2-aminoanthracene 1 Pg/plate (2 Pg/plate) WP2 uvrA methylmethanesulphonate 500 Pg/plate 2-aminoanthracene 10 Pg/plate (20 Pg/plate) Concentrations refer to both treatment methods. When two values are given, the figures in brackets refer to the pre-incubation method assay. 3.3 Media The following growth media were used: Nutrient Broth: Oxoid Nutrient Broth No 2 was prepared at a concentration of 2.5% in distilled water and autoclaved prior to use. This was used for the preparation of liquid cultures of the tester strains. Nutrient Agar: Oxoid Nutrient Broth No 2 (25g) and Difco Bacto-agar (15g) were added to distilled water (1 litre) and autoclaved. The solutions were then poured into 9 cm plastic Petri dishes and allowed to solidify and dry before use. These plates were used for the non-selective growth of the tester strains. Minimal Agar: Minimal medium agar was prepared as 1.5% Difco Bacto-agar in VogelBonner Medium E, with 2% Glucose, and poured into 9 cm plastic Petri dishes. Top Agar: "Top Agar" (overlay agar) was prepared as 0.6% Difco Bacto-agar + 0.5% NaCl in distilled water. Prior to use 10 ml of a sterile solution of 0.5 mM Biotin + 0.5 mM Histidine (or 0.5 mM tryptophan) was added to the top agar (100 ml). RTC Study No.: 76250 Page 11 3.4 S9 tissue homogenate Two MOLTOX rat S9 liver tissue fractions were used in this study 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 : MOLTOX, Molecular Toxicology, Inc. : 05-Dec-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 : MOLTOX, Molecular Toxicology, Inc. : 24-Mar-2009 : Approximately -80C : Sprague Dawley : Male : 37.8 mg/ml Production and quality control certificates can be found in Addendum III of this report The mixture of S9 tissue fraction and cofactors (S9 mix) was prepared as follows (for each 10 ml): S9 tissue fraction NADP (100 mM) G-6-P (100 mM) KCl (330 mM) MgCl2 (100 mM) Phosphate buffer (pH 7.4, 200 mM) Distilled Water 1.0 ml 0.4 ml 0.5 ml 1.0 ml 0.8 ml 5.0 ml 1.3 ml ______ 10.0 ml RTC Study No.: 76250 Page 12 3.5 3.6 3.6.1 3.6.2 Bacterial strains Four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and a strain of Escherichia coli (WP2 uvrA) were used in this study. TA1535 and TA100 are predominantly sensitive to base pair mutagens, TA1537 and TA98 are sensitive to frameshift mutagens. In addition to a mutation in the histidine operon, the Salmonella tester strains contain additional mutations which enhance their sensitivity to some mutagenic compounds. The rfa wall mutation results in the loss of one of the enzymes responsible for the synthesis of part of the lipopolysaccharide barrier that forms the surface of the bacterial cell wall and increases permeability to certain classes of chemicals. All strains are deficient in a DNA excision repair system (uvrB mutation) which enhances the sensitivity to some mutagens. TA98 and TA100 strains contain the pKM101 plasmid which activates an error prone DNA repair system. Tester strain WP2 uvrA is reverted from tryptophan dependence (auxotrophy) to tryptophan independence (prototrophy) bay base substitution mutagens. In addition to the mutation in the tryptophan operon, the tester strain contains an uvrA DNA repair deficiency which enhance its sensitivity to some mutagenic compounds. Permanent stocks of these strains are kept at -80C in RTC. Overnight subcultures of these stocks were prepared for each day's work. Bacteria were taken from vials of frozen cultures, which had been checked for the presence of the appropriate genetic markers, as follows: Histidine requirement Tryptophan requirement uvrA, uvrB rfa pKM101 : No Growth on Minimal plates + Biotin. Growth on Minimal plates + Biotin + Histidine. : No Growth on Minimal agar plates Growth on Minimal plates + Tryptophan. : Sensitivity to UV irradiation. : Sensitivity to Crystal Violet. : Resistance to Ampicillin. Bacterial cultures in liquid and on agar were clearly identified with their identity. Methods Preliminary toxicity test A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in the main assays. In these tests a wide range of dose levels of the test item, set at half-log intervals, were used. Treatments were performed both in the absence and presence of S9 metabolism using the plate incorporation method; a single plate was used at each test point and positive controls were not included. Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation. Main experiments Two experiments were performed including negative and positive controls in the absence and presence of an S9 metabolising system. Three replicate plates were used at each test point. RTC Study No.: 76250 Page 13 3.6.3 In addition, plates were prepared to check the sterility of the test item solutions and the S9 mix, and dilutions of the bacterial cultures were plated on nutrient agar plates to establish the number of bacteria in the cultures. The first experiment was performed using a plate-incorporation method. The components of the assay (the tester strain bacteria, the test item and S9 mix or phosphate buffer) were added to molten overlay agar and vortexed. The mixture was then poured onto the surface of a minimal medium agar plate, and allowed to solidify prior to incubation. The overlay mixture was composed as follows: (i) Overlay agar (held at 45C) (ii) Test or control item solution (iii) S9 mix or phosphate buffer (pH 7.4, 0.1 M) (iv) Bacterial suspension 2 ml 0.1 ml 0.5 ml 0.1 ml The second experiment was performed using a pre-incubation method. The components were added in turn to an empty test-tube: (i) Bacterial suspension (ii) Test item solution or control item solution (iii) S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.1 ml 0.1 ml 0.05 ml 0.5 ml The incubate was vortexed and placed at 37C for 30 minutes. Two ml of overlay agar was then added and the mixture vortexed again and poured onto the surface of a minimal medium agar plate and allowed to solidify. Incubation and scoring The prepared plates were inverted and incubated for approximately 72 hours at 37C. After this period of incubation, the scoring was effected by counting the number of revertant colonies on each plate. RTC Study No.: 76250 Page 14 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 Toxicity test The test item C6O4 CYCLIC was assayed in the toxicity test at a maximum dose level of 5000 Pg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 Pg/plate. Concentrations are expressed in terms of active ingredient. Results are presented in Tables 1 and 2. No precipitation of the test item was noted at the end of the incubation period at any concentration. No toxicity was observed at any dose level with any tester strain, in the absence or presence of S9 metabolic activation. On the basis of these results a maximum concentration of 5000 Pg/plate was selected for the Main Assay. 4.3 Assay for reverse mutation Two experiments were performed; individual plate counts for these tests, and the mean and standard error of the mean for each test point, together with statistical analysis are presented in Tables 3 to 12. In Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels expressed in terms of active ingredient: 5000, 2500, 1250, 625 and 313 Pg/plate. No relevant toxicity was observed with any tester strain at any dose level. As no relevant increases in revertant numbers were observed at any concentration tested, a pre-incubation step was included for all treatments of Main Assay II. The test item was assayed at the same dose levels as in Main Assay I. Toxicity, as indicated by thinning of the background lawn and/or reduction in revertant numbers, was observed at the higher dose levels with all tester strains, both in the absence and presence of S9 metabolic activation. No precipitation of the test item was noted at the end of the incubation period at any concentration tested. No increases in the number of revertant colonies were observed in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism. The sterility of the S9 mix and of the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly. RTC Study No.: 76250 Page 15 5. ANALYSIS OF RESULTS 5.1 Criteria for outcome of the assays For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels. 5.2 Evaluation Results show that mean plate counts for untreated and positive control plates fell within the normal historical range included in Appendices 1 and 2. The estimated numbers of viable bacteria/plate (titre) fell in the range of 100 - 500 million for each strain. No plates were lost through contamination or cracking. The study was accepted as valid. The test item does not induce two-fold increases in the number of revertant colonies, at any dose level, in any tester strain, in the absence or presence of S9 metabolism. On the basis of the stated criteria it must be concluded that the test item C6O4 CYCLIC is not mutagenic to S. typhimurium or E. coli under the reported experimental conditions. RTC Study No.: 76250 Page 16 6. CONCLUSION It is concluded that the test item C6O4 CYCLIC does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions. RTC Study No.: 76250 Page 17 7. KEY TO TABLES 1-12 7.1 Structure of Tables 3-12 These tables show, for each Salmonella typhimurium or Escherichia coli tester strain, the individual plate counts obtained for the negative and positive controls, and at each dose level of the test item. The mean number of revertant colonies and standard error of the mean are also presented. The "untreated" plates received no treatment. The titre of the bacterial cultures is given (million cells/plate). 7.2 Regression line i) The regression analysis fits a regression line to the data by the least squares method, after square root transformation of the plate counts to satisfy normal distribution and homoscedasticity assumptions. The regression equation is expressed as: y = a + bx where y = transformed revertant numbers a = intercept b = slope value x = dose level (in the units given). ii) The regression line includes the untreated control data. iii) Regression lines are calculated using a minimum of the three lowest dose levels, and then including the further dose levels in turn. The correlation co-efficient (r), the value of students "t" statistic, and the p-value for the regression lines are also given. RTC Study No.: 76250 Page 18 Tables C6O4 CYCLIC: BACTERIAL MUTATION ASSAY (S. typhimurium and E. coli) TABLE 1 - Toxicity test without metabolic activation STUDY NO.: 76250 SOLVENT: Sterile distilled water EXPERIMENT: Toxicity test ___________________________________________________________________________ Dose level (g/plate) TA-1535 Rev/pl. TA-1537 Rev/pl. TA-98 Rev/pl. TA-100 Rev/pl. WP2 uvrA Rev/pl. ___________________________________________________________________________ Untreated 25 20 32 151 25 50.0 25 24 30 156 28 158 23 22 34 144 28 500 26 17 31 151 34 1580 27 21 36 140 28 5000 23 16 35 149 27 ___________________________________________________________________________ RTC Study No.: 76250 Page 19 C6O4 CYCLIC: BACTERIAL MUTATION ASSAY (S. typhimurium and E. coli) TABLE 2 - Toxicity test with metabolic activation STUDY NO.: 76250 SOLVENT: Sterile distilled water EXPERIMENT: Toxicity test ___________________________________________________________________________ Dose level (g/plate) TA-1535 Rev/pl. TA-1537 Rev/pl. TA-98 Rev/pl. TA-100 Rev/pl. WP2 uvrA Rev/pl. ___________________________________________________________________________ Untreated 22 27 47 164 34 50.0 19 22 43 162 38 158 21 22 47 166 34 500 24 23 50 175 35 1580 18 25 46 173 35 5000 21 27 40 162 37 __________________________________________________________________________ RTC Study No.: 76250 Page 20 C6O4 CYCLIC: BACTERIAL MUTATION ASSAY (S. typhimurium and E. coli) TABLE 3 - Experiment 1 - Plate incorporation method - TA1535 STUDY NO.: 76250 SOLVENT: Sterile distilled water ________________________________________________________________________ Strain: TA1535 Titre: 252 Dose-level Without metabolic activation With metabolic activation [g/pl] Plate counts Mean S. E. Plate counts Mean S. E. ________________________________________________________________________ Untreated 17 12 18 16 1.9 14 21 20 18 2.2 313 18 17 18 18 0.3 19 21 19 20 0.7 625 18 21 19 19 0.9 16 25 20 20 2.6 1250 23 21 26 23 1.5 19 19 19 19 0.0 2500 19 19 24 21 1.7 21 24 26 24 1.5 5000 19 28 20 22 2.8 23 25 21 23 1.2 ________________________________________________________________________ Regression analysis: Points S9 Intercept Slope Corr. coeff. t P-value 1-3 - 3.954 0.0007 0.64771 2.2493 0.05926 1-4 - 3.961 0.0007 0.83070 4.7186 0.00082 1-5 - 4.164 0.0002 0.54765 2.3600 0.03458 1-6 - 4.252 0.0001 0.48831 2.2383 0.03978 1-3 + 4.284 0.0004 0.27142 0.7461 0.47990 1-4 + 4.359 0.0001 0.08088 0.2566 0.80269 1-5 + 4.294 0.0002 0.52489 2.2234 0.04454 1-6 + 4.366 0.0001 0.53004 2.5003 0.02366 ________________________________________________________________________ Positive and negative controls Treatment S9 Plate counts Mean S. E. Untreated - 17 12 18 16 1.9 Sodium Azide 1 g/pl - 555 533 508 532 13.6 DMSO 100 l/pl + 17 17 19 18 0.7 2-Aminoanthracene 1 g/pl + 121 131 124 125 3.0 ________________________________________________________________________ RTC Study No.: 76250 Page 21 C6O4 CYCLIC: BACTERIAL MUTATION ASSAY (S. typhimurium and E. coli) TABLE 4 - Experiment 1 - Plate incorporation method - TA1537 STUDY NO.: 76250 SOLVENT: Sterile distilled water ________________________________________________________________________ Strain: TA1537 Titre: 229 Dose-level Without metabolic activation With metabolic activation [g/pl] Plate counts Mean S. E. Plate counts Mean S. E. ________________________________________________________________________ Untreated 15 20 12 16 2.3 19 23 21 21 1.2 313 15 22 20 19 2.1 24 25 26 25 0.6 625 26 15 24 22 3.4 19 20 22 20 0.9 1250 23 23 19 22 1.3 28 26 23 26 1.5 2500 21 17 13 17 2.3 17 19 28 21 3.4 5000 17 22 20 20 1.5 21 24 21 22 1.0 ________________________________________________________________________ Regression analysis: Points S9 Intercept Slope Corr. coeff. t P-value 1-3 - 3.958 0.0011 0.53730 1.6855 0.13575 1-4 - 4.094 0.0005 0.50989 1.8744 0.09036 1-5 - 4.328 0.0000 0.00666 0.0240 0.98120 1-6 - 4.317 0.0000 0.07307 0.2931 0.77324 1-3 + 4.731 -0.0001 -0.11334 0.3018 0.77156 1-4 + 4.636 0.0003 0.43631 1.5334 0.15619 1-5 + 4.765 0.0000 -0.04490 0.1621 0.87375 1-6 + 4.763 0.0000 -0.08031 0.3223 0.75142 ________________________________________________________________________ Positive and negative controls Treatment S9 Plate counts Mean S. E. DMSO 100 l/pl - 23 17 18 19 1.9 9-Aminoacridine 50 g/pl - 108 116 121 115 3.8 DMSO 100 l/pl + 28 23 22 24 1.9 2-Aminoanthracene 1 g/pl + 119 105 113 112 4.1 ________________________________________________________________________ RTC Study No.: 76250 Page 22 C6O4 CYCLIC: BACTERIAL MUTATION ASSAY (S. typhimurium and E. coli) TABLE 5 - Experiment 1 - Plate incorporation method - WP2uvrA STUDY NO.: 76250 SOLVENT: Sterile distilled water ________________________________________________________________________ Strain: WP2uvrA Titre: 304 Dose-level Without metabolic activation With metabolic activation [g/pl] Plate counts Mean S. E. Plate counts Mean S. E. ________________________________________________________________________ Untreated 21 28 25 25 2.0 35 34 32 34 0.9 313 20 20 28 23 2.7 31 35 36 34 1.5 625 21 27 28 25 2.2 32 27 29 29 1.5 1250 23 28 31 27 2.3 29 35 33 32 1.8 2500 30 31 20 27 3.5 33 38 29 33 2.6 5000 25 28 19 24 2.6 28 33 30 30 1.5 ________________________________________________________________________ Regression analysis: Points S9 Intercept Slope Corr. coeff. t P-value 1-3 - 4.876 0.0001 0.07522 0.1996 0.84749 1-4 - 4.834 0.0003 0.34752 1.1720 0.26837 1-5 - 4.897 0.0001 0.29491 1.1128 0.28595 1-6 - 4.995 0.0000 0.01307 0.0523 0.95895 1-3 + 5.875 -0.0006 -0.62924 2.1420 0.06943 1-4 + 5.758 -0.0001 -0.26638 0.8739 0.40266 1-5 + 5.695 0.0000 0.00892 0.0322 0.97482 1-6 + 5.724 0.0000 -0.23872 0.9833 0.34010 ________________________________________________________________________ Positive and negative controls Treatment S9 Plate counts Mean S. E. Untreated - 21 28 25 25 2.0 MMS 500 g/pl - 185 169 164 173 6.3 DMSO 100 l/pl + 27 27 33 29 2.0 2-Aminoanthracene 10 g/pl + 204 193 195 197 3.4 ________________________________________________________________________ RTC Study No.: 76250 Page 23 C6O4 CYCLIC: BACTERIAL MUTATION ASSAY (S. typhimurium and E. coli) TABLE 6 - Experiment 1 - Plate incorporation method - TA98 STUDY NO.: 76250 SOLVENT: Sterile distilled water ________________________________________________________________________ Strain: TA98 Titre: 278 Dose-level Without metabolic activation With metabolic activation [g/pl] Plate counts Mean S. E. Plate counts Mean S. E. ________________________________________________________________________ Untreated 24 31 33 29 2.7 35 43 35 38 2.7 313 26 30 33 30 2.0 39 36 33 36 1.7 625 30 26 22 26 2.3 34 32 38 35 1.8 1250 33 35 31 33 1.2 40 37 42 40 1.5 2500 30 34 38 34 2.3 40 41 36 39 1.5 5000 31 34 35 33 1.2 45 46 41 44 1.5 ________________________________________________________________________ Regression analysis: Points S9 Intercept Slope Corr. coeff. t P-value 1-3 - 5.468 -0.0005 -0.36065 1.0230 0.34034 1-4 - 5.288 0.0002 0.30198 1.0017 0.34010 1-5 - 5.299 0.0002 0.48961 2.0246 0.06396 1-6 - 5.385 0.0001 0.45801 2.0609 0.05595 1-3 + 6.126 -0.0004 -0.38130 1.0913 0.31129 1-4 + 5.994 0.0002 0.25503 0.8341 0.42372 1-5 + 6.017 0.0001 0.33136 1.2663 0.22763 1-6 + 6.000 0.0001 0.65894 3.5041 0.00294 ________________________________________________________________________ Positive and negative controls Treatment S9 Plate counts Mean S. E. DMSO 100 l/pl - 33 30 32 32 0.9 2-Nitrofluorene 2 g/pl - 157 169 159 162 3.7 DMSO 100 l/pl + 36 43 39 39 2.0 2-Aminoanthracene 1 g/pl + 459 465 488 471 8.8 ________________________________________________________________________ RTC Study No.: 76250 Page 24 C6O4 CYCLIC: BACTERIAL MUTATION ASSAY (S. typhimurium and E. coli) TABLE 7 - Experiment 1 - Plate incorporation method - TA100 STUDY NO.: 76250 SOLVENT: Sterile distilled water ________________________________________________________________________ Strain: TA100 Titre: 267 Dose-level Without metabolic activation With metabolic activation [g/pl] Plate counts Mean S. E. Plate counts Mean S. E. ________________________________________________________________________ Untreated 150 168 157 158 5.2 150 171 178 166 8.4 313 161 151 154 155 3.0 181 157 167 168 7.0 625 157 169 147 158 6.4 176 165 161 167 4.5 1250 147 144 143 145 1.2 159 173 164 165 4.1 2500 149 156 160 155 3.2 170 166 161 166 2.6 5000 151 164 149 155 4.7 181 173 162 172 5.5 ________________________________________________________________________ Regression analysis: Points S9 Intercept Slope Corr. coeff. t P-value 1-3 - 12.545 0.0000 -0.04005 0.1061 0.91851 1-4 - 12.638 -0.0004 -0.58866 2.3027 0.04405 1-5 - 12.485 -0.0001 -0.21556 0.7959 0.44038 1-6 - 12.438 0.0000 -0.07312 0.2933 0.77308 1-3 + 12.908 0.0001 0.04880 0.1293 0.90079 1-4 + 12.936 0.0000 -0.05952 0.1886 0.85422 1-5 + 12.928 0.0000 -0.07354 0.2659 0.79450 1-6 + 12.883 0.0000 0.18116 0.7368 0.47189 ________________________________________________________________________ Positive and negative controls Treatment S9 Plate counts Mean S. E. Untreated - 150 168 157 158 5.2 Sodium Azide 1 g/pl - 656 611 707 658 27.7 DMSO 100 l/pl + 165 173 171 170 2.4 2-Aminoanthracene 1 g/pl + 1352 1281 1181 1271 49.6 ________________________________________________________________________ RTC Study No.: 76250 Page 25 C6O4 CYCLIC: BACTERIAL MUTATION ASSAY (S. typhimurium and E. coli) TABLE 8 - Experiment 2 - Preincubation method - TA1535 STUDY NO.: 76250 SOLVENT: Sterile distilled water ________________________________________________________________________ Strain: TA1535 Titre: 264 Dose-level Without metabolic activation With metabolic activation [g/pl] Plate counts Mean S. E. Plate counts Mean S. E. ________________________________________________________________________ Untreated 16 23 16 18 2.3 17 20 15 17 1.5 313 16 19 20 18 1.2 23 25 27 25 1.2 625 17 25 20 21 2.3 21 21 18 20 1.0 1250 15 15 20 17 1.7 23 18 22 21 1.5 2500 13 12 16 * 14 1.2 17 14 14 * 15 1.0 5000 13 9 12 * 11 1.2 12 14 17 * 14 1.5 ________________________________________________________________________ Regression analysis: Points S9 Intercept Slope Corr. coeff. t P-value 1-3 - 4.225 0.0004 0.31541 0.8794 0.40836 1-4 - 4.362 -0.0001 -0.18002 0.5787 0.57557 1-5 - 4.419 -0.0003 -0.58176 2.5789 0.02290 1-6 - 4.379 -0.0002 -0.75031 4.5399 0.00033 1-3 + 4.384 0.0005 0.32231 0.9008 0.39761 1-4 + 4.471 0.0001 0.18231 0.5863 0.57064 1-5 + 4.639 -0.0002 -0.49210 2.0382 0.06241 1-6 + 4.591 -0.0002 -0.63518 3.2895 0.00462 ________________________________________________________________________ Positive and negative controls Treatment S9 Plate counts Mean S. E. Untreated - 16 23 16 18 2.3 Sodium Azide 1 g/pl - 495 508 479 494 8.4 DMSO 50 l/pl + 19 17 20 19 0.9 2-Aminoanthracene 1 g/pl + 108 111 107 109 1.2 ________________________________________________________________________ *: thinning of the background lawn RTC Study No.: 76250 Page 26 C6O4 CYCLIC: BACTERIAL MUTATION ASSAY (S. typhimurium and E. coli) TABLE 9 - Experiment 2 - Preincubation method - TA1537 STUDY NO.: 76250 SOLVENT: Sterile distilled water ________________________________________________________________________ Strain: TA1537 Titre: 233 Dose-level Without metabolic activation With metabolic activation [g/pl] Plate counts Mean S. E. Plate counts Mean S. E. ________________________________________________________________________ Untreated 15 21 18 18 1.7 17 26 25 23 2.8 313 23 23 25 24 0.7 24 25 27 25 0.9 625 20 24 17 20 2.0 24 30 28 27 1.8 1250 19 13 15 16 1.8 28 26 24 26 1.2 2500 11 8 7 * 9 1.2 17 25 23 * 22 2.4 5000 11 8 4 * 8 2.0 20 10 12 * 14 3.1 ________________________________________________________________________ Regression analysis: Points S9 Intercept Slope Corr. coeff. t P-value 1-3 - 4.399 0.0004 0.29922 0.8297 0.43410 1-4 - 4.596 -0.0004 -0.41083 1.4250 0.18462 1-5 - 4.719 -0.0007 -0.82886 5.3418 0.00013 1-6 - 4.529 -0.0004 -0.82454 5.8293 0.00003 1-3 + 4.757 0.0008 0.55031 1.7438 0.12472 1-4 + 4.882 0.0003 0.36824 1.2525 0.23889 1-5 + 5.037 -0.0001 -0.23365 0.8664 0.40198 1-6 + 5.159 -0.0003 -0.72443 4.2035 0.00067 ________________________________________________________________________ Positive and negative controls Treatment S9 Plate counts Mean S. E. DMSO 50 l/pl - 20 21 22 21 0.6 9-Aminoacridine 50 g/pl - 101 162 152 138 18.9 DMSO 50 l/pl + 27 27 25 26 0.7 2-Aminoanthracene 1 g/pl + 107 105 92 101 4.7 ________________________________________________________________________ *: thinning of the background lawn RTC Study No.: 76250 Page 27 C6O4 CYCLIC: BACTERIAL MUTATION ASSAY (S. typhimurium and E. coli) TABLE 10 - Experiment 2 - Preincubation method - WP2uvrA STUDY NO.: 76250 SOLVENT: Sterile distilled water ________________________________________________________________________ Strain: WP2uvrA Titre: 313 Dose-level Without metabolic activation With metabolic activation [g/pl] Plate counts Mean S. E. Plate counts Mean S. E. ________________________________________________________________________ Untreated 18 21 25 21 2.0 31 31 32 31 0.3 313 30 32 30 31 0.7 27 27 26 27 0.3 625 27 24 27 26 1.0 33 35 27 32 2.4 1250 31 33 28 31 1.5 27 34 35 32 2.5 2500 22 24 23 * 23 0.6 24 30 22 * 25 2.4 5000 25 32 29 * 29 2.0 22 20 20 * 21 0.7 ________________________________________________________________________ Regression analysis: Points S9 Intercept Slope Corr. coeff. t P-value 1-3 1-4 - 4.836 4.892 0.0008 0.0006 0.46480 0.59215 1.3889 2.3237 0.20744 0.04250 1-5 - 5.151 0.0000 -0.08198 0.2966 0.77148 1-6 - 5.095 0.0000 0.15178 0.6142 0.54770 1-3 + 5.450 0.0000 0.03096 0.0819 0.93699 1-4 + 5.418 0.0002 0.24987 0.8161 0.43348 1-5 + 5.559 -0.0002 -0.38595 1.5084 0.15536 1-6 + 5.590 -0.0002 -0.74254 4.4344 0.00042 ________________________________________________________________________ Positive and negative controls Treatment S9 Plate counts Mean S. E. Untreated - 18 21 25 21 2.0 MMS 500 g/pl - 162 153 139 151 6.7 DMSO 50 l/pl + 23 24 22 23 0.6 2-Aminoanthracene 20 g/pl + 184 184 173 180 3.7 ________________________________________________________________________ *: thinning of the background lawn RTC Study No.: 76250 Page 28 C6O4 CYCLIC: BACTERIAL MUTATION ASSAY (S. typhimurium and E. coli) TABLE 11 - Experiment 2 - Preincubation method - TA98 STUDY NO.: 76250 SOLVENT: Sterile distilled water ________________________________________________________________________ Strain: TA98 Titre: 278 Dose-level Without metabolic activation With metabolic activation [g/pl] Plate counts Mean S. E. Plate counts Mean S. E. ________________________________________________________________________ Untreated 36 36 40 37 1.3 39 39 37 38 0.7 313 33 33 38 35 1.7 36 36 41 38 1.7 625 29 34 37 33 2.3 42 37 36 38 1.9 1250 36 35 28 33 2.5 38 48 38 41 3.3 2500 31 38 36 * 35 2.1 35 30 38 * 34 2.3 5000 30 24 28 * 27 1.8 24 25 24 * 24 0.3 ________________________________________________________________________ Regression analysis: Points S9 Intercept Slope Corr. coeff. t P-value 1-3 - 6.091 -0.0005 -0.53265 1.6651 0.13983 1-4 - 6.025 -0.0003 -0.44374 1.5659 0.14845 1-5 - 5.924 0.0000 -0.14337 0.5223 0.61023 1-6 - 5.992 -0.0001 -0.63457 3.2842 0.00467 1-3 + 6.173 0.0000 -0.00749 0.0198 0.98474 1-4 + 6.122 0.0002 0.36516 1.2404 0.24315 1-5 + 6.258 -0.0001 -0.31406 1.1927 0.25431 1-6 + 6.370 -0.0003 -0.83048 5.9634 0.00002 ________________________________________________________________________ Positive and negative controls Treatment S9 Plate counts Mean S. E. DMSO 50 l/pl - 34 28 33 32 1.9 2-Nitrofluorene 2 g/pl - 171 193 161 175 9.5 DMSO 50 l/pl + 40 45 39 41 1.9 2-Aminoanthracene 2 g/pl + 654 604 647 635 15.6 ________________________________________________________________________ *: thinning of the background lawn RTC Study No.: 76250 Page 29 C6O4 CYCLIC: BACTERIAL MUTATION ASSAY (S. typhimurium and E. coli) TABLE 12 - Experiment 2 - Preincubation method - TA100 STUDY NO.: 76250 SOLVENT: Sterile distilled water ________________________________________________________________________ Strain: TA100 Titre: 264 Dose-level Without metabolic activation With metabolic activation [g/pl] Plate counts Mean S. E. Plate counts Mean S. E. ________________________________________________________________________ Untreated 166 151 167 161 5.2 171 171 162 168 3.0 313 165 166 159 163 2.2 171 162 176 170 4.1 625 157 162 168 162 3.2 167 178 160 168 5.2 1250 119 95 93 * 102 8.4 94 114 108 * 105 5.9 2500 60 78 62 * 67 5.7 75 76 61 * 71 4.8 5000 76 60 71 * 69 4.7 75 68 55 * 66 5.9 ________________________________________________________________________ Regression analysis: Points S9 Intercept Slope Corr. coeff. t P-value 1-3 - 12.719 0.0001 0.08026 0.2130 0.83738 1-4 - 13.259 -0.0022 -0.83593 4.8165 0.00071 1-5 - 13.210 -0.0020 -0.94478 10.3947 0.00000 1-6 - 12.452 -0.0010 -0.84726 6.3802 0.00001 1-3 + 12.980 0.0000 0.01914 0.0506 0.96102 1-4 + 13.529 -0.0022 -0.85193 5.1448 0.00043 1-5 + 13.446 -0.0021 -0.94855 10.8010 0.00000 1-6 + 12.742 -0.0011 -0.87601 7.2655 0.00000 ________________________________________________________________________ Positive and negative controls Treatment S9 Plate counts Mean S. E. Untreated - 166 151 167 161 5.2 Sodium Azide 1 g/pl - 649 625 607 627 12.2 DMSO 50 l/pl + 119 121 134 125 4.7 2-Aminoanthracene 2 g/pl + 945 1021 975 980 22.1 ________________________________________________________________________ *: thinning of the background lawn RTC Study No.: 76250 Page 30 Appendices C6O4 CYCLIC: BACTERIAL MUTATION ASSAY (S. typhimurium and E. coli) APPENDIX 1 - Historical control data without metabolic activation (2000-2008) STUDY NO.: 76250 Untreated TA1535 Mean value SD n TA1537 Mean value SD n TA98 Mean value SD n TA100 Mean value SD n WP2uvrA Mean value SD n Plate incorporation 18 2.6 185 17 1.9 184 31 3.2 187 143 15.4 187 28 3.1 127 Untreated Pre-incubation 18 2.4 190 17 1.8 187 32 3.0 179 142 15.2 186 27 2.9 122 Positive control Plate incorporation 565 76.4 185 169 33.2 184 183 24.3 187 782 123.5 187 180 17.9 127 Positive control Pre-incubation 567 73.5 190 164 41.1 187 180 23.4 179 791 121.9 186 210 42.5 122 SD : n : standard deviation number of experiments RTC Study No.: 76250 Page 31 C6O4 CYCLIC: BACTERIAL MUTATION ASSAY (S. typhimurium and E. coli APPENDIX 2 - Historical control data with metabolic activation (2000-2008) STUDY NO.: 76250 Untreated Untreated Positive control Positive control TA1535 Plate incorporation Mean value 16 SD 1.8 n 185 TA1537 Mean value 21 SD 2.4 n 184 TA98 Mean value 39 SD 3.7 n 187 TA100 Mean value 152 SD 15.7 n 188 WP2uvrA Mean value 35 SD 3.4 n 126 Pre-incubation 16 1.8 189 20 2.1 188 39 2.9 169 150 14.2 184 34 3.1 117 Plate incorporation 142 28.3 185 120 19.9 184 611 137.7 187 1261 196.5 188 205 35.2 126 Pre-incubation 96 12.7 189 97 11.5 188 661 179.7 169 1135 186.9 184 191 32.6 117 SD : n : standard deviation number of experiments RTC Study No.: 76250 Page 32 Addenda C6O4 CYCLIC: BACTERIAL MUTATION ASSAY (S. typhimurium and E. coli ADDENDUM I - Study Protocol STUDY NO.: 76250 RTC Study No.: 76250 Page 33 RESEARCH TOXICOLOGY CENTRE ROMA Version 08/2 C604 CYCLIC BACTERIAL MUTATION ASSAY (S. (vphimurium and E. colt) 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, 12/14 00040 Pomezia (Roma) Italy RTC Enquiry Number: 75839 RTC Study Number: 76250 Commercial Office RTC 5 0.> Ve Tao Sim, 12 00040 Pomona (Ronal ITALY Tot: 3905.91095.1 Fax . 39089105737 ormil:inktencit ww.rIc - 1 of 14 - Head Office and AdmIniatratIon RTC 89 Tao 3074512 03040 Rome Mono ITALY Tel.: 3908.01095.1 Fax 39438.912.2233 P.O. Box 15307-00143 Rem Eto Lawmen> March 2009 RTC SPA Candate 590818 E965 5.184000 C C Aw. 6. 375378 Reg. Soc. Tra. a. Roma n. 2828/72 Coe R.50.. 0065312058,1 Panda len 00920611001 RTC Study No.: 76250 Page 34 Version 08/2 BACTERIAL MUTATION ASSAY (S. typhimurium and E. colt) MANAGEMENT OF STUDY Scientific Director Head of Genetic Toxicology, in vitro Toxicology and Immunology Study Director Sponsor SOLVAY SOLEXIS S.p.A. Viale Lombardia, 20 20021 Bollate (MI) Italy Monitor QUALITY ASSURANCE Quality Assurance Manager LOCATION OF STUDY The study will be performed at Research Toxicology Centre S.p.A. Via Tito Speri, 12/14 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 item will be: C604 CYCLIC RTC Enquiry Number: 75839 RTC Study Number: 76250 -2 of 14 - March 2009 RTC Study No.: 76250 Page 35 Version 08/2 BACTERIAL MUTATION ASSAY (S. typhimurium and E. colt) 1. INTRODUCTION 1.1 Objective To assay the test item for the ability to induce gene mutations in Salmonella typhimurium and Escherichia colt, as measured by reversion of auxotrophic strains to prototrophy. 1.2 Regulatory requirements This study will be conducted in compliance with the GLP regulations of: - US FDA [21 CFR part 58, 22 December 1978] and subsequent revisions; - Directive 2004/10/EC of the European Parliament and of the Council of 11 February 2004; - ENV/MC/CHEM(98)I 7 "OECD principles on Good Laboratory Practice -- as revised in 1997"; - Decreto Legislativo no. 50 of 2 March 2007 and subsequent revisions. In addition, the study is designed to comply with the experimental methods indicated in the guidelines of: - EEC Council Directive 2000/32, Annex 4D. - OECD Guidelines for the testing of chemicals No. 471 (Adopted July 1997). - ICH S2A Genotoxicity: Specific Aspects of Regulatory Tests, Step 5. 1.3 Principles of the method Reverse mutation assays employ bacterial strains which are already mutant at a locus whose phenotypic effects are easily detected. The Salmonella tester strains have mutations causing dependence on a particular amino acid (histidine) for growth. The ability of test items to cause reverse mutations (reversions) to histidine-independence can easily be measured. The E. colt tester strains of the WP2 series are similarly mutant at the tryptophan locus. Since many chemicals only demonstrate mutagenic activity after metabolism to reactive forms, in order to detect these "indirect mutagens" the test is performed in the presence and absence of a rat liver metabolising system. 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 substance, 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 substance will be retained within the archives of the testing facility for a period of ten years after which it will be destroyed. RTC Enquiry Number: 75839 March 2009 RTC Study Number: 76250 -3of14- RTC Study No.: 76250 Page 36 Version 08/2 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. 2.5 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 dilutions of the test item as supplied will be prepared for each day's work at appropriate concentrations. One sterility test, using nutrient broth, will be performed before commencing all experimental treatments. Concentrations of solutions will be expressed in terms of active ingredient. Preferred solvent will be sterile distilled water. 2.8 No assay of 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. 3. MATERIALS 3.1 Bacterial strains Stocks of Salmonella tester strains (TA 1535, TA 1537, TA 1538, TA 98, TA 100, TA 97 and TA 102 and some other related strains) were obtained from Dr. B.N.Ames, University of California. Stocks of E. coli tester strains (WP2, WP2 uvrA and WP2 uvrA pKM101) were obtained from Life Science Research, Occold, Suffolk, UK. Permanent stocks are kept at -80C, and overnight subcultures of these stocks are prepared for each day's work. The cultures used in each experiment contain a high titre of viable bacteria (1-5 x 109 cells per ml). The titre of each tester strain culture will be demonstrated in each experiment through the determination of viable cell numbers using nutrient agar plates. The presence of the appropriate genetic markers in these strains is checked on a monthly basis for those in regular use, and as necessary for other strains, as follows: Histidine requirement Tryptophan requirement uvrA, uvrB rfa pK.M101 No Growth on Minimal plates + Biotin. Growth on Minimal plates + Biotin + Histidine. No Growth on Minimal agar plates Growth on Minimal plates + Tryptophan. Sensitivity to UV irradiation. Sensitivity to Crystal Violet. Resistance to Ampicillin. RTC Enquiry Number: 75839 RTC Study Number: 76250 -4 of 14 - March 2009 RTC Study No.: 76250 Page 37 Version 08/2 Strain identity is also confirmed by reference to the spontaneous reversion levels and responses to mutagens during use. Bacterial cultures in liquid and on agar are clearly identified with their identity. Detailed information about the genetic constitution of the tester strains may be found in the cited publications ofDr. B.N.Ames and Drs. M.H.L. Green and W.J. Muriel. 3.2 Media The following growth media will be used: Nutrient Broth: Oxoid Nutrient Broth No 2 will be prepared at a concentration of 2.5% in distilled water and autoclaved prior to use. This will be used for the preparation of liquid cultures of the tester strains. Nutrient Agar: Oxoid Nutrient Broth No 2 (25g) and Difco Bacto-agar (15g) will be added to one litre of distilled water and autoclaved. The solution will then be poured into plastic Petri dishes and allowed to solidify and dry before use. These plates will be used for the non-selective growth of the tester strains. Incubations on Nutrient Agar will be for approximately 48 or 72 hours. Minimal Agar: Minimal medium agar will be prepared as 1.5% Difco Bacto-agar in VogelBonner Medium E, with 2% Glucose, and poured into plastic Petri dishes. Top Agar: "Top Agar" (overlay agar) will be prepared as 0.6% Difco Bacto-agar + 0.5% NaCI in distilled water. This solution will be autoclaved, and stored. Prior to use 10 ml of a sterile solution of 0.5 mM Biotin + 0.5 mM Histidine (or 0.5 mM tryptophan) will be added to 100 ml of the top agar. All incubations will be at 37C. 3.3 S9 mix The S9 liver 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 betanaphthoflavone (Mixed Induction); induction with Aroclor 1254 will be performed if specifically requested by the Sponsor. Records pertaining to the preparation of the S9 fraction are kept on file at RTC. The mixture of S9 tissue fraction and cofactors (S9 mix) will be prepared as follows (for each 10 ml): S9 tissue fraction NADP (100 mM) G-6-P (100 mM) KCl (330 mM) MgCl2 (100 mM) Phosphate buffer (pH 7.4, 200 mM) Distilled Water 1.0 ml 0.4 ml 0.5 ml 1.0 ml 0.8 ml 5.0 ml 1.3 ml 10.0 ml RTC Enquiry Number: 75839 RTC Study Number: 76250 -5of14- March 2009 RTC Study No.: 76250 Page 38 Version 08/2 3.4 Control items Positive control treatments will be used in each experiment. The positive control agents are obtained commercially and characterised by their labelling, and their stability determined from the scientific literature. Sodium azide and methylmethanesulphonate will usually be dissolved in distilled water; 9-aminoacridine, 2-nitrofluorene and 2-aminoanthracene will usually be dissolved in DMSO. 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. PRELIMINARY TOXICITY TEST 4.1 Experimental design In order to establish the concentrations of test item to be used in the main assay, a preliminary toxicity test will be performed. This test follows the method described in section 6.1, using only one plate per dose level, a single S9 mix concentration (10%) and covering a wide range of concentrations of the test item. The highest dose-level for this preliminary test, unless limited by the solubility of the test item, will be 5 mg/plate, and the lower dose-levels will be spaced at approximately half-log intervals. 4.2 Selection of dose-levels The toxicity will be assessed on the basis of a decline in the number of spontaneous revertants or a thinning of the background lawn. The highest dose-level for the mutation assays will be selected as a concentration which elicits moderate toxicity. If there is no evidence of toxicity following treatment with the test item, then the highest dose-level will be 5 mg/plate. 5. EXPERIMENTAL DESIGN Each experiment will include solvent/vehicle and positive controls, and at least five doses of the test item, tested in the absence and presence of an S9 metabolising system. Three replicate plates will be used at each test point. If a first experiment produces a clear positive response, no further experiments will be undertaken. If negative or equivocal results are obtained in the first experiment a confirmatory experiment will be performed using the pre-incubation method. A further experiment may be undertaken if inconsistent results are obtained. The five bacterial strains S. typhimurium TA1535, TA1537, TA98, TA100 and E. coli WP2 uvrA will be used in this study. RTC Enquiry Number: 75839 RTC Study Number: 76250 -6 of 14- March 2009 RTC Study No.: 76250 Page 39 Version 08/2 Solvent/vehicle controls: untreated and solvent vehicle controls will be prepared for each experiment; when the solvent is distilled water, these will be considered to be equivalent and only one set of controls will be performed. Positive controls: treatments are indicated in the following table: Tester strain Absence ofS9 Presence ofS9 TAI535 sodium azide 1 g/plate 2-aminoanthracene 1 g/plate TA100 sodium azide 1 g/plate 2-aminoanthracene 1 g/plate (2 g/plate) TA1537 9-amino-acridine 50 g/plate 2-aminoanthracene 1 g/plate TA98 2-nitrofluorene 2 g/plate 2-aminoanthracene 1 g/plate (2 g/plate) WP2 uvrA methylmethanesulphonate 500 g/plate 2-aminoanthracene 10 g/plate (20 g/plate) Concentrations refer to both treatment methods. When two values are given, the figures in brackets refer to the pre-incubation method assay. Test item: the highest dose-level of the test item to be used will be selected as described above. Further dose levels will be selected at intervals of a factor of two. Where it seems advisable, further test points or controls may be included in experiments. In addition, plates will be prepared to check the sterility of the test item solutions and the S9 mix, and dilutions of the bacterial cultures will be plated on nutrient agar plates to establish the number of bacteria in the cultures. 6. ASSAY PROCEDURE 6.1 Plate-incorporation The components of the assay (the tester strain bacteria, the test item and S9 mix or phosphate buffer) will be added to molten overlay agar and vortexed. The mixture will then be poured on the surface of a minimal medium agar plate, and allowed to solidify prior to incubation. RTC Enquiry Number: 75839 RTC Study Number: 76250 - 7 of 14 - March 2009 RTC Study No.: 76250 Page 40 Version 08/2 The overlay mixture will be composed as follows: (i) Overlay agar (held at 45C) (ii) Test or control item solution (iii) S9 mix or phosphate buffer (pH 7.4, 0.1 M) (iv) Bacterial suspension 2 ml 0.1 ml 0.5 ml 0.1 nil The volume of test item solution, as indicated, will usually be 0.1 ml; in the event that it is necessary to alter this volume, the quantities used will be carefully recorded. 6.2 Pre-incubation The components will be added in turn to an empty test-tube: (i) Bacterial suspension (ii) Test or control item solution (iii) S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.1 ml 0.05 ml 0.5 ml The volume of test item solution, as indicated, will usually be 0.05 ml. Where control or test items are dissolved in aqueous solvents, the volume used may be 0.1 ml. In the event that it is necessary to alter this volume, the quantities used will be carefully recorded. The treatment mixture will be vortexed and placed at 37C for 30 minutes. Two ml of overlay agar will then be added and the mixture vortexed again and poured onto the surface of a minimal medium agar plate and allowed to solidify. 6.3 Incubation and scoring The prepared plates will be inverted and incubated for approximately 72 hours at 37C. When the test item is a liquid at ambient temperature, the plates will be incubated in separate closed containers for each dose-level. After this period of incubation, the plates may be held at 4C prior to scoring. Scoring is effected by counting the number of revertant colonies on each plate, either manually, or using a Cardinal - Automatic colony counting system (Perceptive Instruments). Contaminated plates will be considered on a case-by-case basis. 7. REPORTING 7.1 Presentation of data The data will be presented in tabular form. The individual plate counts for each experiment will be given, together with the means and standard errors of the means, and regression analyses. RTC Enquiry Number: 75839 RTC Study Number: 76250 -8of14- March 2009 RTC Study No.: 76250 Page 41 Version 08/2 7.2 Evaluation of data For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose-levels or at the highest practicable doselevel only. In addition there must be evidence of a dose- response relationship showing increasing numbers of mutant colonies with increasing dose-levels. Evaluation of Ames test data based on a `doubling rate' has been shown to be as effective as statistical techniques in allowing the correct interpretation of test results (Chu et al. 1981). 7.3 Reporting procedure A Draft Report will be supplied, and a Final Report issued subsequently to include any agreed changes or amendments. If comments have not been received within 6 months of despatch of the audited draft report, the final report will be issued. 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. 7.4 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 article, identified by name, chemical name or chemical number; method used; any unforeseen circumstances that may have affected the quality or integrity of the study; data generated while conducting the study; statistical methods employed for analysing the data; a summary of the data, an analysis of the data and a statement of the conclusions drawn from the analysis; historical untreated and positive control data; the name and signature of the 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. RTC Enquiry Number: 75839 March 2009 RTC Study Number: 76250 - 9 of 14 - RTC Study No.: 76250 Page 42 Version 08/2 7.5 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. 7.6 Archiving All raw data, records and documentation arising from this study, including the original final protocol 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. The original final report will be despatched to and archived by the Sponsor. 8. STUDY CONDUCT 8.1 Language English language and Italian language versions 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 material will be considered to be equivalent. 8.2 Scientific decisions The procedures described in this protocol may not comprehensively cover all the circumstances that can arise in the assay of test items. When the study director considers it advisable to modify the procedures described for the selection of a solvent, selection of dose levels, interpretation of the outcome of the study or other aspects of the study conduct, he/she will record carefully the decision he has reached and the reasoning which led to it. 8.3 Quality assurance According to the RTC quality assurance programme, defined in the RTC QA SOPs, this study will be subjected to the following procedures: the protocol will be inspected, study/process based inspections of procedures/facilities will be carried out at intervals adequate to assure the integrity of the study, the report will be inspected to assure that it accurately describes the methods and Standard Operating Procedures and that the results accurately reflect the raw data. Periodic reports on these activities will be made to management and the Study Director. All raw data pertaining to the study will be available for inspection by the Sponsor's representative and regulatory authorities (following authorisation from the Sponsor). RTC Enquiry Number: 75839 RTC Study Number: 76250 - 10 of 14 - March 2009 RTC Study No.: 76250 Page 43 Version 08/2 9. DEPARTURES FROM REGULATORY REQUIREMENTS Items which are the responsibility of the Sponsor are indicated in sections 2.1, 2.4, 2.8 and 3.3 of this 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 Number: 75839 RTC Study Number: 76250 - 11 of 14 - March 2009 RTC Study No.: 76250 Page 44 Version 08/2 10. REFERENCES Ames,BN, J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test. Mutation Research 31, 347-364. Chu K.C. et al. (1981) Evaluating statistical analyses and reproducibility of microbial mutagenicity assays. Mutation Research 85, 119-132. Claxton L.D. et al. (1987) Guide for the Salmonella typhimurium/mammalian microsome tests for bacterial mutagenicity. Mutation Research 189, 83-91. Gatehouse D. et al. (1994) Recommendations for the performance of bacterial mutation assays. Mutation Research 312, 217-233. Green M.H.L. and W.J. Muriel (1976) Mutagen testing using TRP+ reversion in Escherichia Mutation Research 38, 3-32. Maron D.M. and B.N. Ames (1983). Revised methods for the Salmonella mutagenicity test. Mutation Research 113, 173-215. Venitt S., R. Forster and E. Longstaff (1983). Bacterial Mutation Assays in: Report of the UKEMS Subcommittee on Guidelines for Mutagenicity testing. U.K.E.M.S., Swansea, 1983. Venitt S., C. Croften-Sleigh and R. Forster (1984) Bacterial mutation assays using reverse mutation in: Mutagenicity Testing - a practical approach S. Venitt and J.M. Parry (eds.) IRL Press, Oxford, 1984. RTC Enquiry Number: 75839 RTC Study Number: 76250 - 12 of 14 - March 2009 RTC Study No.: 76250 Page 45 Version 08/2 PROTOCOL APPROVAL PAGE for R.T.C. S.p.A. APPROVED BY RELEASED BY Date IIIMIMIP Date RTC Enquiry Number: 75839 RTC Study Number: 76250 - 13 of 14 - March 2009 RTC Study No.: 76250 Page 46 Version 08/2 PROTOCOL APPROVAL PAGE for SOLVAY SOLEXIS S.p.A. AUTHORISED BY Date Name and Title* Please print or type your name and company status below your signature. S Date ofProtocol Approval: RTC Enquiry Number: 75839 RTC Study Number: 76250 - 14 of 14 - March 2009 RTC Study No.: 76250 Page 47 C6O4 CYCLIC: BACTERIAL MUTATION ASSAY (S. typhimurium and E. coli) ADDENDUM II - Certificate of analysis STUDY NO.: 76250 RTC Study No.: 76250 Page 48 SOLEXIS R,D&T Analytical & Structural Characterization Department %hale Lombarc1.a. 20 - 20021 BoHate tMU Italy Sample Description: C,O., Ammonium Salt. sample 150/28 Analysis Requests: Ammonium chloride and fluoride determination Salt title quantification Organic impurity determination Method (Equipment): Ionic Chromatography Potentiometry NMR Spectroscopy Performance: MDL: na MQL: na Results: The analyzed sample shows the following weight/volume of: NH4F =28 mg/I NH4CI = 601 mg/I O6O4 (NH4 titration) = 160 g/I The organic impurities are dioxolen based and represent 0.8% by mol on total mol of organic phase of sample. Analytical & Structural Characterization Department RTC Study No.: 76250 Page 49 C6O4 CYCLIC: BACTERIAL MUTATION ASSAY (S. typhimurium and E. coli) ADDENDUM III - S9 production and quality control certificates STUDY NO.: 76250 RTC Study No.: 76250 Page 50 POST MITOCHONDRIAL SUPERNATANT (S-9) PRODUCTION & QUALITY CONTROL CERTIFICATE LOT NO.: 2273 SPECIES: Rat PREPARATION DATE: May 1, 2008 PART NO.: 11-105 STRAIN: Sprague Dawlev EXPIRATION DATE: May 1, 2010 VOLUME: 1 & 2 ml SEX: Male BUFFER: 0.154 M KCl TISSUE: Liver INDUCING AGENT(s): Phenobarbital - REFERENCE: Matsushima, et. al.. In: In Vitro Metabolic 5.5-Benzoflavone Activation in Mutagencsis Testing (F.3. de Serres, Ed.), Elsevier, 1976. p. 85. STORAGE: At or below 70C BIOCHEMISTRY: - PROTEIN 37.6 mg/ml Assayed according to the method of Lowry et al., 1BC 193:265, 1951 using bovine serum albumin as the standard. - ALKOXYRESORUFIN-0tDEALKYLASE ACTIVITIES Fold - Activity P450 Induction EROD IA1, IA2 55.5 Assays for ethoxvresoruib-0-deethylase (EROD), pentoxv-, benzyl- and metho,q-resorufin-0-dealleylases (PROD, BROD. & PROD 2B I 19.7 MROD) were conducted using a modification of the methods of Burke, et al., Biocheni Pharm 34:3337, 1985. Fold- BROD 2B1 19.9 inductions were calculated as the ratio of the sample vs. uninduced specific activities (SA's). Control SA's (pmolcs/min/ IvlROD 1A2 11.2 mg protein) were 31.0, 14.2, 57.4, 10.4 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 microtlora by plating 1 .0 ml volumes on Nutrient Agar and Minimal Glucose (Vogel-Bonner E, supplemented with 0.05 mM L-histidine and Dbiotin) media. Triplicate plates were read after 24 - 48 h incubation at 35C. The tested samples met acceptance criteria_ - PROMUTAGEN ACTIVATION No. his-'- Revertants The ability of the sample to activate ethidium bromide (EtBr) EtBr/ CPA/ and cyclophosphamide (CPA) to intermediates mutagenic to TA98 TA 1535 TA98 and TAL535, respectively, was determined according 342.8 1528 to Lesca, et al., Mutation Res 129:299, 1984. Data were expressed as revertants per pg EtBr or per mg CPA. Dilutions of the sample 59, ranging from 0.2 - 10% in S9 mix, were tested for their ability to activate benzo(a)pyrene (BP) and 2-aminoanthracene (2-AA) to intermediates mutagenic to TA100. Assays were conducted using duplicate plates as described by Maron & Ames (Mutat. Res.I13:173, 1983). l S9 per plate/number his* revertants per plate Promutagen BP (5 g) 2-AA (2.5 pg) 0 1 181 297 184 165 5 387 1075 10 650 1727 20 855 2026 50 1065 1283 RTC Study No.: 76250 Page 51 POST MITOCHONDRIAL SUPERNATANT (S-9) PRODUCTION & QUALITY CONTROL CERTIFICATE LOT NO.: 2378 SPECIES: Rat PREPARATION DATE: February 18.2009 PART NO.: 11-105 STRAIN: .Sprague Dawlev EXPIRATION DATE: February 18. 201( VOLUME: I & 2,,,nli SEX: _Male BUFFER: 0.154 MKCI TISSUE: layer._ INDUCING AGENT(a): Phenobarbital - REFERENCE: Matsushima. et. al,. In' In Vitro Metabolig 5.6-Benzoflavone Activation in Mutagenesis Testing (F.J. de Sevres. Ed). Elsevier. 1976. p. 85 STORAGE: at or below - 70C BIOCHEMISTRY: - PROTEIN 1.73-mg/rul Assayed according to the method of Lowry et at., JBC 193:265,1951 using bovine serum albumin as the standard. - ALKOXYRESORUFIN-0-DEALKYLASE ACTIVITIES Fold - Activity FQ EROD 1A1, 1A2 Inductim 60.7 Assays for elhoxyresorufin-0-deethylase (BROD), pentoxy-, benzyl- and methoxyresorufin-0-dealkylases (PROD, BROD, & PROD 2B1 25.9 MROD) were conducted using a modification of the methods of Burke, et al., Biochem Phorm 34:3337, 1985. Fold- BROD 2E1 23.5 inductions were calculated as the ratio of the sample vs. uninduced specific activities (SA's). Control SA's (pmoles/min/ MROD 1A2 34.8 mg protein) were 14.0, 7.5, 22.6, 2.6 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 1.0 ml volumes on - Nutrient AgarAnd Minimal Glucose (Vogel-Bonner E, supplemented with 0.05 niM 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. las+ Revertants The ability of the sample to activate ethidium bromide (EtBr) EtBr/ CPA/ and cyclophosphamide (CPA) to intemiediates mutagenic to TA98 TA1535 TA98 and TA1535, respectively, was determined according 276.8 863 to Lesca, et al., Mutation Res 129:299, 1984. Data were expressed as revertants per pg EtBr or per mg CPA. Dilutions of the sample S9, ranging from 0.2 - 10% in S9 mix, were tested for their ability to activate benzo(a)pyrene (BP) and 2-aminoanthracene (2-AA) to intermediates mutagenic to TAI00. Assays were conducted using duplicate plates as described by Maron & Ames (Mutat. Res.113:173, 1983). pl S9 Per olate/numberh/s* revertants per plate Promutaggn 14 24 BP (5 pg) 113 186 281 431 801 1105 2-AA (2.5 lig) 114 196 486 1249 1433 1497 RTC Study No.: 76250 Page 52